Macromolecular multivalent conjugates and related uses

Abstract

The present disclosure provides multivalent molecules (e.g., compositions of Formula (I), an ionized form thereof, an isomer thereof, or a salt thereof. The present disclosure further provides methods for the use of the compositions, e.g. in the sequencing of poly-nucleic acid molecules.

Description

[0001] MACROMOLECULAR MULTIVALENT CONJUGATES AND RELATED USES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 568,990, filed March 22, 2024, the entire contents of which are incorporated herein by reference.

[0004] BACKGROUND

[0005] Polynucleotide sequencing technology has applications in biomedical research and healthcare settings. Improved methods of polynucleotide sequencing require enhanced surface chemistry, on-support polynucleotide amplification, labeling and detection of nucleobase identities, and base calling. Currently, these elements produce barriers in existing sequencing technology that result in limits in throughput and poor signal-to-noise ratio, and ultimately to increased costs associated with polynucleotide sequencing.

[0006] There exists a need for new polynucleotide sequencing methods with improved surface chemistry, on-support amplification, labeling and detection of nucleobase identities, and base calling. The present disclosure provides methods and compositions to improve sequencing of polynucleotides.

[0007] SUMMARY

[0008] The disclosure provides methods comprising: (a) contacting: (i) a plurality of template nucleic acid molecules comprising two or more copies of a target sequence and two or more copies of a binding sequence for a forward sequencing primer, (ii) a plurality of forward sequencing primers comprising a sequence complementary to the binding sequence for the forward sequencing primer, (iii) a plurality of first polymerases, and (iv) a plurality of multivalent molecules of Formula (I):

[0009] (I) an ionized form thereof, an isomer thereof, or a salt thereof, wherein: C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety or absent; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3- (R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety, wherein each multivalent molecule comprises at least two nucleotide moieties and at one least detectable reporter moiety, wherein the contacting occurs under conditions sufficient to form a plurality of multivalent binding complexes comprising a nucleic acid duplex between a template nucleic acid molecule and forward sequencing primer, a first polymerase, and a nucleotide moiety of a multivalent molecule that is complementary to the nucleotide in the template nucleic acid molecule immediately adjacent to the 3’ end of the forward sequencing primer, and wherein polymerase catalyzed incorporation of a complementary nucleotide moiety into the nucleic acid duplex is inhibited; (b) detecting the detectable reporter moieties; and (c) determining the identities of nucleotides in the nucleic acid template molecules based on the detectable reporter moieties of the multivalent molecules in the plurality of multivalent binding complexes formed in step (a).

[0010] In some embodiments of the methods of the disclosure, the multivalent molecules are of Formula (II):

[0011] (II) an ionized form thereof, an isomer thereof, or a salt thereof, wherein: C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3-(R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety.

[0012] In some embodiments, the multivalent molecules are of Formula (III):

[0013] (III) an ionized form thereof, an isomer thereof, or a salt thereof. In some embodiments, the multivalent molecules are of Formula (IV):

[0014] (IV) an ionized form thereof, an isomer thereof, or a salt thereof.

[0015] In some embodiments, the multivalent molecules are of Formula (V):

[0016] (V) an ionized form thereof, an isomer thereof, or a salt thereof.

[0017] In some embodiments, the two or more copies of a target sequence in an individual of template nucleic acid molecule are the same target sequence. In some embodiments, two or more multivalent binding complexes form on individual template nucleic acid molecules. In some embodiments, the plurality of forward sequencing primers are soluble.

[0018] In some embodiments of the methods of the disclosure, the methods comprise: (d) dissociating the multivalent binding complexes under conditions sufficient to retain the nucleic acid duplexes, thereby generating a plurality of nucleic acid duplexes; (e) contacting the plurality of nucleic acid duplexes with a plurality of second polymerases and a plurality of nucleotides or analogs thereof under conditions sufficient to incorporate nucleotides or analogs thereof complementary to the nucleotides of the nucleic acid template molecules immediately adjacent to the 3’ ends of the forward sequencing primers in a primer extension reaction, thereby generating a plurality of extended nucleic acid duplexes comprising extended forward sequencing primer sequences. In some embodiments, the methods comprise: (f) dissociating the second polymerases from the extended nucleic acid duplexes under conditions sufficient to retain the plurality of extended nucleic acid duplexes.

[0019] In some embodiments, the template nucleic acid molecules comprise concatemers of two or more copies of a sequence comprising (i) the binding sequence for the forward sequencing primer and (ii) the target sequence. In some embodiments, the two or more copies of (i) the binding sequence for the forward sequencing primer hybridize to the forward sequencing primers to form nucleic acid duplexes between the template nucleic acid molecules and the forward sequencing primers.

[0020] In some embodiments, in an individual multivalent molecule, the at least two nucleotide moieties are respectively attached to different X moieties.

[0021] In some embodiments, in an individual multivalent molecule, all nucleotide moieties are the same. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dATP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dTTP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dGTP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dUTP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dCTP.

[0022] In some embodiments, in an individual multivalent molecule, all detectable reporter moieties are the same. In some embodiments, wherein all detectable reporter moieties in an individual multivalent molecule comprise the same fluorescent label. In some embodiments, in an individual multivalent molecule, all detectable reporter moieties are the same and all nucleotide moieties are the same.

[0023] In some embodiments, an individual multivalent molecule comprises two, three, or four nucleotide moieties. In some embodiments, an individual multivalent molecule comprises two, three, or four detectable reporter moieties. In some embodiments, two or more nucleotide moieties in an individual multivalent molecule are associated with two or more different multivalent binding complexes on the same template nucleic acid molecule.

[0024] In some embodiments of the methods of the disclosure, the methods comprise (i) contacting the plurality of extended nucleic acid duplexes with a plurality of first polymerases and a plurality of multivalent molecules of Formula (I), or an ionized form thereof, an isomer thereof, or a salt thereof, wherein the contacting occurs under conditions sufficient to form a plurality of multivalent binding complexes comprising an extended nucleic acid duplex, a first polymerase, and a nucleotide moiety of a multivalent molecule that is complementary to a nucleotide in the template nucleic acid molecule immediately adjacent to the 3’ end of the extended forward sequencing primer, and wherein polymerase catalyzed incorporation of a complementary nucleotide moiety into the extended nucleic acid duplex is inhibited; (ii) detecting the detectable reporter moieties; (iii) determining nucleobase identities of nucleotides in the nucleic acid template sequences complementary to the nucleotide moieties of the multivalent molecules based on the detectable reporter moieties of the multivalent molecules in the plurality of multivalent binding complexes formed in step (a); (iv) dissociating the multivalent binding complexes under conditions sufficient to retain the plurality extended nucleic acid duplexes; and (v) contacting the plurality of extended nucleic acid duplexes with a plurality of second polymerases and a plurality of nucleotides or analogs thereof under conditions sufficient to incorporate nucleotides or analogs thereof complementary to the nucleotides of the nucleic acid template molecules immediately adjacent to the 3’ ends of the extended forward sequencing primers in a primer extension reaction, thereby generating a plurality of extended nucleic acid duplexes comprising extended forward sequencing primers. In some embodiments, in an individual multivalent molecule, the at least two nucleotide moieties are attached to different X moieties.

[0025] In some embodiments, in an individual multivalent molecule, all nucleotide moieties are the same. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dATP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dTTP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dGTP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dUTP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dCTP. In some embodiments, in an individual multivalent molecule, all detectable reporter moieties are the same. In some embodiments, all detectable reporter moieties in an individual multivalent molecule comprise the same fluorescent label. In some embodiments, in an individual multivalent molecule, all detectable reporter moieties are the same and all nucleotide moieties are the same. In some embodiments, an individual multivalent molecule comprises two, three, or four nucleotide moieties. In some embodiments, an individual multivalent molecule comprises two, three, or four detectable reporter moieties.

[0026] In some embodiments, two or more nucleotide moieties in an individual multivalent molecule are associated with two or more different multivalent binding complexes on the same template nucleic acid molecule.

[0027] In some embodiments of the methods of the disclosure, the methods comprise repeating steps (i)-(v) at least 1, 10, 20, 30, 40, 50, 70, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1500 times. In some embodiments, the methods comprise repeating steps (i)-(v) until the identities of the nucleotides in the target sequences have been determined. In some embodiments, the methods comprise, before step (i), dissociating the second polymerases from the extended nucleic acid duplexes under conditions sufficient to retain the plurality of extended nucleic acid duplexes.

[0028] In some embodiments, the template nucleic acid molecules are single-stranded DNA molecules.

[0029] In some embodiments, the nucleotides or analogs thereof comprise a removable chain terminating moiety at the 3’ sugar group. In some embodiments, the removable chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group, and wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3 ’OH moiety on the sugar group. In some embodiments, the nucleotides or analogs thereof comprise a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, the nucleotides or analogs thereof comprise at least one fluorophore-labeled nucleotide analog.

[0030] In some embodiments, the plurality of template nucleic acid molecules are immobilized on a support. In some embodiments, the template nucleic acid molecules are immobilized on the support through hybridization to first surface primers immobilized on the support. In some embodiments, the template nucleic acid molecules are covalently joined to first surface primers immobilized on the support.

[0031] In some embodiments, the template nucleic acid molecules are clonally amplified template nucleic acid molecules. In some embodiments, the template nucleic acid molecules are generated through rolling circle amplification.

[0032] In some embodiments of the methods of the disclosure, sequencing the plurality of template nucleic acid molecules generates a plurality of extended forward sequencing primer strands, and the method comprises: (a) retaining the plurality of template nucleic acid molecules and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands that are hybridized to the plurality of nucleic acid template molecules by conducting a primer extension reaction; (b) removing the plurality of nucleic acid template molecules while retaining the plurality of forward extension strands and retaining the plurality of surface primers; and (c) sequencing the plurality of retained forward extension strands.

[0033] In some embodiments, the template nucleic acid molecules comprise: (i) two or more copies of the target sequence, (ii) two or more copies of the binding sequence for a forward sequencing primer, and (iii) two or more copies of a binding sequence for a reverse sequencing primer. In some embodiments, the template nucleic acid molecules comprise binding sequences for an amplification primer, and wherein conducting the primer extension reaction comprises contacting the plurality of template nucleic acid molecules with a plurality of soluble amplification primers, a plurality of nucleotides and a plurality of polymerases, thereby generating a plurality of forward extension strands that are hybridized to template nucleic acid molecules. In some embodiments, the plurality of amplification primers hybridize to the binding sequences for the amplification primers. In some embodiments, the amplification primers are soluble.

[0034] In some embodiments, the polymerases comprise phi29 DNA polymerases, large fragment of Bst DNA polymerases, large fragment of Bsu DNA polymerases (exo-), Bea DNA polymerases (exo-), KI enow fragment of E. coli DNA polymerases, T5 polymerases, M-MuLV reverse transcriptases, HIV viral reverse transcriptases, Deep Vent DNA polymerases or KOD DNA polymerases.

[0035] In some embodiments, wherein the nucleic acid template molecules comprise at least one nucleotide having a scissile moiety that can be cleaved to generate an abasic site. In some embodiments, the surface primers lack a nucleotide having a scissile moiety. In some embodiments, the nucleotide having a scissile moiety comprises uridine, 8-oxo-7,8- dihydrogunine, or deoxyinosine. In some embodiments, removing the nucleic acid template molecules comprises generating abasic sites in the nucleic acid template molecules, followed by generating gaps at the abasic sites. In some embodiments, the at least one nucleotide having a scissile moiety comprises uracil, and generating abasic sites comprises contacting the nucleic acid template molecules with uracil DNA glycosylase (UDG). In some embodiments, generating gaps at the abasic sites comprises contacting the abasic sites with an endonuclease IV, AP lyase, FPG glycosylase / AP lyase and / or endo VIII glycosylase / AP lyase. In some embodiments, individual template nucleic acid molecules comprise nucleic acid template molecules having up to 30% of thymidines replaced with uridine.

[0036] In some embodiments, sequencing the plurality of retained forward extension strands generates a plurality of extended reverse sequencing primer strands, wherein individual retained forward extension strands have two or more extended reverse sequencing primer strands hybridized thereon. In some embodiments, sequencing the plurality of retained forward extension strands comprises a plurality of soluble reverse sequencing primers and (i) a plurality of a first polymerases and a plurality of multivalent molecules and (ii) a plurality of a second polymerases and a plurality of nucleotides or analogs thereof, thereby generating a plurality of extended reverse sequencing primer strands, wherein individual retained forward extension strands have two or more extended reverse sequencing primer strands hybridized thereon.

[0037] In some embodiments, nucleic acid template molecules comprise one or more copies of a binding sequence for a second surface primer. In some embodiments, the methods comprise a plurality of second surface primers immobilized on the support, whereby binding of the second surface primers to the binding sequence for the second surface primers immobilizes free ends of the plurality nucleic acid template molecules on the support.

[0038] In some embodiments of the methods of the disclosure, sequencing the plurality of retained forward extension strands comprises (a) contacting: (i) the plurality of retained forward extension strands, (ii) a plurality of reverse sequencing primers comprising a sequence complementary to the binding sequence for the reverse sequencing primer, (iii) a plurality of first polymerases, and (iv) a plurality of multivalent molecules of Formula (I):

[0039] (I) an ionized form thereof, an isomer thereof, or a salt thereof, wherein: C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety or absent; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3- (R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety, wherein each multivalent molecule comprises at least two nucleotide moieties and at least one detectable reporter moiety, wherein the contacting occurs under conditions sufficient to form a plurality of multivalent binding complexes comprising a nucleic acid duplex between a retained forward extension strand and a reverse sequencing primer, a first polymerase, and a nucleotide moiety of a multivalent molecule that is complementary to a nucleotide in the retained forward extension strand immediately adjacent to the 3’ end of the reverse sequencing primer, and wherein polymerase catalyzed incorporation of a complementary nucleotide moiety into the nucleic acid duplex is inhibited; (b) detecting the detectable reporter moieties; and (c) determining nucleobase identities of nucleotides in the retained forward extension strands complementary to the nucleotide moieties of the multivalent molecules based on the detectable reporter moieties of the multivalent molecules in the plurality of multivalent binding complexes formed in step (a).

[0040] In some embodiments, individual retained forward extension strands comprise two or more multivalent binding complexes.

[0041] In some embodiments, the plurality of reverse sequencing primers are soluble.

[0042] In some embodiments of the methods of the disclosure, the methods comprise (d) dissociating the multivalent binding complexes under conditions sufficient to retain the nucleic acid duplexes, thereby generating a plurality of nucleic acid duplexes; and (e) contacting the plurality of nucleic acid duplexes with a plurality of second polymerases and a plurality of nucleotides or analogs thereof under conditions sufficient to incorporate nucleotides or analogs thereof complementary to the nucleotides of the retained forward extension strands immediately adjacent to the 3’ ends of the reverse sequencing primers in a primer extension reaction, thereby generating a plurality of extended nucleic acid duplexes comprising extended reverse sequencing primer sequences. In some embodiments, the methods comprise (f) dissociating the second polymerases from the extended nucleic acid duplexes under conditions sufficient to retain the plurality of extended nucleic acid duplexes.

[0043] In some embodiments, the template nucleic acid molecules comprise concatemers of two or more copies of a sequence comprising (i) a sequence for the reverse sequencing primer, (ii) the target nucleic acid sequence, and (iii) a binding sequence for the forward sequencing primer. In some embodiments, wherein the two or more copies of a sequence complementary to (i) the sequence for the reverse sequencing primer hybridize to the reverse sequencing primers to form nucleic acid duplexes between the retained forward extension strands and the reverse sequencing primers.

[0044] In some embodiments, in an individual multivalent molecule, all nucleotide moieties are the same. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dATP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dTTP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dGTP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dUTP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dCTP. In some embodiments, in an individual multivalent molecule, all detectable reporter moieties are the same. In some embodiments, all detectable reporter moieties in an individual multivalent molecule comprise the same fluorescent label. In some embodiments, in an individual multivalent molecule, all detectable reporter moieties are the same and all nucleotide moieties are the same. In some embodiments, an individual multivalent molecule comprises two, three, or four nucleotide moieties. In some embodiments, an individual multivalent molecule comprises two, three, or four detectable reporter moieties.

[0045] In some embodiments of the methods of the disclosure, the methods comprise (a) contacting the plurality of extended nucleic acid duplexes with a plurality of first polymerases and a plurality of multivalent molecules of Formula (I), or an ionized form thereof, an isomer thereof, or a salt thereof, wherein the contacting occurs under conditions sufficient to form a plurality of multivalent binding complexes comprising an extended nucleic acid duplex, a first polymerase, and a nucleotide moiety of a multivalent molecule that is complementary to a nucleotide in the retained forward extension strand immediately adjacent to the 3’ end of the extended reverse sequencing primer, and wherein polymerase catalyzed incorporation of a complementary nucleotide moiety into the extended nucleic acid duplex is inhibited; (b) detecting the detectable reporter moieties; (c) determining nucleobase identities of nucleotides in the retained forward extension strands complementary to the nucleotide moieties of the multivalent molecules based on the detectable reporter moieties of the multivalent molecules in the plurality of multivalent binding complexes formed in step (a); (d) dissociating the multivalent binding complexes under conditions sufficient to retain the plurality extended nucleic acid duplexes; and (e) contacting the plurality of extended nucleic acid duplexes with a plurality of second polymerases and a plurality of nucleotides or analogs thereof under conditions sufficient to incorporate nucleotides or analogs thereof complementary to the nucleotides of the nucleic acid template sequences immediately adjacent to the 3’ ends of the extended reverse sequencing primers in a primer extension reaction, thereby generating a plurality of extended nucleic acid duplexes comprising extended reverse sequencing primers.

[0046] In some embodiments, in an individual multivalent molecule, the at least two nucleotide moieties are respectively attached to different X moieties.

[0047] In some embodiments, in an individual multivalent molecule, all nucleotide moieties are the same. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dATP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dTTP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dGTP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dUTP. In some embodiments, all nucleotide moieties in an individual multivalent molecule are dCTP. In some embodiments, in an individual multivalent molecule, all detectable reporter moieties are the same and all nucleotide moieties are the same. In some embodiments, an individual multivalent molecule comprises two, three, or four nucleotide moieties. In some embodiments, an individual multivalent molecule comprises two, three, or four detectable reporter moieties. In some embodiments, in an individual multivalent molecule, all detectable reporter moieties are the same. In some embodiments, all detectable reporter moieties in an individual multivalent molecule comprise the same fluorescent label.

[0048] In some embodiments, two or more nucleotide moieties in an individual multivalent molecule contact two or more different multivalent binding complexes on the same retained forward extension strand.

[0049] In some embodiments, the methods comprise repeating steps (a)-(e) at least 1, 10, 20, 30, 40, 50, 70, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1500 times. In some embodiments, the methods comprise repeating steps (a)-(e) until the identities of the nucleotides in sequences of the retained forward extension strands complementary to the target sequences have been determined. In some embodiments, the methods comprise, before step (a), dissociating the second polymerases from the extended nucleic acid duplexes under conditions sufficient to retain the plurality of extended nucleic acid duplexes.

[0050] In some embodiments, template nucleic acid molecules comprise concatemers of two or more copies of a sequence comprising: (i) a binding sequence for a forward sequencing primer, (ii) sequence complementary to a binding sequence for a reverse sequencing primer, (iii) a binding sequence for an first surface primer, (iv) a binding sequence for a second surface primer, (v) a binding sequence for a first amplification primer, (vi) a binding sequence for a second amplification primer, (vii) a binding sequence for a soluble compaction oligonucleotide, (viii) a sample barcode sequence, and / or (ix) a unique molecular index sequence.

[0051] In some embodiments, in an individual multivalent molecule, each X moiety comprises one or more nucleotide moiety. In some embodiments, in an individual multivalent molecule, each X moiety comprises one or more detectable reporter moiety. In some embodiments, in an individual multivalent molecule, each X moiety comprises one or more nucleotide moiety and one or more detectable reporter moiety. In some embodiments, each individual multivalent molecule comprises two, three, or four detectable reporter moieties and two, three, or four nucleotide moieties.

[0052] In some embodiments, greater than 90%, greater than 95%, greater than 97%, greater than 98% or greater than 99% of bases have a quality score of Q30. In some embodiments, greater than 80%, greater than 85%, greater than 87%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94% or greater than 95% of bases have a quality score of Q40.

[0053] The disclosure provides a multivalent molecule of Formula (I):

[0054] (I) an ionized form thereof, an isomer thereof, or a salt thereof, wherein: C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety or absent; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3- (R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety.

[0055] In some embodiments of the multivalent molecule of the disclosure, the multivalent molecule is of Formula (II):

[0056] (II) an ionized form thereof, an isomer thereof, or a salt thereof, wherein: C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3-(R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety.

[0057] In some embodiments, the multivalent molecule is of Formula (III):

[0058] (III) an ionized form thereof, an isomer thereof, or a salt thereof.

[0059] In some embodiments, the multivalent molecule is of Formula (IV):

[0060] (IV) an ionized form thereof, an isomer thereof, or a salt thereof.

[0061] In some embodiments, the multivalent molecule is of Formula (V):

[0062] (V) an ionized form thereof, an isomer thereof, or a salt thereof.

[0063] In some embodiments, the multivalent molecule comprises at least two nucleotide moieties and at least one detectable reporter moiety. In some embodiments, the at least two nucleotide moieties are attached to different X moieties. In some embodiments, all the nucleotide moieties are the same. In some embodiments, all the nucleotide moieties are dATP. In some embodiments, all the nucleotide moieties are dTTP. In some embodiments, all the nucleotide moieties are dGTP. In some embodiments, all the nucleotide moieties are dUTP. In some embodiments, all the nucleotide moieties are dCTP. In some embodiments, all the detectable reporter moieties are the same. In some embodiments, all the detectable reporter moieties are the same and all the nucleotide moieties are the same. In some embodiments, the molecule comprises two, three, or four detectable reporter moieties. In some embodiments, the molecule comprises two, three, or four nucleotide moieties. In some embodiments, all detectable reporter moieties comprise the same fluorescent label.

[0064] The disclosure provides a complex comprising: (a) the multivalent molecule of the disclosure; (b) a polymerase; (c) a template nucleic acid molecule comprising at least one of a target sequence and a binding sequence for a sequencing primer; and (d) a sequence complementary to a portion of the template nucleic acid molecule comprising the sequencing primer sequence; wherein the template nucleic acid molecule and the sequence complementary to a portion of the template nucleic acid molecule form a duplex, and wherein a nucleotide moiety of the multivalent molecule binds to a complementary to a nucleotide of the template nucleic acid molecule immediately adjacent to the 3’ end of the sequence complementary to a portion of the template nucleic acid molecule.

[0065] In some embodiments of the complex of the disclosure, the multivalent molecule comprises at least two nucleotide moieties, and wherein the at least two nucleotide moieties are the same.

[0066] In some embodiments, the template nucleic acid molecule comprises a concatemer comprising at least two copies of a sequence comprising the target sequence and the binding sequence for a sequencing primer. In some embodiments, at least two complexes form on the same template nucleic acid molecule. In some embodiments, the at least two nucleotide moieties bind to complementary nucleotides of the template nucleic acid molecule in the at least two complexes. In some embodiments, at least two complexes form on at least two different template nucleic acid molecules. In some embodiments, the at least two different template nucleic acid molecules comprise the same target sequence. In some embodiments, the at least two nucleotide moieties bind to complementary nucleotides of the template nucleic acid molecules in the at least two complexes.

[0067] The disclosure provides methods of sequencing a plurality of template nucleic acid molecules, comprising contacting the plurality of template nucleic acid molecules with a plurality of multivalent molecules of Formula (I):

[0068] C-(X)2-6 (I) an ionized form thereof, an isomer thereof, or a salt thereof, wherein: C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety or absent; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3- (R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety, wherein each multivalent molecule comprises at least one nucleotide moiety and at one least detectable reporter moiety, wherein the contacting occurs under conditions sufficient for the at least one nucleotide moiety of the multivalent molecules in the plurality to bind to complementary nucleotide moieties in template nucleic acid molecules, and wherein the identities of the complementary nucleotide moieties in the template nucleic acid molecules are determined by detecting the detectable reporter moieties.

[0069] In some embodiments, the sequencing comprises polony sequencing, pyrosequencing, chain-terminator sequencing, ion-sensitive sequencing, probe-anchor ligation sequencing DNA nanoball sequencing, nanopore DNA sequencing, sequencing-by-hybridization or sequencing-by-binding.

[0070] BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0072] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0073] FIG. 1 is a schematic of an exemplary low binding support comprising a glass substrate and alternating layers of hydrophilic coatings which are covalently or non-covalently adhered to the glass, and which further comprises chemical ly-reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g , capture oligonucleotides). In an alternative embodiment, the support can be made of any material such as glass, plastic or a polymer material.

[0074] FIG. 2 shows the chemical structure of an exemplary spacer (top), and the chemical structures of various exemplary linkers, including an 11 -atom Linker, a 16-atom Linker, a 23- atom Linker and an N3 Linker (bottom).

[0075] FIG. 3 shows the chemical structures of more exemplary linkers, including Linkers 1- 9.

[0076] FIG. 4A shows the chemical structures of various exemplary linkers joined / attached to nucleotide moieties.

[0077] FIG. 4B shows the chemical structures of various exemplary linkers joined / attached to nucleotide moieties.

[0078] FIG. 4C shows the chemical structures of various exemplary linkers joined / attached to nucleotide moieties.

[0079] FIG. 4D shows the chemical structures of various exemplary linkers joined / attached to nucleotide moieties.

[0080] FIG. 5 shows the chemical structure of an exemplary nucleotide moiety. In this example, the nucleotide moiety comprises a propargyl amine linkage at the 5 position of a pyrimidine base or the 7 position of a purine base.

[0081] FIG. 6 shows the chemical structure of another exemplary nucleotide moiety. In this example, the nucleotide moiety comprises a propargyl amine linkage at the 5 position of a pyrimidine base or the 7 position of a purine base.

[0082] FIG. 7 is a diagram showing an exemplary multivalent molecule of the disclosure.

[0083] FIGS. 8A-8C show representative multivalent molecules of the disclosure. Red ovals indicate the detectable reporter moiety. DOL: degree of labeling; Vai: valency of nucleotide moieties.

[0084] FIG. 9 shows the synthesis of a rigid half multivalent molecule with an azido reactive end, as described in Example 4, in which the Y-shaped linker A comprising the dye and nucleotide moiety (dye-dNTP arm, described in Example 3) is mixed with Y-shaped linker B (described in Example 2) and DIPEA (N,N-Diisopropylethylamine).

[0085] FIG. 10 shows the synthesis of an exemplary flexible half multivalent molecule with an azido reactive end, as described in Example 5, in which the Y-shaped linker A comprising the dye and the nucleotide moiety (dye-dNTP arm, described in Example 3) is reacted with an N-(Azido-PEGn)-N-bis(PEGn-NHS) ester. FIG. 11 shows the synthesis of an exemplary flexible half multivalent molecule with a DBCO reactive end, as described in Example 6, in which the Y-shaped linker A comprising the dye and the nucleotide moiety (dye-dNTP arm, Example 3) is reacted with an N-DBCO- N-bis(PEGn-NHS ester).

[0086] FIG. 12A shows the synthesis of a multivalent molecule comprising two Y-shaped linker A moieties, each comprising a dye and a nucleotide moiety (Dye2-dNTP2), as described in Example 7.

[0087] FIG. 12B shows the conversion of the rigid half multivalent molecule (described in Example 4) into a multivalent molecule with a DOL of 2 and 3 nucleotide moieties (valency of 3), as described in Example 7.

[0088] FIG. 12C shows the conversion of the rigid half multivalent molecule (described in Example 4) into a multivalent molecule with a DOL of 3 and 3 nucleotide moieties (valency of 3), as described in Example 7.

[0089] FIG. 12D shows the conversion of the rigid half multivalent molecule (described in Example 4) into a multivalent molecule with a DOL of 4 and 4 nucleotide moieties (valency of 4), as described in Example 7.

[0090] FIG. 12E shows the conversion of the flexible half multivalent molecules (described in Examples 5-6) into a multivalent molecule with a DOL of 4 and 4 nucleotide moieties (valency of 4), as described in Example 7.

[0091] FIG. 13 A is a table showing results from high throughput sequencing using two representative multivalent molecules of the disclosure, as described in Example 8.

[0092] FIG. 13B is a diagram showing two representative multivalent molecules used in high throughput sequencing, as described in Example 8.

[0093] FIG. 14 is a pair of plots showing cycle number (x axis) versus intensity (CC P90 Intensity, or the 90% percentile extracted intensity for a given image, y axis) on the top plot, and cycle number (x axis) versus mean residual (y axis) on the bottom plot, for high throughput sequencing carried out with the two representative multivalent molecules shown in FIG. 13B. Light blue: molecule 1; dark blue: molecule 2; gray to black: four quality control runs using POR dyes.

[0094] FIG. 15 is a plot showing cycle number (x axis) versus mean percent Q30 (y axis) for high throughput sequencing carried out with the two representative multivalent molecules shown in FIG. 13B. Light blue: molecule 1; dark blue: molecule 2; gray to black: four quality control runs using a POR dyes. FIG. 16 is a pair of plots showing cycle number (x axis) versus mean percent phasing (y axis) on the top plot, and cycle number (x axis) versus mean percent pre-phasing (y axis) on the bottom plot. Light blue: molecule 1; dark blue: molecule 2; gray to black: four quality control runs using POR dyes.

[0095] FIG. 17 is a pair of plots showing cycle number (x axes) versus mean Z-scores for green (GRN, top plot) and red (RED, bottom plot) dyes (y- axes). Light blue: molecule 1; dark blue: molecule 2; gray to black: four quality control runs using POR dyes.

[0096] FIG. 18A is a scatterplot showing polony density (x axis) versus percent error (with respect to base calls). Light blue: molecule 1 ; dark blue: molecule 2; gray to black: four quality control runs using POR dyes.

[0097] FIG. 18B is a pair of bar blots showing the mean error for high error singlets (top), and for high error groups (bottom plot). From left to right: molecule 1, molecule 2, four quality control runs using POR dyes.

[0098] FIG. 19A is a plot showing read number (x-axis) versus average Q-score for molecule 3, compared to three sequencing reactions using POR dyes. The turquoise dotted line shows the results with molecule 3, while the baseline result with the POR dyes is shown by the dark blue line.

[0099] FIG. 19B is a plot showing read number (x-axis) versus percent Q30 for molecule 3, compared to three sequencing reactions using POR dyes. The turquoise dotted line shows the results with molecule 3, while the baseline result with the POR dyes is shown by the dark blue line.

[0100] FIG. 19C is a plot showing read number (x-axis) versus percent Q40 for molecule 3, compared to three sequencing reactions using POR dyes. The turquoise dotted line shows the results with molecule 3, while the baseline result with the POR dyes is shown by the dark blue line.

[0101] FIG. 20A is a series of four plots corresponding to the four channels in a sequencing reaction, showing cycle number (x axis) versus intensity (CC P90 Intensity, or the 90% percentile extracted intensity for a given image, y axis). In channel 2, the POR dye has been replaced with molecule 3 conjugated with far red CF®647 dye. The dotted turquoise line indicates results from the sequencing reaction using molecule 3, while the dark blue line indicates the results from three baseline sequencing reactions using POR dyes.

[0102] FIG. 20B is a series of four plots corresponding to the four channels in a sequencing reaction, showing cycle number (x axis) versus mean residual (y axis). In channel 2, the POR dye has been replaced with molecule 3 conjugated with far red CF®647 dye. POR dyes QC- 0428, QC-0429 and QC-0430 are used in the other channels. The dotted turquoise line indicates results from the sequencing reaction using molecule 3, while the dark blue line indicates the results from three baseline sequencing reactions using POR dyes.

[0103] FIG. 21 is a plot showing the four channels for a representative sequencing run in which channel 2 has been replaced with molecule 3 conjugated with far red CF®647 dye, as described in Example 9.

[0104] FIG. 22 is a series of four plots showing pre-phasing in each of the four channels for a sequencing run in which channel 2 has been replaced with molecule 3 conjugated with far red CF®647 dye. The dotted turquoise line indicates results from the sequencing reaction using molecule 3, while the dark blue line indicates the results from three baseline sequencing reactions using POR dyes.

[0105] FIG. 23A is a plot showing mean Z-score (y-axis) versus cycle number (x-axis) for molecule 3 conjugated with far red dye (dotted turquoise line), versus the red channel using a POR dye (dark blue line).

[0106] FIG. 23B is a plot showing mean Z-score (y-axis) versus cycle number (x-axis) for molecule 3 conjugated with far red dye (dotted turquoise line line), versus the green channel using a POR dye (dark blue line).

[0107] FIG. 24A is a plot showing read number (x-axis) versus percent Q30 for a sequencing run using molecules 3 and 4 in channels 1 and 2, respectively, compared to three baseline sequencing reactions using POR dyes. The dotted line shows the result from the sequencing reaction using molecules 3 and 4, while the sold dark blue line shows the results from the baseline reactions using the POR dyes.

[0108] FIG. 24B is a plot showing read number (x-axis) versus percent Q40 for a sequencing run using molecules 4 and 3 in channels 1 and 2, respectively, compared to three baseline sequencing reactions using POR dyes. The dotted line shows the result from the sequencing reaction using molecules 3 and 4, while the sold dark blue line shows the results from the baseline reactions using the POR dyes.

[0109] FIG. 24C is a plot showing read number (x-axis) versus error rate for a sequencing run using molecules 4 and 3 in channels 1 and 2, respectively, compared to three baseline sequencing reactions using POR dyes. The dotted line shows the result from the sequencing reaction using molecules 3 and 4, while the sold dark blue line shows the results from the baseline reactions using the POR dyes.

[0110] FIG. 25A is a series of four plots corresponding to the four channels in a sequencing run in which channels 1 and 2 use molecules 4 and 3, respectively. Each plot shows cycle number (x axis) versus intensity (CC P90 Intensity, or the 90% percentile extracted intensity for a given image, y axis) for the indicated channel. The dotted line shows the result from the sequencing reaction using molecules 3 and 4, while the sold dark blue line shows the results from the baseline reactions using the POR dyes.

[0111] FIG. 25B is a plot showing cycle number (x axis) versus intensity (CC P90 Intensity, or the 90% percentile extracted intensity for a given image, y axis). The turquoise dotted line indicates results from the sequencing reaction using molecules 3 and 4, while the solid blue line indicates baseline using POR dyes.

[0112] FIG. 26A is a series of four plots showing phasing in each of the four channels for a sequencing run in which channel 1 uses molecule 4 and channel 2 uses molecule 3. Cycle number is shown on the x axis and phasing is shown in on the y axis. The turquoise dotted line indicates results from the sequencing reaction using molecules 3 and 4, while the solid blue line indicates baseline results using POR dyes.

[0113] FIG. 26B is a series of four plots showing prephasing in each of the four channels for a sequencing run in which channel 1 uses molecule 4 and channel 2 uses molecule 3. Cycle number is shown on the x axis and prephasing is shown in on the y axis. The turquoise dotted line indicates results from the sequencing reaction using molecules 3 and 4, while the solid blue line indicates baseline results using POR dyes.

[0114] FIG. 27 is a bar plot showing context error for a sequencing run using molecule 4 in channel 1 and complex 3 in channel 2 (left bar). The bars at right show baseline sequencing reactions using POR dyes (QC-0529, QC-0540 and QC-543).

[0115] FIG. 28 is a schematic showing an exemplary single template stranded nucleic acid molecule, which is immobilized to a support using a first surface primer. The immobilized concatemer template molecule comprises at least one nucleotide having a scissile moiety that can be cleaved to generate an abasic site in the immobilized concatemer template molecule. The immobilized concatemer template molecule can be generated by conducting an on-support rolling circle amplification reaction. The arrangement of the various primer binding sequences is for illustration purposes. The skilled artisan will appreciate that many other arrangements are possible. FIGS. 29-12 show an exemplary workflow for pairwise sequencing the immobilized concatemer template molecule depicted in FIG. 28.

[0116] FIG. 29 is a schematic showing an exemplary forward sequencing reaction conducted on the immobilized concatemer template molecule shown in FIG. 28. The forward sequencing reaction can be conducted with a plurality of soluble forward sequencing primers and generates a plurality of extended forward sequencing primer strands hybridized to the template nucleic acid molecule.

[0117] FIG. 30 is a schematic showing an exemplary method for replacing the extended forward sequencing primer strands by conducting a primer extension reaction with a strand displacing polymerase in the absence of a soluble primer thereby generating a forward extension strand.

[0118] FIG. 31 is a schematic showing an exemplary method for replacing the extended forward sequencing primer strands by conducting a primer extension reaction with a soluble forward sequencing primer thereby generating a forward extension strand.

[0119] FIG. 32 is a schematic showing an exemplary method for replacing the extended forward sequencing primer strands by conducting a primer extension reaction with a soluble amplification primer thereby generating a forward extension strand.

[0120] FIG. 33 is a schematic showing an exemplary method for generating abasic sites in the template nucleic acid molecules at the nucleotides having the scissile moiety, and generating gaps at the abasic sites to generate a plurality of gap-containing template molecules. The plurality of forward extension strands and first surface primers are retained. The forward extension strand can be generated by the method depicted in FIGS. 30 or 31.

[0121] FIG. 34 is a schematic showing an exemplary retained forward extension strand after removal of the gap-containing template molecule as shown in FIG. 33.

[0122] FIG. 35 is an exemplary schematic showing an exemplary method for generating abasic sites in the immobilized single stranded concatemer template molecules at the nucleotides having the scissile moiety and generating gaps at the abasic sites to generate a plurality of gap- containing concatemer template molecules while retaining the plurality of forward extension strands and retaining the plurality of immobilized first surface primers. The forward extension strand can be generated by the method depicted in FIG. 32.

[0123] FIG. 36 is a schematic showing an exemplary retained forward extension strand after removal of the template molecule as shown in FIG. 35.

[0124] FIG. 37 is a schematic showing an exemplary reverse sequencing reaction conducted on the retained forward extension strand shown in FIG. 34. The reverse sequencing reaction can be conducted with a plurality of soluble reverse sequencing primers. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strands are not hybridized to the first surface primer, or covalently joined to the first surface primer. Therefore, the extended reverse sequencing primer strands are not immobilized to the support. For the sake of simplicity, FIGS. 28-37 show an exemplary immobilized concatemer template nucleic acid molecule with one copy of the target and various primer binding sites. The skilled artisan will appreciate that the immobilized concatemer molecule can include two or more tandem copies containing the target and various primer binding sites.

[0125] FIG. 38 is a schematic showing an exemplary reverse sequencing reaction conducted on the retained forward extension strand shown in FIG. 36. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strands are not hybridized to the first surface primer, or covalently joined to the first surface primer. Therefore, the extended reverse sequencing primer strands are not immobilized to the support.

[0126] FIG. 39 is a schematic showing an exemplary support having a first and second surface primer immobilized thereon. A portion of the immobilized concatemer template nucleic acid molecule shown in FIG. 28 is hybridized to the immobilized second surface primer. The immobilized concatemer template molecule has two or more copies of a binding sequence for an immobilized second surface primer. The portion of the immobilized concatemer template molecule that includes the binding sequence for an immobilized second surface primer can hybridize to the immobilized second surface primer.

[0127] DETAILED DESCRIPTION

[0128] Next generation sequencing (NGS) frequently involves the simultaneous sequencing of a large library of target sequences that have been immobilized on a surface, such as a flow cell surface, and clonally amplified to produce PCR colonies (polonies). The polonies are sequenced in parallel by hybridizing sequencing primers to single stranded template strands in the polonies, followed by successive rounds of hybridizing labeled nucleotides to the templates (“trapping”), determining the identity of the labeled nucleotides, followed incorporating a nucleotide at the position of the labeled nucleotide to extend a strand that is the reverse complement of the template (“stepping”). The process is then repeated until the identities of the nucleotides of the target sequence have been determined.

[0129] In some cases, the library of target sequences is amplified in such a manner as to produce PCR colonies (polonies) of concatemerized template molecules which contain multiple copies of the binding sequence for the sequencing primer, the target sequence whose nucleotide identity is to be determined, and optionally, other sequences such as barcodes, which can be used to uniquely identify the source of the target sequence. The disclosure is based, at least in part, on the finding that using multivalent molecules that can simultaneously hybridize to and label multiple copies of a target sequence in a concatemerized template molecule at the trapping step can increase the accuracy of NGS methods.

[0130] Definitions

[0131] The headings provided herein are not limitations of the various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole.

[0132] Unless defined otherwise, technical and scientific terms used herein have meanings that are commonly understood by those of ordinary skill in the art unless defined otherwise. Generally, terminologies pertaining to techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization described herein are those well-known and commonly used in the art. Techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the instant specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclatures utilized in connection with, and the laboratory procedures and techniques described herein are those well-known and commonly used in the art.

[0133] Unless otherwise required by context herein, singular terms shall include pluralities and plural terms shall include the singular. Singular forms “a”, “an” and “the”, and singular use of any word, include plural referents unless expressly and unequivocally limited on one referent.

[0134] It is understood the use of the alternative term (e.g., “or”) is taken to mean either one or both or any combination thereof of the alternatives.

[0135] The term “and / or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include: “A and B”; “A or B”; “A” (A alone); and “B” (B alone). In a similar manner, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: “A, B, and C”; “A, B, or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone); and “C” (C alone).

[0136] As used herein and in the appended claims, terms “comprising”, “including”, “having” and “containing”, and their grammatical variants, as used herein are intended to be non-limiting so that one item or multiple items in a list do not exclude other items that can be substituted or added to the listed items. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0137] As used herein, the terms “about” and “approximately” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (i.e., ±10%) or more depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition. Also, where ranges and / or subranges of values are provided, the ranges and / or subranges can include the endpoints of the ranges and / or subranges.

[0138] As used herein, the term “sequencing” and its variants refers to methods for obtaining sequence information from a nucleic acid strand, typically by determining the ordered identity of at least some nucleotides (including their nucleobase components) within the nucleic acid template molecule. “Sequencing” a given region of a nucleic acid molecule includes identifying each and every nucleotide within the region that is sequenced, as well as methods whereby the identity of only some of the nucleotides in a region are determined, while the identity of some nucleotides remains undetermined or incorrectly determined. Any suitable method of sequencing may be used with the multivalent molecules described herein. Sequencing can include label-free or ion based sequencing methods, as well as labeled or dye-containing nucleotide or fluorescent based nucleotide sequencing methods. Sequencing can include polony-based sequencing or bridge sequencing methods. Sequencing includes massively parallel sequencing platforms that employ sequence-by-synthesis, sequence-by-hybridization or sequence-by-binding procedures. Examples of massively parallel sequence-by-synthesis procedures include polony sequencing, pyrosequencing (e.g., from 454 Life Sciences; U.S. Patent Nos. 7,211,390, 7,244,559 and 7,264,929), chain-terminator sequencing (e.g., from Illumina; U.S. PatentNo. 7,566,537; Bentley 2006 Current Opinion Genetics and Development 16:545-552; and Bentley, et al., 2008 Nature 456:53-59, ion-sensitive sequencing (e.g., from Ion Torrent), probe-anchor ligation sequencing (e.g., Complete Genomics), DNA nanoball sequencing, nanopore DNA sequencing. Examples of single molecule sequencing include Heliscope single molecule sequencing, and single molecule real time (SMRT) sequencing from Pacific Biosciences (Levene, et al., 2003 Science 299(5607):682-686; Eid, et al., 2009 Science 323(5910): 133-138; U.S. patent Nos. 7,170,050; 7,302,146; and 7,405,281). An example of sequence-by-hybridization includes SOLiD sequencing (e.g., from Life Technologies; WO 2006 / 084132). An example of sequence-by-binding includes Omniome sequencing (e.g., U.S patent No. 10,246,744).

[0139] During sequencing by synthesis, each DNA strand in a cluster extends by one base per cycle. A small proportion of strands may become out of phase with the current cycle, either falling a base behind (phasing) or jumping a base ahead (prephasing). The phasing and prephasing rates define the fraction of molecules that become phased or prephased per cycle.

[0140] A Quality score (Q score, or Q) is defined by the equation Q = -101ogl0(e), where e is the estimated probability of the base call being wrong. Higher Q scores indicate a smaller probability of error. Lower Q scores can result in a significant portion of the reads being unusable. They may also lead to increased false-positive variant calls, resulting in inaccurate conclusions. A quality score of Q20 represents an error rate of 1 in 100 (i.e., every 100 bp of sequencing read may contain an error), while a score of Q30 represents an error rate of 1 in 1,000 and a score of Q40 represents an error rate of 1 in 10,000.

[0141] A z-score, also referred to as standard score, z-value, Z score, or normal score, is a dimensionless quantity that is used to indicate the signed, fractional, number of standard deviations by which an event is above the mean value being measured.

[0142] A polony (or PCR colony) refers to a population of molecules fixed to a substrate, such as a microscope slide or acrylamide gel, that have been derived through amplification from a single parental molecule. Amplification of a dilute mixture of single template molecules leads to the formation of distinct polonies. Thus, all molecules within a given polony are amplicons of the same molecule, but molecules in two distinct polonies are amplicons of different single molecules.

[0143] A “concatemer” refers to a contiguous nucleic acid molecule that contains multiple copies of the same polynucleotide sequence linked in a series. Suitable concatemers can be generated by any suitable methods known in the art, including, but not limited to, rolling circle amplification of circular library molecules comprising adaptor sequences and target sequences. Suitable methods of generating library of concatemer template nucleic acid molecules are described, for example, in WO2022 / 266470, WO2023 / 168444 and WO2023 / 168443. The term “polymerase” and its variants, as used herein, comprises any enzyme that can catalyze polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically, but not necessarily, such nucleotide polymerization can occur in a template- dependent fashion. Typically, a polymerase comprises one or more active sites at which nucleotide binding and / or catalysis of nucleotide polymerization can occur. In some embodiments, a polymerase includes other enzymatic activities, such as for example, 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, a polymerase has strand displacing activity. A polymerase can include, without limitation, naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof that retain the ability to catalyze nucleotide polymerization (e.g., catalytically active fragment). Polymerases can be isolated from cells, or generated using recombinant DNA technology or chemical synthesis methods. Polymerases can be expressed in prokaryote, eukaryote, viral, or phage organisms. Polymerases can be post-translationally modified proteins or fragments thereof. A polymerase can be derived from a prokaryote, eukaryote, virus or phage. The term “polymerase” encompasses DNA-directed DNA polymerases and RNA- directed DNA polymerases.

[0144] As used herein, the term “binding complex” refers to a complex formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or a nucleotide moiety (sometimes referred to as nucleotide unit) of a multivalent molecule, where the nucleic acid duplex comprises a template nucleic acid molecule hybridized to a nucleic acid primer. “Multivalent binding complex” refers to a binding complex comprising a nucleic acid duplex, a polymerase, and a nucleotide moiety of a multivalent molecule. Nucleic acid primer sequences, and nucleic acid primer sequences that have undergone one or more rounds of primer extension (or “stepping”) reactions, are both encompassed by these terms. In the multivalent complex, the free nucleotide or the nucleotide moiety of the multivalent molecule may or may not be bound at 3’ end of the nucleic acid primer at a position that is opposite a complementary nucleotide in the template nucleic acid molecule. A “ternary complex” is an example of a binding complex which is formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide moiety of a multivalent molecule, where the free nucleotide or nucleotide moiety is bound at the 3’ end of the nucleic acid primer or extended primer (as part of the nucleic acid duplex) at a position that is opposite a complementary nucleotide in the template nucleic acid molecule. As used herein, “avidity complex” refers to a complex in which two or more nucleotide moieties of a multivalent molecule of the disclosure are associated with two or more multivalent binding complexes.

[0145] The term “persistence time” and related terms refers to the length of time that a binding complex remains stable without dissociation of any of the components. In some cases, the binding complex is a multivalent binding complex as described herein, and the components of the binding complex include a template nucleic acid molecule or its reverse complement and a nucleic acid primer such as a sequence primer or alternatively an extension product, a polymerase, and a nucleotide moiety of a multivalent molecule or a free (e.g., unconjugated) nucleotide. The nucleotide moiety or the free nucleotide can be complementary or non- complementary to a nucleotide residue in the template nucleic acid molecule. The nucleotide moiety or the free nucleotide can bind to the 3’ end of the nucleic acid primer (or extended primer) at a position that is opposite a complementary nucleotide in the template nucleic acid molecule. The persistence time is indicative of the stability of the binding complex and strength of the binding interactions. Persistence time can be measured by observing the onset and / or duration of a binding complex, such as by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides may be present in a binding complex, thus allowing the signal from the label to be detected during the persistence time of the binding complex. One exemplary label is a fluorescent label. The binding complex (e.g., ternary complex) remains stable until subjected to a condition that causes dissociation of interactions between any of the polymerase, template molecule, primer and / or the nucleotide moiety or the free nucleotide. For example, a dissociating condition comprises contacting the binding complex with any one or any combination of a detergent, EDTA and / or water.

[0146] The terms “nucleic acid”, "polynucleotide" and "oligonucleotide" and other related terms used herein are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically-synthesized forms. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids comprise polymers of nucleotides, where the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids comprise naturally-occurring internucleosidic linkages, for example phosphodiester linkages. Nucleic acids comprise non-natural internucleoside linkages, including phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, nucleic acids comprise a one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0147] The term “primer” and related terms used herein refers to an oligonucleotide, either natural or synthetic, that is capable of hybridizing with a DNA and / or RNA polynucleotide template to form a duplex (double stranded) molecule. Primers may have any length, but typically range from 4-50 nucleotides. A typical primer comprises a 5’ end and 3’ end. The 3’ end of the primer can include a 3’ OH moiety which serves as a nucleotide polymerization initiation site in a polymerase-mediated primer extension reaction. Alternatively, the 3’ end of the primer can lack a 3’ OH moiety, or can include a terminal 3’ blocking group that inhibits nucleotide polymerization in a polymerase-mediated reaction. Any one nucleotide, or more than one nucleotide, along the length of the primer can be labeled with a detectable reporter moiety. A primer can be in solution (e.g., a soluble primer) or can be immobilized on a support (e.g., a surface or capture primer).

[0148] The term “target sequence” or “target polynucleotide”, sometimes also referred to herein as “sequence of interest” refers to a sequence whose nucleotide identity is to be determined by the sequencing methods described herein. The term “template nucleic acid”, “template polynucleotide”, “template strand” and other variations refer to a nucleic acid strand that serves as the basis nucleic acid molecule for generating a complementary nucleic acid strand. The template nucleic acid can be single-stranded or double-stranded, or the template nucleic acid can have single-stranded or double-stranded portions. The sequence of the template nucleic acid can be partially or wholly complementary to the sequence of the complementary strand. The template nucleic acid can be obtained from a naturally-occurring source, recombinant form, or chemically synthesized to include any type of nucleic acid analog. The template nucleic acid can be linear, circular, or other forms. The template sequence can be single-stranded, or double-stranded, for example a single-stranded DNA molecule. Template nucleic acid molecules of the disclosure can include an insert region having an insert sequence comprising the target sequence. In addition, the template nucleic acids can also include at least one adaptor sequence, such as an adaptor sequencing comprising a primer binding sequence. The template nucleic acid can be a concatemer having two or tandem copies of a target sequence and at least one adaptor sequence. The target sequence can be isolated in any form, including chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned, amplified, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotides, whole genomic DNA, obtained from fresh frozen paraffin embedded tissue, needle biopsies, cell free circulating DNA, or any type of nucleic acid library. The target sequence can be isolated from any source including from organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, viruses cells, tissues, normal or diseased cells or tissues, body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, semen, environmental samples, culture samples, or synthesized nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. The target sequence can be isolated from any organ, including head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs. The template nucleic acid can be subjected to nucleic acid analysis, including sequencing and composition analysis.

[0149] When used in reference to nucleic acid molecules, the terms “hybridize” or “hybridizing” or “hybridization” or other related terms refers to hydrogen bonding between two different nucleic acids to form a duplex (double-stranded) nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule having a duplex region. Hybridization can comprise Watson- Crick or Hoogstein binding to form a duplex double-stranded nucleic acid, or a double-stranded region within a nucleic acid molecule. The double-stranded nucleic acid, or the two different regions of a single nucleic acid, may be wholly complementary, or partially complementary. Complementary nucleic acid strands need not hybridize with each other across their entire length. The complementary base pairing can be the standard A-T or C-G base pairing, or can be other forms of base-pairing interactions. Duplex nucleic acids can include mismatched base- paired nucleotides.

[0150] The term “nucleotides” and related terms refers to a molecule comprising an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Canonical and non-canonical nucleotides are consistent with use of the term. The phosphate, in some cases, comprises a monophosphate, diphosphate, or triphosphate, or corresponding phosphate analog. In some cases, the nucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate groups. The term “nucleoside” refers to a molecule comprising an aromatic base and a sugar.

[0151] Nucleotides (and nucleosides) typically comprise a heterocyclic base including a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring which are commonly found in nucleic acids, including naturally-occurring, substituted, modified, or engineered variants, or analogs of the same. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and / or Hoogstein hydrogen bonds with an appropriate complementary base. Exemplary bases include, but are not limited to, purines and pyrimidines such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N6-A2- isopentenyladenine (6iA), N6-A2-isopentenyl-2-methylthioadenine (2ms6iA), N6- methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7mG), 2 -thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O6-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6- dihydrothymine, O4-methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines; hydroxymethylcytosines; 5-methycytosines; base (Y); as well as methylated, glycosylated, and acylated base moieties; and the like. Additional exemplary bases can be found in Fasman, 1989, in “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Boca Raton, Fla.

[0152] Nucleotides (and nucleosides) typically comprise a sugar moiety, such as carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Letters vol. 7: 3013-3016), and other sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No. 5,558,991). Exemplary sugar moieties comprise ribosyl; 2'- deoxyribosyl; 3 '-deoxyribosyl; 2', 3 '-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'- alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'- alkylthioribosyl; 3 '-alkoxyribosyl; 3 '-azidoribosyl; 3 '-aminoribosyl; 3 '-fluororibosyl; 3'- mercaptoriboxyl; 3 '-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[0153] In some cases, nucleotides comprise a chain of one, two or three phosphorus atoms. The chain is typically attached to the 5’ carbon of the sugar moiety via an ester or phosphoramide linkage. In some cases, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene or ethylene. The phosphorus atoms in the chain can include substituted side groups, including O, S or BH3. In some cases, the chain includes phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphordithioate, and O- methylphosphoroamidite groups.

[0154] When used in reference to nucleic acids, the terms “extend”, “extending”, “extension” and other variants, refers to incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation comprises polymerization of one or more nucleotides into the terminal 3’ OH end of a nucleic acid strand, resulting in extension of the nucleic acid strand. Nucleotide incorporation can be conducted with natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent fashion. Any suitable method of extending a nucleic acid molecule may be used, including primer extension catalyzed by a DNA polymerase or RNA polymerase.

[0155] The terms “reporter moiety”, “reporter moieties”, “detectable reporter moieties,” and related terms refer to a compound that generates, or causes to generate, a detectable signal. A reporter moiety is sometimes called a “label.” Any suitable reporter moiety may be used, including luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent, chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten, affinity tag, atom, or enzymatic. A reporter moiety generates a detectable signal resulting from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events). A proximity event includes two reporter moieties approaching each other, or associating with each other, or binding each other. It is well known to one skilled in the art to select multiple reporter moieties so that each absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from the other reporter moieties to permit monitoring of the presence of different reporter moieties in the same reaction, or in different reactions. Two or more different reporter moieties can be selected having spectrally distinct emission profiles, or having minimal overlapping spectral emission profiles. Reporter moieties can be linked (e.g., operably linked) to the multivalent molecules described herein, as well as nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerases or reverse transcriptases), or supports (e.g., surfaces) by any suitable methods.

[0156] Reporter moieties, or labels, include fluorescent labels and fhiorophores. Exemplary fluorescent moieties which may serve as fluorescent labels or fluorophores include, but are not limited to fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA- fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR- iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE- hydrazide, BODIPY™ and derivatives such as BODIPY™ FL C3-SE, BODIPY™ 530 / 550 C3, BODIPY™ 530 / 550 C3-SE, BODIPY™ 530 / 550 C3 hydrazide, BODIPY™ 493 / 503 C3 hydrazide, BODIPY™ FL C3 hydrazide, BODIPY™ FL IA, BODIPY™ 530 / 551 IA, Br- BODIPY™ 493 / 503, Cascade Blue™ and derivatives such as Cascade Blue™ acetyl azide, Cascade Blue™ cadaverine, Cascade Blu™e ethylenediamine, Cascade Blue™ hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanine and derivatives such as indolium based cyanine dyes, benzo-indolium based cyanine dyes, pyridium based cyanine dyes, thiozolium based cyanine dyes, quinolinium based cyanine dyes, imidazolium based cyanine dyes, Cy 3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, Europium chelates, Terbium chelates, Alexa Fluor® dyes, DyLight™ dyes, Atto dyes, LightCycler® Red dyes, CAL Flour dyes, JOE and derivatives thereof, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, Malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes and others known in the art such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. Cyanine dyes may exist in either sulfonated or non- sulfonated forms, and consist of two indolenin, benzo-indolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3, (which may comprise 1 - [6-(2, 5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3 - { 1 - [6-(2, 5-dioxopyrrolidin-1-yloxy)-6- oxohexyl]-3,3-dimethyl-l,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H- indolium or l-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{ l-[6-(2,5-dioxopyrrolidin- l-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-

[0157] 3.3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may comprise 1-(6-((2,5- dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1- yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-l,3-dien-1-yl)-3,3-dimethyl-3H- indol-1-ium or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-1-(6- ((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-

[0158] 1.3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate), and Cy7 (which may comprise 1-(5- carboxypentyl)-2-[(lE, 3E,5E,7Z)-7-(l -ethyl-l,3-dihydro-2H-indol-2-ylidene)hepta- 1,3,5- trien-1-yl]-3H-indolium or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(l-ethyl-5-sulfo-l,3- dihydro-2H-indol-2-ylidene)hepta-l,3,5-trien-1-yl]-3H-indolium-5-sulfonate), where “Cy” stands for 'cyanine', and the first digit identifies the number of carbon atoms between two indolenine groups. Cy2 which is an oxazole derivative rather than indolenin, and the benzo- derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule.

[0159] Reporter moieties include fluorescence resonance energy transfer (FRET) pairs, such that multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET may comprise excitation exchange (Forster) transfers, or electron-exchange (Dexter) transfers.

[0160] The terms “linked”, “joined”, “attached”, and variants thereof comprise any type of fusion, bond, adherence or association between any combination of compounds or molecules that is of sufficient stability to withstand use in the particular procedure. The procedure can include but are not limited to: nucleotide transient-binding; nucleotide incorporation; de- blocking; washing; removing; flowing; detecting; imaging and / or identifying. Such linkage can comprise, for example, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonds or associations involving van der Waals forces, mechanical bonding, and the like. Linkage can occur intramolecularly, for example linking together the ends of a single-stranded or double-stranded linear nucleic acid molecule to form a circular molecule. Linkage can also occur between a combination of different molecules, or between a molecule and a non-molecule, including but not limited to: linkage between a nucleic acid molecule and a solid surface; linkage between a protein and a detectable reporter moiety; linkage between a nucleotide and detectable reporter moiety; and the like. Some examples of linkages can be found, for example, in Hermanson, G., “Bioconjugate Techniques”, Second Edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).

[0161] The term “adaptor” and related terms refers to oligonucleotides that can be operably linked (appended) to a target polynucleotide, where the adaptor confers a function to the co- joined adaptor-target molecule. Adaptors comprise DNA, RNA, chimeric DNA / RNA, or analogs thereof. Adaptors can include at least one ribonucleoside residue. Adaptors can be single-stranded, double-stranded, or have single-stranded and / or double-stranded portions. Adaptors can be configured to be linear, stem-looped, hairpin, or Y-shaped forms. Adaptors can be any length, including 4-100 nucleotides or longer. Adaptors can have blunt ends, overhang ends, or a combination of both. Overhang ends include 5’ overhang and 3’ overhang ends. The 5’ end of a single-stranded adaptor, or one strand of a double-stranded adaptor, can have a 5’ phosphate group or lack a 5’ phosphate group. Adaptors can include a 5’ tail that does not hybridize to a target polynucleotide (e.g., tailed adaptor), or adaptors can be non- tailed. An adaptor can include a sequence that is complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., soluble or immobilized capture primers). Adaptors can include a random sequence or degenerate sequence. Adaptors can include at least one inosine residue. Adaptors can include at least one phosphorothioate, phosphorothiolate and / or phosphoramidate linkage. Adaptors can include a barcode sequence which can be used to distinguish polynucleotides (e.g., target sequences) from different sample sources in a multiplex assay such as high throughput sequencing. Adaptors can include a unique identification sequence (e.g., unique molecular index, UMI; or a unique molecular tag) that can be used to uniquely identify a nucleic acid molecule to which the adaptor is appended. In some cases, a unique identification sequence can be used to increase error correction and accuracy, reduce the rate of false-positive variant calls and / or increase sensitivity of variant detection. Adaptors can include at least one restriction enzyme recognition sequence, including any one or any combination of two or more selected from a group consisting of type I, type II, type III, type IV, type Hs or type IIB.

[0162] The term “universal sequence”, “universal adaptor sequences” and related terms refers to a sequence in a nucleic acid molecule that is common among two or more polynucleotide molecules, for example sequences shared amongst nucleic acid molecules in a library. For example, adaptors having the same universal sequence can be joined to a plurality of polynucleotides so that the population of co-joined molecules carry the same universal adaptor sequence. Examples of universal adaptor sequences include binding sequences amplification primers, sequencing primers or capture primers (e.g., soluble or support-immobilized capture primers).

[0163] As used herein, “support” refers to a substrate that is solid, semi-solid, or a combination of both, to which a plurality of nucleic acid molecules (e.g., template nucleic acid molecules and / or capture primers) can be affixed. The support can be porous, semi-porous, non-porous, or any combination of porosity. The support can be substantially planar, concave, convex, or any combination thereof. The support can be cylindrical, for example comprising a capillary or interior surface of a capillary. The support can be a surface of a flow cell. The surface of the support can be substantially smooth. Alternatively, the support can be regularly or irregularly textured, including bumps, etched, pores, three-dimensional scaffolds, or any combination thereof. The term “support” also encompasses beads having any shape, including spherical, hemi- spherical, cylindrical, barrel-shaped, toroidal, disc-shaped, rod-like, conical, triangular, cubical, polygonal, tubular or wire-like. The support can be fabricated from any material, including but not limited to glass, fused-silica, silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[0164] The surface of the support can be coated with one or more compounds to produce a passivated layer on the support. Supports comprising a low non-specific binding surface that enable improved nucleic acid hybridization and amplification performance on the support are envisaged as within the scope of the instant disclosure. In general, the support may comprise one or more layers of a covalently or non-covalently attached low-binding, chemical modification layers, e.g., silane layers, polymer films, and one or more covalently or non- covalently attached oligonucleotides that may be used for immobilizing a plurality of nucleic acid template molecules to the support.

[0165] The degree of hydrophilicity (or “wettability” with aqueous solutions) of the surface coatings of the support may be assessed, for example, through the measurement of water contact angles in which a small droplet of water is placed on the surface and its angle of contact with the surface is measured using, e.g., an optical tensiometer. In some cases, a static contact angle may be determined. In some cases, an advancing or receding contact angle may be determined. The water contact angle for the hydrophilic, low-binding support surfaced disclosed herein may range from about 0 degrees to about 30 degrees, for example the water contact angle for the hydrophilic, low-binding support surface disclosed herein may no more than 50 degrees, 40 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. In many cases the contact angle is no more than 40 degrees. Those of skill in the art will realize that a given hydrophilic, low-binding support surface of the present disclosure may exhibit a water contact angle having a value of anywhere within this range.

[0166] The present disclosure provides a plurality (e.g., two or more) of template nucleic acid molecules immobilized to a support. The immobilized plurality of template nucleic acid molecules have the same sequence or have different sequences (e.g., template nucleic acid molecules in the plurality have different target sequences). Individual nucleic acid template molecules in the plurality of nucleic acid templates can be immobilized to a different site on the support, for example in an array. The term “array” refers to a support comprising a plurality of sites located at pre-determined locations on the support to form an array of sites. The sites can be discrete and separated by interstitial regions. The pre-determined sites on the support can be arranged in one dimension in a row or a column, or arranged in two dimensions in rows and columns. The plurality of pre-determined sites can be arranged on the support in an organized fashion, for example in any organized pattern, including rectilinear, hexagonal patterns, grid patterns, patterns having reflective symmetry, patterns having rotational symmetry', or the like. The pitch between different pairs of sites can be that same or can vary. The support can have template nucleic acid molecules immobilized at a plurality of sites at a surface density of about 102-• 1015sites per mm2, or more, to form a template nucleic acid array. For example, the support comprises at least 102sites, at least 103sites, at least 104sites, at least 105sites, at least 106sites, at least 107sites, at least 108sites, at least 109sites, at least 1010sites, at least 1011sites, at least 1012sites, at least 1013sites, at least 1014sites, at least 1015sites, or more, where the sites are located at pre-determined locations on the support. In some cases, a plurality of pre-determined sites on the support (e.g., 102- 1015sites or more) are immobilized with nucleic acid templates to form a nucleic acid template array. The nucleic acid templates can be immobilized at a plurality of pre-determined sites by any methods known in the art, including hybridization to immobilized surface capture primers, or covalent attachment to immobilized surface capture primers. In some cases, the template nucleic acid molecules are immobilized at a plurality of pre-determined sites, for example 102- 1015sites or more. The template nucleic acid molecules that are immobilized at a plurality of sites on the support can comprise linear or circular molecules, or a mixture of both linear and circular molecules. The immobilized template nucleic acid molecules can be clonally-amplified to generate immobilized polonies at the plurality of pre-determined sites. In some embodiments, individual immobilized template nucleic acid molecules comprise one copy of a target sequence of interest, or comprise concatemers having two or more tandem copies of a target sequence of interest. Optionally, the concatemerized sequence can include additional sequences, such as binding sequences for sequencing primers, amplification primers, and / or barcodes.

[0167] Alternatively, a support can comprise a plurality of sites located at random locations (referred to herein as a support having randomly located sites thereon). In a support having randomly located sites, the locations of the sites on the support are not pre-determined. The plurality of randomly-located sites is arranged on the support in a disordered and / or unpredictable fashion. A support with randomly located sites can comprise at least 102sites, at least 103sites, at least 104sites, at least 105sites, at least 106sites, at least 107sites, at least 108sites, at least 109sites, at least 1010sites, at least 1011sites, at least 1012sites, at least 1013sites, at least 1014sites, at least 1015sites, or more. In some cases, a plurality of template nucleic acid molecules are randomly located on the support (e.g., at 102- 1015sites or more) and are immobilized to form a support comprising immobilized template nucleic acid molecules. Template nucleic acid molecules can be immobilized randomly on a support by hybridization to immobilized, randomly-located surface capture primers, or by covalently attached to the surface capture primers. The template nucleic acid molecules can be immobilized at a plurality of randomly located sites on the support, for example immobilized at 102- 1015sites or more. The nucleic acid templates that are immobilized at a plurality of sites on the support can comprise linear or circular molecules, or a mixture of both linear and circular molecules. The immobilized template nucleic acid molecules can be clonally-amplified to generate immobilized nucleic acid polonies at the plurality of randomly located sites. In some embodiments, individual immobilized template nucleic acid molecules comprise one copy of a target sequence of interest, or comprise concatemers having two or more tandem copies of a target sequence of interest. Optionally, the concatemerized sequence can include additional sequences, such as binding sequences for sequencing primers, amplification primers, and / or barcodes.

[0168] In general, during NGS methods described herein, nucleic acid molecules immobilized to the support are in fluid communication with each other to permit flowing a solution of reagents (e.g., enzymes including polymerases, multivalent molecules, nucleotides, divalent cations and / or buffers and the like) onto the support so that the plurality nucleic acid molecules on the support can be reacted with the reagents in a massively parallel manner. For example, a library of template nucleic acid molecules, or the reverse complements thereof, depending upon the sequencing protocol, can be immobilized to a surface of a flow cell, and are in fluid communication with each other as reagents are flowed into and out of the flow cell. The fluid communication of the plurality of immobilized nucleic acid molecules can be used to conduct nucleotide binding assays and / or conduct nucleotide polymerization reactions (e.g., primer extension or sequencing) on the plurality of immobilized nucleic acid template molecules, and to conduct detection and imaging for massively parallel sequencing.

[0169] The term “immobilized” and related terms, refers to nucleic acid molecules or enzymes (e.g., polymerases) that are attached to the support. The attachment can be directly to the support through covalent bond or non-covalent interaction, or indirectly to a coating on the support.

[0170] As used herein, the term “clonally amplified” and it variants refers to a nucleic acid molecule that has been subjected to one or more amplification reactions either in-solution or on-support. In the case of in-solution amplified template nucleic acid molecules, the resulting amplicons are distributed onto the support. Prior to amplification, the template nucleic acid molecule comprises a target sequence and at least one universal adaptor sequence (e.g., an adaptor sequence comprising binding sequence for a forward sequencing primer). Clonal amplification comprises the use of a polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence- based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-stranded binding (SSB) protein-dependent amplification, or any combination thereof.

[0171] As used herein, “alkyl”, “C1, C2, C3, C4, C5C6C7C8C9C10C11C12C13C14C15C16C17C18C19C20alkyl” or “C1-C20alkyl” is intended to include C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20straight chain (linear) saturated aliphatic hydrocarbon groups and C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20branched saturated aliphatic hydrocarbon groups. For example, C1-C6alkyl is intends to include C1, C2, C3, C4, C5and C6alkyl groups. Examples of alkyl include, moieties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, i -propyl, n-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, or n-hexyl. In some embodiments, a straight chain or branched alkyl has twenty or fewer carbon atoms (e.g., C1-C20for straight chain, C3-C20for branched chain). In some embodiments, a straight chain or branched alkyl has six or fewer carbon atoms (e.g., C1-C6for straight chain, C3-C6for branched chain), and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms. The term “alkylene” refers to a multivalent alkyl group, e.g., a bivalent, trivalent, or tetravalent alkyl group. In some embodiments, “alkylene” refers to a bivalent alkyl group.

[0172] As used herein, the term “optionally substituted alkyl” refers to unsubstituted alkyl or alkyl having designated substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0173] As used herein, the term “alkenyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term “alkenyl” includes straight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenyl groups. In certain embodiments, a straight chain or branched alkenyl group has twenty or fewer carbon atoms in its backbone (e.g., C2-C20for straight chain, C3-C20for branched chain). In certain embodiments, a straight chain or branched alkenyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6for straight chain, C3-C6for branched chain). The term “ C2-C6” includes alkenyl groups containing two to six carbon atoms. The term “ C3-C6” includes alkenyl groups containing three to six carbon atoms. The term “alkenylene” refers to a multivalent (e.g., bivalent, trivalent, tetravalent) alkenyl group. In some embodiments, “alkenylene” refers to a bivalent alkenyl group.

[0174] As used herein, the term “optionally substituted alkenyl” refers to unsubstituted alkenyl or alkenyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0175] As used herein, the term “alkynyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, “alkynyl” includes straight chain alkynyl groups (e.g, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl), and branched alkynyl groups. In certain embodiments, a straight chain or branched alkynyl group has twenty or fewer carbon atoms in its backbone (e.g, C2-C20for straight chain, C3-C20for branched chain). In certain embodiments, a straight chain or branched alkynyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6for straight chain, C3-C6for branched chain). The term “C2-C6” includes alkynyl groups containing two to six carbon atoms. The term “C3-C6” includes alkynyl groups containing three to six carbon atoms. The term “alkynylene” refers to a multivalent (e.g., bivalent, trivalent, tetravalent) alkynyl group. In some embodiments, “alkynylene” refers to a bivalent alkynyl group.

[0176] As used herein, the term “optionally substituted alkynyl” refers to unsubstituted alkynyl or alkynyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0177] Other optionally substituted moieties (such as optionally substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both the unsubstituted moieties and the moieties having one or more of the designated substituents. For example, substituted heterocycloalkyl includes those substituted with one or more alkyl groups, such as 2,2,6,6-tetramethyl- piperidinyl and 2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.

[0178] As used herein, the term “cycloalkyl” refers to a saturated or partially unsaturated hydrocarbon monocyclic or polycyclic (e.g., fused, bridged, or spiro rings) system having 3 to 30 carbon atoms (e.g., C3-C12, C3-C10, or C3-C8). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 1,2,3,4-tetrahydronaphthalenyl, and adamantyl. In the case of polycyclic cycloalkyl, only one of the rings in the cycloalkyl needs to be non- aromatic. The term “cycloalkylene” refers to a multivalent (e.g., bivalent, trivalent, tetravalent) cycloalkylene group.

[0179] As used herein, the term “heterocycloalkyl” refers to a saturated or partially unsaturated 3-8 membered monocyclic, 7-12 membered bicyclic (fused, bridged, or spiro rings), or 11-14 membered tricyclic ring system (fused, bridged, or spiro rings) having one or more heteroatoms (such as O, N, S, P, or Se), e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g. , 1, 2, 3, 4, 5, or 6 heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulfur, unless specified otherwise. Examples of heterocycloalkyl groups include, but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl, indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl, dihydropyranyl, pyranyl, morpholinyl, tetrahydrothiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl, 2-oxa-5- azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 2-oxa-6-azaspiro[3.3]heptanyl, 2,6-diazaspiro[3.3]heptanyl, 1,4-dioxa-8-azaspiro[4.5]decanyl, 1,4-dioxaspiro[4.5]decanyl, 1- oxaspiro [4.5 ] decany 1 , 1 -azaspiro [4.5 ] decany 1 , 3 'H- spiro [cy cl ohexane- 1 , 1' -i sob enzofuran] -y 1 , 7'H-spiro[cyclohexane- 1 ,5'-furo[3 ,4-b]pyridin]-yl, 3 'H-spiro[cy cl ohexane- 1 , 1 '-furo[3 ,4- c]pyridin]-yl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[3.1.0]hexan-3-yl, 1, 4,5,6- tetrahydropyrrolo[3,4-c]pyrazolyl, 3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidinyl, 4, 5,6,7- tetrahydro-lH-pyrazolo[3,4-c]pyridinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, 2- azaspiro[3.3]heptanyl, 2-methyl-2-azaspiro[3.3]heptanyl, 2-azaspiro[3.5]nonanyl, 2-methyl-2- azaspiro[3.5]nonanyl, 2-azaspiro[4.5]decanyl, 2-methyl-2-azaspiro[4.5]decanyl, 2-oxa- azaspiro[3 ,4]octanyl, 2-oxa-azaspiro[3 ,4]octan-6-yl, 5,6-dihydro-4H- cyclopenta[b]thiophenyl, and the like. In the case of multicyclic heterocycloalkyl, only one of the rings in the heterocycloalkyl needs to be non-aromatic (e.g., 4, 5,6,7- tetrahydrobenzo[c]isoxazolyl). The term “heterocycloalkylene” refers to a multivalent (e.g., bivalent, trivalent, tetravalent) heterocycloalkyl group.

[0180] It is understood that when a variable has two attachments to the rest of the formula of the compound, the two attachments could be at the same atom or different atoms of the variable. For example, when a variable (e.g., variable X) is cycloalkyl or heterocycloalkyl, and has two attachments to the rest of the formula of the compound, the two attachments could be at the same atom or different atoms of the cycloalkyl or heterocycloalkyl.

[0181] As used herein, the term “aryl” includes groups with aromaticity, including “conjugated,” or multicyclic systems with one or more aromatic rings and do not contain any heteroatom in the ring structure. The term aryl includes both monovalent species and divalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl and the like. For example, an aryl is phenyl. The term “aryl” refers to a multivalent (e.g., bivalent, trivalent, or tetravalent) aryl group.

[0182] As used herein, the term “heteroaryl” is intended to include a stable 5-, 6-, or 7- membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromatic heterocyclic ring which consists of carbon atoms and one or more heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g. , 1, 2, 3, 4, 5, or 6 heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulfur. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR wherein R is H or other substituents, as defined). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O)P, where p = 1 or 2). It is to be noted that total number of S and O atoms in the aromatic heterocycle is not more than 1. Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like. Heteroaryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multi cyclic system (e.g., 4,5,6,7-tetrahydrobenzo[c]isoxazolyl). In some embodiments, the heteroaryl is thiophenyl or benzothiophenyl. In some embodiments, the heteroaryl is thiophenyl. In some embodiments, the heteroaryl benzothiophenyl. The term “heteroarylene” refers to a multivalent (e.g., bivalent, trivalent, or tetravalent) heteroaryl group.

[0183] Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodi oxazole, benzothiazole, benzoimidazole, benzothiophene, quinoline, isoquinoline, naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.

[0184] The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring can be substituted at one or more ring positions (e.g., the ring-forming carbon or heteroatom such as N) with such substituents as described above, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and heteroaryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., tetralin, methylenedioxyphenyl such as benzo[d][l,3]dioxole-5-yl).

[0185] As used herein, the term “substituted,” means that any one or more hydrogen atoms on the designated atom is replaced with a selection from the indicated groups, provided that the designated atom’s normal valency is not exceeded, and that the substitution results in a stable compound. When a substituent is oxo or keto (i.e., =O), then 2 hydrogen atoms on the atom are replaced. Keto substituents are not present on aromatic moieties. Ring double bonds, as used herein, are double bonds that are formed between two adjacent ring atoms (e.g., C=C, C=N or N=N). “Stable compound” and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0186] When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then such substituent may be bonded to any atom in the ring. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent may be bonded via any atom in such formula. Combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0187] When any variable (e.g., R) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R moieties, then the group may optionally be substituted with up to two R moieties and R at each occurrence is selected independently from the definition of R. Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0188] As used herein, the term “hydroxy” or “hydroxyl” includes groups with an -OH or -O'

[0189] As used herein, the term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

[0190] The term “haloalkyl” or “haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or more halogen atoms.

[0191] As used herein, the term “optionally substituted haloalkyl” refers to unsubstituted haloalkyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. As used herein, the term “alkoxy” or “alkoxyl” includes substituted and unsubstituted alkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups or alkoxyl radicals include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

[0192] As used herein, the term “alkyl aryl” refers to an alkyl group, as defined herein, substituted with an aryl group, as defined herein. A “C1-6alkyl C6-10aryl” group refers to a C1-6 alkyl group, as defined herein, substituted with a C6-10aryl group, as defined herein. Unless otherwise specified, alkyl group and aryl group may be optionally substituted as described herein.

[0193] As used herein, the term “alkyl cycloalkyl” refers to an alkyl group, as defined herein, substituted with a cycloalkyl group, as defined herein. A “C1-6alkyl C3-10aryl” group refers to a C1-6alkyl group, as defined herein, substituted with a C3-10cycloalkyl group, as defined herein. Unless otherwise specified, alkyl group and cycloalkyl group may be optionally substituted as described herein.

[0194] As used herein, the term “alkyl heteroaryl” refers to an alkyl group, as defined herein, substituted with a heteroaryl group, as defined herein. A “C1-6alkyl C2-9heteroaryl” group refers to a C1-6alkyl group, as defined herein, substituted with a C2-9heteroaryl group, as defined herein. Unless otherwise specified, alkyl group and heteroaryl group may be optionally substituted as described herein.

[0195] As used herein, the term “alkyl heterocycloalkyl” refers to an alkyl group, as defined herein, substituted with a heteroaryl group, as defined herein. A “C1-6alkyl C2-9heterocycloalkyl” group refers to a C1-6alkyl group, as defined herein, substituted with a C2-9 heterocycloalkyl group, as defined herein. Unless otherwise specified, alkyl group and heterocycloalkyl group may be optionally substituted as described herein.

[0196] As used herein, the term “heteroalkyl” refers to an alkyl group, as defined herein, that further comprises at least one heteroatom (e.g., at least one nitrogen, oxygen, or sulfur atom). In some embodiments, a C1-20heteroalkyl group has twenty or fewer carbon atoms and twenty or fewer heteroatoms. In some embodiments, a straight branched alkyl has six or fewer carbon atoms and six or fewer heteroatoms, and in another embodiment, a straight chain or branched chain heteroalkyl has four or fewer carbon atoms and four or fewer heteroatoms. A C1heteroalkyl comprises one carbon atom and at least one heteroatom. The term “heteroalkylene” refers to a multivalent heteroalkyl group, e.g., a bivalent, trivalent, or tetraval ent heteroalkyl group.

[0197] As used herein, the term “amino acid” refers to an organic molecule that comprises an amino group and a carboxylic acid that separated by one, two ,or three methylene units, wherein the methylene units are optionally substituted with a C1-6alkyl group, C1-6heteroalkyl group, C1-6alkyl C6-10aryl group, C1-6alkyl C2-9heteroaryl group, C1-6alkyl C3-10cycloalkyl group, or C1-6alkyl C2-9heterocycloalkyl group, wherein the C1-6alkyl group, C1-6heteroalkyl group, C1-6alkyl C6-10aryl group, C1-6alkyl C2-9heteroaryl group, C1-6alkyl C3-10cycloalkyl group, or C1-6alkyl C2-9heterocycloalkyl group is optionally substituted with a hydroxy, halo, cyano, or nitro group. Amino acids in which the carboxylic acid and amino group are separated by one optionally substituted methylene unit are referred to herein as “alpha amino acids.” Amino acids in which the carboxylic acid and amino group are separated by two optionally substituted methylene units are referred to herein as “beta amino acids.” Amino acids in which the carboxylic acid and amino group are separated by optionally substituted methylene units are referred to herein as “gamma amino acids.” This disclosure contemplates the use of naturally occurring and non-naturally occurring amino acids. This disclosure contemplates the use of (L) and (D) amino acids. Exemplary and nonlimiting amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolocysteine, selenocysteine, pyrrolysine, 2-naphthyl-alanine, statine, homoalanine, 3-pyridyl-alanine, 4-fluorophenyl-alanine, cyclohexyl-alanine, homo-cysteine, penicillamine, 3 -nitro-tyrosine, homo-phenyl-alanine, t-leucine, and hydroxy -proline. As used herein, the term “amino acid moiety” refers to the portion of a chain of linked amino acids that would correspond to the atoms in one individual amino acid, were the amino acids unlinked. Scheme AA-1 illustrates the relationship between a linked chain of amino acids an amino acid moiety: Scheme AA-1

[0198] This disclosure contemplates embodiments in which amino acids are linked to one or more moieties that are not amino acids. In such instance, the term “amino acid moiety” refers to the atoms that would correspond to atoms within the individual amino acid, were the amino acid unlinked from the moiety or moieties that are not amino acids.

[0199] Unless defined otherwise, technical and scientific terms used herein have meanings that are commonly understood by those of ordinary skill in the art unless defined otherwise. Generally, terminologies pertaining to techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization described herein are those well-known and commonly used in the art. Techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the instant specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclatures utilized in connection with, and the laboratory procedures and techniques described herein are those well-known and commonly used in the art.

[0200] Throughout this application various publications, patents, and / or patent applications are referenced. The disclosures of the publications, patents and / or patent applications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0201] Multivalent Molecules

[0202] The disclosure provides multivalent molecules (e.g., compositions comprising formula (I), below), and methods of using same in high throughput sequencing. In some embodiments, the methods comprising contacting a plurality of template nucleic acid molecules comprising two or more copies of a target sequence, or its complement, and two or more copies of a binding sequence for a sequencing primer, wherein the binding sequences of the template nucleic acid molecules comprise duplexes with sequencing primers or extended strands thereof, a plurality of polymerases, and a plurality of multivalent molecules of Formula (I) below, or an ionized form thereof, an isomer thereof, or a salt thereof, under conditions sufficient to form a plurality of multivalent binding complexes comprising a nucleic acid duplex between a template nucleic acid molecule and sequencing primer or extended sequencing primer strand, a first polymerase, and a nucleotide moiety of a multivalent molecule that is complementary to a nucleotide in the template nucleic acid molecule immediately adjacent to a 3’ end of the sequencing primer or extended sequencing primer strand. In some embodiments, the methods comprise detecting the detectable reporter moieties of the multivalent molecules, and determining nucleobase identities of nucleotides in the nucleic acid template molecules complementary to the nucleotide moieties of the multivalent molecules based on the detectable reporter moieties of the multivalent molecules in the plurality of multivalent binding complexes. In some embodiments, the multivalent molecule comprises at least two nucleotide moieties and at least one detectable reporter moiety, and two or more nucleotide moieties in an individual multivalent molecule contact two or more different multivalent binding complexes. In some embodiments, the two or more different multivalent binding complexes are on the same template nucleic acid molecule. In some embodiments, the two or more multivalent binding complexes are on different template nucleic acid molecules.

[0203] In some aspects, the present disclosure provides a multivalent molecule, e.g., a composition of Formula (I):

[0204] (I) an ionized form thereof, an isomer thereof, or a salt thereof, wherein:

[0205] C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety or absent; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3-(R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety.

[0206] In some aspects, the present disclosure provides a multivalent molecule, e.g., a composition of Formula (II):

[0207] (II) an ionized form thereof, an isomer thereof, or a salt thereof, wherein:

[0208] C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6) each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3-(R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety.

[0209] In some embodiments, the composition is of Formula (III):

[0210] (III) an ionized form thereof, an isomer thereof, or a salt thereof.

[0211] In some embodiments, the composition is of Formula (IV): (IV) an ionized form thereof, an isomer thereof, or a salt thereof.

[0212] In some embodiments, the composition is of Formula (V):

[0213] (V) an ionized form thereof, an isomer thereof, or a salt thereof.

[0214] In some embodiments, the multivalent molecule comprises at least two nucleotide moieties.

[0215] In some embodiments, the multivalent molecule comprises at least one detectable reporter moiety.

[0216] In some embodiments, the multivalent molecule comprises at least two nucleotide moieties and at least one detectable reporter moiety. In some embodiments, the at least two nucleotide moieties are respectively attached to different X moieties.

[0217] In some embodiments, the multivalent molecule comprises two nucleotide moieties. In some embodiments, the multivalent molecule comprises three nucleotide moieties. In some embodiments, the multivalent molecule comprises four moieties.

[0218] In some embodiments, all the nucleotide moieties in the multivalent molecule are the same.

[0219] In some embodiments, all detectable reporter moieties in the multivalent molecule comprise the same fluorescent label.

[0220] In some embodiments, all detectable reporter moieties in the multivalent molecule are the same.

[0221] In some embodiments, the multivalent molecule comprises two detectable reporter moieties. In some embodiments, the multivalent molecule comprises three detectable reporter moieties. In some embodiments, the multivalent molecule comprises four detectable reporter moieties.

[0222] In some embodiments, all detectable reporter moieties in the multivalent molecule are the same and all the detectable reporter moieties in the multivalent molecule are the same.

[0223] The present disclosure contemplates that multivalent molecules comprising a specific nucleotide moiety, e.g., a nucleotide moiety comprising dATP, dTTP, dGTP, dUTP, or dCTP,

[0224] Central Moiety

[0225] In some embodiments, the Central Moiety is bivalent, trivalent, tetravalent, pentavalent, or hexavalent. In some embodiments, the Central Moiety is a bond.

[0226] In some embodiments, the Central Moiety is a cyclic moiety. In some embodiments, the Central Moiety is a 5- to 10-membered heteroarylene. In some embodiments, the Central Moiety is an C6-10arylene. In some embodiments, the Central Moiety is a 3- to 10-membered heterocyclene. In some embodiment, the Central Moiety is a C3-10cycloalkylene.

[0227] In some embodiments, the Central Moiety is an acyclic moiety. In some embodiments, the Central Moiety is a C1-20alkyl moiety. In some embodiments, the central moiety is a C1-20heteroalkyl moiety.

[0228] Bivalent Linking Moiety

[0229] In some embodiments, each Bivalent Linking Moiety independently comprises from 1 to 20 cyclic moieties, wherein the cyclic moieties are linked to one another by way of chemical bonds. In some embodiments, the cyclic moieties are directly bonded to one another. In some embodiments, the cyclic moieties are linked by way of amide, sulfonamide, ester, carbonate, urea, imide, ether, or thioether bonds. In some embodiments, the cyclic moieties comprise 5- to 10-membered heteroarylene moieties. In some embodiments, the cyclic moieties comprise 3-to 10-membered heterocycloalkylene moieties. In some embodiments, the cyclic moieties comprise C6-10arylene moieties. In some embodiments, the cyclic moieties comprise C3-10cycloalkylene moieties. In some embodiments, the cyclic moieties comprise a mixture of 5- to 10-membered heteroarylene moieties, 3-to 10-membered heterocycloalkylene moieties, C6-10arylene moieties, and C3-10cycloalkylene moieties. In some embodiments, the series of 1-20 linked cyclic moieties further comprises a series of polyethylene glycol units.

[0230] In some embodiments, the Bivalent Linking Moiety comprises a poly-ether or poly- amine group, e.g., a poly-ethylene glycol or polyethylenimine group.

[0231] In some embodiments, the Bivalent Linking Moiety comprises a linear or branched C1-20alkyl group.

[0232] In some embodiments, the Bivalent Linking Moiety comprises a linear or branched C1-20heteroalkyl group.

[0233] In some embodiments, the Bivalent Linking Moiety comprises a plurality of linked amino acids. In some embodiments, the amino acids are naturally occurring. In some embodiments, the amino acids are not naturally occurring. In some embodiments, the Bivalent Linking Moiety comprises naturally occurring and non-naturally occurring amino acids. In some embodiments, the plurality of linked amino acids further comprises a series of polyethylene glycol units. Multivalent Branching Moiety

[0234] In some embodiments, the Multivalent Branching is trivalent, tetravalent, pentavalent, or hexavalent.

[0235] In some embodiments, the Multivalent Branching Moiety is a cyclic moiety. In some embodiments, the Multivalent Branching Moiety is a 5 to 10 membered heteroarylene. In some embodiments, the Multivalent Branching Moiety is an C6-10arylene. In some embodiments, the Multivalent Branching Moiety is a 3 to 10 membered heterocyclene. In some embodiment, the Multivalent Branching Moiety is a C3-10cycloalkylene.

[0236] In some embodiments, the Multivalent Branching Moiety is an acyclic moiety. In some embodiments, the Multivalent Branching Moiety is a C1-20alkyl moiety. In some embodiments, the Multivalent Branching Moiety is a C1-20heteroalkyl moiety.

[0237] Core / Arm

[0238] In some aspects, the multivalent molecule (e.g., the composition of Formula (I), an ionized form thereof, an isomer thereof, or a salt thereof) comprises a Core and one or more Arms.

[0239] In some embodiments, the Core is of Formula (F)

[0240] (I ) wherein:

[0241] C is a Central Moiety; each X independently is -(L1-C1-(Rla)1-6) each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety or absent; each Rlaindependently is -T, L2-*, L2-T’, or -L2-C2-(R2a)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2aindependently is L3-*, L3-T’or -L3-C3-(R3a)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3aindependently is L4-*, L4-T’ or L4-C4-(R4a)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4aindependently is L5-* or L5-T’; each L5independently is a Bivalent Linking Moiety; each T’ independently is a Terminal Moiety comprising a detectable reporter moiety; and

[0242] * indicates the attachment point to an Arm.

[0243] In some embodiments, each Arm independently is a Terminal Moiety comprising a nucleotide moiety.

[0244] In some embodiments:

[0245] C is a bond, C1-6alkylene, C2-6alkenylene, C2-6alkynylene, C1-6heteroalkylene, C6-10arylene, 5- to 10-membered heteroarylene, C3-10cycloalkylene, or 3- to 10-membered heterocycloalkylene;

[0246] C1is absent, C1-6alkylene, C2-6alkenylene, C2-6alkynylene, C 1-6 heteroalkylene, C6-10arylene, 5- to 10-membered heteroarylene, C3-10cycloalkylene, or 3- to 10-membered heterocycloalkylene;

[0247] C2is C1-6alkylene, C2-6alkenylene, C2-6alkynylene, C 1-6 heteroalkylene, C6-10arylene, 5- to 10-membered heteroarylene, C3-10cycloalkylene, or 3- to 10-membered heterocycloalkylene;

[0248] C3is C1-6alkylene, C2-6alkenylene, C2-6alkynylene, C 1-6 heteroalkylene, C6-10arylene, 5- to 10-membered heteroarylene, C3-10cycloalkylene, or 3- to 10-membered heterocycloalkylene;

[0249] C4is C1-6alkylene, C2-6alkenylene, C2-6alkynylene, C 1-6 heteroalkylene, C6-10arylene, 5- to 10-membered heteroarylene, C3-10cycloalkylene, or 3- to 10-membered heterocycloalkylene;

[0250] L1is C3-10cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-10arylene, 5- to 10-membered heteroarylene, -{Ra1-C(=O)-NH-Rb1}1-20-, -{Ra1-C(=O)-O-Rb1}1-20-, -{Ra1- C(=O)-S-Rb1}1-20-, -{Ra1-C(=O)2-S-Rb1}1-20-, -{Ra1-Rb1}1-20-, PEG1-12, PEI1-12, C1-20alkylene, C1-20heteroalkylene, C1-20alkenylene, C1-20alkynylene, or -{Rcl-Rd1}1-20-; Ra1and Rb1each independently are C3-10cycloalkylene, 3- to 10-membered heterocyclene, C6-10arylene, or 5- to 10-membered heteroarylene;

[0251] RC1and Rd1each independently are an amino acid moiety;

[0252] L2is C3-10cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-10arylene, 5- to 10-membered heteroarylene, -{Ra2-C(=O)-NH-Rb2}1-20-, -{Ra2-C(=O)-O-Rb2}1-20-, -{Ra2- C(=O)-S-Rb2}1-20-, -{Ra2-C(=O)2-S-Rb2}1-20-, -{Ra2-Rb2}1-20-, -{ Ra2-C(=O)-NH-Rb2}1-20, -{Ra2- C(=O)-O-Rb2}1-20, -{Ra2-C(=O)-S-Rb2}1-20, -{Ra2-C(=O)2-S-Rb2}1-20, PEG1-12, PEI1-12, C1-20 branched or linear alkylene, C1-20heteroalkylene, C1-20alkenylene; C1-20alkynylene, or -{Rc2- Rd2}1-20-.

[0253] Ra2and Rb2each independently are C3-10cycloalkylene, 3- to 10-membered heterocyclene, C6-10arylene, or 5- to 10-membered heteroarylene;

[0254] Rc2and Rd2each independently are an amino acid moiety;

[0255] L3is C3-10cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-10arylene, 5- to 10-membered heteroarylene, -{Ra3-C(=O)-NH-Rb3}1-20-, -{Ra3-C(=O)-O-Rb3}1-20-, -{Ra3- C(=O)-S-Rb3}1-20-, -{Ra3-C(=O)2-S-Rb3}1-20-, -{Ra3-Rb3}1-20-, -{Ra3-Rb3}1-20, -{ Ra3-C(=O)-NH- Rb3}1-20, -{Ra3-C(=O)-O-Rb3}1-20, -{Ra3-C(=O)-S-Rb3}1-20, -{Ra3-C(=O)2-S-Rb3}1-20, PEG1-12, PEI1-12, C1-20branched or linear alkylene, C1-20heteroalkylene, C2-20alkenylene; C2-20alkynylene, or -{Rc3-Rd3}1-20-;

[0256] Ra3and Rb3each independently are C3-10cycloalkylene, 3- to 10-membered heterocycylene, C6-10arylene, or 5- to 10-membered heteroarylene;

[0257] Rc3and Rd3each independently are an amino acid moiety;

[0258] L4is C3-10cycloalkylene, 3- to 10-membered heterocycloalkylene, C6-10arylene, 5- to 10-membered heteroarylene,, -{Ra4-C(=O)-NH-Rb4}1-20-, -{Ra4-C(=O)-0-Rb4}1-20-, -{Ra4- C(=O)-S-Rb4}1-20-, -{Ra4-C(=O)2-S-Rb4}1-20-, -{Ra4-Rb4}1-20-, -{Ra4-Rb4}1-20, -{ Ra4-C(=O)-NH- Rb4}1-20, -{Ra4-C(=O)-O-Rb4}1-20, -{Ra4-C(=O)-S-Rb4}1-20, -{Ra4-C(=O)2-S-Rb4}1-20, PEG1-12, PEI1-12, C1-20branched or linear alkylene, C1-20heteroalkylene, C2-20alkenylene; C2-20alkynylene, or -{Rc4-Rd4}1-20-;

[0259] Ra4and Rb4each independently are C3-10cycloalkylene, 3- to 10-membered heterocycylene, C6-10arylene, or 5- to 10-membered heteroarylene;

[0260] R4Cand R4deach independently are an amino acid moiety; and

[0261] T is adenosine triphosphate, -PEG{3000-7000}-adenosine triphosphate, guanosine triphosphate, -PEG{3000-7000}-guanosine triphosphate, thymidine triphosphate, -PEG{3000-7000}- thymidine triphosphate, cytidine triphosphate, -PEG{3000-7000}-cytidine triphosphate, fluor ophore, or -fluorophore.

[0262] Core moiety C

[0263] In some embodiments, C is a bond.

[0264] In some embodiments, C is C1-6alkylene. In some embodiments, C is methylene, ethylene, propylene, butylene, pentylene, or hexylene.

[0265] In some embodiments, C is C2-6alkynylene. In some embodiments, C is ethynylene, propynylene, butynylene, pentynylene, or hexynylene. In some embodiments, C is C2-6alkenylene. In some embodiments, C is ethenylene, propenylene, butenylene, pentenylene, or hexenylene.

[0266] In some embodiments, C is C1-6heteroalkylene. In some embodiments, C is,

[0267] In some embodiments, C is C6-10arylene. In some embodiments, C is phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene.

[0268] In some embodiments, C is 5- to 10-membered heteroarylene. In some embodiments, C is pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene.

[0269] In some embodiments, C is C3-10cycloalkylene. In some embodiments, C is cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene.

[0270] In some embodiments, C is C5-10cycloalkenylene. In some embodiments, C is cyclopentenylene, cyclohexenylene, cycloheptenylene, or 1,2,3,4-tetrahydronaphthalenylene.

[0271] In some embodiments, C is 3- to 10-membered heterocycloalkylene. In some embodiments, C is piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{4.5}decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene,

[0272] 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro {3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro{4.5}decanylene, 2-oxa- azaspiro {3.4} octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenyl ene.

[0273] Multivalent branching moiety C1

[0274] In some embodiments, C1is absent.

[0275] In some embodiments, C1is C1-6alkylene. In some embodiments, C1is methylene, ethylene, propylene, butylene, pentylene, or hexylene.

[0276] In some embodiments, C1is C2-6alkenylene. In some embodiments, C1is ethenylene, propenylene, butenylene, pentenylene, or hexenylene.

[0277] In some embodiments, C1is C2-6alkynylene. In some embodiments, C1is ethynylene, propynylene, butynylene, pentynylene, or hexynylene.

[0278] In some embodiments, C1is C1-6heteroalkylene. In some embodiments, C1is

[0279] In some embodiments, C1is C6-10arylene. In some embodiments, C1is phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene.

[0280] In some embodiments, C1is 5- to 10-membered heteroarylene. In some embodiments, C1is pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene.

[0281] In some embodiments, C1is C3-10cycloalkylene. In some embodiments, C1is cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene. In some embodiments, C1is 3- to 10-membered heterocycloalkylene. In some embodiments, C1is piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenylene. Multivalent Branching Moiety C2

[0282] In some embodiments, C2is C1-6alkylene. In some embodiments, C2is methylene, ethylene, propylene, butylene, pentylene, or hexylene.

[0283] In some embodiments, C2is C2-6alkenylene. In some embodiments, C2is ethenylene, propenylene, butenylene, pentenylene, or hexenylene.

[0284] In some embodiments, C2is C2-6alkynylene. In some embodiments, C2is ethynylene, propynylene, butynylene, pentynylene, or hexynylene.

[0285] In some embodiments, C2is C1-6heteroalkylene. In some embodiments, C2is

[0286] In some embodiments, C2is C6-10arylene. In some embodiments, C2is phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene. In some embodiments, C2is 5- to 10-membered heteroarylene. In some embodiments, C2is pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene.

[0287] In some embodiments, C2is C3-10cycloalkylene. In some embodiments, C2is cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene.

[0288] In some embodiments, C2is 3- to 10-membered heterocycloalkylene. In some embodiments, C2is piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5}nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenyl ene. Multivalent Branching Moiety C3

[0289] In some embodiments, C3is C1-6alkylene. In some embodiments, C3is methylene, ethylene, propylene, butylene, pentylene, or hexylene.

[0290] In some embodiments, C3is C2-6alkenylene. In some embodiments, C3is ethenylene, propenylene, butenylene, pentenylene, or hexenylene. In some embodiments, C3is C2-6alkynylene. In some embodiments, C3is ethynylene, propynylene, butynylene, pentynylene, or hexynylene.

[0291] In some embodiments, C3is C1-6heteroalkylene. In some embodiments, C3is

[0292] In some embodiments, C3is C6-10arylene. In some embodiments, C3is phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene.

[0293] In some embodiments, C3is 5- to 10-membered heteroarylene. In some embodiments, C3is pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene.

[0294] In some embodiments, C3is C3-10cycloalkylene. In some embodiments, C3is cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene.

[0295] In some embodiments, C3is 3- to 10-membered heterocycloalkylene. In some embodiments, C3is piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{4.5}decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenylene.

[0296] Multivalent Branching Moiety C4

[0297] In some embodiments, C4is C1-6alkylene. In some embodiments, C4is methylene, ethylene, propylene, butylene, pentylene, or hexylene.

[0298] In some embodiments, C4is C2-6alkenylene. In some embodiments, C4is ethenylene, propenylene, butenylene, pentenylene, or hexenylene.

[0299] In some embodiments, C4is C2-6alkynylene. In some embodiments, C4is ethynylene, propynylene, butynylene, pentynylene, or hexynylene.

[0300] In some embodiments, C4is C1-6heteroalkylene. In some embodiments, C4is

[0301] In some embodiments, C4is C6-10arylene. In some embodiments, C4is phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene.

[0302] In some embodiments, C4is 5- to 10-membered heteroarylene. In some embodiments, C4is pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene.

[0303] In some embodiments, C4is C3-10cycloalkylene. In some embodiments, C4is cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene.

[0304] In some embodiments, C4is 3- to 10-membered heterocycloalkylene. In some embodiments, C4is piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5}nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenylene.

[0305] Bivalent Linking Moiety L1In some embodiments, L1is C3-10cycloalkylene. In some embodiments, L1is cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene.

[0306] In some embodiments, L1is 3- to 10-membered heterocycloalkylene. In some embodiments, L1is piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro {3.5 } non any 1 ene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro{4.5 }decanylene, 2-oxa- azaspiro {3.4} octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenylene.

[0307] In some embodiments, L1is C6-10arylene. In some embodiments, L1is phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene.

[0308] In some embodiments, L1is 5- to 10-membered heteroarylene. In some embodiments, L1is pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene.

[0309] In some embodiments, L1is -{Ra1-C(=O)-NH-Rb1}1-20-. In some embodiments, Li is -{ C3-10cycloalkylene -C(=O)-NH- C3-10cycloalkylene }1-20-, -{ C3-10cycloalkylene -C(=O)-NH- 3- to 10-membered heterocyclene}1-20-, -{ C3-10cycloalkylene -C(=O)-NH- C6-10arylene }1-20- , -{ C3-10cycloalkylene -C(=O)-NH- 5- to 10-membered heteroarylene }1-20-, -{3- to 10- membered heterocycloalkylene-C(=O)-NH- C3-10cycloalkylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-NH-3- to 10-membered heterocycloalkylene }1-20-, -{3- to 10- membered heterocycloalkylene-C(=O)-NH- C6-10arylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-NH-5- to 10-membered heteroarylene }1-20-, -{C6-10arylene- C(=O)-NH- C3-10cycloalkylene }1-20-, -{C6-10arylene-C(=O)-NH-3- to 10-membered heterocycloalkylene }1-20-, -{C6-10arylene-C(=O)-NH- C6-10arylene }1-20-, -{C6-10arylene- C(=O)-NH-5- to 10-membered heteroarylene }1-20-, -{5- to 10-membered heteroaryl ene- C(=O)-NH- C3-10cycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-NH-3- to 10- membered heterocycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-NH- C6-10arylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-NH-5- to 10-membered heteroarylene }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-NH- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-NH- (piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5}decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 }decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0 }hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5}nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-NH- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-NH- }pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro {4.5 Jdecanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-NH- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b }thiophenylene)-C(=O)-NH-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3 }heptanylene, 2, 6-diazaspiro{3.3 }heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene,

[0310] 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-cJpyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3 }heptanylene, 2-methylene-2-azaspiro{3.3 }heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, di oxany 1 ene, tetrahydrofuranyl ene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2, 6-diazaspiro{3.3 }heptanylene, 1,4-dioxa-8- azaspiro {4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1,4, 5, 6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-NH- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 {heptanylene, 2,5-diazabicyclo{2.2.1 {heptanylene, 2-oxa-6-azaspiro{3.3 {heptanylene, 2, 6-diazaspiro{3.3 {heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5{decanylene, l-oxaspiro{4.5{decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran{-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c{pyridin{-ylene, 3-azabicyclo{3.1.0{hexanylene, 3-azabicyclo{3.1.0{hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c{pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d{pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c{pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3 {heptanylene, 2-methylene-2-azaspiro{3.3 {heptanylene, 2- azaspiro{3.5 {nonanylene, 2-methylene-2-azaspiro{3.5 {nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-NH-5- to 10-membered heteroarylene }1-20-, {(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-NH- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-NH- (piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene) }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-NH- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, - {(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-NH-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-NH- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-NH-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene) }1-20-, -{ }pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)- NH- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, or -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolyl ene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-NH-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-.

[0311] In some embodiments, L1is -{Ra1-C(=O)-0-Rb1}1-20-. In some embodiments, L1is -{ C3-10cycloalkylene -C(=O)-O- C3-10cycloalkylene}1-20-, -{ C3-10cycloalkylene -C(=O)-O- 3- to 10-membered heterocyclene}1-20-, -{ C3-10cycloalkylene -C(=O)-O- C6-10arylene }1-20-, -{ C3-10cycloalkylene -C(=O)-O- 5- to 10-membered heteroarylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-O- C3-10cycloalkylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-O-3- to 10-membered heterocycloalkylene }1-20-, -{3- to 10- membered heterocycloalkylene-C(=O)-O- C6-10arylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-O-5- to 10-membered heteroarylene }1-20-, -{C6-10arylene-C(=O)- O- C3-10cycloalkylene }1-20-, -{C6-10arylene-C(=O)-O-3- to 10-membered heterocycloalkylene }1-20-, -{C6-10arylene-C(=O)-O- C6-10arylene }1-20-, -{C6-10arylene-C(=O)-O-5- to 10- membered heteroarylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-O- C3-10cycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-O-3- to 10-membered heterocycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-O- C6-10arylene }1-20-, - {5- to 10-membered heteroarylene-C(=O)-O-5- to 10-membered heteroarylene }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-O- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-O- (piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{4.5}decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-O- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-O- }pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 {nonanylene, 2-methylene-2-azaspiro{3.5 {nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-O- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b }thiophenylene)-C(=O)-O-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro {3.4} octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, di oxany 1 ene, tetrahydrofuranyl ene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-O- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-O-5- to 10-membered heteroarylene }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-O- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, - {(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-O-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{4.5}decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro {3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro{4.5}decanylene, 2-oxa- azaspiro {3.4} octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cy cl openta{b {thiophenylene) }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-O- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, - {(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-O-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene) -C(=O)-O- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene) -C(=O)-O-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 {nonanylene, 2-methylene-2-azaspiro{3.5 {nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene) }1-20-, -{}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-O- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, or -{ }pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolyl ene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-O-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-.

[0312] In some embodiments, L1is -{Ra1-C(=O)-S-Rb1}1-20-. In some embodiments, L1is -{ C3-10cycloalkylene -C(=O)-S- C3-10cycloalkylene}1-20-, -{ C3-10cycloalkylene -C(=O)-S- 3- to 10-membered heterocyclene}1-20-, -{ C3-10cycloalkylene -C(=O)-S- C6-10arylene }1-20-, -{ C3- 10 cycloalkylene -C(=O)-S- 5- to 10-membered heteroarylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-S- C3-10cycloalkylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-S-3- to 10-membered heterocycloalkylene }1-20-, -{3- to 10- membered heterocycloalkylene-C(=O)-S- C6-10arylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-S-5- to 10-membered heteroarylene }1-20-, -{C6-10arylene-C(=O)- S- C3-10cycloalkylene }1-20-, -{C6-10arylene-C(=O)-S-3- to 10-membered heterocycloalkylene }1-20-, -{C6-10arylene-C(=O)-S- C6-10arylene }1-20-, -{C6-10arylene-C(=O)-S-5- to 10- membered heteroarylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-S- C3-10cycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-S-3- to 10-membered heterocycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-S- C6-10arylene }1-20-, - {5- to 10-membered heteroarylene-C(=O)-S-5- to 10-membered heteroarylene }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-S- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-S- (piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3 }heptanylene, 2, 6-diazaspiro{3.3 }heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 }decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 }decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3 }heptanylene, 2-methylene-2-azaspiro{3.3 }heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-S- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-S- }pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 }decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 }decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyndin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-S- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b }thiophenylene)-C(=O)-S-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3 {heptanylene, 2, 6-diazaspiro{3.3 {heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3 {heptanylene, 2-methylene-2-azaspiro{3.3 {heptanylene, 2- azaspiro{3.5 {nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro {3.4} octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, di oxany 1 ene, tetrahydrofuranyl ene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahy drothi opy rany 1 ene, 1 ,4-diazepanylene, 1 ,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro {3.3} heptanylene, 2, 6 - di azaspiro {3.3} heptanylene, 1,4-dioxa-8- azaspiro {4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-S- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 {heptanylene, 2,5-diazabicyclo{2.2.1 {heptanylene, 2-oxa-6-azaspiro{3.3 {heptanylene, 2, 6-diazaspiro{3.3 {heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5{decanylene, l-oxaspiro{4.5{decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran{-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c{pyridin{-ylene, 3-azabicyclo{3.1.0{hexanylene, 3-azabicyclo{3.1.0{hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c{pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d{pyrimidinylene,

[0313] 4.5.6.7-tetrahydro-lH-pyrazolo{3,4-c{pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3 {heptanylene, 2-methylene-2-azaspiro{3.3 {heptanylene, 2- azaspiro{3.5 {nonanylene, 2-methylene-2-azaspiro{3.5 {nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-S-5- to 10-membered heteroarylene }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-S- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, - {(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-S-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene,

[0314] 4.5.6.7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro {3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro{4.5 {decanylene, 2-oxa- azaspiro {3.4} octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene) }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-S- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, - {(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-S-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-S- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-S-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro {4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene) }1-20-, -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene) -C(=O)-S- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, or -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolyl ene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-S-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-.

[0315] In some embodiments, L1is -{Ra1-C(=O)2-S-Rb1}1-20-. In some embodiments, L1is -{ C3-10cycloalkylene -C(=O)2-S- C3-10cycloalkylene}1-20-, -{ C3-10cycloalkylene -C(=O)2-S- 3- to 10-membered heterocyclene}1-20-, -{ C3-10cycloalkylene -C(=O)2-S- C6-10arylene }1-20-, -{ C3-10cycloalkylene -C(=O)2-S- 5- to 10-membered heteroarylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)2-S- C3-10cycloalkylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)2-S-3- to 10-membered heterocycloalkylene }1-20-, -{3- to 10- membered heterocycloalkylene-C(=O)2-S- C6-10arylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)2-S-5- to 10-membered heteroarylene }1-20-, -{C6-10arylene- C(=O)2-S- C3-10cycloalkylene }1-20-, -{C6-10arylene-C(=O)2-S-3- to 10-membered heterocycloalkylene }1-20-, -{C6-10arylene-C(=O)2-S- C6-10arylene }1-20-, -{C6-10arylene- C(=O)2-S-5- to 10-membered heteroarylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)2- S- C3-10cycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)2-S-3- to 10-membered heterocycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)2-S- C6-10arylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)2-S-5- to 10-membered heteroarylene }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)2-S- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)2-S- (piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{4.5}decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)2-S- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)2-S- }pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3 }heptanylene, 2, 6-diazaspiro{3.3 }heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5 {decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3 }heptanylene, 2-methylene-2-azaspiro{3.3 }heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)2-S- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)2-S-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro {4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, di oxany 1 ene, tetrahydrofuranyl ene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)2-S- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)2-S-5- to 10-membered heteroarylene }1-20-, {(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)2-S- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)2-S- (piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene,

[0316] 1.2.3.6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{4.5}decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1,4, 5, 6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene,

[0317] 4.5.6.7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene) }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)2-S- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, - {(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)2-S-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene) -C(=O)2-S- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene) -C(=O)2-S-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{4.5}decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro {3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro{4.5}decanylene, 2-oxa- azaspiro {3.4} octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cy cl openta{b {thiophenylene) }1-20-, -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene) -C(=O)2- S- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, or -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)2-S-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-.

[0318] In some embodiments, L1is -{Ra1-Rb1}1-20-. In some embodiments, L1is -{ C3-10cycloalkylene - C3-10cycloalkylene}1-20-, -{ C3-10cycloalkylene - 3- to 10-membered heterocyclene}1-20-, -{ C3-10cycloalkylene - C6-10arylene }1-20-, -{ C3-10cycloalkylene - 5- to 10-membered heteroarylene }1-20-, -{3- to 10-membered heterocycloalkylene- C3-10cycloalkylene }1-20-, -{3- to 10-membered heterocycloalkylene-3- to 10-membered heterocycloalkylene }1-20-, -{3- to 10-membered heterocycloalkylene- C6-10arylene }1-20-, -{3- to 10-membered heterocycloalkylene-5- to 10-membered heteroarylene }1-20-, -{C6-10arylene- C3-10cycloalkylene }1-20-, -{C6-10arylene-3- to 10-membered heterocycloalkylene }1-20-, -{C6-10 arylene- C6-10arylene }1-20-, -{C6-10arylene-5- to 10-membered heteroarylene }1-20-, -{5- to 10-membered heteroarylene- C3-10cycloalkylene }1-20-, -{5- to 10-membered heteroarylene-3- to 10-membered heterocycloalkylene }1-20-, -{5- to 10-membered heteroarylene- C6-10arylene }1-20-, -{5- to 10-membered heteroaryl ene- 5- to 10-membered heteroarylene }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) - (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) - (piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro {4.5 }decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 }decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) - (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) - }pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 }decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 }decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro{4.5}decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenyl ene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, di oxany 1 ene, tetrahydrofuranyl ene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro {3.3} heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro {4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, - {(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene,

[0319] 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 {nonanylene, 2-methylene-2-azaspiro{3.5 {nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-5- to 10-membered heteroarylene }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 {nonanylene, 2-methylene-2-azaspiro{3.5 {nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cy cl openta{b {thiophenylene) }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene) }1-20-, - {}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene) - (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, - {}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene) -(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 {heptanylene, 2,5-diazabicyclo{2.2.1 {heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2, 6-diazaspiro{3.3 {heptanylene, 1,4-dioxa-8- azaspiro{4.5}decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene) }1-20-, -{}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene) - (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, or -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolyl ene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-.

[0320] In some embodiments, L1is PEG1-12.

[0321] In some embodiments, L1is PEI1-12.

[0322] In some embodiments, L1is C1-20alkylene. In some embodiments, L1is methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene, or icosanylene.

[0323] In some embodiments, L1is C1-20heteroalkylene. In some embodiments, L1is In some embodiments, L1is C1-20alkenylene. In some embodiments, L1is ethenylene, propenylene, butenylene, pentenylene, hexenylene, heptenylene, octenylene, nonenylene, decenylene, undecenylene, dodecenylene, tridecenylene, tetradecenylene, pentadecenylene, hexadecenylene, heptadecenylene, octadecenylene, nonadecenylene, or icosenylene.

[0324] In some embodiments, L1is C1-20alkynylene. In some embodiments, L1is ethynylene, propynylene, butynylene, pentynylene, hexynylene, heptynylene, octynylene, nonynylene, decynylene, undecynylene, dodecynylene, tridecynylene, tetradecynylene, pentadecynylene, hexadecynylene, heptadecynylene, octadecynylene, nonadecynylene, or icosynylene.

[0325] In some embodiments, L1is -{ {Rc1}0-1-{Rd1}0-1}1-20, wherein there is at least one of RC1or Rd1. In some embodiments, L1is -{ {Rc1}0-1-{Rd1}0-1}, -{ {Rc1}0-1-{Rd1}0-1}2, -{{Rc1}0-1- {Rd1}0-1}3, -{ {Rc1}0-1-{Rd1}0-1}4, -{ {Rc1}0-1-{Rd1}0-1} , -{ {Rc1}0-1-{Rd1}0-1}6, -{{Rc1}0-1- {Rd1}0-1}7, -{ {Rc1}0-1-{Rd1}0-1}8, -{ {Rc1}0-1-{Rd1}0-1}9, -{ {Rc1}0-1-{Rd1}0-1}10, -{{Rc1}0-1- {Rd1}0-1}11, -{ {Rc1}0-1-{Rd1}0-1} 12, -{ {Rc1}0-1-{Rd1}0-1}13, -{ {Rc1}0-1-{Rd1}0-1} 14, -{{Rc1}0-1- {Rd1}0-1}i5, -{ {Rc1}0-1-{Rd1}0-1}16, -{ {Rc1}0-1-{Rd1}0-1}17, -{ {Rc1}0-1-{Rd1}0-1}18, -{{Rc1}0-1- {Rd1}0-1} 19, or -{ {Rc1}0-1-{Rd1}0-1}20.

[0326] Bivalent Linking Moiety L2

[0327] In some embodiments, L2is C3-10cycloalkylene. In some embodiments, L2is cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene.

[0328] In some embodiments, L2is 3- to 10-membered heterocycloalkylene. In some embodiments, L2is piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{4.5}decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro {3.5 } non any 1 ene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro{4.5 }decanylene, 2-oxa- azaspiro {3.4} octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenylene.

[0329] In some embodiments, L2is C6-10arylene. In some embodiments, L2is phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene.

[0330] In some embodiments, L2is 5- to 10-membered heteroarylene. In some embodiments, L2is pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene.

[0331] In some embodiments, L2is -{Ra2-C(=O)-NH-Rb2}1-20-. In some embodiments, L2is -{ C3-10cycloalkylene -C(=O)-NH- C3-10cycloalkylene }1-20-, -{ C3-10cycloalkylene -C(=O)-NH- 3- to 10-membered heterocyclene}1-20-, -{ C3-10cycloalkylene -C(=O)-NH- C6-10arylene }1-20- , -{ C3-10cycloalkylene -C(=O)-NH- 5- to 10-membered heteroarylene }1-20-, -{3- to 10- membered heterocycloalkylene-C(=O)-NH- C3-10cycloalkylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-NH-3- to 10-membered heterocycloalkylene }1-20-, -{3- to 10- membered heterocycloalkylene-C(=O)-NH- C6-10arylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-NH-5- to 10-membered heteroarylene }1-20-, -{C6-10arylene- C(=O)-NH- C3-10cycloalkylene }1-20-, -{C6-10arylene-C(=O)-NH-3- to 10-membered heterocycloalkylene }1-20-, -{C6-10arylene-C(=O)-NH- C6-10arylene }1-20-, -{C6-10arylene- C(=O)-NH-5- to 10-membered heteroarylene }1-20-, -{5- to 10-membered heteroaryl ene- C(=O)-NH- C3-10cycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-NH-3- to 10- membered heterocycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-NH- C6-10arylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-NH-5- to 10-membered heteroarylene }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-NH- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-NH- (piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 }decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 }decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-NH- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-NH- }pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro {4.5 Jdecanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-NH- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b }thiophenylene)-C(=O)-NH-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 {heptanylene, 2,5-diazabicyclo{2.2.1 {heptanylene, 2-oxa-6- azaspiro{3.3 {heptanylene, 2, 6-diazaspiro{3.3 {heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene,

[0332] 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c{pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3 {heptanylene, 2-methylene-2-azaspiro{3.3 {heptanylene, 2- azaspiro{3.5 {nonanylene, 2-methylene-2-azaspiro{3.5 {nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, di oxany 1 ene, tetrahydrofuranyl ene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 {heptanylene, 2,5-diazabicyclo{2.2.1 {heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2, 6-diazaspiro{3.3 {heptanylene, 1,4-dioxa-8- azaspiro {4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1,4, 5, 6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-NH- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 {heptanylene, 2,5-diazabicyclo{2.2.1 {heptanylene, 2-oxa-6-azaspiro{3.3 {heptanylene, 2, 6-diazaspiro{3.3 {heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5{decanylene, l-oxaspiro{4.5{decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran{-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c{pyridin{-ylene, 3-azabicyclo{3.1.0{hexanylene, 3-azabicyclo{3.1.0{hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c{pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d{pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c{pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3 {heptanylene, 2-methylene-2-azaspiro{3.3 {heptanylene, 2- azaspiro{3.5 {nonanylene, 2-methylene-2-azaspiro{3.5 {nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-NH-5- to 10-membered heteroarylene }1-20-, {(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-NH- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-NH- (piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene) }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-NH- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, - {(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-NH-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-NH- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-NH-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene) }1-20-, -{ }pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)- NH- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, or -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolyl ene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-NH-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-.

[0333] In some embodiments, L2is -{Ra2-C(=O)-O-Rb2}1-20-. In some embodiments, L2is -{ C3-10cycloalkylene -C(=O)-O- C3-10cycloalkylene}1-20-, -{ C3-10cycloalkylene -C(=O)-O- 3- to 10-membered heterocyclene}1-20-, -{ C3-10cycloalkylene -C(=O)-O- C6-10arylene }1-20-, -{ C3-10cycloalkylene -C(=O)-O- 5- to 10-membered heteroarylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-O- C3-10cycloalkylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-O-3- to 10-membered heterocycloalkylene }1-20-, -{3- to 10- membered heterocycloalkylene-C(=O)-O- C6-10arylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-O-5- to 10-membered heteroarylene }1-20-, -{C6-10arylene-C(=O)- O- C3-10cycloalkylene }1-20-, -{C6-10arylene-C(=O)-O-3- to 10-membered heterocycloalkylene }1-20-, -{C6-10arylene-C(=O)-O- C6-10arylene }1-20-, -{C6-10arylene-C(=O)-O-5- to 10- membered heteroarylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-O- C3-10cycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-O-3- to 10-membered heterocycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-O- C6-10arylene }1-20-, - {5- to 10-membered heteroarylene-C(=O)-O-5- to 10-membered heteroarylene }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-O- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-O- (piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{4.5}decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-O- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-O- }pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 {nonanylene, 2-methylene-2-azaspiro{3.5 {nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-O- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b }thiophenylene)-C(=O)-O-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro {3.4} octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, di oxany 1 ene, tetrahydrofuranyl ene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-O- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-O-5- to 10-membered heteroarylene }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-O- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, - {(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-O-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{4.5}decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro {3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro{4.5}decanylene, 2-oxa- azaspiro {3.4} octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cy cl openta{b {thiophenylene) }1-20-, -{(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-O- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, - {(phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene)-C(=O)-O-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{}pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene) -C(=O)-O- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranyl ene, deazapurinylene, or indolizinylene) -C(=O)-O-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 {nonanylene, 2-methylene-2-azaspiro{3.5 {nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene) }1-20-, -{}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-O- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, or -{ }pyrrolyl ene, furanylene, thiophenylene, thiazolylene, isothiazolyl ene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolyl ene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) -C(=O)-O-}pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodi oxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranylene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-.

[0334] In some embodiments, L2is -{Ra2-C(=O)-S-Rb2}1-20-. In some embodiments, L2is -{ C3-10cycloalkylene -C(=O)-S- C3-10cycloalkylene}1-20-, -{ C3-10cycloalkylene -C(=O)-S- 3- to 10-membered heterocyclene}1-20-, -{ C3-10cycloalkylene -C(=O)-S- C6-10arylene }1-20-, -{ C3- 10 cycloalkylene -C(=O)-S- 5- to 10-membered heteroarylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-S- C3-10cycloalkylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-S-3- to 10-membered heterocycloalkylene }1-20-, -{3- to 10- membered heterocycloalkylene-C(=O)-S- C6-10arylene }1-20-, -{3- to 10-membered heterocycloalkylene-C(=O)-S-5- to 10-membered heteroarylene }1-20-, -{C6-10arylene-C(=O)- S- C3-10cycloalkylene }1-20-, -{C6-10arylene-C(=O)-S-3- to 10-membered heterocycloalkylene }1-20-, -{C6-10arylene-C(=O)-S- C6-10arylene }1-20-, -{C6-10arylene-C(=O)-S-5- to 10- membered heteroarylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-S- C3-10cycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-S-3- to 10-membered heterocycloalkylene }1-20-, -{5- to 10-membered heteroarylene-C(=O)-S- C6-10arylene }1-20-, - {5- to 10-membered heteroarylene-C(=O)-S-5- to 10-membered heteroarylene }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-S- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-S- (piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4- oxazepanylene, 2-oxa-5-azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6-azaspiro{3.3 }heptanylene, 2, 6-diazaspiro{3.3 }heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 }decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 }decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3 }heptanylene, 2-methylene-2-azaspiro{3.3 }heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)}1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-S- (phenylene, napthylene, anthracenylene, phenanthrenylene, chrysenylene, pyrenylene, corannulenylene, coronenylene, or hexahelicenylene) }1-20-, -{ (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) -C(=O)-S- }pyrrolylene, furanylene, thiophenylene, thiazolylene, isothiazolylene, imidazolylene, triazolylene, tetrazolylene, pyrazolylene, oxazolylene, isoxazolylene, isothiazolylene, pyridinylene, pyrazinylene, pyridazinylene, pyrimidinylene, benzoxazolylene, benzodioxazolylene, benzothiazolylene, benzoimidazolylene, benzothiophenylene, quinolinylene, isoquinolinylene, naphthrydinylene, indolylene, benzofuranyl ene, purinylene, benzofuranylene, deazapurinylene, or indolizinylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 }decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 }decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyndin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nomanylene, 2-methylene-2-azaspiro{3.5 }nomanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b}thiophenylene)-C(=O)-S- (cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, or adamantylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1, 2,3,6- tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3}heptanylene, 2,6-diazaspiro{3.3}heptanylene, 1,4-dioxa-8- azaspiro{ 4.5} decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5}decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3}heptanylene, 2-methylene-2-azaspiro{3.3}heptanylene, 2- azaspiro{3.5 }nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro{3.4}octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b }thiophenylene)-C(=O)-S-(piperidinylene, piperazinylene, pyrrolidinylene, dioxanylene, tetrahydrofuranylene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahydrothiopyranylene, 1,4-diazepanylene, 1,4-oxazepanylene, 2-oxa-5- azabicyclo{2.2.1 }heptanylene, 2,5-diazabicyclo{2.2.1 }heptanylene, 2-oxa-6- azaspiro{3.3 {heptanylene, 2, 6-diazaspiro{3.3 {heptanylene, 1,4-dioxa-8- azaspiro{ 4.5 {decanylene, 1,4-dioxaspiro{4.5}decanylene, l-oxaspiro{4.5}decanylene, 1- azaspiro{4.5 {decanylene, 3'H-spiro{cyclohexane-1,1'-isobenzofuran}-ylene, 7'H- spiro{cyclohexane-1,5'-furo{3,4-b}pyridin}-ylene, 3'H-spiro{cyclohexane-1,1'-furo{3,4- c}pyridin}-ylene, 3-azabicyclo{3.1.0}hexanylene, 3-azabicyclo{3.1.0}hexan-3-ylene, 1, 4,5,6- tetrahydropyrrolo{3,4-c}pyrazolylene, 3,4,5,6,7,8-hexahydropyrido{4,3-d}pyrimidinylene, 4,5,6,7-tetrahydro-lH-pyrazolo{3,4-c}pyridinylene, 5,6,7,8-tetrahydropyrido{4,3- d}pyrimidinylene, 2-azaspiro{3.3 {heptanylene, 2-methylene-2-azaspiro{3.3 {heptanylene, 2- azaspiro{3.5 {nonanylene, 2-methylene-2-azaspiro{3.5 }nonanylene, 2- azaspiro {4.5} decanylene, 2-methylene-2-azaspiro {4.5} decanylene, 2-oxa- azaspiro {3.4} octanylene, 2-oxa-azaspiro{3.4}octan-6-ylene, or 5,6-dihydro-4H- cyclopenta{b } thiophenylene) }1-20-, -{(piperidinylene, piperazinylene, pyrrolidinylene, di oxany 1 ene, tetrahydrofuranyl ene, isoindolinylene, indolinylene, imidazolidinylene, pyrazolidinylene, oxazolidinylene, isoxazolidinylene, triazolidinylene, oxiranylene, azetidinylene, oxetanylene, thietanylene, 1,2,3,6-tetrahydropyridinylene, tetrahydropyranylene, dihydropyranylene, pyranylene, morpholinylene, tetrahy drothi opy rany 1 ene, 1 ,4-diazepanylene, 1 ,4-oxazepanylene, 2-oxa-5- azabicyc...

Claims

CLAIMSWhat is claimed is:

1. A method comprising: a. contacting:(i) a plurality of template nucleic acid molecules comprising two or more copies of a target sequence and two or more copies of a binding sequence for a forward sequencing primer,(ii) a plurality of forward sequencing primers comprising a sequence complementary to the binding sequence for the forward sequencing primer,(iii) a plurality of first polymerases, and(iv) a plurality of multivalent molecules of Formula (I):(I) an ionized form thereof, an isomer thereof, or a salt thereof, wherein:C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety or absent; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3-(R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety, wherein each multivalent molecule comprises at least two nucleotide moieties and at one least detectable reporter moiety,wherein the contacting occurs under conditions sufficient to form a plurality of multivalent binding complexes comprising a nucleic acid duplex between a template nucleic acid molecule and forward sequencing primer, a first polymerase, and a nucleotide moiety of a multivalent molecule that is complementary to the nucleotide in the template nucleic acid molecule immediately adjacent to the 3’ end of the forward sequencing primer, and wherein polymerase catalyzed incorporation of a complementary nucleotide moiety into the nucleic acid duplex is inhibited; b. detecting the detectable reporter moi eties; and c. determining the identities of nucleotides in the nucleic acid template molecules based on the detectable reporter moieties of the multivalent molecules in the plurality of multivalent binding complexes formed in step (a).

2. The method of claim 1, wherein the multivalent molecules are of Formula (II):(II) an ionized form thereof, an isomer thereof, or a salt thereof, wherein:C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3-(R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety.

3. The method of claim 1 or 2, wherein the multivalent molecules are of Formula (III):(III) an ionized form thereof, an isomer thereof, or a salt thereof.

4. The method of claim 1 or 2, wherein the multivalent molecules are of Formula (IV):(IV) an ionized form thereof, an isomer thereof, or a salt thereof.

5. The method of claim 1 or 2, wherein the multivalent molecules are of Formula (V):(V) an ionized form thereof, an isomer thereof, or a salt thereof.

6. The method of any one of claims 1-5, wherein the two or more copies of a target sequence in an individual of template nucleic acid molecule are the same target sequence.

7. The method of any one of claims 1-6, wherein two or more multivalent binding complexes form on individual template nucleic acid molecules.

8. The method of any one of any one of claims 1-7, wherein the plurality of forward sequencing primers are soluble.

9. The method of any one of claims 1-8, comprising: d. dissociating the multivalent binding complexes under conditions sufficient to retain the nucleic acid duplexes, thereby generating a plurality of nucleic acid duplexes; e. contacting the plurality of nucleic acid duplexes with a plurality of second polymerases and a plurality of nucleotides or analogs thereof under conditions sufficient to incorporate nucleotides or analogs thereof complementary to the nucleotides of the nucleic acid template molecules immediately adjacent to the 3’ ends of the forward sequencing primers in a primer extension reaction, thereby generating a plurality of extended nucleic acid duplexes comprising extended forward sequencing primer sequences.

10. The method of claim 9, comprising: f. dissociating the second polymerases from the extended nucleic acid duplexes under conditions sufficient to retain the plurality of extended nucleic acid duplexes.

11. The method of any one of claims 1-10, wherein the template nucleic acid molecules comprise concatemers of two or more copies of a sequence comprising (i) the binding sequence for the forward sequencing primer and (ii) the target sequence.

12. The method of claim 11, wherein the two or more copies of (i) the binding sequence for the forward sequencing primer hybridize to the forward sequencing primers to form nucleic acid duplexes between the template nucleic acid molecules and the forward sequencing primers.

13. The method of any one of claims 1-12, wherein, in an individual multivalent molecule, the at least two nucleotide moieties are respectively attached to different X moieties.

14. The method of any one of claims 1-13 wherein, in an individual multivalent molecule, all nucleotide moieties are the same.

15. The method of claim 14, wherein all nucleotide moi eties in an individual multivalent molecule are dATP.

16. The method of claim 14, wherein all nucleotide moieties in an individual multivalent molecule are dTTP.

17. The method of claim 14, wherein all nucleotide moieties in an individual multivalent molecule are dGTP.

18. The method of claim 14, wherein all nucleotide moieties in an individual multivalent molecule are dUTP19. The method of claim 14, wherein all nucleotide moieties in an individual multivalent molecule are dCTP.

20. The method of any one of claims 1-19, wherein, in an individual multivalent molecule, all detectable reporter moieties are the same.

21. The method of claim 20, wherein all detectable reporter moieties in an individual multivalent molecule comprise the same fluorescent label.

22. The method of any one of claims 1-21, wherein, in an individual multivalent molecule, all detectable reporter moieties are the same and all nucleotide moieties are the same.

23. The method of any one of claims 1-22, wherein an individual multivalent molecule comprises two, three, or four nucleotide moieties.

24. The method of any one of claims 1-22, wherein an individual multivalent molecule comprises two, three, or four detectable reporter moieties.

25. The method of any one of claims 1-24, wherein two or more nucleotide moieties in an individual multivalent molecule are associated with two or more different multivalent binding complexes on the same template nucleic acid molecule.

26. The method of any one of claims 10-25, comprising i. contacting the plurality of extended nucleic acid duplexes with a plurality of first polymerases and a plurality of multivalent molecules of Formula (I), or an ionized form thereof, an isomer thereof, or a salt thereof, wherein the contacting occurs under conditions sufficient to form a plurality of multivalent binding complexes comprising an extended nucleic acid duplex, a first polymerase, and a nucleotide moiety of a multivalent molecule that is complementary to a nucleotide in the template nucleic acid molecule immediately adjacent to the 3’ end of the extended forward sequencing primer, and wherein polymerase catalyzed incorporation of a complementary nucleotide moiety into the extended nucleic acid duplex is inhibited; ii. detecting the detectable reporter moi eties; iii. determining nucleobase identities of nucleotides in the nucleic acid template sequences complementary to the nucleotide moieties of the multivalent molecules based on the detectable reporter moieties of the multivalent molecules in the plurality of multivalent binding complexes formed in step (a); iv. dissociating the multivalent binding complexes under conditions sufficient to retain the plurality extended nucleic acid duplexes; and v. contacting the plurality of extended nucleic acid duplexes with a plurality of second polymerases and a plurality of nucleotides or analogs thereof under conditions sufficient to incorporate nucleotides or analogs thereof complementary to the nucleotides of the nucleic acid template molecules immediately adjacent to the 3’ ends of the extended forward sequencing primers in a primer extension reaction, thereby generating a plurality of extended nucleic acid duplexes comprising extended forward sequencing primers.

27. The method of claim 26, wherein, in an individual multivalent molecule, the at least two nucleotide moieties are attached to different X moieties.

28. The method of claim 26 or 27, wherein, in an individual multivalent molecule, all nucleotide moieties are the same.

29. The method of claim 28, wherein all nucleotide moieties in an individual multivalent molecule are dATP.

30. The method of claim 28, wherein all nucleotide moieties in an individual multivalent molecule are dTTP.

31. The method of claim 28, wherein all nucleotide moieties in an individual multivalent molecule are dGTP.

32. The method of claim 28, wherein all nucleotide moieties in an individual multivalent molecule are dUTP.

33. The method of claim 28, wherein all nucleotide moieties in an individual multivalent molecule are dCTP.

34. The method of any one of claims 26-33, wherein, in an individual multivalent molecule, all detectable reporter moieties are the same.

35. The method of claim 34, wherein all detectable reporter moieties in an individual multivalent molecule comprise the same fluorescent label.

36. The method of any one of claims 26-35, wherein, in an individual multivalent molecule, all detectable reporter moieties are the same and all nucleotide moieties are the same.

37. The method of any one of claims 26-36, wherein an individual multivalent molecule comprises two, three, or four nucleotide moieties.

38. The method of any one of claims 26-37, wherein an individual multivalent molecule comprises two, three, or four detectable reporter moieties.

39. The method of any one of claims 26-38, wherein two or more nucleotide moieties in an individual multivalent molecule are associated with two or more different multivalent binding complexes on the same template nucleic acid molecule.

40. The method of any one of claims 26-39, comprising repeating steps (i)-(v) at least 1, 10, 20, 30, 40, 50, 70, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1500 times.

41. The method of any one of claims 26-40, comprising repeating steps (i)-(v) until the identities of the nucleotides in the target sequences have been determined.

42. The method of any one of claims 26-41, comprising, before step (i), dissociating the second polymerases from the extended nucleic acid duplexes under conditions sufficient to retain the plurality of extended nucleic acid duplexes.

43. The method of any one of claims 26-42, wherein the template nucleic acid molecules are single-stranded DNA molecules.

44. The method of any one of claims 26-43, wherein the nucleotides or analogs thereof comprise a removable chain terminating moiety at the 3’ sugar group.

45. The method of claim 44, wherein the removable chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group, and wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3 ’OH moiety on the sugar group.

46. The method of any one of claims 1-45, wherein the nucleotides or analogs thereof comprise a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

47. The method of any one of claims 1-46, wherein the nucleotides or analogs thereof comprise at least one fluorophore-labeled nucleotide analog.

48. The method of any one of claims 1-47, wherein the plurality of template nucleic acid molecules are immobilized on a support.

49. The method of claim 48, wherein the template nucleic acid molecules are immobilized on the support through hybridization to first surface primers immobilized on the support.

50. The method of claim 48, wherein the template nucleic acid molecules are covalently joined to first surface primers immobilized on the support.

51. The method of any one of claims 1-50, wherein the template nucleic acid molecules are clonally amplified template nucleic acid molecules.

52. The method of any one of claims 1-51, wherein the template nucleic acid molecules are generated through rolling circle amplification.

53. The method of any one of claims 1-52, wherein sequencing the plurality of template nucleic acid molecules generates a plurality of extended forward sequencing primer strands, and wherein the method comprises: a. retaining the plurality of template nucleic acid molecules and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands that are hybridized to the plurality of nucleic acid template molecules by conducting a primer extension reaction; b. removing the plurality of nucleic acid template molecules while retaining the plurality of forward extension strands and retaining the plurality of surface primers; and c. sequencing the plurality of retained forward extension strands.

54. The method of claim 53, wherein the template nucleic acid molecules comprise: i. two or more copies of the target sequence, ii. two or more copies of the binding sequence for a forward sequencing primer, and iii. two or more copies of a binding sequence for a reverse sequencing primer.

55. The method of claim 54, wherein the template nucleic acid molecules comprise binding sequences for an amplification primer, and wherein conducting the primer extension reaction comprises contacting the plurality of template nucleic acid molecules with a plurality of soluble amplification primers, a plurality of nucleotides and a plurality of polymerases, thereby generating a plurality of forward extension strands that are hybridized to template nucleic acid molecules.

56. The method of claim 55, wherein the plurality of amplification primers hybridize to the binding sequences for the amplification primers.

57. The method of claim 55 or 56, wherein the amplification primers are soluble.

58. The method of any one of claims 55-57, wherein the polymerases comprise phi29 DNA polymerases, large fragment of Bst DNA polymerases, large fragment of Bsu DNA polymerases (exo-), Bea DNA polymerases (exo-), Klenow fragment of E. coli DNA polymerases, T5 polymerases, M-MuLV reverse transcriptases, HIV viral reverse transcriptases, Deep Vent DNA polymerases or KOD DNA polymerases.

59. The method of any one of claims 1-58, wherein the nucleic acid template molecules comprise at least one nucleotide having a scissile moiety that can be cleaved to generate an abasic site.

60. The method of any one of claims 49-59, wherein the surface primers lack a nucleotide having a scissile moiety.

61. The method of any one of claims 59 or 60, wherein the nucleotide having a scissile moiety comprises uridine, 8-oxo-7,8-dihydrogunine, or deoxyinosine.

62. The method of any one of claims 59-61, wherein removing the nucleic acid template molecules comprises generating abasic sites in the nucleic acid template molecules, followed by generating gaps at the abasic sites.

63. The method of claim 62, wherein the at least one nucleotide having a scissile moiety comprises uracil, and generating abasic sites comprises contacting the nucleic acid template molecules with uracil DNA glycosylase (UDG).

64. The method of claim 62 or 63, wherein generating gaps at the abasic sites comprises contacting the abasic sites with an endonuclease IV, AP lyase, FPG glycosylase / AP lyase and / or endo VIII glycosylase / AP lyase.

65. The method of any one of claims 1-64, wherein individual template nucleic acid molecules comprise nucleic acid template molecules having up to 30% of thymidines replaced with uridine.

66. The method of any one of claims 53-65, wherein sequencing the plurality of retained forward extension strands generates a plurality of extended reverse sequencing primer strands, wherein individual retained forward extension strands have two or more extended reverse sequencing primer strands hybridized thereon.

67. The method of any one of claims 53-66, wherein sequencing the plurality of retained forward extension strands comprises a plurality of soluble reverse sequencing primers and (i) a plurality of a first polymerases and a plurality of multivalent molecules and (ii) a plurality of a second polymerases and a plurality of nucleotides or analogs thereof, thereby generating a plurality of extended reverse sequencing primer strands, wherein individual retained forward extension strands have two or more extended reverse sequencing primer strands hybridized thereon.

68. The method of any one of claims 53-67, wherein the nucleic acid template molecules comprise one or more copies of a binding sequence for a second surface primer.

69. The method of claim 68, comprising a plurality of second surface primers immobilized on the support, whereby binding of the second surface primers to the binding sequence for the second surface primers immobilizes free ends of the plurality nucleic acid template molecules on the support.

70. The method of any one of claims 53-69, wherein sequencing the plurality of retained forward extension strands comprises a. contacting:(i) the plurality of retained forward extension strands,(ii) a plurality of reverse sequencing primers comprising a sequence complementary to the binding sequence for the reverse sequencing primer,(iii) a plurality of first polymerases, and(iv) a plurality of multivalent molecules of Formula (I):(I)an ionized form thereof, an isomer thereof, or a salt thereof, wherein:C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety or absent; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3-(R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety, wherein each multivalent molecule comprises at least two nucleotide moieties and at least one detectable reporter moiety, wherein the contacting occurs under conditions sufficient to form a plurality of multivalent binding complexes comprising a nucleic acid duplex between a retained forward extension strand and a reverse sequencing primer, a first polymerase, and a nucleotide moiety of a multivalent molecule that is complementary to a nucleotide in the retained forward extension strand immediately adjacent to the 3’ end of the reverse sequencing primer, and wherein polymerase catalyzed incorporation of a complementary nucleotide moiety into the nucleic acid duplex is inhibited; b. detecting the detectable reporter moieties; and c. determining nucleobase identities of nucleotides in the retained forward extension strands complementary to the nucleotide moieties of the multivalent molecules based on the detectable reporter moieties of the multivalent molecules in the plurality of multivalent binding complexes formed in step (a).

71. The method of claim 70, wherein individual retained forward extension strands comprise two or more multivalent binding complexes.

72. The method of claim 70 or 71, wherein the plurality of reverse sequencing primers are soluble.

73. The method of any one of claims 70-72, comprising: d. dissociating the multivalent binding complexes under conditions sufficient to retain the nucleic acid duplexes, thereby generating a plurality of nucleic acid duplexes; and e. contacting the plurality of nucleic acid duplexes with a plurality of second polymerases and a plurality of nucleotides or analogs thereof under conditions sufficient to incorporate nucleotides or analogs thereof complementary to the nucleotides of the retained forward extension strands immediately adjacent to the 3’ ends of the reverse sequencing primers in a primer extension reaction, thereby generating a plurality of extended nucleic acid duplexes comprising extended reverse sequencing primer sequences.

74. The method of claim 73, comprising: g. dissociating the second polymerases from the extended nucleic acid duplexes under conditions sufficient to retain the plurality of extended nucleic acid duplexes.

75. The method of any one of claims 70-74, wherein the template nucleic acid molecules comprise concatemers of two or more copies of a sequence comprising (i) a sequence for the reverse sequencing primer, (ii) the target nucleic acid sequence, and (iii) a binding sequence for the forward sequencing primer.

76. The method of claim 75, wherein the two or more copies of a sequence complementary to (i) the sequence for the reverse sequencing primer hybridize to the reverse sequencing primers to form nucleic acid duplexes between the retained forward extension strands and the reverse sequencing primers.

77. The method of any one of claims 70-76 wherein, in an individual multivalent molecule, all nucleotide moieties are the same.

78. The method of claim 77, wherein all nucleotide moieties in an individual multivalent molecule are dATP.

79. The method of claim 77, wherein all nucleotide moieties in an individual multivalent molecule are dTTP.

80. The method of claim 77, wherein all nucleotide moieties in an individual multivalent molecule are dGTP.

81. The method of claim 77, wherein all nucleotide moieties in an individual multivalent molecule are dUTP82. The method of claim 77, wherein all nucleotide moieties in an individual multivalent molecule are dCTP.

83. The method of any one of claims 70-82, wherein, in an individual multivalent molecule, all detectable reporter moieties are the same.

84. The method of claim 83, wherein all detectable reporter moieties in an individual multivalent molecule comprise the same fluorescent label.

85. The method of any one of claims 70-84, wherein, in an individual multivalent molecule, all detectable reporter moieties are the same and all nucleotide moieties are the same.

86. The method of any one of claims 70-85, wherein an individual multivalent molecule comprises two, three, or four nucleotide moieties.

87. The method of any one of claims 70-86, wherein an individual multivalent molecule comprises two, three, or four detectable reporter moieties.

88. The method of any one of claims 70-87, comprising a. contacting the plurality of extended nucleic acid duplexes with a plurality of first polymerases and a plurality of multivalent molecules of Formula (I), or an ionized form thereof, an isomer thereof, or a salt thereof, wherein the contacting occurs under conditions sufficient to form a plurality of multivalent binding complexes comprising an extended nucleic acid duplex, a first polymerase, and a nucleotide moiety of a multivalent molecule that is complementary to a nucleotide in the retained forward extension strand immediately adjacent to the 3’ end of the extended reverse sequencing primer, and wherein polymerase catalyzed incorporation of a complementary nucleotide moiety into the extended nucleic acid duplex is inhibited; b. detecting the detectable reporter moieties; c. determining nucleobase identities of nucleotides in the retained forward extension strands complementary to the nucleotide moieties of the multivalent molecules based on the detectable reporter moieties of the multivalent molecules in the plurality of multivalent binding complexes formed in step (a);d. dissociating the multivalent binding complexes under conditions sufficient to retain the plurality extended nucleic acid duplexes; and e. contacting the plurality of extended nucleic acid duplexes with a plurality of second polymerases and a plurality of nucleotides or analogs thereof under conditions sufficient to incorporate nucleotides or analogs thereof complementary to the nucleotides of the nucleic acid template sequences immediately adjacent to the 3’ ends of the extended reverse sequencing primers in a primer extension reaction, thereby generating a plurality of extended nucleic acid duplexes comprising extended reverse sequencing primers.

89. The method of claim 88, wherein, in an individual multivalent molecule, the at least two nucleotide moieties are respectively attached to different X moieties.

90. The method of claim 88 or 89, wherein, in an individual multivalent molecule, all nucleotide moieties are the same.

91. The method of claim 90, wherein all nucleotide moieties in an individual multivalent molecule are dATP.

92. The method of claim 90, wherein all nucleotide moieties in an individual multivalent molecule are dTTP.

93. The method of claim 90, wherein all nucleotide moieties in an individual multivalent molecule are dGTP.

94. The method of claim 90, wherein all nucleotide moieties in an individual multivalent molecule are dUTP.

95. The method of claim 90, wherein all nucleotide moieties in an individual multivalent molecule are dCTP.

96. The method of any one of claims 88-95, wherein, in an individual multivalent molecule, all detectable reporter moieties are the same and all nucleotide moieties are the same.

97. The method of any one of claims 88-96, wherein an individual multivalent molecule comprises two, three, or four nucleotide moieties.

98. The method of any one of claims 88-97, wherein an individual multivalent molecule comprises two, three, or four detectable reporter moieties.

99. The method of any one of claims 88-98, wherein, in an individual multivalent molecule, all detectable reporter moieties are the same.

100. The method of claim 99, wherein all detectable reporter moieties in an individual multivalent molecule comprise the same fluorescent label.

101. The method of any one of claims 88-100, wherein two or more nucleotide moieties in an individual multivalent molecule contact two or more different multivalent binding complexes on the same retained forward extension strand.

102. The method of any one of claims 88-101, comprising repeating steps (a)-(e) at least 1, 10, 20, 30, 40, 50, 70, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1500 times.

103. The method of any one of 88-102, comprising repeating steps (a)-(e) until the identities of the nucleotides in sequences of the retained forward extension strands complementary to the target sequences have been determined.

104. The method of any one of claims 88-103, comprising, before step (a), dissociating the second polymerases from the extended nucleic acid duplexes under conditions sufficient to retain the plurality of extended nucleic acid duplexes.

105. The method of any one of claims 1-104, wherein template nucleic acid molecules comprise concatemers of two or more copies of a sequence comprising:(i) a binding sequence for a forward sequencing primer,(ii) sequence complementary to a binding sequence for a reverse sequencing primer,(iii) a binding sequence for an first surface primer,(iv) a binding sequence for a second surface primer,(v) a binding sequence for a first amplification primer,(vi) a binding sequence for a second amplification primer,(vii) a binding sequence for a soluble compaction oligonucleotide,(viii) a sample barcode sequence, and / or(ix) a unique molecular index sequence.

106. The method of any of claims 1-105, wherein, in an individual multivalent molecule, each X moiety comprises one or more nucleotide moiety.

107. The method of any one of claims 1-106, wherein, in an individual multivalent molecule, each X moiety comprises one or more detectable reporter moiety.

108. The method of any one of claims 1-107, wherein, in an individual multivalent molecule, each X moiety comprises one or more nucleotide moiety and one or more detectable reporter moiety.

109. The method of any one of claims 1-108, wherein each individual multivalent molecule comprises two, three, or four detectable reporter moieties and two, three, or four nucleotide moieties.

110. The method of any one of claims 1-109, wherein greater than 90%, greater than 95%, greater than 97%, greater than 98% or greater than 99% of bases have a quality score of Q30.

111. The method of any one of claims 1-109, wherein greater than 80%, greater than 85%, greater than 87%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94% or greater than 95% of bases have a quality score of Q40.

112. A multivalent molecule of Formula (I):(I) an ionized form thereof, an isomer thereof, or a salt thereof, wherein:C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety or absent; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3-(R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety.

113. The multivalent molecule of claim 112, wherein the multivalent molecule is of Formula (II):(II) an ionized form thereof, an isomer thereof, or a salt thereof, wherein:C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety;each C1independently is a Multivalent Branching Moiety; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3-(R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety; each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety.

114. The multivalent molecule of claim 112 or 113, wherein the multivalent molecule is of Formula (III):(III) an ionized form thereof, an isomer thereof, or a salt thereof.

115. The multivalent molecule of claim 112 or 113, wherein the multivalent molecule is of Formula (IV):(IV) an ionized form thereof, an isomer thereof, or a salt thereof.

116. The multivalent molecule of claim 112 or 113, wherein the multivalent molecule is of Formula (V):(V) an ionized form thereof, an isomer thereof, or a salt thereof.

117. The multivalent molecule of any one of claims 107-111, wherein the multivalent molecule comprises at least two nucleotide moieties and at least one detectable reporter moiety.

118. The multivalent molecule of claim 112, wherein the at least two nucleotide moieties are attached to different X moieties.

119. The multivalent molecule of any one of claims 112-118, wherein all the nucleotide moieties are the same.

120. The multivalent molecule of any one of claims 112-119, wherein all the nucleotide moieties are dATP.

121. The multivalent molecule of any one of claims 112-119, wherein all the nucleotide moieties are dTTP.

122. The multivalent molecule of any one of claims 112-119, wherein all the nucleotide moieties are dGTP.

123. The multivalent molecule of any one of claims 112-119, wherein all the nucleotide moieties are dUTP.

124. The multivalent molecule of any one of claims 112-119, wherein all the nucleotide moieties are dCTP.

125. The multivalent molecule of any one of claims 112-119, wherein all the detectable reporter moieties are the same.

126. The multivalent molecule of any one of claims 112-119, wherein all the detectable reporter moieties are the same and all the nucleotide moieties are the same.

127. The multivalent molecule of any one of claims 112-119, the molecule comprises two, three, or four detectable reporter moieties.

128. The multivalent molecule of any one of claims 112-119, the molecule comprises two, three, or four nucleotide moieties.

129. The multivalent molecule of any one of claims 112-119, wherein all detectable reporter moieties comprise the same fluorescent label.

130. A complex compri sing : a. the multivalent molecule of any one of claims 112-129; b. a polymerase; c. a template nucleic acid molecule comprising at least one of a target sequence and a binding sequence for a sequencing primer; and d. a sequence complementary to a portion of the template nucleic acid molecule comprising the sequencing primer sequence; wherein the template nucleic acid molecule and the sequence complementary to a portion of the template nucleic acid molecule form a duplex, and wherein a nucleotide moiety of the multivalent molecule binds to a complementary to a nucleotide of the template nucleic acid moleculeimmediately adjacent to the 3’ end of the sequence complementary to a portion of the template nucleic acid molecule.

131. The complex of claim 130, wherein the multivalent molecule comprises at least two nucleotide moieties, and wherein the at least two nucleotide moieties are the same.

132. The complex of claim 130 or 131, wherein the template nucleic acid molecule comprises a concatemer comprising at least two copies of a sequence comprising the target sequence and the binding sequence for a sequencing primer.

133. The complex of claim 132, wherein at least two complexes form on the same template nucleic acid molecule.

134. The complex of claim 132 or 133, wherein the at least two nucleotide moieties bind to complementary nucleotides of the template nucleic acid molecule in the at least two complexes.

135. The complex of claim 131, wherein at least two complexes form on at least two different template nucleic acid molecules.

136. The complex of claim 135, wherein the at least two different template nucleic acid molecules comprise the same target sequence.

137. The complex of claim 136, wherein the at least two nucleotide moieties bind to complementary nucleotides of the template nucleic acid molecules in the at least two complexes.

138. A method of sequencing a plurality of template nucleic acid molecules, comprising contacting the plurality of template nucleic acid molecules with a plurality of multivalent molecules of Formula (I):(I) an ionized form thereof, an isomer thereof, or a salt thereof, wherein:C is a Central Moiety; each X independently is -(L1-C1-(R1)1-6); each L1independently is a Bivalent Linking Moiety; each C1independently is a Multivalent Branching Moiety or absent; each R1independently is -T, L2-T or -L2-C2-(R2)2-6; each L2independently is a Bivalent Linking Moiety; each C2independently is a Multivalent Branching Moiety; each R2independently is L3-T or -L3-C3-(R3)2-6; each L3independently is a Bivalent Linking Moiety; each C3independently is a Multivalent Branching Moiety;each R3independently is L4-T or L4-C4-(R4)2-6; each L4independently is a Bivalent Linking Moiety; each C4independently is a Multivalent Branching Moiety; each R4independently is L5-T; each L5independently is a Bivalent Linking Moiety; and each T independently is a Terminal Moiety comprising a nucleotide moiety or a detectable reporter moiety, wherein each multivalent molecule comprises at least one nucleotide moiety and at one least detectable reporter moiety, wherein the contacting occurs under conditions sufficient for the at least one nucleotide moiety of the multivalent molecules in the plurality to bind to complementary nucleotide moieties in template nucleic acid molecules, and wherein the identities of the complementary nucleotide moieties in the template nucleic acid molecules are determined by detecting the detectable reporter moieties.

139. The method of claim 138, wherein the sequencing comprises polony sequencing, pyrosequencing, chain-terminator sequencing, ion-sensitive sequencing, probe-anchor ligation sequencing DNA nanoball sequencing, nanopore DNA sequencing, sequencing-by- hybridization or sequencing-by-binding.

140. The method or complex of any one of the previous claims, wherein the multivalent molecule is shown in FIG. 8A, 8B, or 8C.

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