Sequencing reagent conjugates and uses thereof

Abstract

Provided herein are conjugates of modified nucleotides comprising reactive groups configured to react with N-terminal amino acids, polymers (e.g., oligonucleotides) comprising the modified nucleotides, and methods of single molecule proteins sequencing using the same.

Description

WSGR Docket No. 60652-721601SEQUENCING REAGENT CONJUGATES AND USES THEREOFCROSS REFERENCE

[0001] This application claims the benefit of U. S. Provisional Patent App. No. 63 / 879,299, filed September 10, 2025, U. S. Provisional Patent App. No. 63 / 816,216, filed June 2, 2025, U. S. Provisional Patent App. No. 63 / 777,272, filed March 25, 2025, and U. S. Provisional Patent App. No. 63 / 737,996, filed December 23, 2024, each of which applications is incorporated by reference herein in its entirety.BACKGROUND

[0002] Proteins serve an important role at a cellular level, carrying out a variety of integral functions. Having the technology required to quantify and identify proteins is crucial to understanding their contributions to biological function. Advancements in proteomics have lagged behind while DNA sequencing has rapidly advanced the study of genomics primarily due to technologies that allow for high-throughput sequencing.

[0003] Mass spectrometry (MS) has enabled protein identification and quantification based on the mass / charge ratio of peptide fragments, which can be bioinformatically mapped back to a genomic database. However, MS has yet to quantify a complete set of proteins from a biological system, despite significant advancements. MS exhibits attomole detection for whole proteins and subattomole sensitives after fractionation. Yet, functionally-important, low copy-number proteins that make up about 10% of mammalian protein expression remain undetected.SUMMARY

[0004] Considering the present need for improved methods of single molecule protein sequencing, presented herein are conjugates of polymers (e.g., nucleotide compositions) and reacting groups configured to react with N-terminal residues of amino acids to enable single molecule protein sequencing. In some instances, these sequencing reagents allow for sequencing of polymeric analytes, such as polypeptides. Also provided herein are methods of sequencing polymeric analytes using the sequencing reagents provided herein. A brief summary of various exemplary embodiments is presented. Some simplifications and omissions may be made in the following summary, which is intended to highlight and introduce aspects of certain embodiments disclosed herein, but not to limit the scope of the disclosure. Detailed descriptions of various embodiments adequate to allow those of ordinary skill in the art to make and use the concepts disclosed herein will follow in later sections.WSGR Docket No. 60652-721601

[0005] In some embodiments, provided herein is a composition comprising a polymer substituted with -L-B, wherein L is a linker, wherein the linker comprises one or more electron-withdrawing or electron-donating groups, and B is a reactive group configured to react with an N-terminus of a peptide. In some embodiments, the polymer is a polynucleotide, a polypeptide, a polymeric material, or a combination thereof. In some embodiments, the composition is a polynucleotide composition comprising a modified nucleotide substituted with -L-B.

[0006] In some embodiments, provided herein is a polynucleotide composition comprising a modified nucleotide of Formula (IA):X 9n L^B°- yoO=p-O‘I°yFormula (I A)wherein,L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups; andB is a reactive group configured to react with an N-terminus of a peptide.

[0007] In some embodiments, the polynucleotide composition comprises a modified nucleotide of Formula (II A):X 9 L^Bo-p-o— \0®0\ / oO=P-O- I°yFormula (II A)wherein,Ring A is a nitrogenous base or modified version thereof;L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups; andB is a reactive group configured to react with an N-terminus of a peptide.WSGR Docket No. 60652-721601

[0008] In some embodiments, the polynucleotide composition comprises a modified nucleotide of Formula (IB), (IB-a), (IB-b), (IB-c), (IB-d), (IIB), or (IIB-a):OiO=P— L-BFormula (IB) Formula (IB-a) Formula (IB-b)O IO=P-O— L-BlFormula (IB-c)Formula (IB-d) Formula (vIIB)7Formula (IIB-a), orwherein,L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups;B is a reactive group configured to react with an N-terminus of a peptide;Xais O, CH(L-B), or CH2;Xbis O, CH(L-B), or CH2;Xcis absent, CH(L-B), or CH2;WSGR Docket No. 60652-721601Xdis absent, H, O, OH, halogen, CH(L-B), or OMe;Xeis absent, O, CH(L-B), or CH2.

[0009] In some embodiments, the polynucleotide composition comprises a modified nucleotide of Formula (IVA-A), (IVA-B), (IVA-C), (IVA-D), or (IVA-E):Formula (IVA- A) Formula (IVA-B) Formula (IVA-C)Formula (I VA-D) Formula (IVA-E)wherein,L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups; andB is a reactive group configured to react with an N-terminus of a peptide;L1and L2are each individually substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or a bond; andL3and L4are each individually substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or H.WSGR Docket No. 60652-721601

[0010] In some embodiments, the polynucleotide composition comprises a modified nucleotide of Formula (IVB-A), (IVB-B), or (IVB-C):Formula (IVB-A) Formula (IVB-B) Formula (IVB-C)wherein,L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups;B is a reactive group configured to react with an N-terminus of a peptide; and w )is cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, each optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2.[OH] In some embodiments, B is or comprises isothiocyanate (ITC). In some embodiments, the isothiocyanate is formed by conversion of an amine or a protected aminegroup. In some embodiments, B is or comprises the structure: N=C=S. In some embodiments, B is or comprises phenylisothiocyanate (PITC).

[0012] In some embodiments, B is or comprises -NRX(C=S)-LG; wherein Rxis independently H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl; and wherein LG is a leaving group. In some embodiments, B is or comprises -NH(C=S)-aryl or -NH(C=S)-heteroaryl. In some embodiments, the -NRX(C=S)-LG is -N(CH3)(C=S)-imidazole or -N(cyclopentyl)(C=S)-imidazole. In some embodiments, the nitrogen atom of the -NRX(C=S)-LG group is a member of the heterocycloalkyl or the heteroaryl. In some embodiments, Rxis five- or six-membered aryl or heteroaryl. In some embodiments, Rxis configured to facilitate an interaction with a peptide.WSGR Docket No. 60652-721601

[0013] In some embodiments, the polynucleotide composition further comprises one or more abasic nucleotides. In some embodiments, at least one of the one or more abasic nucleotides is immediately adjacent to the modified nucleotide.

[0014] In some embodiments, B has a structure selected from Table 3 A.

[0015] In some embodiments, -L-B has a structure selected from Table 3B.

[0016] In some embodiments, the modified nucleotide has a structure selected from Table 3C.

[0017] In some embodiments, the -L-B is or comprises the structure of Formula (V):LGFormula (V),wherein:LG is a leaving group;R1and R2are each independently hydrogen, 5- or 6- membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6- membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl, wherein the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0018] In some embodiments, the -L-B has the structure of Formula (V)

[0019] In some embodiments, the -L-B is or comprises the structure of Formula (VI):LGFormula (VI)WSGR Docket No. 60652-721601wherein:LG is a leaving group;R1is hydrogen, 5- or 6-membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl, wherein the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2, wherein each R” is independently H or C1-C3 alkyl; andn is an integer from 0 to 2.

[0020] In some embodiments, the -L-B has the structure of Formula (VI)

[0021] In some embodiments, the -L-B is or comprises the structure of any one of Formula (VILA), (VII-B), or (VILC):LG LG■' / , 7, orFormula (VILA) Formula (VILB) Formula (VILC)wherein,LG is a leaving group;L3 and L4 are each individually linkers; or— represents L3 and L4 being taken together to form Cs-Cs heterocycle;andR1is hydrogen, or 5- or 6-membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci- Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-memberedWSGR Docket No. 60652-721601heteroaryl, wherein the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0022] In some embodiments, the -L-B has the structure of any one of Formula (VILA), (VII-B), or (VII-C).

[0023] In some embodiments, the leaving group is: / YI-Y2X3-X4X3-X4 / / w / / " 4Y3x2 Xx5X2. XX5'YXi *14or0wherein,each of X1-X5 is independently selected from N, CH, or C-EWG; each of Y1-Y4 is independently selected from N, CH, or C-EWG; and EWG is an electron-withdrawing group;wherein at least one of X1-X5 is N.

[0024] In some embodiments, the modified polynucleotide has the structure selected from Table 4C.

[0025] In some embodiments, the -L-B is or comprises the structure of Formula (VIII):S AL S'R3Formula (VIII)wherein,R3is hydrogen or R’, wherein R’ is 5 or 6-membered aryl, 5 or 6- membered heteroaryl, or Ci-Ce alkyl optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6- membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0026] In some embodiments, the -L-B has the structure of Formula (VIII).WSGR Docket No. 60652-721601

[0027] In some embodiments, the -L-B is or comprises the structure of Formula (IX):SFormula (IX)wherein,R4is Ci-Ce alkylene, 5- or 6-membered cycloalkylene, or fused polycyloalkylene;R5is hydrogen or R’, wherein R’ is 5 or 6-membered aryl, 5 or 6- membered heteroaryl, or Ci-Ce alkyl optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6- membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0028] In some embodiments, the -L-B has the structure of Formula (IX).

[0029] In some embodiments, the modified polynucleotide has a structure selected from Table 5C.

[0030] In some embodiments, the -L-B is or comprises the structure of Formula (XI):Formula (XI),wherein,X6is O, S, Se, or NR10;R8is a bond, alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, or heteroarylene, each optionally substituted with one or more electron-donating or electron-withdrawing groups;R9is a protecting group configured for cleavage from said Formula (XI); andWSGR Docket No. 60652-721601R10is independently selected from a group comprising hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, aryl, or heteroaryl, each optionally substituted with one or more electron-donating or electron-withdrawing groups.

[0031] In some embodiments, the -L-B has the structure of Formula (XI).

[0032] In some embodiments, the -L-B is or comprises the structure of Formula (XII):R13Formula (XII),wherein,X7is O, S, SO, SR14, Se, SeO, SeR14, or NR14;X8is O, S, SR14, SOR14, SO2R14, Se, SeR14, SeOR14, SeO2R14or NR14; X9is O, S, Se, or NR14;R11is independently selected from a group comprising hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with one or more electron-donating or electron-withdrawing groups;R12is a protecting group configured for cleavage from said Formula (XII);R13is a bond, alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, or heteroarylene, each optionally substituted with one or more electron-donating or electron-withdrawing groups; andR14is independently selected from a group comprising hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, aryl, or heteroaryl, each optionally substituted with one or more electron-donating or electron-withdrawing groups.WSGR Docket No. 60652-721601

[0033] In some embodiments, the -L-B has the structure of Formula (XII).

[0034] In some embodiments, the modified polynucleotide has a structure selected from Table 6C.

[0035] In some embodiments, the linker comprises a moiety that is derived from a click chemistry moiety.

[0036] In some embodiments, the linker comprises a moiety that is derived from bicyclononyne (BCN), an alkyne, an azide, dibenzocyclooctyne (DBCO), tetrazine, transcyclooctene (TCO), or a structure of Formula (X):Formula (X)whereinR6is coupling moiety;R7is Ci-Ce alkylene or Ci-Ce heteroalkylene optionally substituted with oxo or absent;X is CH or N; andeach m is independently an integer from 1 to 5.

[0037] In some embodiments, the compound of Formula (X) has the structure of Formula (X-A):Formula (X-A)whereinR7is Ci-Ce alkylene optionally substituted with oxo or absent;X is CH or N;p is an integer from 0 to 6; andeach m is independently an integer from 1 to 5.WSGR Docket No. 60652-721601

[0038] In some embodiments, the compound of Formula (X) is or comprises the compound of Formula (X-A).

[0039] In some embodiments, the compound of Formula (X-A) is selected from:

[0040] In some embodiments, the linker comprises a bond, alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, arylene, or heteroarylene, each optionally substituted with one or more electron-withdrawing or electron-donating groups.

[0041] In some embodiments, the linker comprises one or more structures selected from Table 7.

[0042] In some embodiments, the polynucleotide composition is a sequencing reagent.

[0043] In some embodiments, provided herein are methods of using the sequencing reagents provided herein, the method comprising:(a) providing the sequencing reagent and a polymeric analyte comprising a plurality of monomers, wherein the sequencing reagent optionally comprises a catalytic group;(b) contacting the sequencing reagent with a monomer of the plurality of monomers, thereby generating a sequencing reagent-monomer complex;(c) cleaving a monomer of the polymeric analyte, thereby providing a modified monomer; and(d) detecting the modified monomer.

[0044] In some embodiments, the method further comprises prior to (a), converting a primary amine group or a protected primary amine group to an isothiocyanate, thereby generating the sequencing reagent.

[0045] In some embodiments, the polymeric analyte comprises a polypeptide, and wherein the monomer is an N-terminal amino acid. In some embodiments, the detecting of (d) comprises contacting the modified monomer complex with a binding agent. In some embodiments, the binding agent comprises a polymerizable molecule, and wherein the polymerizable molecule comprises a nucleic acid molecule.WSGR Docket No. 60652-721601

[0046] In some embodiments, the method further comprises coupling the sequencing reagent or the sequencing reagent-monomer complex to a capture moiety.

[0047] In some embodiments, the detecting is performed using a nanopore sequencer.

[0048] In some embodiments, the method further comprises:(e) contacting the polymeric analyte with an additional sequencing reagent, wherein the additional sequencing reagent binds to an additional monomer of the plurality of monomers; wherein binding of the additional sequencing reagent to the additional monomer of the polymeric analyte provides an additional sequencing reagent-monomer complex;(f) coupling the additional sequencing reagent to the modified monomer; and (g) cleaving the additional sequencing reagent-monomer complex from the polymeric analyte, thereby providing a stacked sequencing reagent-monomer complex.

[0049] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0050] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:WSGR Docket No. 60652-721601

[0052] FIG. 1A schematically shows an example workflow for processing polymeric analytes molecules (e.g., peptides) described herein. FIG. 1B schematically shows another example workflow for processing polymeric analytes in solution or on a substrate. FIG. 1C schematically shows another example workflow for processing polymeric analytes in solution or on a substrate. FIG. 1D schematically shows yet another example workflow for processing polymeric analytes in solution or on a substrate.

[0053] FIG.2A schematically shows an example workflow for processing polymeric analytes molecules (e.g., peptides) described herein. FIG.2B schematically shows another example workflow for processing polymeric analytes in solution. FIG.2C schematically shows another example workflow for processing polymeric analytes and detection. FIG.2D schematically shows another example workflow for processing polymeric analytes and detection using a nanopore sequencing system.

[0054] FIG. 3 schematically shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0055] FIG. 4 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0056] FIG. 5 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0057] FIG. 6 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0058] FIG. 7 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0059] FIG. 8 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0060] FIG. 9 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0061] FIG. 10A shows an exemplary scheme for preparation of a modified nucleotide provided herein. FIG. 10B shows another exemplary scheme for preparation of a modified nucleotide provided herein.

[0062] FIG. 11 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0063] FIG. 12 shows an exemplary scheme for the preparation and use of a modified nucleotide provided herein.WSGR Docket No. 60652-721601

[0064] FIG. 13 shows an exemplary scheme for the preparation and use of a modified nucleotide provided herein.

[0065] FIG. 14A shows an exemplary scheme for the preparation and use of a modified nucleotide provided herein. FIG. 14B shows liquid chromatography and mass spectrometry spectra of a modified nucleotide provided herein.

[0066] FIG. 15 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0067] FIG. 16A shows an exemplary scheme for preparation of a modified nucleotide provided herein. FIG. 16B shows another exemplary scheme for preparation of a modified nucleotide provided herein.

[0068] FIG. 17 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0069] FIG. 18A shows an exemplary scheme for preparation of a modified nucleotide provided herein. FIG. 18B shows an exemplary scheme for an Edman-like degradation process using a modified nucleotide with a -(CH2CH2)-(2,5-pyridindiyl )- structure in a controlled removal of a terminal amino acid from a peptide.

[0070] FIG. 19 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0071] FIG. 20 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0072] FIG. 21 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0073] FIG. 22 shows an exemplary scheme for preparation of a modified nucleotide provided herein.

[0074] FIG. 23 shows an exemplary scheme for an Edman-like degradation process using a modified nucleotide with an N-methyl thiocarbamoyl (N-methyl-N-phenyl-lH-imidazole-1-carbothioamide) in a controlled removal of a terminal amino acid from a peptide.

[0075] FIG. 24 shows an exemplary scheme for preparation of a modified nucleotide provided herein and an exemplary scheme for an Edman-like degradation process in the controlled removal of a terminal amino acid from a peptide.

[0076] FIG. 25A shows an exemplary scheme for preparation of a modified nucleotide provided herein. FIG. 25B shows an exemplary scheme for an Edman-like degradation process in the controlled removal of a terminal amino acid from a peptide.WSGR Docket No. 60652-721601

[0077] FIG. 26A shows an exemplary scheme for preparation of a modified nucleotide provided herein. FIG. 26B shows an exemplary scheme for an Edman-like degradation process in the controlled removal of a terminal amino acid from a peptide.

[0078] FIG. 27A shows an exemplary scheme for preparation of a modified nucleotide provided herein. FIG. 27B shows an exemplary scheme for an Edman-like degradation process in the controlled removal of a terminal amino acid from a peptide.

[0079] FIG. 28A schematically shows an example workflow using a modified nucleotide to remove and characterize a terminal amino acid from a peptide. FIG. 28B shows linker structures of six modified nucleotides. FIG. 28C is a classification matrix showing the impact of linker structure on the accuracy and specificity of amino acid identification using modified nucleotides.FIG. 28D is a t-SNE analysis showing a visual representation of the classification performance for modified amino acids using modified nucleotides.

[0080] FIG. 29A schematically shows an example using two different sequencing reagents having the same modified nucleotide to characterize a terminal amino acid from a peptide. FIG.29B shows exemplary classification performance for a panel of modified amino acids and control oligonucleotides using RC25 in normalized confusion matrices. FIG. 29C shows exemplary classification performance for a panel of modified amino acids and control oligonucleotides using RC96 in normalized confusion matrices.DETAILED DESCRIPTION

[0081] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.Definitions

[0082] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0083] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,”WSGR Docket No. 60652-721601“less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0084] References to “one embodiment,” “an embodiment,” “example embodiment,” “some embodiments,” “certain embodiments,” “various embodiments,” etc., indicate that the embodiment(s) of the disclosed technology so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment” does not necessarily refer to the same embodiment, although it may.

[0085] Ranges may be expressed herein as from “about” or “approximately” or “substantially” one particular value and / or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and / or to the other particular value. Further, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.

[0086] By “comprising” or “containing” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named.

[0087] Throughout this description, various components may be identified having specific values or parameters, however, these items are provided as exemplary embodiments. Indeed, the exemplary embodiments do not limit the various aspects and concepts of the present disclosure as many comparable parameters, sizes, ranges, and / or values may be implemented. The terms “first,” “second,” and the like, “primary,” “secondary,” and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.WSGR Docket No. 60652-721601

[0088] As used herein, the term “protein” generally refers to a molecule comprising two or more amino acids joined by a peptide bond. A protein may also be referred to as a “polypeptide”, “oligopeptide”, or “peptide”. A protein can be a naturally occurring molecule, or a synthetic molecule (e.g., an artificial protein, peptide, enzyme). A protein may include one or more non-natural amino acids, modified amino acids, or non-amino acid linkers. A protein may contain D-amino acid enantiomers, L- amino acid enantiomers or both. Amino acids of a protein may be modified naturally or synthetically, such as by post-translational modifications or by chemical modification. In some circumstances, different proteins may be distinguished from each other based on different genes from which they are expressed in an organism, different primary sequence length or different primary sequence composition. Proteins expressed from the same gene may nonetheless be different proteoforms, for example, being distinguished based on non-identical length, non-identical amino acid sequence or non-identical post-translational modifications. Different proteins can be distinguished based on one or both of gene of origin and proteoform state.

[0089] As used herein, the term “peptide” may refer to any short, single peptide chain. A peptide may be no more than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less than about 5 amino acids in length. A peptide may have a known or unknown biological function or activity. Peptides can include natural, synthetic, modified, or degraded proteins or peptides, or a combination thereof. Peptides can include proteinogenic, natural, synthetic, or modified amino acids or amino acid residues, or a combination thereof.

[0090] As used herein, the term “single analyte” may refer to an analyte that is individually manipulated or distinguished from other analytes. A single analyte may comprise a biomolecule or a synthetic molecule. A single analyte may comprise a small molecule. A single analyte can be a single molecule (e.g., a single biomolecule such as a single protein, nucleic acid molecule, affinity reagent, lipid, carbohydrate, metabolite, hapten, small molecule, pharmaceutical compound, nanoparticle, amino acid derivative, synthetic amino acid, etc.), a single complex of two or more molecules (e.g., a multimeric protein having two or more separable subunits, a single protein attached to a nucleic acid molecule or a single protein attached to an affinity reagent), a single particle, or the like. Reference herein to a “single analyte” in the context of a composition, system or method herein does not necessarily exclude application of the composition, system or method to multiple single analytes that are manipulated or distinguished individually, unless indicated contextually or explicitly to the contrary.

[0091] As used herein, “polypeptide” refers to two or more amino acids linked together by a peptide bond. The term “polypeptide” includes proteins that have a C-terminal end and an N-WSGR Docket No. 60652-721601terminal end as generally known in the art and may be synthetic in origin or naturally occurring. As used herein “at least a portion of the polypeptide” refers to 2 or more amino acids of the polypeptide. A polypeptide may comprise one or more peptides. Optionally, a portion of the polypeptide includes at least: 1, 5, 10, 20, 30 or 50 amino acids, either consecutive or with gaps, of the complete amino acid sequence of the polypeptide, or the full amino acid sequence of the polypeptide.

[0092] As used herein, “affixed” refers to a connection between two molecules that are held in physical proximity. The term “affixed” encompasses both an indirect or direct connection and may be reversible or irreversible, for example the connection is optionally a covalent bond or a non-covalent bond.

[0093] As used herein, the term “sample” refers to a collected substance or material that comprises or is suspected to comprise one or more analytes of interest (e.g., biomolecules, e.g., polypeptides). A sample may be modified for purposes such as storage or stability. A sample may be naturally occurring or synthetic. A sample may be processed to separate or remove unwanted fractions or impurities from the analyte(s) of interest. A sample may be enriched or purified. For example, a sample may comprise a fraction of a separation process (e.g., chromatography, fractionation, electrophoresis, etc.). Alternatively, a sample may not be subjected to processing that separates or removes any unwanted fractions or impurities from the analyte(s) of interest. A sample may be obtained from any suitable source or location, including from organisms, cells, tissues, cell preparations, cell-free compositions, the environment (e.g., air, water, dirt, soil, agriculture, soil, dust, sewage). A sample may be obtained from an organism or part of an organism, such as from a fluid, tissue, or cell. A sample may include biological and / or non-biological components. As used herein, the terms “biological sample” or “biological source” refer to a sample that is derived from a predominantly biological system or organism, such as one or more viral particles, cells (e.g. individualized cells), organelles (e.g. individualized organelles), tissues, organs, bodily fluids, bone, cartilage, and exoskeleton. A biological sample may comprise a prokaryotic cell (e.g., bacteria) or eukaryotic cell (e.g., fungus, protist, algae, plant, animal). A biological sample may comprise a majority of biological material on a mass basis, excluding the weight of fluid within the sample. Biological samples may comprise one or more proteins, referred to herein as protein samples. Biological samples can be acquired from various sources, e.g., from a clinical patient sample, such as blood, serum, plasma, Cerebral Spinal Fluid (CSF), saliva, mucosal secretions, sputum, urine, lymph, perspiration, vaginal fluid, semen, fecal matter, amniotic fluid, perspiration, synovial fluid, fine needle aspirates, a tissue biopsy, a tumor biopsy, etc. A biological sample may be processed toWSGR Docket No. 60652-721601purify and retain one or more biomolecules (e.g., proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, metabolites, etc.) from the biological sample. A biological sample (e.g., a protein sample) may be derived from cultured cells, which may be treated or untreated. A biological sample (e.g., a protein sample) can also result from tissue specimens, such as biopsy samples, which may optionally be processed to liberate biomolecules (e.g., proteins) contained therein. Tissue samples may also be derived from in vivo specimens, including fresh, frozen, acute, and fixed tissues. A sample or a biological sample may comprise non-biological molecules, including but not limited to nanoparticles, polymers, haptens, small molecules, chemicals, fluorescent reagents, inert materials, pharmaceuticals, food additives, environmental contaminants, solvents, industrial chemicals, nanomaterials, radioisotopes, by-products from non-biological molecules.

[0094] As used herein, the term “hydrogel” refers to a three-dimensional polymeric structure that is substantially insoluble in water, but which is capable of absorbing and retaining large quantities of water to form a substantially stable, often soft and pliable, structure. In some embodiments, water can penetrate in between polymer chains of a polymer network, subsequently causing swelling and the formation of a hydrogel. In some embodiments, hydrogels are super-absorbent (e.g., containing more than about 90% water) and can be included of natural or synthetic polymers. Examples of hydrogels include but are not limited to, hyaluronans, chitosans, agar, heparin, sulfate, cellulose, alginates (including alginate sulfate), collagen, dextrans (including dextran sulfate), pectin, carrageenan, polylysine, gelatins (including gelatin type A), agarose, (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, PEO — PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, polyethylene imine), polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N, N'-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations thereof. A detailed description of suitable hydrogels may be found in published U. S. Patent Publication US 2010 / 0055733, herein specifically incorporated by reference. As used herein, the terms “hydrogel subunits” or “hydrogelWSGR Docket No. 60652-721601precursors” mean hydrophilic monomers, prepolymers, or polymers that can be crosslinked, or “polymerized”, to form a three-dimensional (3D) hydrogel network. It is believed that this fixation of the biological specimen in the presence of hydrogel subunits crosslinks the components of the specimen to the hydrogel subunits, thereby securing molecular components in place, preserving the tissue architecture and cell morphology.

[0095] As used herein, the terms “antibody” and “immunoglobulin” may generally refer to proteins that can recognize and bind to a specific antigen. An antibody or immunoglobulin may refer to an antibody isotype, fragments of antibodies including, but not limited to, Fab, Fv, scFv, vHH, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins including an antigen-binding portion of an antibody and a non-antibody protein. The antibodies may be detectably labeled, e.g, with a fluorophore, radioisotope, enzyme (e.g., a peroxidase), epitope tag, which generates a detectable product, fluorescent protein, nucleic acid barcode sequence, and the like. The antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. Also encompassed by the terms are nanobodies, Fab', Fv, F(ab')2, scFv, and other antibody fragments that retain specific binding to antigen. Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and (Fab)2, diabodies, monobodies, single domain antibodies (sdAb), as well as bi-functional (z.e., bi-specific, e.g., bi-specific T-cell engager) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U. S. A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference). (See, generally, Hood et al., Immunology, Benjamin, N. Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are herein incorporated by reference). Naturally occurring immunoglobulins or antibody types include immunoglobulin A, immunoglobulin G, immunoglobulin D, immunoglobulin E, immunoglobulin M, or other immunoreactive components.

[0096] “Binding” or “coupling” as used herein generally refers to a covalent or non-covalent interaction between two molecules (referred to herein as “binding partners”, e.g., a substrate and an enzyme or an antibody and an epitope). Binding between binding partners may be specific or non-specific. Binding between binding partners may involve one or more additional molecules (e.g, biomolecules) or enhancer molecules or substrates.

[0097] As used herein, “specifically binds” or “binds specifically” generally refers to an interaction between binding partners (e.g., a binding partner and a cognate molecule) such that the binding partners bind to one another, but do not bind to other molecules that may be presentWSGR Docket No. 60652-721601in the environment (e.g., in a biological sample, in tissue, in an in vitro assay) under a set of conditions. A specific binding interaction may entail a binding partner that binds to a cognate molecule. The specific binding interaction may entail the binding of the binding partner to its cognate molecule at a significantly or substantially higher level or with greater affinity as compared to the binding of the binding partner to a non-cognate molecule. A specific binding interaction may entail a first binding partner that has greater selectivity of binding to the cognate molecule as compared to a non-cognate molecule.

[0098] The terms “nucleic acid”, “nucleic acid molecule”, “oligonucleotide” and “polynucleotide” may be used interchangeably herein and generally refer to a polymeric form of naturally occurring or synthetic nucleotides, or analogs thereof, of any length. A nucleic acid molecule may comprise one or more deoxyribonucleotides, deoxynucleotide triphosphates, dideoxynucleotide triphosphates, deoxynucleotide hexaphosphates, dideoxynucleotide hexaphosphates, ribonucleotides, hexitol nucleotides, cyclohexane nucleotides, or analogs or combinations thereof. A nucleic acid molecule may comprise, e.g., DNA, RNA, HNA, LNA, GNA, PNA, CeNA, and modified forms thereof. A nucleic acid molecule may comprise nucleotides that are linked by phosphodiester bonds or other types of linkages, including but not limited to phosphate-alkyl backbones, phosphorothioate linkages, boronophosphate linkages, peptide nucleic acid (PNA) backbones, and other synthetic or modified internucleotide linkages. In some embodiments, a modified nucleic acid molecule may comprise a modified nucleotide in which one or more structural components — such as the base, sugar, or phosphate backbone — are chemically altered or replaced with synthetic or non-naturally occurring analogs. In some embodiments, a modified nucleotide may comprise a non-canonical base, such as a purine or pyrimidine analog. In some embodiments, a modified nucleotide may comprise a sugar modification, such as a LNA or GNA. In some embodiments, a modified nucleotide may lack a conventional sugar moiety and instead possess an alternative backbone structure, such as a phosphate-alkyl backbone, a phosphate-sugar backbone, or a peptide backbone, as found in PNAs. In some embodiments, a modified nucleotide may comprise a modified phosphate backbone without a conventional sugar or base component. The modified nucleotides described herein may be incorporated into polynucleotides to modulate stability, hybridization properties, enzymatic recognition, or other functional characteristics. A nucleic acid molecule may have any two- or three-dimensional structure, and may perform any function, known or unknown. A nucleic acid molecule may be single stranded, double stranded, or partially double stranded. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, noncoding RNA, small interferingWSGR Docket No. 60652-721601RNA, short hairpin RNA, micro RNA, scaRNA, ribozymes, riboswitches, viral RNA, complementary DNA (cDNA), cosmid DNA, mitochondrial DNA, chromosomal or genomic DNA, viral DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, nucleic acid adapters, and primers. The nucleic acid molecule may be linear, circular, or any other geometry. Examples of polynucleotide analogs include but are not limited to xeno nucleic acid (XNA), bridged nucleic acid (BNA), glycol nucleic acid (GNA), hexitol nucleic acid (HNA), cyclohexane nucleic acid (CeNA), 2’-F-Arabinonucleic acids (2’-F-ANA), peptide nucleic acids (PNAs), yPNAs, morpholino polynucleotides, locked nucleic acids (LNAs), nonconstrained nucleic acid (NNA), threose nucleic acid (TNA), D-threoninol nucleic acid (D-aTNA), L-threoninol nucleic acid (L-aTNA), 2'-O-Methyl polynucleotides, 2'-O-alkyl ribosyl substituted polynucleotides, phosphorothioate polynucleotides, and boronophosphate polynucleotides. A polynucleotide analog may possess purine or pyrimidine analogs, including for example, 7-deaza purine analogs, 8-halopurine analogs, 5-halopyrimidine analogs, inverted base, or universal base analogs that can pair with any base, including hypoxanthine, nitroazoles, isocarbostyril analogues, azole carboxamides, and aromatic triazole analogues, or base analogs with additional functionality, such as a biotin moiety for affinity binding. In addition, a nucleic acid molecule may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

[0099] As used herein, the term “amino acid” generally refers to an organic compound that combines to form a protein or peptide. An amino acid generally comprises an amine group, a carboxylic acid group, and a side-chain specific to each amino acid, which serve as a monomeric subunit of a peptide. An amino acid may include the 20 standard, naturally occurring or canonical amino acids as well as non-standard or non-canonical amino acids. The standard, naturally-occurring or canonical amino acids include Alanine (A or Ala), Cysteine (C or Cys), Aspartic Acid (D or Asp), Glutamic Acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or He), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Vai), Tryptophan (W or Trp), and Tyrosine (Y or Tyr). An amino acid may be an L-amino acid or a D-amino acid. Non-standard amino acids may be modified amino acids, amino acid analogs, amino acid mimetics, non-standard proteinogenic amino acids, or non-proteinogenic amino acids that occur naturally or are chemically synthesized. Examples of non-standard amino acids include, but are not limited to, selenocysteine, pyrrolysine, and N-formylmethionine, (3 -amino acids, Homo-WSGR Docket No. 60652-721601amino acids, Proline and Pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, and N-methyl amino acids.

[0100] As used herein, the term “amino acid type” generally refers to one of the standard, naturally-occurring or canonical amino acids, e.g., one member of the group consisting of Alanine (A or Ala), Cysteine (C or Cys), Aspartic Acid (D or Asp), Glutamic Acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or He), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Vai), Tryptophan (W or Trp), Tyrosine (Y or Tyr), derivatives thereof, and modified forms of any of the aforementioned amino acids. The term “amino acid type” may be used herein to distinguish a plurality of amino acids that comprise different side chain groups, rather than a plurality of amino acids that are identical (e.g., different positional amino acids of a single peptide that have the same side chain). An amino acid type may comprise a modified version of one of the standard, naturally-occurring or canonical amino acids e.g., post translational modifications, an epigenetic modification, or chemical or enzymatic modifications. In some instances, an amino acid type can include non-canonical amino acids.

[0101] As used herein, the term “post-translational modification” refers to modifications that occur on a peptide subsequent to translation. A post-translational modification may be a covalent modification or enzymatic modification. Examples of post-translation modifications include, but are not limited to, acylation, acetylation, alkylation (including methylation), benzoylation, biotinylation, butyrylation, carb amyl ati on, carbonylation, carboxylation, crotonylation, deamidation, deiminiation, dimethylation, diphthamide formation, disulfide bridge formation, eliminylation, flavin attachment, formylation, gamma-carboxylation, glutamyl ati on, glutarylation, glycylation, glycosylation, glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, isoprenylation, lipidation, lipoylation, malonylation, methylation, myristolylation, nitration, oxidation, transglutamination, palmitoylation, pegylation, phosphopantetheinylation, phosphorylation, prenylation, propionylation, pyroglutamate formation, retinylidene Schiff base formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, S-adenosylation, sulfation, selenation, stearoyl ati on, succinylation, sulfination, trimethylation, ubiquitination, and C-terminal amidation. A post-translational modification includes modifications of the amino terminus and / or the carboxyl terminus of a peptide. Modifications (both naturally occurring and synthetic) of the terminal amino group include, but are not limited to, des-amino, N-lower alkyl, N-di-WSGR Docket No. 60652-721601lower alkyl, and N-acyl modifications, N-terminal cyclization, deamination, oxidation, ubiquitination, SUMOylation, Neddylation, ISGylation, pupylation, eliminylation, biotinylation, lipidation, N-terminal methylation, N-terminal acetylation, N-terminal propionylation, N-terminal butyrylation, N-terminal crotonylation, N-terminal myristoylation, N-terminal palmitoylation, N-terminal stearoyl ati on, and N-terminal benzoylation. Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., wherein lower alkyl is C1-C4 alkyl). A post-translational modification also includes modifications, such as but not limited to those described above, of amino acids falling between the amino and carboxy termini. The term post-translational modification can also include peptide modifications that include one or more detectable labels. A post-translational modification may be naturally occurring or synthetic.

[0102] As used herein, the term “binding agent” refers to a molecule, e.g., a nucleic acid molecule, a peptide, a polypeptide, a protein, carbohydrate, a synthetic molecule, or a small molecule that binds to, associates with, unites with, recognizes, or combines with another molecule. The binding agent may bind to a macromolecule or a component or feature of a macromolecule. A binding agent may form a covalent association or non-covalent association with a molecule, a macromolecule, or a component or feature of a macromolecule. A binding agent may also be a chimeric binding agent, composed of two or more types of molecules, such as a nucleic acid molecule-peptide chimeric binding agent, a carbohydrate-peptide chimeric binding agent, or a lipid-peptide chimeric binding agent. A binding agent may be a naturally occurring, synthetically produced, or recombinantly expressed molecule. A binding agent may bind to a single monomer or subunit of a polymeric analyte, such as a macromolecule (e.g., a single amino acid of a peptide) or bind to a plurality of linked subunits of a macromolecule (e.g., a di-peptide, tri-peptide, or higher order peptide of a longer peptide, polypeptide, or protein molecule). A binding agent may bind to a linear molecule or a molecule having a three-dimensional structure (also referred to as conformation). For example, an antibody binding agent may bind to linear peptide, polypeptide, or protein, or bind to a conformational peptide, polypeptide, or protein. A binding agent may bind to an N-terminal peptide, a C-terminal peptide, or an intervening peptide of a peptide, polypeptide, or protein molecule. A binding agent may bind to an N-terminal amino acid, C-terminal amino acid, or an intervening amino acid of a peptide molecule. A binding agent may preferably bind to a chemically modified or labeled amino acid over a non-modified or unlabeled amino acid. For example, a binding agent may preferably bind to an amino acid that has been modified with an acetyl moiety, guanyl moiety, dansyl moiety, PTC moiety, DNP moiety, SNP moiety, etc., over an amino acid thatWSGR Docket No. 60652-721601does not possess such a moiety. A binding agent may bind to a post-translational modification, either naturally occurring or synthetic, of a peptide molecule. A binding agent may exhibit selective binding to a component or feature of a macromolecule (e.g., a binding agent may selectively bind to one of the 20 possible natural amino acid residues and bind with very low affinity or not at all to the other 19 natural amino acid residues). A binding agent may exhibit less selective binding, where the binding agent is capable of binding a plurality of components or features of a macromolecule (e.g., a binding agent may bind with similar affinity to two or more different amino acid residues). A binding agent may comprise a tag, which may be coupled to the binding agent via a linker.

[0103] As used herein, the term “linker” generally refers to a molecule or moiety that is involved in joining two or more molecules. A linker may facilitate a covalent or noncovalent interaction of two or more molecules. A linker may be a crosslinker. The linker can be unifunctional, bifunctional, trifunctional, quadrifunctional, or polyfunctional. A linker can be chiral or achiral, or may contain one or more chiral centers, to influence enzymatic or chemical reactivity, conformational dynamics, steric interactions, or electronic properties of the linked molecules. A linker can be or comprise a nucleotide, a nucleotide analog, an amino acid, a peptide, a polypeptide, or a non-nucleotide chemical moiety, such as an organic or inorganic compound. A linker may comprise a polymer, such as a polyethylene glycol (PEG), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), poly-L-lysine (PLL), poly (DL-lactic acid) (PLA), poly (DL-lactide-co-glycoside) (PLGA), polyomithine, polyarginine, or other organic or inorganic polymer. A linker may comprise a homopolymer, which contains a single type of repeating monomer unit. A linker may comprise a copolymer, which contains two or more different types of monomer units. In some embodiments, the copolymer is a random copolymer, in which different monomer units are distributed randomly along the polymer chain. In some embodiments, the copolymer is an alternating copolymer, in which the monomers alternate in a regular sequence (e.g., ABABAB...). In some embodiments, the copolymer is a block copolymer, comprising extended sequences or blocks of one monomer type followed by blocks of another (e.g., AAA.. BBB...). In some embodiments, the copolymer is a graft copolymer, in which side chains of one monomer type are grafted onto the backbone of a different polymer. In some embodiments, the choice of homopolymer or copolymer structure may influence the physical, chemical, or biological properties of the linker. A linker may comprise one or more reactive ends, e.g., an amine-reactive group, a carboxyl-reactive group, a sulfhydryl-reactive group, a hydroxylreactive group, etc. Alternatively, a linker may not comprise a reactive end. In some examples, aWSGR Docket No. 60652-721601linker may be used to join different molecule types, e.g., different biomolecule types such as a peptide with a nucleic acid molecule, a lipid with a peptide, a carbohydrate with a peptide, etc.; non-biomolecule types; or a biomolecule to a non-biomolecule. For example, a linker may be used to join a binding agent with a tag, a tag with a macromolecule (e.g., peptide, nucleic acid molecule), a macromolecule with a solid support, a tag with a solid support, etc. A linker may join two molecules via enzymatic reaction or chemistry reaction e.g., click chemistry). A linker may join more than two molecules, e.g., via enzymatic or chemical reactions. A linker may influence reaction kinetics, yield, or product specificity by modulating molecular proximity, steric hindrance, or electronic environment. For example, a linker may enhance or inhibit reaction rates by controlling the spatial arrangement of reactants, stabilizing intermediates, or facilitating catalytic interactions. In some reactions, a linker may stabilize or position reaction intermediates in a favorable orientation, thereby influencing reaction efficiency, pathway selection, or product distribution. In chemical synthesis, a linker may dictate regioselectivity or stereoselectivity by constraining molecular conformation. Additionally, a linker may participate in dynamic structural changes, enabling conformational flexibility or rigidity to promote or suppress specific reaction pathways. A linker may modulate product stability, for example, by reducing susceptibility to hydrolysis or degradation. In some cases, a linker may introduce functional groups that participate in subsequent transformations, thereby influencing multi-step reaction cascades. A linker can be relatively linear or non-linear, e.g., cyclic or circularized, branched, polygonal, etc.

[0104] The term “conjugated” as used herein generally refers to a covalent or ionic interaction between two entities, e.g., molecules, compounds, or combinations thereof.

[0105] As used herein, the term “tag” generally refers to a molecule or moiety that is conjugated to a molecule. A tag may comprise a detectable label, e.g., a fluorophore or fluorescent protein, a radioactive isotope, an enzyme (e.g., a chromogenic or fluorescent protein, proteins that can catalyze chromogenic substrates), a mass tag, a hapten (e.g., biotin, digoxigenin, urushiol, fluorescein), a vibrational or FTIR tag (e.g., alkyne group). A tag may comprise a biomolecule, such as a nucleic acid molecule, a protein, a lipid, a carbohydrate, or a combination thereof. A tag may comprise one or more nucleic acid molecules, which may optionally encode information regarding the tag or the molecule onto which a tag is conjugated (e.g., a binding agent, such as an antibody). For example, a tag may comprise a nucleic acid barcode molecule. A tag may comprise an organic compound or an inorganic compound. As used herein, the term “tag” may also refer to a patterned sequence of signals, wherein signals appear, skip, or disappear at defined intervals, creating a recognizable marker or referenceWSGR Docket No. 60652-721601within the signal data. This patterned appearance may encompass periodic or non-periodic intervals between signals, selective omissions, or complex structured combinations of signal presence and absence that collectively form a unique, detectable signature. For example, a tag may comprise a nucleic acid barcode molecule that has a series of distinct vibrational signatures detectable by techniques such as Raman or FTIR spectroscopy.

[0106] As used herein, the term “barcode” generally refers to an identifying feature that may be used to distinguish similar items. A barcode may comprise a nucleic acid molecule of about 2 to about 150 bases. A barcode may comprise a nucleic acid molecule of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 or more bases, which may provide a unique identifier tag or origin information for a molecule (e.g., protein, polypeptide, peptide), a binding agent, a set of binding agents from a binding cycle, a sample molecule, a set of samples, molecules within a compartment (e.g., droplet, bead, partition or separated location), macromolecules within a set of compartments, a fraction of macromolecules, a set of macromolecule fractions, a spatial region or set of spatial regions, a library of macromolecules, or a library of binding agents. A barcode can be an artificial sequence or a naturally occurring sequence including peptides, proteins, protein complexes, carbohydrates, and synthetic polymeric materials such as peptoids, polysaccharides, polymers, fluorescent tags, chemical tags, magnetic tags, isobaric tags, Raman spectroscopic tags, quantum dots, etc. In certain embodiments, each barcode within a population of barcodes is different. In other embodiments, a portion of barcodes in a population of barcodes is different, e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the barcodes in a population of barcodes is different. A population of barcodes may be randomly generated or non-randomly generated. A population of barcodes may comprise error correcting barcodes. Barcodes can be used to computationally deconvolute sequence reads derived from an individual molecule, partition, sample, library, etc. Barcodes may comprise multiplexed information, e.g., arising from different samples, compartments, individual molecules, etc. A barcode can also be used for deconvolution of a collection of molecules that have been distributed into small compartments for enhanced mapping. For example, rather than mapping a peptide back to the proteome, the peptide can be mapped back to its originating protein molecule or protein complex, a sample or partition from which it originated, etc. A barcode may comprise any useful structure moiety orWSGR Docket No. 60652-721601motif, e.g., hairpins, loop sequences, or spacers. Barcodes can comprise artificial or modified nucleic acids, e.g., locked nucleic acids (LNA), protein nucleic acids (PNA), hexitol nucleic acids (HNA), cyclohexane nucleic acids (CeNA), or a combination thereof. Barcodes may comprise or be generated using a protein, e.g., Tai effector, Cas protein (e.g., Cas9), Argonaut, or coiled coils. A barcode may comprise any useful sequence, including repeat sequences (e.g., a poly-A, poly-T, poly-C, poly-G region) or the barcode may comprise non-repeat sequences. A barcode may encode for information including, but not limited to time, lineage, sample types, cell number, beads, single molecule information, meta data, space / location (e.g., a slide, well, tissue), proximity (e.g., to other molecules, cells, metabolites, DNA, RNA), patient info, biological sample information, library information computer data, weather, physical parameters such as temperature, humidity, precipitation.

[0107] As used herein, the term “fluorescent label,” “fluorescent tag,” or “fluorophore” comprises a signaling moiety that conveys information through the fluorescent absorption and / or emission properties of one or more molecules. Exemplary fluorescent properties comprise fluorescence intensity, fluorescence lifetime, emission spectrum characteristics and energy transfer. Fluorophores available for post-synthetic attachment comprise, but are not limited to, ALEXA FLUOR™ 350, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethyl rhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg.), Cy2, Cy3.5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway, N. J.). FRET tandem fluorophores may also be used, comprising, but not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE- Alexa dyes (610, 647, 680), and APC-Alexa dyes. Examples of fluorescent nucleotide analogues readily incorporated into nucleotide and / or polynucleotide sequences comprise, but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, N. J.), fluorescein- 12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHOD AMINE GREEN™-5-dUTP, OREGON GREENR™ 488-5-dUTP, TEXAS RED™-12-dUTP, BODIPY™ 630 / 650-14-dUTP, BODIPY™ 650 / 665-14-dUTP, ALEXA FLUOR™ 488-5-dUTP, ALEXA FLUOR™ 532-5-dUTP, ALEXA FLUOR™ 568-5-dUTP, ALEXA FLUOR™ 594-5-dUTP, ALEXA FLUOR™WSGR Docket No. 60652-721601546-14-dUTP, fluorescein- 12-UTP, tetramethylrhodamine-6-UTP, TEXAS RED™-5-UTP, mCherry, CASCADE BLUE™-7-UTP, BODIPY™ FL-14-UTP, BODIPY TMR-14-UTP, BODIPY™ TR-14-UTP, RHOD AMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, and ALEXA FLUOR™ 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg.). For exemplary methods for custom synthesis of nucleotides having other fluorophores, see, Henegariu et al. (2000) Nature Biotechnol. 18:345. More examples of fluorescent labels and nucleotides and / or polynucleotides conjugated to such fluorescent labels comprise those described in, for example, Hoagland, Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991). In some embodiments, exemplary techniques and methods methodologies applicable to the provided embodiments comprise those described in, for example, U. S. Pat. Nos. 4,757,141, 5,151,507 and 5,091,519. In some embodiments, one or more fluorescent dyes are used as labels for labeled target sequences, for example, as described in U. S. Pat. No.5,188,934 (4,7-dichlorofluorescein dyes); U. S. Pat. No. 5,366,860 (spectrally resolvable rhodamine dyes); U. S. Pat. No. 5,847,162 (4,7-dichlororhodamine dyes); U. S. Pat. No.4,318,846 (ether- substituted fluorescein dyes); U. S. Pat. No. 5,800,996 (energy transfer dyes); U. S. Pat. No. 5,066,580 (xanthine dyes); and U. S. Pat. No. 5,688,648 (energy transfer dyes). Labelling can also be carried out with quantum dots, as described in U. S. Pat. Nos. 6,322,901, 6,576,291, 6,423,551, 6,251,303, 6,319,426, 6,426,513, 6,444,143, 5,990,479, 6,207,392, US 2002 / 0045045 and US 2003 / 0017264. All references are herein incorporated by reference in their entireties.

[0108] As used herein, the term “FRET” refers to fluorescence resonance energy transfer, a process in which chemical moi eties (e.g., fluorophores) transfer energy among themselves, or from a fluorophore to a non-fluorophore (e.g, a quencher molecule). In some circumstances, FRET involves an excited donor fluorophore transferring energy to an acceptor fluorophore via a short-range (e.g, about 10 nm or less) dipole-dipole interaction. In other circumstances, FRET involves a loss of fluorescence energy from a donor and an increase in fluorescence in an acceptor fluorophore. In still other forms of FRET, energy can be exchanged from an excited donor fluorophore to a non-fluorescing molecule (e.g., a quenching molecule). FRET includes Time-Resolved FRET (or TR-FRET), which combines the use of long-lived fluorophores and time-resolved detection (a delay between excitation and emission detection) to minimize fluorescent interference due to any inherent fluorescence of, e.g., target molecules or target-WSGR Docket No. 60652-721601selective binding agents (see, e.g., Klostermeier et al. (2001-2002) Biopolymers 61(3): 159-79). FRET is known to those of skill in the art and has been described (See Stryer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods EnzymoL, 246:300; Orpana, 2004 Biomol Eng 21, 45-50; Olivier, 2005 Mutant Res 573, 103-110, each of which is incorporated herein by reference in its entirety).

[0109] As used herein, “chemiluminescence” means light resulting from a chemical reaction in which one or more reagents of the reaction undergo a chemical change. The term “chemiluminescence” is intended to encompass electrochemiluminescence (e.g., a chemiluminescent reaction that occurs subsequently to an electrochemical reaction), bioluminescence (e.g., light resulting from biological reactions), phosphorescence (e.g., a type of photoluminescence similar to fluorescence, but it involves a longer-lived excited state, resulting in delayed light emission after the excitation source is removed), as well as light resulting from other types of reactions. Non-limiting examples of chemiluminescent reagents include luminol, isoluminol, acridinium esters, lucigenin, peroxyoxalates, firefly luciferin, coelenterazine, dioxetanes, benzoyl peroxide, oxalyl chloride derivatives, 1,2-dioxetanone, and pyrogallol derivatives. Non-limiting examples of electrochemiluminescent reagents include luminol, acridan ester, ruthenium, ruthenium chelate, and ruthenium tribipyridine, and NHS ester. Nonlimiting examples of bioluminescence reagents include firefly luciferin, Renilla luciferin (coelenterazine), bacterial luciferin, vargulin, and Cypridina luciferin. Non-limiting examples of phosphorescence reagents include Ruthenium(II) complexes, iridium(III) complexes, europium(III) complexes, terbium(III) complexes, erythrosin, eosin, halogenated aromatic compounds (e.g., bromo- or iodo- substituted benzenes), strontium aluminate, zinc sulfide, calcium tungstate, anthracene derivatives, carbazole compounds.

[0110] As used herein, the term “nucleic acid sequence” or “oligonucleotide sequence” generally refers to a contiguous string of nucleotide bases and may refer to the particular placement of nucleotide bases in relation to each other as they appear in an oligonucleotide. Similarly, the term “polypeptide sequence” or “amino acid sequence” refers to a contiguous string of amino acids and may refer to the particular placement of amino acids in relation to each other as they appear in a polypeptide.

[0111] A “nucleic acid molecule” according to the present disclosure may include any polymer or oligomer of nucleotides such as pyrimidine and purine bases, such as cytosine, thymine, and uracil, and adenine and guanine, respectively and combinations thereof. The nucleotide sequence may comprise any deoxyribonucleotide, ribonucleotide, hexitol -nucleotide, cyclohexane-nucleotide, peptide nucleic acid component, and any chemical variants thereof,WSGR Docket No. 60652-721601such as methylated, 7-deaza purine analogs, 8-halopurine analogs, hydroxymethylated or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, a nucleotide sequence may be DNA, RNA, HNA, CeNA or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

[0112] The terms “complementary” or “complementarity” refer to polynucleotides ( / .<., a sequence of nucleotides) related by base-pairing rules. For example, the sequence “5'-AGT-3',” is complementary to the sequence “5'-ACT-3'”. Complementarity may be “partial,” in which only some of the nucleic acids’ bases are matched according to the base pairing rules, or there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands can have significant effects on the efficiency and strength of hybridization between nucleic acid strands under defined conditions.

[0113] As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (e.g., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the melting temperature of the formed hybrid. Hybridization methods involve the annealing of one nucleic acid to another, complementary nucleic acid, e.g., based on Watson-Crick base pairing.

[0114] As used herein, the term “proteomics” generally refers to quantitative and / or qualitative analysis of the proteome within a sample, such as biological sample, e.g., from cells, tissues, or bodily fluids. Proteomics may include the analysis of spatial distributions of proteins within a sample (e.g., cell and / or tissues). Proteomics may include studies of the dynamic state of the proteome, e.g., how one or more proteins change in time. A proteome may comprise multiple “-omes”, e.g., a kinome; a secretome; a receptome (e.g., GPCRome); an immunoproteome; a nutriproteome; a proteome subset defined by a post-translational modification (e.g., phosphorylation, ubiquitination, methylation, acetylation, glycosylation, oxidation, lipidation, and / or nitrosylation), such as a phosphoproteome (e.g., phosphotyrosine-proteome, tyrosine-kinome, and tyrosine-phosphatome), a glycoproteome, etc.; a proteome subset associated with a tissue or organ, a developmental stage, or a physiological or pathological condition e.g., cancer, disease); a proteome subset associated a cellular process, such as cell cycle, differentiation (or de-differentiation), cell death, senescence, cell migration, transformation, or metastasis; or any combination thereof.WSGR Docket No. 60652-721601

[0115] The terminal amino acid at one end of the peptide chain that has a free amino group may be referred to herein as the “N-terminal amino acid” (NTAA). The terminal amino acid at the other end of the chain that has a free carboxyl group may be referred to herein as the “C-terminal amino acid” (CTAA). The amino acids making up a peptide may be numbered in order, with the peptide being “n” amino acids in length. As used herein, in some instances, NTAA may be considered the nth amino acid (also referred to herein as the “n NTAA”). In such cases, the next amino acid is the n-1 amino acid, then the n-2 amino acid, and so on down the length of the peptide from the N-terminal end to C-terminal end. Alternatively, CTAA may be considered the nth amino acid (also referred to herein as the “n CTAA”). In such cases, the next amino acid is the n-1, then the n-2 amino acid, and so on down the length of the peptide from the C-terminal end to N-terminal end. An NTAA, CTAA, or both may be modified or labeled with a chemical moiety.

[0116] As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

[0117] As used herein, the term “unique molecular identifier” or “UMI” generally refers to a molecule barcode comprising indexing information. A UMI may comprise a nucleic acid molecule of about 3 to about 150 bases (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 bases) in length. A UMI may provide a unique identifier tag for each molecule (e.g., peptide, binding agent, a nucleic acid molecule) that comprises or is coupled to a UMI. A UMI may comprise a random sequence (e.g. a random N-mer).

[0118] As used herein, a “derivative” of a nucleic acid molecule generally refers to a nucleic acid molecule that is derived from an originating nucleic acid molecule. The derivative may have the same or substantially the same nucleotide sequence as the originating nucleic acid molecule, or the derivative may comprise a complement or partial complement as the originating nucleic acid molecule. A derivative may be the same type of nucleic acid (e.g., DNA or RNA) as the originating nucleic acid molecule, or the derivative may be a different type of nucleic acid (e.g., cDNA generated from an RNA molecule). A nucleic acid molecule derivative may display sequence identity as the originating nucleic acid molecule. The derivative nucleic acid molecule may also be subjected to additional processing from the originating nucleic acid molecule, e.g.,WSGR Docket No. 60652-721601chemical or enzymatic modification, splicing, ligation, polymerization, fragmentation, tagmentation (e.g., using a transposase), digestion, etc.

[0119] A derivative polypeptide or peptide may be derived from an originating polypeptide (or peptide). A derivative may comprise the same amino acid sequence as the originating polypeptide, or the sequence may be different. The derivative polypeptide may result from or be subjected to additional processing from the originating polypeptide, e.g., chemical or enzymatic modification. The derivative polypeptide may comprise one or more tags, nucleic acid molecules, barcode molecules, labels (e.g., detectable labels), fluorophores, probes, linkers, post-translational modifications, chemical protecting groups, or other chemical moieties.

[0120] As used herein, the term “compartment” or “partition” generally refers to a physical area or volume that separates or isolates a subset of molecules from a sample of molecules. For example, a compartment or partition may separate an individual cell from other cells, or a subset of a sample’s proteome from the rest of the sample’s proteome. A compartment or partition may be an aqueous compartment (e.g., microfluidic droplet), a solid compartment (e.g., picotiter well or microtiter well on a plate, tube, vial, gel bead), a liquid-liquid phase separation, a liquid condensate, a subcellular region, or a separated region on a surface. A compartment may comprise one or more beads to which macromolecules may be immobilized. A compartment may be transient.

[0121] In some embodiments, the partitions as described herein are droplets. The terms “drop,” “droplet,” and “microdroplet” are used interchangeably herein, to refer to small, generally spherically structures, containing at least a first fluid phase, e.g., an aqueous phase (e.g., water), bounded by a second fluid phase (e.g., oil) which is immiscible with the first fluid phase. In some embodiments, droplets according to the present disclosure may contain a first fluid phase, e.g., oil, bounded by a second immiscible fluid phase, e.g., an aqueous phase fluid (e.g., water). In some embodiments, the second fluid phase will be an immiscible phase carrier fluid. Thus, droplets according to the present disclosure may be provided as aqueous-in-oil emulsions or oil-in-aqueous emulsions. Droplets may be sized and / or shaped as described herein for discrete entities. For example, droplets according to the present disclosure generally range from 1 pm to 1000 pm, inclusive, in diameter. Droplets according to the present disclosure may be used to encapsulate cells, nucleic acids (e.g., DNA), enzymes, reagents, and a variety of other components. The term droplet may be used to refer to a droplet produced in, on, or by a microfluidic device and / or flowed from or applied by a microfluidic device.

[0122] As used herein, the term “solid support”, “solid surface”, or “solid substrate” or “substrate” refers to any solid material, including porous and non-porous materials, to which aWSGR Docket No. 60652-721601molecule can be associated directly or indirectly. The molecule may be associated with the substrate by covalent or non-covalent interactions, or a combination thereof. A substrate may be two-dimensional (e.g., planar surface) or three-dimensional (e.g., gel matrix or bead). A solid support may comprise, in non-limiting examples, a bead, a microbead, an array, a glass surface, a silicon surface, a plastic surface, a filter, a membrane, nylon or other polymer, a silicon wafer chip, a flow through chip, a flow cell, a microfluidic device or chip or a surface thereof, a biochip including signal transducing electronics, a channel, a microtiter well, an ELISA plate, a spinning interferometry disc, a nitrocellulose membrane, a nitrocellulose-based polymer surface, a polymer matrix, a nanoparticle, or a microsphere. Materials for a solid support include but are not limited to acrylamide, agarose, cellulose, nitrocellulose, glass, gold, quartz, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, Teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, polyamino acids, dextran, or any combination thereof. Solid supports further include thin film, membrane, bottles, dishes, fibers, woven fibers, shaped polymers such as tubes, (e.g., nanotubes), particles, beads, DNA origami, microspheres, microparticles, or any combination thereof. For example, when solid surface is a bead, the bead can include, but is not limited to, a ceramic bead, polystyrene bead, a polymer bead, a methylstyrene bead, an agarose bead, an acrylamide bead, a solid core bead, a porous bead, a magnetic or paramagnetic bead, a glass bead, or a controlled pore bead, or a combination thereof. A magnetic bead may comprise or be composed of any useful material and may respond to an applied magnetic field. A bead may be spherical or an irregularly shaped. A bead’s size may range from nanometers, e.g., 1 nm, 10 nm, 100 nm, to millimeters, e.g., 1 mm. In certain embodiments, beads range in size from about 0.2 micron to about 200 microns, or from about 0.5 micron to about 5 microns. In some embodiments, beads can be about 1, 1.5, 2, 2.5, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pm in diameter. In certain embodiments, “a bead” solid support may refer to an individual bead or a plurality of beads. A solid support may assume any useful geometry, e.g., pyramid, cube, cylinder, helix, sphere, spheroid, rod, disc, arrow, spring, teardrop, prism, tetrapod, or any other useful geometry. A bead may be coated or treated with a range of substances or surface modifications to alter its physical, chemical, or biological properties.WSGR Docket No. 60652-721601

[0123] As used herein, “sequencing” generally refers to determining the order and identity of: (A) nucleotides (base sequences) in a nucleic acid sample, e.g., DNA or RNA; (B) amino acids in all or part of a polymer, such as a protein, peptide, or other multimeric molecule; or (C) monomers in all or part of a polymer (e.g., a synthetic or naturally occurring polymer, e.g., with more than one type of monomer). Many techniques are available for nucleic acid sequencing, such as Sanger sequencing or High Throughput Sequencing technologies (HTS). Sanger sequencing may involve sequencing via detection through (capillary) electrophoresis, in which up to 384 capillaries may be sequence analyzed in one run. High throughput sequencing involves the parallel sequencing of thousands or millions or more sequences at once. HTS can be defined as Next Generation sequencing (NGS), i.e. techniques based on solid phase pyrosequencing or as Next-Next Generation sequencing based on single nucleotide real time sequencing (SMRT). HTS technologies are available such as offered by Roche, Illumina and Applied Biosystems (Life Technologies). Further high throughput sequencing technologies are described by and / or available from Helicos, Pacific Biosciences, Complete Genomics, Ion Torrent Systems, Oxford Nanopore Technologies, Nabsys, ZS Genetics, GnuBio. Additional sequencing methods include Raman sequencing and Infrared (IR) sequencing, which utilizes Raman spectroscopy or IR spectroscopy to detect molecular vibrations associated with specific nucleotide or amino acid sequences, enabling label-free sequencing based on unique vibrational energy signatures. Tunneling current sequencing identifies base sequences or amino acid sequences through electronic signal variations as nucleotides or amino acids pass through a nanoscale gap, detecting characteristic tunneling currents specific to each molecular component.

[0124] As used herein, “next generation sequencing” refers to high-throughput sequencing methods that allow the sequencing of millions to billions of molecules in parallel. Examples of next generation sequencing methods include sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, nanopore sequencing, and pyrosequencing. By attaching primers to a solid substrate and a complementary sequence to a nucleic acid molecule, a nucleic acid molecule can be hybridized to the solid substrate via the primer and then multiple copies can be generated in a discrete area on the solid substrate by using polymerase to amplify (these groupings are sometimes referred to as polymerase colonies or polonies). Consequently, during the sequencing process, a nucleotide at a particular position can be sequenced multiple times (e.g., hundreds or thousands of times) — this depth of coverage is referred to as “deep sequencing.” Examples of high throughput nucleic acid sequencing technology include platforms provided by Illumina, BGI, Qiagen, ThermoFisher, and Roche, including formats such as parallel bead arrays, sequencing byWSGR Docket No. 60652-721601synthesis, sequencing by ligation, capillary electrophoresis, electronic microchips, “biochips,” microarrays, parallel microchips, and single-molecule arrays, zero mode waveguide based sequencing, some of which are reviewed by Service (Science 311:1544-1546, 2006).

[0125] As used herein, “analyzing” the macromolecule means to quantify, characterize, distinguish, or a combination thereof, all or a portion of the components of a molecule (e.g., a macromolecule, a biological molecule such as a protein, amino acid, nucleic acid molecule, etc.). For example, analyzing a peptide, polypeptide, or protein may comprise determining all or a portion of the amino acid sequence (contiguous or non-continuous) of the peptide. Analyzing a macromolecule may include partial identification of a component of the macromolecule. For example, partial identification of amino acids in a protein sequence can identify an amino acid in the protein as belonging to a subset of possible amino acids. Analysis may be performed sequentially, e.g., beginning with analysis of the n NTAA, and then proceeding to the next amino acid of the peptide (i.e., n-1, n-2, n-3, and so forth). In such instances, sequencing may be performed by cleavage of the n NTAA, thereby converting the n-1 amino acid of the peptide to an N-terminal amino acid (referred to herein as the “n-1 NTAA”). Similarly, analysis of a peptide may begin from C-terminus towards the N-terminus with each round of cleavage from the C-terminus creating a new CTAA. Cleavage of the n CTAA converts the n-1 amino acid of the peptide to a C-terminal amino acid, referred to herein as an “n-1 CTAA”. Analyzing the peptide may also include determining a presence and frequency of post-translational modifications on the peptide, which may or may not include information regarding the sequential order of the post-translational modifications on the peptide. Analyzing the peptide may also include determining the presence and frequency of epitopes in the peptide, which may or may not include information regarding the sequential order or location of the epitopes within the peptide. Analyzing the peptide may include combining different types of analysis, for example obtaining epitope information, amino acid sequence information, post-translational modification information, or any combination thereof.

[0126] As used herein, the term “analyte” generally refers to a substance that is of interest to be further identified, characterized, or measured. An analyte can be, in non-limiting examples, an ion, chemical, compound, small molecule, element, particle, metal, biomolecule, macromolecule, metabolite, lipid, carbohydrate, peptide or protein, nucleic acid molecule, organelle, or cell. An analyte may be naturally occurring or synthetic. The analyte may be a solid, semi-solid, liquid, semi-liquid, gas, or plasma. The analyte may be characterized qualitatively or quantitatively. A portion of an analyte may be analyzed. For example, an analyte may be a peptide and the constituent amino acids may be analyzed. The analyte may comprise aWSGR Docket No. 60652-721601polymer, also referred to herein as “polymeric analyte”, which generally refers to an analyte of interest that comprises one or more monomers. A polymeric analyte can be, in non-limiting examples, a group of ions, chemicals, compounds, small molecules, elements, particles, metals, or a biomolecule, macromolecule, metabolite, lipid, carbohydrate, peptide or protein, nucleic acid molecule, organelle, or cell.

[0127] As used herein, the term “array” generally refers to a population of molecules that is attached to one or more solid supports such that the molecules at one address can be distinguished from molecules at other addresses. An array can include different molecules that are each located at different addresses on a solid support. Alternatively, an array can include separate solid supports each functioning as an address that bears a different molecule, wherein the different molecules can be identified according to the locations of the solid supports on a surface to which the solid supports are attached, or according to the locations of the solid supports in a liquid such as a fluid stream. The molecules of the array can be, for example, nucleic acids such as SNAPs, polypeptides, proteins, peptides, oligopeptides, enzymes, ligands, or receptors such as antibodies, functional fragments of antibodies or aptamers. The addresses of an array can optionally be optically observable and, in some configurations, adjacent addresses can be optically distinguishable when detected using a method or apparatus set forth herein.

[0128] As used herein, the term “functionalized” refers to any material or substance that has been modified to include a functional group. A functionalized material or substance may be naturally or synthetically functionalized. For example, a polypeptide can be naturally functionalized with a phosphate group, oligosaccharide (e.g., glycosyl, glycosylphosphatidylinositol or phosphoglycosyl), nitrosyl, methyl, acetyl, lipid (e.g., glycosyl phosphatidylinositol, myristoyl or prenyl), ubiquitin or other naturally occurring post-translational modification. A functionalized material or substance may be functionalized for any given purpose, including altering chemical properties (e.g., altering hydrophobicity or changing surface charge density) or altering reactivity (e.g., capable of reacting with a moiety or reagent to form a covalent bond to the moiety or reagent).

[0129] As used herein, the term “click reaction,” “click chemistry,” or “bioorthogonal reaction” refers to single-step, thermodynamically favorable conjugation reaction utilizing biocompatible reagents. A click reaction may utilize no toxic or biologically incompatible reagents (e.g., acids, bases, heavy metals) or generate no toxic or biologically incompatible byproducts. A click reaction may utilize an aqueous solvent or buffer (e.g., phosphate buffer solution, Tris buffer, saline buffer, MOPS, etc.). A click reaction may be thermodynamicallyWSGR Docket No. 60652-721601favorable if it has a negative Gibbs free energy of reaction, for example a Gibbs free energy of reaction of less than about -5 kiloJoules / mole (kJ / mol), -10 kJ / mol, -25 kJ / mol, -50 kJ / mol, -100 kJ / mol, -200 kJ / mol, -300 kJ / mol, -400 kJ / mol, or less than -500 kJ / mol. Exemplary bioorthogonal and click reactions are described in detail in WO 2019 / 195633A1, which is herein incorporated by reference in its entirety. Exemplary click reactions may include metal-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted azide-nitrone cycloaddition, strained alkene reactions, thiolene reaction, Diels- Alder reaction, inverse electron demand Di els- Alder reaction, [3+2] cycloaddition, [4+1] cycloaddition, nucleophilic substitution, dihydroxylation, thiolyne reaction, photoclick, nitrone dipole cycloaddition, norbornene cycloaddition, oxanobomadiene cycloaddition, tetrazine ligation, and tetrazole photoclick reactions. Exemplary functional groups or reactive handles utilized to perform click reactions (also referred to herein as “click chemistry moieties”) may include alkenes (e.g., linear alkenes or cyclic alkenes such as trans-cyclooctene (TCO)), alkynes (e.g., linear alkynes or cycloalkynes (e.g., cyclooctynes or derivatives thereof, e.g, aza-dimethoxy cyclooctyne (DIMAC), symmetrical pyrrolocyclooctyne (SYPCO), pyrrolocyclooctyne (PYRROC), difluorocyclooctyne (DIFO), a,a-bis(trifluoromethyl)pyrrolocyclooctyne(TRIPCO), bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctyne (DBCO), difluorinated cyclooctyne (DIFO), difluorobenzocyclooctyne (DIFBO), dibenzoazacyclo-octyne (DBACO), difluoro-aza-dibenzocyclooctyne (F2-DIBAC), biaryl-azacyclooctynone (BARAC), difluorodimethoxydibenzocyclooctynol (FMDIBO), difluorodimethoxydibenzocyclooctynone (keto-FMDIBO), and 3,3,6,6-tetramethylthiacycloheptyne (TMTH)), TMTH-sulfoximine (TMTHSI), azides, epoxides, amines, thiols, nitrones, isonitriles, isocyanides, aziridines, activated esters, and tetrazines, triazoles, and combinations, variations, or derivatives thereof. The click chemistry moieties may be subjected to conditions sufficient to react the first click chemistry moiety to the second click chemistry moiety, e.g., provision of metal catalysts, appropriate solvents, pH, temperature, ionic concentration, or light / energy, for any useful duration of time.

[0130] As used herein, the terms “group” and “moiety” are intended to be synonymous when used in reference to the structure of a molecule. The terms refer to a component or part of the molecule. The terms do not necessarily denote the relative size of the component or part compared to the molecule, unless indicated otherwise. The terms do not necessarily denote the relative size of the component or part compared to any other component or part of the molecule, unless indicated otherwise. A group or moiety can contain one or more atoms.WSGR Docket No. 60652-721601

[0131] As used herein, “primers” generally refer to nucleic acid molecules which can prime the synthesis of a nucleic acid molecule (e.g., DNA or RNA). A primer may be single stranded. A primer may comprise one or more recognition sites for a protein (e.g., a polymerizing enzyme, a restriction enzyme, a cleaving enzyme, a nuclease, etc.) to bind to the primer or a primer hybridized to a template strand. A primer may comprise DNA, RNA, or other nucleic acid analogs or noncanonical bases (e.g., spacer moieties, uracils, abasic sites). A primer may optionally comprise any number of functional sequences such as sequencing primer sequences (e.g., P5 or P7 sequences), sequencing primer-binding sequences, read sequences (e.g., R1 or R2 sequences), restriction sites, nuclease-recognition sites, abasic sites, cleavage sites, transposition sites e.g., mosaic end sequences), a barcode sequence, a unique molecular identifier (UMI), etc.

[0132] “Amplification” or “amplifying” generally refers to a polynucleotide amplification reaction, namely, a population of polynucleotides that are replicated from one or more starting sequences. Amplifying may refer to a variety of amplification reactions, including but not limited to polymerase chain reaction (PCR), linear polymerase reactions, nucleic acid sequencebased amplification, rolling circle amplification and similar reactions. An amplification reaction may generate an amplicon. Amplification may be performed isothermally or by cycling of temperatures. Amplification or amplifying may refer to an increase in quantity of a measurable output, for example, signal amplification.

[0133] An “adapter” as referred to herein, generally refers to a short nucleic acid molecule (e.g., about 10 to about 100 base pairs in length). An adapter may comprise a short doublestranded DNA molecule. An adapter may be attached, e.g., via polymerization or ligation, to an end of a DNA fragments or amplicons. Adapters may comprise synthetic oligonucleotides, e.g., oligonucleotides that have nucleotide sequences which are at least partially complementary to each other. An adapter may have blunt ends, may have staggered ends (also referred to herein as a 3’ or 5’ “overhang sequence” or “sticky end”, or a blunt end and a staggered end. Adapters may be attached (e.g., via ligation) to fragments to provide an adapter-ligated fragment; the adapter-ligated fragment may serve as a starting point for subsequent manipulation e.g., for amplification or sequencing. An adapter may be functionalized, e.g., conjugated with a tag, probe, detectable label, affinity capture reagent (e.g., biotin or streptavidin). An adapter may comprise one of more functional sequences, e.g., a primer sequence (e.g., for sequencing, reading, or amplification), a restriction site, a recombination site, a transposition site, an abasic site, a barcode sequence, a translational control element, a splice site, an origin of replication, a promoter, an enhancer, a silencer, an insulator, and operator, a polyadenylation (poly A) site, or other functional sequence or a combination thereof.WSGR Docket No. 60652-721601

[0134] The term “capture moiety” as used herein generally refers to a molecule that is configured to be coupled to another moiety or molecule. A capture moiety can be a biomolecule, e.g., a lipid, carbohydrate, sugar, amino acid, peptide or protein, nucleotide, nucleic acid molecule, metabolite, or a combination thereof (e.g., glycoproteins, lipoproteins, glycosaminoglycans, etc.). A capture moiety can be a small molecule, organic compound, inorganic compound, metal, polymer, ion, or other molecule or molecular compound. A capture moiety may comprise a macromolecule. A capture moiety may comprise an enzyme, antibody, antibody fragment, nanobody, aptamer, biotin, streptavidin, avidin, neutravidin, or analogs or derivatives thereof. A capture moiety may comprise more than one molecule, e.g., a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, etc. A capture moiety can be a solid substrate or part of a solid substrate, or the capture moiety can be separate from a substrate, e.g., in a fluidic medium (e.g., air, in a liquid solution). A capture moiety may have specificity to a binding partner or a plurality of binding partners. A capture moiety may be able to bind to one molecule or moiety (univalent), or a plurality of molecules or moieties (multivalent).

[0135] The term “translocating” and “translocation,” as used herein, generally refers to the movement of a molecule through a medium (e.g., a gas, a liquid, a solid, or a multiphase medium). Translocation of a molecule may occur spontaneously e.g., through diffusion, Brownian motion, etc.). Alternatively, or in addition to, translocation of a molecule may occur with an application of force or pressure, e.g., using frictional force, tension force, a normal force, air resistance force, spring force, a temperature gradient, gravitational force, electrical force, magnetic force, acoustic force (e.g., acoustophoresis) etc. In some examples, translocation of a molecule may be achieved by application of pressure-driven flow or electrophoretic forces. Translocation may occur through a liquid or through a solid or semi-solid substrate (e.g., through a pore or gap) or adjacent or in proximity to the solid or semi-solid substrate.

[0136] As used herein, the term “nanopore” or “nanogap” generally refers to a pore, hole, aperture, gap, or channel of nanometer scale. The nanopore, nanogap, or nanochannel may be generated from an organic material, e.g., a pore-forming protein or a transmembrane protein. Such a protein may be naturally occurring, synthetic, or engineered. Examples of naturally occurring organic nanopores include wild-type aerolysin, alpha-hemolysin, mycobacterial porins (e.g., MspA porin), Phi29 connector channels, Fragaceatoxin C, Cytolysin A, Ferric hydroxyamate uptake component A, Curb specific gene G, outer membrane porin G, and viral DNA packaging motors. The nanopore, nanochannel, or nanogap may comprise an engineered variant of a naturally occurring nanopore. The nanopore, nanogap, or nanochannel may comprise an inorganic material. For example, solid-state nanopores may be made from dielectricWSGR Docket No. 60652-721601materials such as a silicon compound (e.g., silicon nitride, silicon dioxide), an aluminum compound (e.g., aluminum oxide), a titanium compound (e.g., titanium oxide), a molybdenum compound (e.g., molybdenum disulfide), hafnium, graphene, etc. The nanopore, nanochannel, or nanogap may assume any useful form factor or geometry, e.g., gaps or channels within membranes, capillaries, etc., and may be generated using any suitable process, e.g., ion-beam sculpting, electron beam exposure. The nanopore, nanochannel, or nanogap may comprise an elastomeric material.

[0137] As used herein, the abbreviations for the natural 1 -enantiomeric amino acids are conventional and can be as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Vai). Unless otherwise specified, X can indicate any amino acid. In some aspects, X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R). References to these amino acids are also in the form of “[amino acid] [residues / residues]” (e.g., lysine residue, lysine residues, leucine residue, leucine residues, etc.).

[0138] “Amino” refers to the -NH2 radical.

[0139] “Cyano” refers to the -CN radical.

[0140] “Nitro” refers to the -NO2 radical.

[0141] Oxo” refers to the =0 radical.

[0142] “Hydroxyl” refers to the -OH radical.

[0143] “Alkyl” generally refers to an acyclic (e.g., straight or branched) or cyclic hydrocarbon (e.g., chain) radical consisting of carbon and hydrogen atoms, such as having from one to fifteen carbon atoms (e.g., C1-C15 alkyl). Unless otherwise state, alkyl is saturated or unsaturated (e.g., an alkenyl, which comprises at least one carbon-carbon double bond; or an alkynyl, which comprises at least one carbon-carbon triple bond). Disclosures provided herein of an “alkyl” are intended to include independent recitations of a saturated “alkyl,” unless otherwise stated. Alkyl groups described herein are generally monovalent, but may also be divalent (which may also be described herein as “alkylene,” “alkylenyl,” “alkenylene,” “alkenylenyl,” “alkynylene,” “alkynylenyl,” groups). In certain embodiments, an alkyl comprises one to thirteen carbon atoms (e.g., C1-C13 alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (e.g., Ci-Cs alkyl). In other embodiments, an alkyl comprises one to five carbon atoms (e.g., C1-C5 alkyl). In other embodiments, an alkyl comprises one to four carbon atoms (e.g., C1-C4 alkyl). In other embodiments, an alkylWSGR Docket No. 60652-721601comprises one to three carbon atoms (e.g., C1-C3 alkyl). In other embodiments, an alkyl comprises one to two carbon atoms (e.g., C1-C2 alkyl). In other embodiments, an alkyl comprises one carbon atom (e.g., Ci alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (e.g., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., Cs-Cs alkyl). In other embodiments, an alkyl comprises two to five carbon atoms (e.g., C2-C5 alkyl). In other embodiments, an alkyl comprises three to five carbon atoms (e.g., C3-C5 alkyl). In other embodiments, the alkyl group is selected from methyl, ethyl, 1 -propyl (n-propyl), 1 -methylethyl (iso-propyl), 1 -butyl (n-butyl), 1 -methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1 -dimethylethyl (tert-butyl), 1 -pentyl (n-pentyl). The alkyl is attached to the rest of the molecule by a single, double, or triple bond. In general, alkyl groups are each independently substituted or unsubstituted. Each recitation of “alkyl” provided herein, unless otherwise stated, includes a specific and explicit recitation of an unsaturated “alkyl” group. Similarly, unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, -ORa, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -C(O)N(Ra)2, -N(Ra)C(O)ORa, -OC(O)-N(Ra)2, -N(Ra)C(O)Ra, -N(Ra)S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tRa(where t is 1 or 2) and -S(O)tN(Ra)2 (where t is 1 or 2) where each Rais independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).

[0144] “Alkylene” or “alkylene chain” generally refers to a straight or branched divalent alkyl group linking the rest of the molecule to a radical group, such as having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, / -propylene, w-butylene, and the like. In some embodiments, alkylene may encompass unsaturated divalent hydrocarbons, such as alkenylene (e.g., -CH=CH-) or alkynylene (e.g., -C=C-) groups. Unless stated otherwise specifically in the specification, an alkylene chain is optionally substituted as described for alkyl groups herein.WSGR Docket No. 60652-721601

[0145] “Alkoxy” refers to a radical bonded through an oxygen atom of the formula -O-alkyl, where alkyl is an alkyl chain as defined above.

[0146] “Alkenyl” refers to a straight or branched hydrocarbon chain radical group consisting of carbon and hydrogen atoms, containing at least one carbon-carbon double bond, and having from two to twelve carbon atoms. In certain embodiments, an alkenyl comprises two to eight carbon atoms. In other embodiments, an alkenyl comprises two to four carbon atoms. The alkenyl is optionally substituted as described for “alkyl” groups. In some embodiments, two or more carbon-carbon double bonds may form a conjugated system (e.g., alternating single (c) and double (jt) bonds such as -CH=CH-CH=CH-). In some embodiments, the alkenyl is optionally substituted with an alkynyl group. In some embodiments, a carbon-carbon single bond (o), a carbon-carbon double bond (K), and a carbon-carbon triple bond (JC), may form a conjugated system (e.g., -CH=CH-C=C-).

[0147] “Alkenylene” or “alkenylene chain” generally refers to a straight or branched divalent alkenyl group linking the rest of the molecule to a radical group, such as having from one to twelve carbon atoms, for example, ethenylene (-CH=CH-), propenylene (-CH2CH=CH-), butenylene (-CH=CHCH2CH2-, or -C1UCH=CHCH2-), and the like. Unless stated otherwise specifically in the specification, an alkenylene chain is optionally substituted as described for alkyl groups herein.

[0148] “Alkynyl” refers to a straight or branched hydrocarbon chain radical group consisting of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and having from two to twelve carbon atoms. In certain embodiments, an alkynyl comprises two to eight carbon atoms. In other embodiments, an alkynyl comprises two to four carbon atoms. The alkynyl is optionally substituted as described for “alkyl” groups. In some embodiments, two or more carbon-carbon triple bonds may form a conjugated system (e.g., alternating single (c) and double (jt) bonds such as -C=C-C=C-). In some embodiments, an alkynyl is optionally substituted with an alkenyl group. In some embodiments, a carbon-carbon single bond (o), a carbon-carbon double bond (K), and a carbon-carbon triple bond (K), may form a conjugated system (e.g., -CH=CH-C=C-).

[0149] “Alkynylene” or “alkynylene chain” generally refers to a straight or branched divalent alkynyl group linking the rest of the molecule to a radical group, such as having from one to twelve carbon atoms, for example, ethynylene (-C=C-), propynylene (-CH2OC-), butynylene (-OCCH2CH2-, or -CH2OCCH2-), and the like. Unless stated otherwise specifically in the specification, an alkynylene chain is optionally substituted as described for alkyl groups herein.WSGR Docket No. 60652-721601

[0150] “Aryl” refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or multicyclic hydrocarbon ring system contains hydrogen and carbon from five to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, / .<., it contains a cyclic, delocalized (4n+2) 7t-electron system in accordance with the Hiickel theory. The ring system from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin and naphthalene. Unless stated otherwise specifically in the specification, the term "aryl" or the prefix "ar-" (such as in "aralkyl") is meant to include aryl radicals optionally substituted by one or more substituents independently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -Rb-ORa, -Rb-OC(O)-Ra, -Rb-OC(O)-ORa, -Rb-OC(O)-N(Ra)2, -Rb-N(Ra)2, -Rb-C(O)Ra, -Rb-C(O)ORa, -Rb-C(O)N(Ra)2, -Rb-O-Rc-C(O)N(Ra)2, -Rb-N(Ra)C(O)ORa, -Rb-N(Ra)C(O)Ra, -Rb-N(Ra)S(O)tRa(where t is 1 or 2), -Rb-S(O)tRa(where t is 1 or 2), -Rb-S(O)tORa(where t is 1 or 2) and -Rb-S(O)tN(Ra)2 (where t is 1 or 2), where each Rais independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each Rbis independently a direct bond, or a straight or branched alkylene, alkenylene, or alkynylene chain, and Rcis a straight or branched alkylene, alkenylene, or alkynylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

[0151] “Aralkyl” or “aryl-alkyl” refers to a radical of the formula -Rc-aryl where Rcis an alkylene chain, an alkenylene chain, or an alkynylene chain as defined above, for example, methylene, ethylene, butylene, ethenylene, butenylene, ethynylene, butynylene, and the like. The alkylene chain part of the aralkyl radical is optionally substituted as described above for anWSGR Docket No. 60652-721601alkylene chain. The alkenylene chain part of the aralkyl radical is optionally substituted as described above for an alkenylene chain. The alkynylene chain part of the aralkyl radical is optionally substituted as described above for an alkynylene chain. The aryl part of the aralkyl radical is optionally substituted as described above for an aryl group.

[0152] “Carbocyclyl” or “cycloalkyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting of carbon and hydrogen atoms, which includes fused, bridged, or spiro ring systems, having from three to fifteen carbon atoms. In certain embodiments, a carbocyclyl comprises three to ten carbon atoms. In other embodiments, a carbocyclyl comprises five to seven carbon atoms. The carbocyclyl is attached to the rest of the molecule by a single bond, a double bond, or a triple bond. Carbocyclyl or cycloalkyl is saturated ( / .<., containing single C-C bonds) or unsaturated (i.e., containing one or more double bonds or triple bonds). Examples of saturated cycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. An unsaturated carbocyclyl is also referred to as "cycloalkenyl" or "cycloalkynyl." Examples of monocyclic cycloalkenyls include, e.g, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Examples of monocyclic cycloalkynyls include, e.g, cyclooctynyl, cyclononynyl, and cyclodecynyl. Polycyclic carbocyclyl radicals include, for example, adamantyl, norbornyl (i.e., bicyclo[2.2.1]heptanyl), norbornenyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise stated specifically in the specification, the term “carbocyclyl” is meant to include carbocyclyl radicals that are optionally substituted by one or more substituents independently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -Rb-0Ra, -Rb-OC(O)-Ra, -Rb-OC(O)-ORa, -Rb-OC(O)-N(Ra)2, -Rb-N(Ra)2, -Rb-C(O)Ra, -Rb-C(O)ORa, -Rb-C(O)N(Ra)2, -Rb-O-Rc-C(O)N(Ra)2, -Rb-N(Ra)C(O)ORa, -Rb-N(Ra)C(O)Ra, -Rb-N(Ra)S(O)tRa(where t is 1 or 2), -Rb-S(O)tRa(where t is 1 or 2), -Rb-S(O)tORa(where t is 1 or 2) and -Rb-S(O)tN(Ra)2 (where t is 1 or 2), where each Rais independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, orWSGR Docket No. 60652-721601trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each Rbis independently a direct bond, or a straight or branched alkylene, alkenylene, or alkynylene chain, and Rcis a straight or branched alkylene, alkenylene, or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

[0153] “Carbocyclylalkyl” refers to a radical of the formula -Rc-carbocyclyl where Rcis an alkylene chain, an alkenylene chain, or an alkynylene chain as defined above. The alkylene chain, the alkenylene chain, the alkynylene chain, or the carbocyclyl radical is optionally substituted as defined above.

[0154] “Carbocyclylalkenyl” refers to a radical of the formula -Rc-carbocyclyl where Rcis an alkenylene chain as defined above. The alkenylene chain and the carbocyclyl radical is optionally substituted as defined above.

[0155] “Carbocyclylalkynyl” refers to a radical of the formula -Rc-carbocyclyl where Rcis an alkynylene chain as defined above. The alkynylene chain and the carbocyclyl radical is optionally substituted as defined above.

[0156] “Carbocyclylalkoxy” refers to a radical bonded through an oxygen atom of the formula -O-Rc-carbocyclyl where Rcis an alkylene chain, an alkenylene chain, or an alkynylene chain as defined above. The alkylene chain, the alkenylene chain, the alkynylene chain, or the carbocyclyl radical is optionally substituted as defined above.

[0157] “Halo” or “halogen” refers to fluoro, bromo, chloro, or iodo substituents.

[0158] “Haloalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more halogen radicals, as defined above, for example, trihalomethyl, dihalomethyl, halomethyl, and the like. In some embodiments, the haloalkyl is a fluoroalkyl, such as, for example, trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl,l-fluoromethyl-2-fluoroethyl, and the like. In some embodiments, the alkyl part of the fluoroalkyl radical is optionally substituted as defined above for an alkyl group.

[0159] “Heteroalkyl” refers to an alkyl group as defined above in which one or more skeletal carbon atoms of the alkyl are substituted with one to six heteroatoms selected from boron, nitrogen, oxygen, silicon, phosphorus, and sulfur (with the appropriate number of substituents or valencies - for example, -CH2- may be replaced with -NH- or -O-). For example, each substituted carbon atom is independently substituted with a heteroatom, such as wherein the carbon is substituted with nitrogen, oxygen, sulfur, or other suitable heteroatom. In someWSGR Docket No. 60652-721601instances, each substituted carbon atom is independently substituted for an oxygen, nitrogen (e.g. -NH-, -N(alkyl)-, or -N(aryl)- or having another substituent contemplated herein), or sulfur (e.g. -S-, -S(=O)-, or -S(=O)2-). In some embodiments, a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl. In some embodiments, a heteroalkyl is attached to the rest of the molecule at a heteroatom of the heteroalkyl. In some embodiments, a heteroalkyl is a Ci-Cis heteroalkyl. In some embodiments, a heteroalkyl is a C1-C12 heteroalkyl. In some embodiments, a heteroalkyl is a Ci-Ce heteroalkyl. In some embodiments, a heteroalkyl is a Ci-C4 heteroalkyl. In some embodiments, heteroalkyl includes alkylamino, alkylaminoalkyl, aminoalkyl, heterocycloalkyl, heterocyclyl, and heterocycloalkylalkyl, as defined herein. Unless stated otherwise specifically in the specification, a heteroalkyl group is optionally substituted as defined above for an alkyl group.

[0160] “Heteroalkylene” refers to a divalent heteroalkyl group defined above which links one part of the molecule to another part of the molecule. Unless stated specifically otherwise, a heteroalkylene is optionally substituted, as defined above for an alkyl group.

[0161] “Heterocyclyl” or “heterocycloalkyl” refers to a stable 3- to 18-membered nonaromatic ring radical that comprises two to twelve carbon atoms and from one to six heteroatoms selected from boron, nitrogen, oxygen, silicon, phosphorus, and sulfur (with the appropriate number of substituents or valencies - for example, -CH2- may be replaced with -NH- or -O-). Unless stated otherwise specifically in the specification, the heterocyclyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which optionally includes fused, bridged, or spiro ring systems. The heteroatoms in the heterocyclyl radical are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized ( / .<., a nitrogen atom forms four covalent bonds, resulting in a positively charged quaternary ammonium species N+). The heterocyclyl radical is partially or fully saturated. The heterocyclyl radical is saturated ( / .<., containing single C-C bonds) or unsaturated e.g., containing one or more double bonds or triple bonds in the ring system). In some instances, the heterocyclyl radical is saturated. In some instances, the heterocyclyl radical is saturated and substituted. In some instances, the heterocyclyl radical is unsaturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, the termWSGR Docket No. 60652-721601“heterocyclyl” is meant to include heterocyclyl radicals as defined above that are optionally substituted by one or more substituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -Rb-0Ra, -Rb-0C(0)-Ra, -Rb-0C(0)-0Ra, -Rb-0C(0)-N(Ra)2, -Rb-N(Ra)2, -Rb-C(0)Ra, -Rb-C(0)0Ra, -Rb-C(0)N(Ra)2, -Rb-0-Rc-C(0)N(Ra)2, -Rb-N(Ra)C(0)0Ra, -Rb-N(Ra)C(0)Ra, -Rb-N(Ra)S(O)tRa(where t is 1 or 2), -Rb-S(O)tRa(where t is 1 or 2), -Rb-S(O)tORa(where t is 1 or 2) and -Rb-S(0)tN(Ra)2 (where t is 1 or 2), where each Rais independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each Rbis independently a direct bond, or a straight or branched alkylene, alkenylene, or alkynylene chain, and Rcis a straight or branched alkylene, alkenylene, or alkynylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

[0162] ‘W-heterocyclyl” or “N-attached heterocyclyl” refers to a heterocyclyl radical as defined above containing at least one nitrogen and where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a nitrogen atom in the heterocyclyl radical. An ^'-heterocyclyl radical is optionally substituted as described above for heterocyclyl radicals. Examples of such A-heterocyclyl radicals include, but are not limited to, 1-morpholinyl, 1-piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl.

[0163] “C-heterocyclyl” or “C-attached heterocyclyl” refers to a heterocyclyl radical as defined above containing at least one heteroatom and where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a carbon atom in the heterocyclyl radical. A C-heterocyclyl radical is optionally substituted as described above for heterocyclylWSGR Docket No. 60652-721601radicals. Examples of such C-heterocyclyl radicals include, but are not limited to, 2-morpholinyl, 2- or 3- or 4-piperidinyl, 2-piperazinyl, 2- or 3-pyrrolidinyl, and the like.

[0164] “Heterocyclylalkyl” refers to a radical of the formula -Rc-heterocyclyl where Rcis an alkylene, alkenylene, or alkynylene chain as defined above. If the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene, the alkenylene, or the alkynylene chain of the heterocyclylalkyl radical is optionally substituted as defined above. The heterocyclyl part of the heterocyclylalkyl radical is optionally substituted as defined above for a heterocyclyl group.

[0165] “Heterocyclylalkoxy” refers to a radical bonded through an oxygen atom of the formula -O-Rc-heterocyclyl where Rcis an alkylene chain, an alkenylene chain, or an alkynylene chain as defined above. If the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain of the heterocyclylalkoxy radical is optionally substituted as defined above for an alkylene chain. The heterocyclyl part of the heterocyclylalkoxy radical is optionally substituted as defined above for a heterocyclyl group.

[0166] “Heteroaryl” refers to a radical derived from a 3 - to 18-membered aromatic ring radical that comprises two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur (with the appropriate number of substituents or valencies - for example, -CEh- may be replaced with -NH- or -O-). As used herein, the heteroaryl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, / .<., it contains a cyclic, delocalized (4n+2) 7t-electron system in accordance with the Huckel theory. Heteroaryl includes fused, bridged, or spiro ring systems. The heteroatom(s) in the heteroaryl radical is optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized ( / .<., a nitrogen atom forms four covalent bonds, resulting in a positively charged quaternary ammonium species N+). The heteroaryl is attached to the rest of the molecule through any atom of the ring(s). Examples of heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][l,4]dioxepinyl, benzo[b][l,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodi oxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotri azolyl,benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl,6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[l,2-c]pyridazinyl,WSGR Docket No. 60652-721601dibenzofuranyl, dibenzothiophenyl, furanyl, furan onyl, furo[3,2-c]pyridinyl,5.6.7.8.9.10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5.6.7.8.9.10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl,5.8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyri dinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl,1 -phenyl- IH-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5.6.7.8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl,6.7.8.9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl,5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl ( / .<. thienyl). Unless stated otherwise specifically in the specification, the term "heteroaryl" is meant to include heteroaryl radicals as defined above which are optionally substituted by one or more substituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, haloalkenyl, haloalkynyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -Rb-0Ra, -Rb-OC(O)-Ra, -Rb-0C(0)-0Ra, -Rb-0C(0)-N(Ra)2, -Rb-N(Ra)2, -Rb-C(0)Ra, -Rb-C(0)0Ra, -Rb-C(0)N(Ra)2, -Rb-0-Rc-C(0)N(Ra)2, -Rb-N(Ra)C(0)0Ra, -Rb-N(Ra)C(0)Ra, -Rb-N(Ra)S(O)tRa(where t is 1 or 2), -Rb-S(O)tRa(where t is 1 or 2), -Rb-S(O)tORa(where t is 1 or 2) and -Rb-S(0)tN(Ra)2 (where t is 1 or 2), where each Rais independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, orWSGR Docket No. 60652-721601trifluoromethyl), each Rbis independently a direct bond, or a straight or branched alkylene, alkenylene or alkynylene chain, and Rcis a straight or branched alkylene, alkenylene or alkynylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

[0167] ‘W-heteroaryl” refers to a heteroaryl radical as defined above containing at least one nitrogen and where the point of attachment of the heteroaryl radical to the rest of the molecule is through a nitrogen atom in the heteroaryl radical. An A-heteroaryl radical is optionally substituted as described above for heteroaryl radicals.

[0168] C-heteroaryl” refers to a heteroaryl radical as defined above and where the point of attachment of the heteroaryl radical to the rest of the molecule is through a carbon atom in the heteroaryl radical. A C-heteroaryl radical is optionally substituted as described above for heteroaryl radicals.

[0169] “Heteroarylalkyl” refers to a radical of the formula -Rc-heteroaryl, where Rcis an alkylene chain, an alkenylene chain, or an alkynylene chain as defined above. If the heteroaryl is a nitrogen-containing heteroaryl, the heteroaryl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain, the alkenylene chain, or the alkynylene chain of the heteroarylalkyl radical is optionally substituted as defined above. The heteroaryl part of the heteroarylalkyl radical is optionally substituted as defined above for a heteroaryl group.

[0170] “Heteroarylalkoxy” refers to a radical bonded through an oxygen atom of the formula -O-Rc-heteroaryl, where Rcis an alkylene chain, an alkenylene chain, or an alkynylene chain as defined above. If the heteroaryl is a nitrogen-containing heteroaryl, the heteroaryl is optionally attached to the alkyl radical at the nitrogen atom. The alkylene chain, the alkenylene chain, or the alkynylene chain of the heteroarylalkoxy radical is optionally substituted as defined above. The heteroaryl part of the heteroarylalkoxy radical is optionally substituted as defined above for a heteroaryl group.

[0171] “Leaving group” or “LG” as used herein may refer to an atom or a group that departs with a pair of electrons in heterolytic bond cleavage. In some instances, a leaving group is capable of being displaced by a nucleophile. Such a leaving group may include, but is not limited to halogen such as chloro, bromo, fluoro, and iodo; alkyl; aryl; aralkyl; carbocyclyl; heteroalkyl; heterocyclyl; and the like. Additional examples may be found in Smith, March’s Advanced Organic Chemistry, 8thEdition (2020), which is incorporated herein by reference. In some instances, a leaving group may stabilize the negative charge upon departure, such leaving group may include, but is not limited to halogen, tosylate (OTs), mesylate (OMs), triflate (OTf), nonaflate (ONf), besylate (OBs), sulfate (-SO4), and phosphate (-PO4). In some instances, aWSGR Docket No. 60652-721601leaving group may dissociate after undergoing chemical modification (e.g., transformation of -OH into a more favorable leaving group -OTs) or under specific reaction conditions (e.g., protonation in acidic conditions), such leaving group may include, but is not limited to, hydroxyl (-OH), amine (-NH2), and alkoxide (-OR). In some instances, the leaving group is an intramolecular leaving group, which participates in a reaction where bond cleavage and new bond formation occur within the same molecule. Examples of such intramolecular leaving groups include phenylthiocarbamoyl (PTC) group elimination in Edman degradation, where the PTC-peptide intermediate undergoes intramolecular cyclization and subsequent cleavage to release the N-terminal amino acid as an anilinothiazolinone (ATZ) derivative.

[0172] “Protecting group” or “PG” refers to a group that can temporarily block a particular functional moiety, e.g., O, S, or N, so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. A protecting group can be added and removed at specific stages during the synthesis of a compound or when the compound participates in a reaction. Exemplary protecting groups are described in detail in Theodora W. Greene et. al. Protecting Groups in Organic Synthesis, 4thedition (2007), which is herein incorporated by reference in its entirety. Protection of functional groups of a compound may alter other physical properties besides the reactivity of the protected functional group, such as the polarity, solubility, lipophilicity (hydrophobicity), and other properties.

[0173] “Bulky group” or “BG” refers to a substituent or functional group that occupy spatial volume around a molecular center, often impeding steric access to reactive sites. In some embodiments, the bulky group can influence molecular conformations, reaction pathways, and / or steric hindrance effects in chemical reactions. Examples may include, but are not limited to, tert-butyl (-C(CH3)3), triphenylmethyl (-C(C6Hs)3), isopropyl (-CH(CH3)2), silyl protecting groups (e.g., trimethylsilyl, -Si(CH3)3), etc. In some embodiments, a bulky group comprises a methyl group. In systems with constrained geometries (e.g., bridgehead positions, cyclic structures, or sterically crowded environments), the presence of a methyl group can significantly influence molecular conformation and restrict access to reactive sites.

[0174] “Electron-withdrawing group” or “EWG” as used herein is a moiety, e.g., an atom or group, which draws electron density from the neighboring atoms towards itself, usually by resonance or inductive effects. Non-limiting examples of EWG are halogen, haloalkyl, -C(O)R', -COOR', -C(O)NH2, -NHC(O)R', -C(O)NR'R", carbonyl, -CF3, -CN, sulfonate, amino, alkylamino, -SO3H, -SO2CF3, -SO2R', SO2NR'R", oxo (=0), alkyl ammonium, and -NO2, wherein R' and R" are independently H, alkyl, heteroalkyl, aryl, heteroaryl, each optionally substituted with one or more EWGs.WSGR Docket No. 60652-721601

[0175] “Electron-donating group” or “EDG” as used herein is a moiety, e.g., an atom or group, which releases electron density to the neighboring atoms from itself, usually by resonance or inductive effects. Non-limiting examples of EDG are -NRN1RN2, -OR', -NHC(O)R', -OC(O)R', phenyl, and vinyl, wherein RN1, RN2, and R' are independently H, alkyl, heteroalkyl, aryl, heteroaryl, each optionally substituted with one or more EDGs.

[0176] Solvent” refers to a substance that dissolves one or more solutes, resulting in a solution. A solvent may serve as a medium for any reaction or transformation described herein. The solvent may dissolve one or more reactants or reagents in a reaction mixture. The solvent may facilitate the mixing of one or more reagents or reactants in a reaction mixture. The solvent may also serve to increase or decrease the rate of a reaction relative to the reaction in a different solvent. Solvents can be organic solvents and aqueous (water-based) solvents. Organic solvents comprise carbon-based compounds that dissolve organic and inorganic substances and are commonly used in chemical synthesis, extraction, and purification processes. Aqueous solvents comprise water-miscible organic solvents or buffers to adjust pH, ionic strength, or solubility properties. Solvents can be polar or non-polar, protic or aprotic. Common solvents useful in the methods described herein include, but are not limited to, acetone, acetonitrile, benzene, benzonitrile, 1 -butanol, 2-butanone, butyl acetate, tert-butyl methyl ether, carbon disulfide carbon tetrachloride, chlorobenzene, 1 -chlorobutane, chloroform, cyclohexane, cyclopentane, 1,2-di chlorobenzene, 1,2-dichloroethane, dichloromethane (DCM), N, N-dimethylacetamide N, N-dimethylformamide (DMF), l,3-dimethyl-3,4,5,6-tetrahydro-2-pyrimidinone (DMPU), 1,4-di oxane, 1,3 -di oxane, di ethylether, 2-ethoxy ethyl ether, ethyl acetate, ethyl alcohol, ethylene glycol, dimethyl ether, heptane, n-hexane, hexanes, hexamethylphosphoramide (EIMP A), 2-methoxyethanol, 2-methoxyethyl acetate, methyl alcohol, 2-methylbutane, 4-methyl-2-pentanone, 2-methyl-l -propanol, 2-methyl-2-propanol, l-methyl-2-pyrrolidinone, dimethylsulfoxide (DMSO), nitromethane, 1-octanol, pentane, 3-pentanone, 1-propanol, 2-propanol, pyridine, tetrachloroethylene, tetrahydrofuran (THF), 2-methyltetrahydrofuran, toluene, tri chlorobenzene, 1, 1,2-tri chlorotrifluoroethane, 2,2,4-trimethylpentane, trimethylamine, triethylamine, N, N-diisopropylethylamine, diisopropylamine, water, o-xylene, and p-xylene.

[0177] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.WSGR Docket No. 60652-721601

[0178] Provided herein are sequencing reagents. The sequencing reagents provide a novel approach to sequencing polymeric analytes, such as peptides, wherein monomers, such as terminal amino acids, of the polymeric analytes are isolated using a sequencing reagent provided herein. In some instances, isolation occurs by tethering of a monomer, such as a terminal amino acid, to a capture moiety (e.g., a capture moiety bound to a substrate, the polymeric analyte of interest, or in solution) using the sequencing reagent and cleavage of the monomer from the polymeric analyte (e.g., peptide). In some instances, sequencing comprises coupling the sequencing reagent to a monomer, thereby generating a sequencing reagent-monomer complex, and detecting the sequencing reagent-monomer complex or derivative thereof. The detection of the sequencing reagent-monomer complex or derivative thereof may be performed using specific binding agents (e.g., antibody, nanobody, scFv) to the sequencing reagent-monomer complexes or derivatives thereof, such as sequencing reagent-amino acid complexes. Alternatively, or in addition to, the detection may be performed using a nanopore device or instrument (e.g., nanopore sequencer). In some embodiments, the isolation of amino acids provided herein avoids a local environment problem in which adjacent amino acids can impact the detection of an amino acid, e.g., by altering the binding (e.g., selectivity or affinity) of a binding agent or detection of convoluted signals arising from multiple amino acids present in the detection area of a nanopore device or instrument.

[0179] Provided herein, in some embodiments, are conjugates of sequencing reagents and polymers (e.g., nucleic acids). The conjugation of nucleic acids and sequencing reagents which have the capability of reacting with N-terminal amino acids may enable more rapid and more facile single molecule protein sequencing. The methods provided herein may enable single molecule protein sequencing without the need for a step of conjugation of the sequencing reagent and the (e.g., linking) nucleic acid. This may have benefits of decreasing error, decreasing cost, decreasing waste, and decreasing the time required for protein sequencing.Sequencing Reagents

[0180] Provided herein in some embodiments, are compositions that can be used as sequencing reagents. The compositions may comprise a polymer substituted with a linker and a reactive group. The reactive group may be configured to react with a monomer of a polymeric analyte, such as an N-terminus of a peptide.

[0181] In some embodiments, provided herein are compositions comprising a polymer substituted with -L-B, wherein L is a linker and B is a reactive group configured to react with an N-terminus of a peptide. In some embodiments, the linker comprises one or more electron-WSGR Docket No. 60652-721601withdrawing or electron-donating groups. In some embodiments, the linker comprises one or more electron-withdrawing groups. In some embodiments, the linker comprises one or more electron-donating groups.

[0182] In some embodiments, the polymer is a polynucleotide, a polypeptide, a polymeric material (e.g., a natural polymeric material, synthetic polymeric material), or a combination thereof. In some embodiments, the polymer is a polynucleotide. In some embodiments, the polymer is a polypeptide. In some embodiments, the polymer is a polymeric material. In some embodiments, the polymeric material is a natural polymeric material. In some embodiments, the polymeric material is a synthetic polymeric material.

[0183] In some embodiments, the polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), a locked nucleic acid (LNA), a parallel-stranded DNA (p-DNA), a nonconstrained nucleic acid (NNA), messenger RNA (mRNA), transfer RNA (tRNA), micro RNA (miRNA), small interfering RNA (siRNA), long non-coding RNA (IncRNA), CRISPR RNA (crRNA), guide RNA (gRNA), complementary DNA (cDNA), single stranded DNA (ssDNA), or double stranded DNA (dsDNA). In some embodiments, the polynucleotide is DNA or RNA. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the polynucleotide is a locked nucleic acid (LNA). In some embodiments, the polynucleotide is a parallel-stranded DNA (p-DNA). In some embodiments, the polynucleotide is a non-constrained nucleic acid (NNA). In some embodiments, the polynucleotide comprises one or more sugar modifications. In some embodiments, the sugar ring of the nucleotide is replaced or modified. In some embodiments, the sugar is substituted with a cyclopentane ring. In some embodiments, the sugar is substituted with a cyclohexane, cycloheptane, or other six- or more-membered carbocyclic or heterocyclic ring. In some embodiments, the sugar moiety comprises one or more substitutions at the 2’-position, 3’-position, or 4’ -position. In some embodiments, the sugar moiety comprises a 2’-O-methyl, 2’-fluoro, 2’-amino, or 2’-azido modification. In some embodiments, the sugar moiety comprises a 3’-fluoro or 4’-thio substitution. In some embodiments, the sugar moiety is a hexose or pentose analog. In some embodiments, the sugar ring is replaced with a morpholino ring. In some embodiments, the sugar ring is replaced with a bridged structure. In some embodiments, the sugar ring is replaced with a locked structure. In some embodiments, the sugar ring is replaced with a bridged or locked structure, such as in a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a hexitol nucleic acid (HNA), or a threose nucleic acid (TNA). In some embodiments, the sugar is replaced with a constrained ethylene bridge, as in ethylene-bridged nucleic acids (ENAs). In some embodiments, the sugar is replaced with a cyclohexenyl orWSGR Docket No. 60652-721601bicyclo[2.2.1]heptane structure. In some embodiments, the sugar ring is replaced with a piperidine, pyrrolidine, or azetidine ring. In some embodiments, the sugar moiety is substituted with a six-membered nitrogen-containing ring, such as in hexitol nucleic acids (HNAs). In some embodiments, the sugar is replaced with a threose moiety, as in threose nucleic acids (TNAs). In some embodiments, the sugar is replaced with a glycol or other acyclic linker. In some embodiments, the sugar is replaced with an acyclic linker comprising a 2-carbon backbone. In some embodiments, the sugar is replaced with an acyclic linker comprising a 3-carbon backbone. In some embodiments, the sugar is replaced with an acyclic linker comprising a 4-carbon backbone. In some embodiments, the sugar is replaced with an acyclic linker comprising a 5-carbon backbone. In some embodiments, the sugar is replaced with an acyclic linker comprising a 6-carbon backbone. In some embodiments, the acyclic linker comprises a substituted or unsubstituted alkylene chain. In some embodiments, the acyclic linker comprises ethylene glycol, propylene glycol, butylene glycol, pentylene glycol, or hexylene glycol moieties. In some embodiments, the acyclic linker comprises one or more ether, thioether, amine, or alkyl groups. In some embodiments, the acyclic linker comprises polyethylene glycol (PEG) or related oligomeric or polymeric units. In some embodiments, the acyclic linker comprises one or more functional groups that enable phosphodiester or phosphorami date linkage to adjacent nucleotides. In some embodiments, the acyclic linker comprises a substituted or unsubstituted alkenylene or alkynylene chain. In some embodiments, the acyclic linker is linear, branched, or cyclic. In some embodiments, the acyclic linker maintains base-pairing geometry and backbone spacing compatible with Watson-Crick hybridization. In some embodiments, the sugar is replaced with a non-sugar, non-cyclic linker as part of a synthetic nucleotide analog. In some embodiments, the sugar ring is replaced with a non-natural scaffold capable of maintaining base-pairing and backbone spacing. In some embodiments, the sugar is replaced with a peptide-like scaffold, as in peptide nucleic acids (PNAs). In some embodiments, the sugar does not comprise adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), or any abasic derivative thereof (e.g., abasic nucleotides). In some embodiments, the polynucleotide is a nucleic acid as described elsewhere herein.

[0184] In some embodiments, the polynucleotide counterion is monovalent. Non-limiting examples of monovalent counterions include Sodium (Na+), Potassium (K+), Lithium (Li+), Ammonium (NHL), Cesium (Cs+), Rubidium (Rb+), Hydrogen (H+) (e.g., protonation of the phosphate group at low pH), etc. In some embodiments, the polynucleotide counterion is divalent. Non-limiting examples of divalent counterions include Magnesium (Mg2+), Calcium (Ca2+), Barium (Ba2+), Strontium (Sr2+), Zinc (Zn2+), Cobalt (Co2+), Manganese (Mn2j, CopperWSGR Docket No. 60652-721601(Cu2+), Nickel (Ni2+), etc. In some embodiments, the polynucleotide counterion is trivalent or higher valence counterions. Non-limiting examples of trivalent or higher valence counterions include Aluminum (Al3+), Lanthanides (e.g., La3+, Ce3+, Eu3+), Chromium (Cr3+), Iron (Fe3+), etc. In some embodiments, the polynucleotide counterion is organic. Non-limiting examples of organic counterions include Spermine4, Spermidine34, Protonated Amines (e.g., ethanolamine4, trimethylamine4), Histidine-derived cations, Choline (CSHMNO ), etc. In some embodiments, the polynucleotide counterions are complex or specialized counterions. Non-limiting examples of the complex or specialized counterions include Polyethyleneimine (PEI), Polycations (e.g., poly-L-lysine), Ionic liquid components (e.g., imidazolium-based cations), Transition metal complexes (e.g., Ru24, Pt24), Artificially modified cations (e.g., N-methylimidazolium), etc.

[0185] In some embodiments, the natural polymeric material is cellulose, starch, chitin, lignin, pectin, alginate, agar, or a modified version thereof. In some embodiments, the synthetic polymer is polyethylene, polypropylene, polystyrene, polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), polyurethane, polycarbonate, or nylon.

[0186] In some embodiments, the composition is a polynucleotide composition comprising a modified nucleotide. In some embodiments, the modified nucleotide is substituted with a linker and a reactive group configured to react with an N-terminus of a peptide. In some embodiments, the modified nucleotide is substituted with a reactive group configured to react with an N-terminus of a peptide. In some embodiments, the modified nucleotide is substituted with -L-B, such as described elsewhere herein.

[0187] In some embodiments, the modified nucleotide is structurally analogous to a corresponding naturally occurring nucleotide (e.g., adenosine, guanosine, cytidine, thymidine, or uridine). For example, in some embodiments, the modified nucleotide comprises a sugarphosphate backbone that is the same as, or substantially the same as, the sugar-phosphate backbone of the corresponding naturally occurring nucleotide and / or comprises the same nucleobase identity as the corresponding naturally occurring nucleotide. In some embodiments, the modification is located at a position that does not substantially disrupt the overall nucleotide geometry, such as at the 5-position of a pyrimidine base, the 7-position of a 7-deazapurine base, the 2'-position of the sugar, and / or the phosphate group. In some embodiments, the modified nucleotide has a ribose or deoxyribose sugar that is the same as, or substantially the same as, the sugar of the corresponding naturally occurring nucleotide (e.g., 2 '-deoxyribose for DNA or ribose for RNA), and a phosphate group capable of forming a phosphodiester linkage within a nucleic acid backbone.WSGR Docket No. 60652-721601

[0188] In some embodiments, the modified nucleotide is functionally analogous to a corresponding naturally occurring nucleotide. For example, in some embodiments, the modified nucleotide participates in Watson-Crick base pairing with a complementary nucleotide (e.g., A: T or A: U, G: C) that is the same as, or not substantially different from, that of the corresponding naturally occurring nucleotide. In some embodiments, the modified nucleotide is recognized and incorporated by a polymerase (e.g., a DNA polymerase, an RNA polymerase, or a reverse transcriptase) and / or serves as a template for replication, transcription, amplification, or sequencing, substantially as does the corresponding naturally occurring nucleotide. In some embodiments, the modified nucleotide is introducible into, or incorporable within, a polynucleotide by synthetic nucleic acid synthesis methods, for example solid-phase synthesis, enzymatic synthesis, or other template-directed or non-template-directed chemical coupling methods. In some embodiments, the modified nucleotide is compatible with one or more nucleic-acid-processing enzymes, such as ligases, nucleases, and / or repair enzymes, such that the presence of the modification does not substantially interfere with primer extension, ligation, cleavage, or other enzymatic processing relative to the corresponding naturally occurring nucleotide.

[0189] In some embodiments, the modified nucleotide is comprised in a hairpin nucleic acid molecule. For example, in some embodiments, the hairpin nucleic acid molecule is or comprises a linking nucleic acid molecule (e.g., 111 or 211). In some embodiments, the hairpin nucleic acid molecule comprising the modified nucleotide is compatible with one or more nucleic-acid-processing enzymes, such as ligases, nucleases, and / or repair enzymes.

[0190] Provided herein, in some embodiments, is a polynucleotide composition comprising a modified nucleotide of Formula (IA):OO=P-O’i°yFormula (I A)wherein,L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups;B is a reactive group configured to react with an N-terminus of a peptide.WSGR Docket No. 60652-721601

[0191] In some embodiments, L is a linker, such as a linker described elsewhere herein. In some embodiments, the linker may additionally comprise one or more electron-donating groups. In some embodiments, the linker may additionally comprise one or more electron-withdrawing groups. In some embodiments, the electron-donating or electron-withdrawing groups are positioned to modulate the local electronic or stereochemical environment. In some embodiments, the electron-donating or electron-withdrawing groups are positioned to stabilize transient charge distributions in Edman degradation reactions. In some embodiments, the electron-donating or electron-withdrawing groups are positioned to facilitate Edman degradation reactions.

[0192] In some embodiments, B is a reactive group. In some embodiments, the reactive group is configured to react with an N-terminus of a peptide. In some embodiments, the reactive group is a reactive group described elsewhere herein.

[0193] In some embodiments, -L-B is attached at a stereochemically defined position on a ribose or ribose analog. In some embodiments, -L-B is attached at the 1’-, 2’-, 3’-, or 5’-position of a ribose or ribose analog. In some embodiments, the stereochemistry at the carbon bearing -L-B is in the R or S configuration. In some embodiments, -L-B is positioned at an axial orientation on a ribose or sugar analog. In some embodiments, -L-B is positioned at an equatorial orientation on a ribose or ribose analog. In some embodiments, the axial or equatorial orientation of -L-B is preserved during synthesis by using stereoselective or stereospecific chemistry. In some embodiments, the equatorial or axial disposition of -L-B influences the accessibility of adjacent functional groups (e.g., the reactive group B). In some embodiments, -L-B comprises one or more stereocenters. In some embodiments, the stereochemistry of the -L-B is selected to reduce conformational flexibility, increase molecular rigidity, or enhance structural stability.

[0194] In some embodiments, one or both of the phosphate of the modified nucleotide of Formula (IA) is a boranophosphate, thiophosphate, or alkylated phosphate. In some embodiments, one of the phosphates of the modified nucleotide of Formula (IA) is a boranophosphate. In some embodiments, one of the phosphates of the modified nucleotide of Formula (IA) is a thiophosphate. In some embodiments, one of the phosphates of the modified nucleotide of Formula (IA) is an alkylated phosphate.

[0195] In some embodiments, provided herein is a polynucleotide composition comprising a modified nucleotide of Formula (lA-a), Formula (lA-b), Formula (IA-c), or Formula (lA-d):WSGR Docket No. 60652-721601Formula (lA-a) Formula (lA-b) Formula (IA-c) Formula (IA- d), wherein,Ring A is a nitrogenous base or modified version thereof,L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups; andB is a reactive group configured to react with an N-terminus of a peptide.

[0196] In some embodiments, L and B are as described elsewhere herein.

[0197] In some embodiments, Ring A is a nitrogenous base or modified version thereof. In some embodiments, Ring A is a nitrogenous base. Ring A may be adenine. Ring A may be guanine. Ring A may be thymine. Ring A may be cytosine. Ring A may be uracil. In some embodiments, Ring A is adenine, guanine, thymine, or cytosine. In some embodiments, Ring A is adenine, guanine, uracil, or cytosine.

[0198] In some embodiments, provided herein is a polynucleotide composition comprising a modified nucleotide of Formula (IIA):Formula (IIA),wherein,Ring A is a nitrogenous base or modified version thereof;L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups; andB is a reactive group configured to react with an N-terminus of a peptide.

[0199] In some embodiments, Ring A, L, and B are as described elsewhere herein.WSGR Docket No. 60652-721601

[0200] In some embodiments, -Ring A-L-B is attached at a stereochemically defined position on a ribose or ribose analog. In some embodiments, -Ring A-L-B is attached at the 1’-, 2’-, 3’-, or 5’-position of a ribose or ribose analog. In some embodiments, the stereochemistry at the carbon bearing -Ring A-L-B is in the R or S configuration. In some embodiments, -Ring A-L-B is positioned at an axial orientation on a ribose or sugar analog. In some embodiments, -Ring A-L-B is positioned at an equatorial orientation on a ribose or sugar analog. In some embodiments, the axial or equatorial orientation of -Ring A-L-B is preserved during synthesis by using stereoselective or stereospecific chemistry. In some embodiments, the equatorial or axial disposition of -Ring A-L-B influences the accessibility of adjacent functional groups (e.g., the reactive group B). In some embodiments, -Ring A-L-B comprises one or more stereocenters. In some embodiments, the stereochemistry of the -Ring A-L-B is selected to reduce conformational flexibility, increase molecular rigidity, or enhance structural stability.

[0201] In some embodiments, the polynucleotide composition comprises a modified nucleotide of Formula (IIA-a):O=P-O’i°yFormula (IIA-a),wherein,Ring A is a nitrogenous base or modified version thereof;L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups; andB is a reactive group configured to react with an N-terminus of a peptide.

[0202] In some embodiments, Ring A, L, and B are as described elsewhere herein.

[0203] In some embodiments, -Ring A-L-B is attached at a stereochemically defined position on a ribose or ribose analog. In some embodiments, -Ring A-L-B is attached at the 1’-position of a ribose or ribose analog.

[0204] Provided herein in some embodiments, is a polynucleotide composition comprising a modified nucleotide of Formula (IIIA):WSGR Docket No. 60652-721601Formula (III A),wherein,Ring A is a nitrogenous base or modified version thereof;L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups; andB is a reactive group configured to react with an N-terminus of a peptide.

[0205] In some embodiments, Ring A, L, and B are as described elsewhere herein.

[0206] In some embodiments, Ring A is attached at a stereochemically defined position on a ribose or ribose analog. In some embodiments, Ring A is attached at the l’-position of a ribose or ribose analog. In some embodiments, -L-B is attached at a stereochemically defined position on a ribose or ribose analog. In some embodiments, - L-B is attached at the 2’ -position of a ribose or ribose analog.

[0207] In some embodiments, the modified nucleotide of Formulas (IA), (lA-a), (lA-b), (IA-c), (lA-d), (IIA), or (IIIA), comprises a structure selected from Table 1 A.Table 1AStructuralStructureBackboneNH2" O Cr I'MO=P-O.TlA-la 6- Vo-J0OP-O- VWSGR Docket No. 60652-721601NH2X A0 O^N'\-B O=P-O. 1TlA-lb d- V-Q-J0O=P-O- V HN-L-B X X O O NO=P-O. 1TlA-lc 6- vyoO=P-O- Oy- 0 HNX^L-B o O^N'JO=p-O. ITlA-2a 6- V-Q-J0O=P-O- V0> HN^X0 O^N^L-B O=P-O. ITlA-2b O’ V°^l0O=P-O- dyB1 0k A / x yr0 O^NO=p-O. I TlA-2c o- Vy0O=P-O- VWSGR Docket No. 60652-7216010X* 0 cHXNV'bT-BO=P-O. 1 TlA-3a O’0O=P-O- V0XHN10 O^N^L-B O=P-(X ITlA-3b O’ V°^l0O=p-crOyBl 0 XL'^0 cr N O=p-O. 1 TlA-3c 0- Vo^l 0OP-CrOyNH2Bl / TlA-4a O=P-CkO’ V-0^0OP-O- VB-L NH2Z<bTlA-4b O=p-Oxxb 6- 0O=P-O’VWSGR Docket No. 60652-721601NH2B JITlA-4c O=P-O.6- VO'-J0OP-O- V01'J^'N^NHs TlA-5a O^p-O.d- \-0^0O=P-O- VB0J'^N'X^NH2TlA-5b O=p-O.d- V-0-^0O=P-CT0^ / 0Bi z ^^rj^NHz<b1TlA-5c OP-O.0- 00=P-0’0y>4 / 0=P-0. I0- T1A-600=P-0’0y>WSGR Docket No. 60652-721601O=p-O.O’T1A-70 F-BO=P-O- Oy® O=p-O. ® 16- TlA-8a0O=p-O- L- BV® ®O=p-O. 16"TlA-8b0O=P-L-BOO=P-O.d- TlA-9a0O=P-O-L-BO^p-O.6’ V0"?TlA-9b0O=p- L- B0

[0208] Provided herein, in some embodiments, is a polynucleotide composition comprising a modified nucleotide of Formula (IB):WSGR Docket No. 60652-721601oO=p-OI°yFormula (IB)wherein,L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups;B is a reactive group configured to react with an N-terminus of a peptide;Xais O, CH(L-B), or CH2;Xbis O, CH(L-B), or CH2;Xcis absent, CH(L-B), or CH2;Xdis absent, H, O, OH, halogen, CH(L-B), OMe, or CH2; andXeis absent, O, CH(L-B), or CH2.

[0209] In some embodiments, L is a linker, such as a linker described elsewhere herein. In some embodiments, the linker may additionally comprise one or more electron-donating groups. In some embodiments, the linker may additionally comprise one or more electron-withdrawing groups. In some embodiments, the electron-donating or electron-withdrawing groups are positioned to modulate the local electronic or stereochemical environment. In some embodiments, the electron-donating or electron-withdrawing groups are positioned to stabilize transient charge distributions in Edman degradation reactions. In some embodiments, the electron-donating or electron-withdrawing groups are positioned to facilitate Edman degradation reactions.

[0210] In some embodiments, B is a reactive group. In some embodiments, the reactive group is configured to react with an N-terminus of a peptide. In some embodiments, the reactive group is a reactive group described elsewhere herein.

[0211] In some embodiments, -L-B is attached at a stereochemically defined position on a ribose or ribose analog. In some embodiments, -L-B is attached at the 1 ’(i.e., Xa)-, 2’-, 3’-, Xb-, Xc-, Xd-, or 5 ’(i.e., Xe)-position of a ribose or ribose analog. In some embodiments, the stereochemistry at the carbon bearing -L-B is in the R or S configuration. In some embodiments,WSGR Docket No. 60652-721601-L-B is positioned at an axial orientation on a ribose or sugar analog. In some embodiments, -L-B is positioned at an equatorial orientation on a ribose or sugar analog. In some embodiments, the axial or equatorial orientation of -L-B is preserved during synthesis by using stereoselective or stereospecific chemistry. In some embodiments, the equatorial or axial disposition of -L-B influences the accessibility of adjacent functional groups (e.g., the reactive group B). In some embodiments, -L-B comprises one or more stereocenters. In some embodiments, the stereochemistry of the -L-B is selected to reduce conformational flexibility, increase molecular rigidity, or enhance structural stability.

[0212] In some embodiments, Xais O, CH(L-B), or CH2. In some embodiments, Xais O. In some embodiments, Xais CH(L-B). In some embodiments, Xais CH2.

[0213] In some embodiments, Xbis O, CH(L-B), or CH2. In some embodiments, Xbis O. In some embodiments, Xbis CH(L-B). In some embodiments, Xbis CH2.

[0214] In some embodiments, Xcis absent, CH2, or CH(L-B). In some embodiments, Xcis absent. In some embodiments, Xcis CH2. In some embodiments, Xcis CH(L-B).

[0215] In some embodiments, Xdis absent, H, O, OH, halogen, CH(L-B), OMe, or CH2. In some embodiments, Xdis absent. In some embodiments, Xdis H. In some embodiments, Xdis O. In some embodiments, Xdis OH. In some embodiments, Xdis halogen. In some embodiments, Xdis F. In some embodiments, Xdis Br. In some embodiments, CH(L-B). In some embodiments, Xdis OMe. In some embodiments, Xdis CH2.

[0216] In some embodiments, Xeis absent, O, CH2, or CH(L-B). In some embodiments, Xeis absent. In some embodiments, Xeis O. In some embodiments, Xeis CH2. In some embodiments, Xeis CH(L-B).

[0217] In some embodiments, one or both of the phosphate of the modified nucleotide of Formula (IB) is a boranophosphate, thiophosphate, or alkylated phosphate. In some embodiments, one of the phosphates of the modified nucleotide of Formula (IB) is a boranophosphate. In some embodiments, one of the phosphates of the modified nucleotide of Formula (IB) is a thiophosphate. In some embodiments, one of the phosphates of the modified nucleotide of Formula (IB) is an alkylated phosphate. In some embodiments, one of the phosphates of the modified nucleotide of Formula (IB) is attached at a stereochemically defined position on a ribose or ribose analog. In some embodiments, one of the phosphates of the modified nucleotide of Formula (IB) is attached at the 1 ’(i.e., Xa)-, 2’-, 3’-, Xb-, Xc-, Xd-, or 5’(i.e., Xe)-position of a ribose or ribose analog. In some embodiments, one of the phosphates of the modified nucleotide of Formula (IB) is attached at the 1 ’(i.e., Xa)-position of a ribose or ribose analog. In some embodiments, one of the phosphates of the modified nucleotide ofWSGR Docket No. 60652-721601Formula (IB) is attached at the 2’ -position of a ribose or ribose analog. In some embodiments, one of the phosphates of the modified nucleotide of Formula (IB) is attached at the 3’-position of a ribose or ribose analog. In some embodiments, one of the phosphates of the modified nucleotide of Formula (IB) is attached at the Xb-position of a ribose or ribose analog.

[0218] In some embodiments, provided herein is a polynucleotide composition comprising a modified nucleotide of Formula (IB-a), Formula (IB-b), Formula (IB-c), or Formula (IB-d):Formula (IB-a) Formula (IB-b) Formula (IB-c)OiO=p— L-BIFormula (IB-d)wherein,Ring A is a nitrogenous base or modified version thereof,L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups; andB is a reactive group configured to react with an N-terminus of a peptide.

[0219] In some embodiments, L and B are as described elsewhere herein.

[0220] In some embodiments, Ring A is a nitrogenous base or modified version thereof. In some embodiments, Ring A is a nitrogenous base. Ring A may be adenine. Ring A may be guanine. Ring A may be thymine. Ring A may be cytosine. Ring A may be uracil. In some embodiments, Ring A is adenine, guanine, thymine, or cytosine. In some embodiments, Ring A is adenine, guanine, uracil, or cytosine.WSGR Docket No. 60652-721601

[0221] In some embodiments, provided herein is a polynucleotide composition comprising a modified nucleotide of Formula (IIB):OO=P-O"i°yFormula (IIB)wherein,Ring A is a nitrogenous base or modified version thereof;L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups; andB is a reactive group configured to react with an N-terminus of a peptide.

[0222] In some embodiments, Ring A, L, and B are as described elsewhere herein.

[0223] In some embodiments, -Ring A-L-B is attached at a stereochemically defined position on a ribose or ribose analog. In some embodiments, -Ring A-L-B is attached at the 1 ’(i.e., Xa)-, 2’-, 3’-, Xb-, Xc-, Xd-, or 5 ’ (i.e., Xe)-position of a ribose or ribose analog. In some embodiments, the stereochemistry at the carbon bearing -Ring A-L-B is in the R or S configuration. In some embodiments, -Ring A-L-B is positioned at an axial orientation on a ribose or sugar analog. In some embodiments, -Ring A-L-B is positioned at an equatorial orientation on a ribose or sugar analog. In some embodiments, the axial or equatorial orientation of -Ring A-L-B is preserved during synthesis by using stereoselective or stereospecific chemistry. In some embodiments, the equatorial or axial disposition of -Ring A-L-B influences the accessibility of adjacent functional groups (e.g., the reactive group B). In some embodiments, -Ring A-L-B comprises one or more stereocenters. In some embodiments, the stereochemistry of the -Ring A-L-B is selected to reduce conformational flexibility, increase molecular rigidity, or enhance structural stability.

[0224] In some embodiments, the polynucleotide composition comprises a modified nucleotide of Formula (IIB-a):WSGR Docket No. 60652-721601Formula (IIB-a)wherein,Ring A is a nitrogenous base or modified version thereof;L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups; andB is a reactive group configured to react with an N-terminus of a peptide.

[0225] In some embodiments, Ring A, L, and B are as described elsewhere herein.

[0226] In some embodiments, -Ring A-L-B is attached at a stereochemically defined position on a ribose or ribose analog. In some embodiments, -Ring A-L-B is attached at the 1 ’(i.e., Xa)-position of a ribose or ribose analog.

[0227] In some embodiments, Xais CH2, Xbis O, Xcis absent, Xdis OH, and Xeis CH2. In some embodiments, the modified nucleotide of Formulas (IB), (IB-a), (IB-b), (IB-c), (IB-d),(IIB), or (IIB-a) comprises a structureof

[0228] In some embodiments, Xais CH2, Xbis O, Xcis absent, Xdis F, and Xeis CH2. In some embodiments, the modified nucleotide of Formulas (IB), (IB-a), (IB-b), (IB-c), (IB-d),(IIB), or (IIB-a) comprises a structureofWSGR Docket No. 60652-721601

[0229] In some embodiments, Xais CH2, Xbis O, Xcis absent, Xdis OMe, and Xeis CH2. In some embodiments, the modified nucleotide of Formulas (IB), (IB-a), (IB-b), (IB-c), (IB-d),(IIB), or (IIB-a) comprises a structureof

[0230] In some embodiments, Xais CH2, Xbis CH2, Xcis absent, Xdis H, and Xeis CH2. In some embodiments, the modified nucleotide of Formulas (IB), (IB-a), (IB-b), (IB-c), (IB-d),°\(IIB), or (IIB-a) comprises a structureof.

[0231] In some embodiments, Xais CH2, Xbis O, Xcis CH2, Xdis O, and Xeis CH2. In some embodiments, the modified nucleotide of Formulas (IB), (IB-a), (IB-b), (IB-c), (IB-d),(IIB), or (IIB-a) comprises a structureof.

[0232] In some embodiments, Xais O, Xbis CH2, Xcis absent, Xdis H, and Xeis O. In some embodiments, the modified nucleotide of Formulas (IB), (IB-a), (IB-b), (IB-c), (IB-d), (IIB), or(IIB-a) comprises a structureof f

[0233] In some embodiments, the modified nucleotide of Formulas (IB), (IB-a), (IB-b), (IB-c), (IB-d), (IIB), or (IIB-a) comprises a structure selected from Table IB.Table IBStructuralStructureBackboneWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601MH2kXL—BT'ATIB-leH2\LV / - ■s— B M? X{\ O J \ OJ 000— \ X? —- TIIOQ-—-- II V1 6 o ° ° ~o x OOCL-, 1,o \TIB-lf A ii\ V IIo H „ z OOCL-- - X V iiO OTJ-O- po x O"0-- ' 1 ' 1°° O o / / _7A- V\O OTJo O-—T--- / x i o X 4 o ok / J O W—JH2XJ— B TlB-2a1JH2" XJ— B TlB-2bWSGR Docket No. 60652-721601\IH2jX^L — B TlB-2c / leSIH27\Z. < Z1— -^ ■Ji\ O JO0- \ O—- O- JOQ- A (—-,- x?A II r II-S I'^^L — B / < °O O X XTl OC--, i, 1B-2d A A ii II OV II OO0---LOO O OTITJ-- O X o X V II -- ■ ■ i i°° o o / / _A ' ii II / y\ / / X°° V o Pz, / k / * / J 2 J w w—x _\IH2— B TlB-2eH2x— B TlB-2f,0WSGR Docket No. 60652-721601— L — B TlB-3at— L — B v / °\ \ O O JO JO0-0-? -- ---- A? x?" ° °II II A1,, i O o ° °TlB-3b o o \ \ O OOOCLCL---- x A A V ii III xzo o xI I IOO O OTlU---- ' 11i°° o O / / AA II II / / \ rOO O OTITJ- —- —-- ) y % Xi X ° qz x— L — B )TlB-3c1e— L — B " J] ATlB-3dWSGR Docket No. 60652-721601IN — L — B J]‘N^TlB-3e / )CD_ l< o z T= - V Oooi Z--- M ( I '1\ O JO0-—-- A T ' x ° °IIo x3 AI IxOO, 6L- °, 1OC- TlB- f Xo OO OO xTI0- A---- V ii i II■ iV II O OTI-- o x ° ' 1 O / A II / rxO O-D- —- x Pi V Ol°°T / vxo>oTlB-4ajT^L - BTlB-4bWSGR Docket No. 60652-7216017 V^L-B XHN>0 0^O=p-CkTlB-4c4V7o or / leiO=p-O’1°y7\ rnJ< z=- \ O / JO0- L —- O O-O XQ-l A (—-- ' x (? xO \TlB-4d AI IT-, 16 ° ° IIO O OO0-- 1-- OO0.--,X X ii xz V II ' 1o o ° o / _AO OTH-NxV^-L-B TlB-4ey^L-BTlB-4f PWSGR Docket No. 60652-721601)J— B TlB-5at)Y / ■°T H X jTO J— A X? XC x?? °\ \ \ O OOL-- ° °OO J JQ-Q-—--—-- Z r H II II-^7^7J— B O 6 ° °Tl - b o X,, 1 1 1,B 5 A IIxOOO OO OCLQ-CL------ ii xzO OT-- o o x x V V II IIo x ■ i° O / IIA / \O f O-0-—- _1 Xx / )J— B TlB-5c / le) Y"J— B TlB-5dWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601) L ZL— B TrTlB-7ai)r / ■° l^ zL — Bx£A x X i? i ° - \ \ O OO JOQ-0-5TJ—- —--- r A II II~^ r6 c> ° °O o x X,, 1 iTlB-7b I I O OOO0-0.---- A A V V H H iiO o X x OO O O "0TJ——-- ' ' 11°° O O / / AA II II / \ r (0xO 0 O-0-U —---- —X 4° M / ) 1 VxL °) L / l. — B TrTlB-7c1e) U / L — B TTlB-7dWSGR Docket No. 60652-7216013—BTTlB-7eV / ■s— B o A A VC? X H? X? x\L0° ° T\ \ O 0 JO J00-0—O—--—--- II 11 IIO 0 6TlB-7f OOC,, l 1, 1000^0 X O OOOCLCLL------ PV II A XZ 1100 X V 11110 x X O OT-- ' 1° 0 / II\ r0 o 0T —-- _v>jlJ— B TlB-8a1)— B TlB-8bWSGR Docket No. 60652-721601Ji I^^L — B TlB-8cVie)r / ■°jl\ \ O O (OO J J0-0-—-—--- L H X f x? °II II — B TlB-8d O X o X,, i iA ii O O O O CL CLH II xz X A II————ZO OT-- o o x x O OTJ-- ' 1 ■ i°° o / O / _' IIA / \ / Z II\ / o oy-- f-O c Ot>-U- —- _ / Xi J o) H x 4A / J o \)Jl I'^L — B TlB-8e— B TlB-8f 0WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601NH2? 1 ^0O=p-CLTIB-lOc 0- n \ / le 0=P-0- V NH2? 1L-^ Z'O=p-O.TIB-lOd o- 0O=p-crdy>NH2? i ^xb O=p-O.TIB-lOeO=p-O’Oy- NH2B1,Nxb OP-CkTIB-lOf o- 00O=P-O- VWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-7216010L-^1O=P-O.TlB-13a 0- VoJn HO=p-O’Oy0 / b. U- —T x 9 r oJ^N^NH2\9 CO O -11OD-— —-- TlB-13b, 9 °iixb ocL-- o \0q'^rp 'NH ^01N^NHs O=p-O- T1B-13C O’ Vx0 0 VieO=p-O’Oy0^01O=P-O- TlB-13d 0- 0O=p-O’VWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-7216010B UO=p-O.TlB-15e' w00O=p-O’Oyo / ], o—BJ-L? X o AX \ O JO0- --- QH<3 °NXXH2O=P-O., 1TlB-15f Q OQ.-- 6’ II XZ VXoo xO o X X OA AI ii I OO o O oTI u----=P-O’■ ■ 11°° o O / / A IIA II / / \\ < Oo o O ("uti- —-- -- Xi X o o o B X / ’ o— _ TlB-16aBTlB-16bBT1B-16CWSGR Docket No. 60652-721601TlB-16dTlB-16eTlB-16fTlB-17aWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601TlB-20bCD CD CD co 1 1 1 J.1. —1 Y / S / / 10V 11- " / x O\ / A i? X? X? X ° o °\ ) \ \ O J O O JOCL —— --- rft-^r O VO0- —oo----- TlB-20c II II6 ° r H-^, 1, l, 1, i 6 6 o ° ° °7xOO0- OOxOOCLCL--O O--LC---- o X V V ii V xzo o X o X Xo XA O OT-- ' 1° O / X / \ (O OT-- —\ 1N / o1 V TlB-20d1TlB-20eTlB-20fWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601

[0234] In some embodiments, provided herein is a polynucleotide composition comprising a modified nucleotide of Formula (IVA-A), (IVA-B), (IVA-C), (IVA-D), or (IVA-E):Formula (IVA-A) Formula (IVA-B) Formula (I VA-C) Formula (IVA-D)Formula (IVA-E)WSGR Docket No. 60652-721601wherein,L is a linker, wherein the linker comprises one or more electron-withdrawing or electron-donating groups;B is a reactive group configured to react with an N-terminus of a peptide; L1and L2are each individually substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or a bond; andL3and L4are each individually substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or H.

[0235] In some embodiments, L and B are as described elsewhere herein.

[0236] In some embodiments, L1, and L2are each individually substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or a bond. In some embodiments, L1, L2, L3, and L4may encompass unsaturated divalent hydrocarbons, such as alkenylene (e.g., -CH=CH-) or alkynylene (e.g., -C=C-) groups.

[0237] In some embodiments, L1is substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or a bond. In some embodiments, L1is a bond. In some embodiments, L1is substituted or unsubstituted alkylene. In some embodiments, L1is substituted or unsubstituted Ci-Ce alkylene. In some embodiments, L1is substituted or unsubstituted heteroalkylene. In some embodiments, L1is substituted or unsubstituted Ci-Ce heteroalkylene. In some embodiments, L1is substituted or unsubstituted cycloalkylene. In some embodiments, L1is substituted or unsubstituted heteroarylene. In some embodiments, L1is substituted or unsubstituted arylene. In some embodiments, L1is methylene (-CH2-).

[0238] In some embodiments, L2is substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or a bond. In some embodiments, L2is a bond. In someWSGR Docket No. 60652-721601embodiments, L2is substituted or unsubstituted alkylene. In some embodiments, L2is substituted or unsubstituted Ci-Ce alkylene. In some embodiments, L2is substituted or unsubstituted heteroalkylene. In some embodiments, L2is substituted or unsubstituted Ci-Ce heteroalkylene. In some embodiments, L2is substituted or unsubstituted cycloalkylene. In some embodiments, L2is substituted or unsubstituted heteroarylene. In some embodiments, L2is substituted or unsubstituted arylene. In some embodiments, L2is methylene (-CH2-).

[0239] In some embodiments, L3and L4are each individually substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or H. In some embodiments, L3and L4may encompass unsaturated divalent hydrocarbons, such as alkenylene (e.g., -CH=CH-) or alkynylene (e.g., -C=C-) groups.

[0240] In some embodiments, L3is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or H. In some embodiments, L3is substituted or unsubstituted alkyl. In some embodiments, L3is substituted or unsubstituted Ci-Ce alkyl. In some embodiments, L3is substituted or unsubstituted heteroalkyl. In some embodiments, L3is substituted or unsubstituted Ci-Ce heteroalkyl. In some embodiments, L3is substituted or unsubstituted cycloalkyl. In some embodiments, L3is substituted or unsubstituted heteroaryl. In some embodiments, L3is substituted or unsubstituted aryl. In some embodiments, L3is H.

[0241] In some embodiments, L4is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or H. In some embodiments, L4is substituted or unsubstituted alkyl. In some embodiments, L4is substituted or unsubstituted Ci-Ce alkyl. In some embodiments, L4is substituted or unsubstituted heteroalkyl. In some embodiments, L4is substituted or unsubstituted Ci-Ce heteroalkyl. In some embodiments, L4is substituted or unsubstituted cycloalkyl. In some embodiments, L4is substituted or unsubstituted heteroaryl. In some embodiments, L4is substituted or unsubstituted aryl. In some embodiments, L4is H.

[0242] In some embodiments, -L, -L1, -L2, -L3, -L4, or -L-B is attached at a stereochemically defined position. In some embodiments, the stereochemistry at the carbon bearing -L, -L1, -L2, -L3, -L4, or -L-B is in the R or S configuration. In some embodiments, the stereochemistry at the carbon bearing -L, -L1, -L2, -L3, -L4, or -L-B is preserved during synthesis by using stereoselective or stereospecific chemistry. In some embodiments, the stereochemistry at theWSGR Docket No. 60652-721601carbon bearing -L, -L1, -L2, -L3, -L4, or -L-B influences the accessibility of adjacent functional groups (e.g., the reactive group B). In some embodiments, -L, -L1, -L2, -L3, -L4, or -L-B comprises one or more stereocenters. In some embodiments, the stereochemistry of the -L, -L1, -L2, -L3, -L4, or -L-B is selected to reduce conformational flexibility, increase molecular rigidity, or enhance structural stability.

[0243] In some embodiments, L1is methylene (-CH2-). In some embodiments, L2is methylene (-CH2-). In some embodiments, L3is H. In some embodiments, the modified nucleotide of Formulas (IVA-A), (IVA-B), or (IVA-C) comprises a derivative of 2-amino-l,3-propanediol (serinol) or 2-methyl- 1,3 -propanediol (MPD).

[0244] In some embodiments, L1is a bond. In some embodiments, L2is methylene (-CH2-). In some embodiments, L3is H. In some embodiments, the modified nucleotide of (IVA-A), (IVA-B), or (IVA-C) comprises a derivative of ethane- 1, 1,2-triol or 1 -aminoethane- 1,2-diol.

[0245] In some embodiments, L1is methylene (-CH2-). In some embodiments, L2is a bond. In some embodiments, L3is H. In some embodiments, the modified nucleotide of Formulas (IVA-A), (IVA-B), or (IVA-C) comprises a derivative of ethane- 1, 1,2-triol or 1-aminoethane-1,2-diol.

[0246] In some embodiments, L1and L2are bonds. In some embodiments, L3is H.

[0247] In some embodiments, L1is CH2. In some embodiments, L2is CH(CH3). In some embodiments, L3is H.

[0248] In some embodiments, L1is CH(CH3). In some embodiments, L2is CH2. In some embodiments, L3is H.

[0249] In some embodiments, L1is CH2CH2. In some embodiments, L2is CH2. In some embodiments, L3is H.

[0250] In some embodiments, one or both of the phosphate of the modified nucleotide of Formulas (IVA-A), (IVA-B), (IVA-C), (IVA-D), or (IVA-E) is a boranophosphate, thiophosphate, or alkylated phosphate. In some embodiments, one of the phosphates of the modified nucleotide of Formulas (IVA-A), (IVA-B), (IVA-C), (IVA-D), or (IVA-E) is a boranophosphate. In some embodiments, one of the phosphates of the modified nucleotide of Formulas (IVA-A), (IVA-B), (IVA-C), (IVA-D), or (IVA-E) is a thiophosphate. In some embodiments, one of the phosphates of the modified nucleotide of Formulas (IVA-A), (IVA-B), (IVA-C), (IVA-D), or (IVA-E) is an alkylated phosphate.

[0251] In some embodiments, the modified nucleotide of Formulas (IVA-A), (IVA-B), (IVA-C), (IVA-D), or (IVA-E) comprises a structure selected from Table 2A.WSGR Docket No. 60652-721601Table 2AStructural Backbone StructureT2A-la co 1 co co CO 111 —1 11 I -1 -1 -1 _ _ \ O \ o I 1_ \ O oPOOo0---^ OOO O POOOof O CXQ-Q-CL '------^'^X'^K K >A ° o o o o o o O > >*n » / vw % / vw 1 1 1 >v / VW 1 / wT2A-lbT2A-1CT2A-2aWSGR Docket No. 60652-721601T2A-2btn 1 _i I i\ OOOoQ.-- '00* ° >T2A-2c x / VW 1 / wv,o°° zO?x <P\' 0 OC3 oV 3Ti-- k 11O \o-L-B ' 1l T2A-3a I- <3 1 co o=p-o“ Y°o“zpxo' 0Vo-L-B T2A-3b0o=p-o“ Y°^-Os,o~zpx0' 0Vo-L-B T2A-3c0o=p-o“Y°WSGR Docket No. 60652-721601T2A-4acn _ \ / o / 1zO O OQ--- / \ 11o o X >T2A-4b K / WV 1 / VW / VW' AAAX1ko ° °\K z0°° V Ax °O? < < < X' \II) 0_ i OOO o OO o OO o o-U-0-0--—----. RL / / \ \ \1111 1 1 _ _\ oA ° \ 1 z, T2A-4cAoX / ZZ- ’" 7 7 7 00 0003 O=p-O“1Y°T2A-5aT2A-5bWSGR Docket No. 60652-721601wherein RLis H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, wherein Ring A is a nitrogenous base or modified version thereof.

[0252] In some embodiments, RLis H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In some embodiments, RLis H. In some embodiments, RLis alkyl. In some embodiments, RLis heteroalkyl. In some embodiments, RLis cycloalkyl. In some embodiments, RLis heterocycloalkyl. In some embodiments, RLis aryl. In some embodiments, RLis heteroaryl.

[0253] In some embodiments, provided herein is a polynucleotide composition comprising a modified nucleotide of Formula (IVB-A), (IVB-B), or (IVB-C):Formula (I VB- A) Formula (IVB-B) Formula (IVB-C), 5 U1, wherein,L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups;B is a reactive group configured to react with an N-terminus of a peptide; and w"is cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, each optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2.

[0254] In some embodiments, L and B are as described elsewhere herein.WSGR Docket No. 60652-721601( W )

[0255] In some embodiments, is cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, each optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2. In someembodiments,is cycloalkylene, heterocycloalkylene, arylene, or heteroarylene. In someembodiments,is cycloalkylene, substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2. Insome embodiments,is heterocycloalkylene, substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and1 w;C0N(R”)2. In some embodiments, isarylene, substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”,and C0N(R”)2. In some embodiments,is heteroarylene, substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2.

[0256] In some embodiments,comprises a structureof. In someembodiments,comprises a structureof, wherein Ring A is a nitrogenous base or modified version thereof.( w ) H /

[0257] In some embodiments, comprises a structureof. In someembodiments,comprises a structureof, wherein Ring A is a nitrogenous base or modified version thereof.WSGR Docket No. 60652-721601O=P— N.i°\y

[0258] In some embodiments, ( ') comprises a structureofe. In someO=P— Niw'')embodiments, comprises a structureof, wherein Ring A is a nitrogenous ( w Jbase or modified version thereof. In some embodiments, comprises a structure of0=P-0 0=P-0( w')?. In some embodiments, comprises a structureofe, wherein Ring A is a nitrogenous base or modified version thereof.• W ' <-> z

[0259] In some embodiments, comprises a structureof. In someembodiments, Iw) comprises a structureof, wherein Ring A is a nitrogenous base( W ) or modified version thereof. In some embodiments, comprises a structureof. Insome embodiments.comprises a structureof, wherein Ring A is anitrogenous base or modified version thereof. In some embodiments,comprises aWSGR Docket No. 60652-721601 / Hxvv7 structureof. In some embodiments,comprises a structure of wherein Ring A is a nitrogenous base or modified version thereof.( W;•

[0260] In some embodiments, k / comprises a structureof. In somei W iembodiments, comprises a structureof, wherein Ring A is a nitrogenous base or modified version thereof.NH2O O 'v. / II IIOO OOTTJ-- —-- / ( w i / \ / 11 „ _) < X o o —,

[0261] In some embodiments, comprises a structureof. In some1° \NH2I- 1embodiments,comprises a structure of, wherein Ring A is a nitrogenous base or modified version thereof.

[0262] In some embodiments, the modified nucleotide of Formulas (IVB-A), (IVB-B), or (IVB-C) comprises a structure selected from Table 2B.Table 2BStructural Backbone StructureT2B-laWSGR Docket No. 60652-721601T2B-lb,? I O O OO Q-0.-- — '-- / -II IIO O X,T2B-1Co o O O 'H O O 'H. / ii ii. / II II / II II OO OOTJT OO OO" D" D-- / —-- OO OOTT-- / —---- / —-- / 1 \ 1 / , _X < / o o —, _ / V111.° z \>. / \=.X ( >°1—A -z. / Z / . I CD 03 N> T2B-ldT2B-leWSGR Docket No. 60652-721601T2B-lfCD / \ 1 - ( O \ O -O1i °" / \ 11 / OO OOQ-CL---- — ' / „ II IIo oT2B-lgO y OO f" D O O 'h —--,\ / II 1 / II II ) OO (" D o OO OO --- —TTJ-- / —-- / \I / 1< t o1,<1o \ CD / I- _ 1 DDT2B-2aO=p-OT2B-2boXo- 1 °yWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601T2B-2g- - / \ >, ° CL— sO=p-O T2B-3aYr N? > 1 / OOT / —-- O=P-l\k IO o / -1O H OT>—- °v M k < pT2B-3brO=p-O H 0T2B-3cYr r 1 / O=P-|\k1WSGR Docket No. 60652-721601^00=P1-0 - Lx0 )~—N T2B-3dkY°YNy\-NH2LNJ N< JN I zO=P-N.1°vT2B-3eO 'h-.A II OOT ^-- o o o / — / II1O O ZOOTJ-0—- ^-- ° - / \ r- I o O zT—- O=p-O—ro\ / 1 / L- _ BI X — z, 0> Hz\z / X z w— T2B-3f V I YJ o N1 / O=P-|\L1°vT2B-3gWSGR Docket No. 60652-721601T2B-4a^M. CCDCD f o H, O \ O \ O O11<\ / ) <\ n OO OOCL 1CL — —-- / -- Oo v / V / ' ' H11'OO OOCLCL--^--^ / / .. II IIO OT2B-4bOH ", / IIOOT ^-- ° O II 1 (ZOO yT —-- \< i <=\1I- 1 CDT2B-4c^01 -. B 0=P-0 Lx0VY y^yNH2T2B-4dN<^N0o=p-o“1°vWSGR Docket No. 60652-7216011 - 0=P-06T2B-4e w w kss JJ N<y, NH o NH2O=P-O“ CD I 1_l 1 / \?:>=<\ / ) OOCL —--nV / '11'OOCL--^<T2B-4f? >OOT-- L ° o n—ii iz x<> OOu —-- < \ i <= \ 1I- s 1 O=p-O CD 10T2B-4g0O=p— L-B 1°vT2B-5aWSGR Docket No. 60652-721601T2B-5b^M CCD f O H)\ / \? / OQ- —? > >== °)) <\O OOCL —---- °o O V / V / ' / 1111OO OOCLD----- / . IIT2B-5c o O11oA II OOTJ-- ( O o / —\ / / II 1 OO-U v y —-- S. £o=P-oVV y^NH2T2B-5d \\ i O0o=p-o“i°vT2B-5eWSGR Docket No. 60652-721601T2B-5fO=p-O1T2B-5g0? > O OOT-- O=p— L-B II 1OO OU 'H--.A o ii II.zOO ( °TI OO zT-- —-- °vIIlC< < C o OO Z-U —= H-- O / r~ O\ / / II i< i<1z= - ZI ( 00 y-U —--1ZI< o y 1 I- 1 CDT2B-6aH JMCDT2B-6bWSGR Docket No. 60652-721601O=P-O“H1 YT2B-6c VNY-NA<y-N0o=p-o“i°YT2B-6dO / IIOOT-- ° o.(zOO zT —-- i <<= -AO1<O=p-O1 H N 1 r> °-^NYN- / "ik 'i” r Y <" JZ^ T2B-6e NVNNYz\NHT NH z 022I N> o=p-o“1°vsO=p-OVYT2B-6f N^bJ0O=P-O— L-B1YWSGR Docket No. 60652-721601O=p-O YYT2B-6g N<^N0 ^CD O=p— L-B o r CD11Y 45 T2B-7a? > L -- z? YOOC \- / °Y1 o o XO nCL-- A iio YZ IIz l o 1 o\I OO-Q--Y oY11O Y, Y OO0-11-- O Y,O'h ■- / IIOOu-- rOo^*|Y II z OO \T>-- T2B-7boc ° YMCDT2B-7cWSGR Docket No. 60652-721601T2B-7dCD1f >T2B-7e 0 OD.-- IIX oAu ' ° ° / 1OOCL-- IIo A,O 'S O- / Ilii OOT-- O O"0-— O / i / IIA o rC J °° OOu' o-- o / 1 OO AT--.T2B-7f O NI / OOT--1A O o ZI o,< i^1^5,' OOTI-- i <~i"fA 1Z / I- 103M z<\ —1z o IT2B-7g

[0263] In some embodiments, the reactive group (B) is or comprises isothiocyanate (ITC).In some embodiments, the reactive group (B) is represented by the structure: N=C=S As used herein, “ITC” and “NCS” refer to the same isothiocyanate reactive group and may be used interchangeably. Accordingly, reference to “NCS” includes isothiocyanate (ITC), and reference to “ITC” includes the “NCS” structure.WSGR Docket No. 60652-721601

[0264] In some embodiments, the isothiocyanate is formed by conversion of an amine or a protected amine group. In some embodiments, the amine is a primary amine or a secondary amine. In some embodiments, the protected amine group comprises tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), 2-nitrobenzenesulfonyl (Nosyl), 5-dimethylaminonaphthalene-1 -sulfonyl (Dansyl), phthaloyl (Pht), or l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)ethyl (Dde). In some embodiments, the conversion is carried out using carbon disulfide. The conversion is described in Techapanalai et al. One-Pot Synthesis of Isothiocyanates from Amines Mediated by Carbon Tetrabromide ChemistrySelect 2023, 8, e202302045, which is incorporated by reference herein in its entirety. In some embodiments, the conversion is carried out using triflic chloride. The conversion is described in Wei et al.Synthesis of Thiocarbamoyl Fluorides and Isothiocyanates Using Amines with CF3SO2CI J. Org. Chem., 2020, 85, 12374-12381, which is incorporated by reference herein in its entirety. In some embodiments, the conversion is carried out using a thiocarbonyl transfer reagent comprising 1,1’ -carbonyldiimidazole (CDI), thiophosgene, di-2-pyridyl thiocarbonate, 1,1’-thiocarbonyldiimidazole (TCDI), or N, N’ -thiocarbonyldiimidazole. Additional thiocarbonyl transfer reagents for conversion of an amine or a protected amine group are described in Larsen et al. Organic sulfur chemistry. 25. Thiocarbonyl transfer reagents J. Org. Chem. 1978, 43, 2, 337-339, and Katritzky et al. l-(Alkyl / Arylthiocarbamoyl)benzotriazoles as Stable Isothiocyanate Equivalents: Synthesis of Di- and Tri substituted Thioureas J. Org. Chem. 2004, 69, 9, 2976-2982, each of which is incorporated by reference herein in its entirety.

[0265] In some embodiments, B is alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl substituted with isothiocyanate. In some embodiments, B is alkyl substituted with isothiocyanate. In some embodiments, B is heteroalkyl substituted with isothiocyanate. In some embodiments, B is heterocycloalkyl substituted with isothiocyanate. In some embodiments, B is aryl substituted with isothiocyanate. In some embodiments, B is heteroaryl substituted with isothiocyanate. In some embodiments, B is a cubane susbtitued with isothiocyanate.

[0266] In certain embodiments, B is phenylisothiocyanate (PITC).

[0267] In some embodiments, B is or comprises -NRX(C=S)-LG. In some embodiments, Rxis independently H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl. In some embodiments, Rxis H. In some embodiments, Rxis alkyl substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3WSGR Docket No. 60652-721601haloalkyl, NO2, CN, COOR”, and C0N(R”)2, wherein each R” is independently H or C1-C3 alkyl. In some embodiments, Rxis heteroalkyl substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2, wherein each R” is independently H or C1-C3 alkyl. In some embodiments, Rxis cycloalkyl substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2, wherein each R” is independently H or C1-C3 alkyl. In some embodiments, Rxis heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2, wherein each R” is independently H or Ci-C3 alkyl. In some embodiments, Rxis aryl substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2, wherein each R” is independently H or C1-C3 alkyl. In some embodiments, Rxis heteroaryl substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2, wherein each R” is independently H or Ci-C3 alkyl.

[0268] In some embodiments, B is or comprises -NRX(C=S)-LG. In some embodiments, LG is a leaving group. In some embodiments, LG is aryl or heteroaryl. In some embodiments, LG is aryl. In some embodiments, LG is heteroaryl. In some embodiments, the heteroaryl is a monocyclic, fused bicyclic, or fused tricyclic heteroaryl group. In some embodiments, the heteroaryl is a monocyclic heteroaryl group. In some embodiments, the heteroaryl is a fused bicyclic heteroaryl group. In some embodiments, the heteroaryl is a fused tricyclic heteroaryl group. In some embodiments, the heteroaryl is a five-, six-, seven-, eight-, nine-, or tenmembered ring containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl is a five-membered ring containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl is a six-membered ring containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl is a seven-membered ring containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl is an eight-membered ring containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl is a nine-membered ring containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl is a ten-membered ring containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl is selected from thiophene, furan, pyrrole, imidazole, thiazole, oxazole, pyrazole, triazole, tetrazole, oxadiazole, and thiadiazole. InWSGR Docket No. 60652-721601some embodiments, the heteroaryl is selected from pyridine, pyridazine, pyrimidine, pyrazine thiopyran, and benzodioxane. In some embodiments, the heteroaryl is imidazole.

[0269] In some embodiments, Rxin -NRX(C=S)-LG is H. In some embodiments, B is or comprises -NH(C=S)-aryl or -NH(C=S)-heteroaryl. In some embodiments, B is or comprises -NH(C=S)-aryl. In some embodiments, B is or comprises -NH(C=S)-heteroaryl. In some embodiments, B is or comprises -NH(C=S)-benzothiazole.

[0270] In some embodiments, Rxin -NRX(C=S)-LG is straight-chain or branched alkyl or heteroalkyl, each optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2. In some embodiments, Rxis straight-chain alkyl, optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2. In some embodiments, Rxin -NRX(C=S)-LG is straight-chain heteroalkyl, optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2. In some embodiments, Rxin -NRX(C=S)-LG is branched alkyl, optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2. In some embodiments, Rxin -NRX(C=S)-LG is branched heteroalkyl, optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2. In some embodiments, Rxin -NRX(C=S)-LG is CH3. In some embodiments, B is or comprises -N(CH3)(C=S)-imidazole or -N(cyclopentyl)(C=S)-imidazole. In some embodiments, B is or comprises -N(CH3)(C=S)-imidazole. In some embodiments, B is or comprises -N(cyclopentyl)(C=S)-imidazole.

[0271] In some embodiments, Rxin -NRX(C=S)-LG is five-, six-, seven-, eight-, nine-, or ten-membered cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2. In some embodiments, Rxis five-, six-, seven-, eight-, nine-, or ten-membered cycloalkyl. In some embodiments, Rxis five-, six-, seven-, eight-, nine-, or tenmembered heterocycloalkyl. In some embodiments, Rxis five-, six-, seven-, eight-, nine-, or tenmembered aryl. In some embodiments, Rxis five-, six-, seven-, eight-, nine-, or ten-membered heteroaryl. In some embodiments, the five-, six-, seven-, eight-, nine-, or ten-membered cycloalkyl is optionally substituted with one or two members selected from oxo, halo, OH, Ci-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2. In some embodiments, the five-, six-, seven-, eight-, nine-, or ten-membered heterocycloalkyl is optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3WSGR Docket No. 60652-721601alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2. In some embodiments, the five-, six-, seven-, eight-, nine-, or ten-membered aryl is optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2. In some embodiments, the five-, six-, seven-, eight-, nine-, or tenmembered heteroaryl is optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2. In some embodiments, Rxis five- or six-membered cycloalkyl or heterocycloalkyl. In some embodiments, the nitrogen atom of the -NRX(C=S)-LG group is a member of the heterocycloalkyl or the heteroary. In some embodiments, Rxis five- or six-membered cycloalkyl. In some embodiments, Rxis five- or six-membered heterocycloalkyl. In some embodiments, Rxis five- or six-membered aryl or heteroaryl. In some embodiments, Rxis five-or six-membered aryl. In some embodiments, Rxis five- or six-membered heteroaryl.

[0272] In some embodiments, Rxin -NRX(C=S)-LG is a fused bicyclic or tricyclic cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group. In some embodiments, Rxis a fused bicyclic or tricyclic cycloalkyl group. In some embodiments, Rxis a fused bicyclic or tricyclic heterocycloalkyl group. In some embodiments, Rxis a fused bicyclic or tricyclic aryl group. In some embodiments, Rxis a fused bicyclic or tricyclic heteroaryl group.

[0273] In some embodiments, Rxin -NRX(C=S)-LG is configured to facilitate an interaction with a peptide. In some embodiments, the interaction comprises a proximity-mediated interaction, a charge-mediated interaction, a hydrogen bond-mediated interaction, a metal ion-mediated interaction, a 7t-cation interaction, a 7t-stacking interaction, a van der Waals-mediated interaction, a hydrophobic interaction, an allosteric-induced interaction, or any combination thereof. In some embodiments, Rxis configured to facilitate a cyclization reaction with an N-terminal amino acid of the peptide under physiological conditions. A proximity-mediated Edman or Edman-like degradation process enabling a controlled removal of a terminal amino acid from a peptide under acidic or basic conditions is illustrated in Scheme 1.Scheme 1WSGR Docket No. 60652-721601

[0274] In some embodiments, B is or comprises -NH(C=S)-R. In some embodiments, R is aryl or heteroaryl. In some embodiments, R is aryl or heteroaryl substituted with one or more electron-withdrawing groups. In some embodiments, R is aryl or heteroaryl substituted with one or more electron-donating groups. In some embodiments, B is -NH(C=S)-aryl. In some embodiments, B is -NH(C=S)-heteroaryl.

[0275] In some embodiments, B is alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl substituted with -NH(C=S)-heteroaryl. In some embodiments, B is alkyl substituted with -NH(C=S)-heteroaryl. In some embodiments, B is heteroalkyl substituted with -NH(C=S)-heteroaryl. In some embodiments, B is cycloalkyl substituted with -NH(C=S)-heteroaryl. In some embodiments, B is heterocyloalkyl substituted with -NH(C=S)-heteroaryl. In some embodiments, B is aryl substituted with -NH(C=S)-heteroaryl. In some embodiments, B is heteroaryl substituted with -NH(C=S)-heteroaryl. In some embodiments, the heteroaryl is imidazole. In some embodiments, -NH(C=S)-heteroaryl is -NH(C=S)-imidazole.

[0276] In some embodiments, the reactive group has a structure selected from Table 3 A.Table 3AReactive Group StructureT3A-1 ■5— NCST3A-2ascr^—T3A-2b^NCST3A-2c— C —ncsT3A-3aT3A-3b\ics / ==\ / - NCST3A-3cWSGR Docket No. 60652-721601NT3A-4a I JL Y^^NCST3A-4b7?NCST3A-4c yONCST3A-4d XI \^^NCST3A-4e yONCS / •^NCS T3A-4fNCST3A-5 N J _ ( / \\ _ / 0 NCST3A-6 / “(N=\ f-NH ' — dzNT3A-7a ^-NZ^N^CS 0 NCST3A-7b Y~( N=N f-NH / Nij-NZ^NCS T3A-7cNxT3A-7d NCSWSGR Docket No. 60652-721601T3A-8^NCS T3A-9axAA / VT3A-9bJWV^^. NCS T3A-10aT3A-10bH3C^ _ / NCS T3A-11H3CT3A-12 } — e V- NCST3A-13osIIT3A-14a s ANI — N NXNH \=JS IIT3A-14bI — <Z— N NXN \= / H \=J S IIT3A-15a|— N NXN\ \=JST3A-15bNxN ST3A-16a J^N^CIWSGR Docket No. 60652-721601iioIIz

[0277] In some embodiments, -L-B, as described herein has a structure selected from Table 3B.Table 3BL-B (Linker- Reactive StructureGroup)T3B-1T3B-2a Y^^'NCST3B-2b•i^^NCST3B-3aT3B-3b^^NCST3B-4aT3B-4bT3B-4cOxNCST3B-5a If-NHOKNCST3B-5b, ^0(-NHWSGR Docket No. 60652-721601NCS HN~^T3B-5c0^ NCS T3B-6NH0 / — NCS T3B-6aNH NCS HN^T3B-6cHoT3B-7a ^O^NCS T3B-7b A / \ / NCSeGT3B-8asci^—T3B-8b^NCS T3B-8c — C —ncsT3B-9aT3B-9b\ics / — T3B-9cNCSl\kT3B-10a\ J^L^ JX^NCST3B-10bY J^U^ANCSWSGR Docket No. 60652-721601T3B-10cX3NCS^^NCS T3B-10dT3B-llaNCS O NCST3B-llb s |-N MH ' — / N=\ >zNT3B-12a^CS NxN^T3B-12b ^j= / NCSO NCST3B-12b / — (N=NNHT3B-13,v^^ / NCSH3C NCS T3B-14 — oH3CT3B-15 ) — \ / NCS00T3B-16a Y^N^^^ NCSxH0T3B-16bxHWSGR Docket No. 60652-7216010T3B-16c Y^N^Q A^NCS0T3B-16d0 NCS T3B-16e0 NCS T3B-16fH H \ OT3B-17aH^-^NCS T3B-17bT3B-17cxNCS OT3B-17d N >|H0T3B-18a A_ „A^NCS OA ^ ^ A^^ T3B-18bH0T3B-18cH\^\ / NCSWSGR Docket No. 60652-721601NA1N— \ / =\T3B-18d / ) —NCSNAT3B-18eT3B-18f £. / N'N"? — < / X=\ z>— NCSNAA / NA< 0T3B-18g >° O 0> _z O^NCS NCS / N- N < T3B-18h _ _ N j \NCSz 1 a 0 o z c> T3B- 9A 0 / CLNHH CH30T3B-19bH\ / AN / ^^^NCST3B-19cWSGR Docket No. 60652-721601T3B-20aNCSoT3B-20b_ JL-HN^N^S 1, NYViV / Z'"NIZy°=NCSo\=z o / / C0O \= — yT3B-20co \ / 1"| Z V-HN^N^S 1z z- z,N'k—Q NCSzo JH Ho / coT3B-20dY Y Y \T )S O 1 / ^^NNCSH 0, 0T3B-20eY JsVZ i / \ V-No NCS H HT3B-21a0H5 YWSGR Docket No. 60652-721601o NCS II HT3B-22bn coz_ I \T3B-22c H A J z^ ^v-x / x^y^ o \= QO NCST3B-22d z—NCST3B-23ao < VN'VH0 0NCST3B-23b r° < VN'H° oNCST3B-23co < VN'H° oWSGR Docket No. 60652-721601NCST3B-23di N H?OSHHs o o Z \0 OH H \— / T3B-23e ° k ANCShu OII 1 NT3B-23f0>=<N?NCS OT3B-24x H^^ NCST3B-25 / / N^NCSos N=N> L N.T3B-26a0OL ^^NCS,N=NT3B-26b N A / T A^^NCS

[0278] In some embodiments, the modified nucleotide is selected from Table 3C.Table 3CModifiedStructureNucleotideWSGR Docket No. 60652-721601t JH2hr XNCST<o oX ■j O=P-O.T3C-la d- 0O=P-O-NH2X / l 0 CT N"^NCS O=p-CLT3C-lb 0- 0O=P-O- V NCS HNZxN<k] O O^N' O=P-O. 1 T3C-1C o- 0O=P-O"Vr JCS XNCST<o o^r ■j O=P-O.T3C-ld d- 0O=p-O" °x0 X^NCS xHN>< S9 CXN' O=P-O. 1 T3C-2a d- 0O=P-O"VWSGR Docket No. 60652-721601o o O^N^NCSO=P-O.T3C-2c d- oO=P-O' dy- SON Q x O O^NC 7\ O=p-O. 1O=-^ T3C-2d o- 1 o X,^ o 7zVOO^0- ■---uf I ° 0 x ObO-- 0=P-0’ \ Oy0 A -NCSHNjf 0 O^N O=P-O. 1 T3C-3a O’oO=P-O" y0HNjl 0 O^N^NCS O=P-CLT3C-3b o- 0O=P-O" dy>T3C-3cWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601SCN oXo1N^^NHa T3C-5b O=p-O.d- oO=P-O"yO^yi 'NH SCN — CXoJN<^NH2T3C-5c O=F>-O- d- oO=p-O"0J~>|X'NH SCN —Xo N^^NCS T3C-5d O=p-O.o- 0O=P-O-O=p-O^ NCS O- VoJ T3C-6aoO=P-O" yO=P-(X NCS O- T3C-6boO = P-O"WSGR Docket No. 60652-721601O=P-CLd- V°>T3C-6cO NCSO=P-O"NCSO=p-CL oT3C-6d O’ V'"'>oO=P-O- V4 ® O=p-O. 1 d- VodT3C-7ao NCS O=P-O"O^ / 0 A> [|nlhO=P-O“ <NA0°>T3C-7bV ®or^ NCS O=p— 01NH2> _ AO=P-O A1 N^O T3C-7co0NCS O=P-O“ovWSGR Docket No. 60652-721601T3C-7do c o o c o 0 z NH2 / 7v * -°\ - fSO=p— 0 11 X| ° N Y? > ° ^ 'OX H 1 XTT3C-7e °> Y? o A o o >—— o r o,oO " U oj —- '\ OO 1 J0- —--, o X \ _77 N=N? II H oV / \1OO (Ti-- —v+°°, o X? - O=p— O X < L H 17NCSl 20 z= -O'V^M“ o ' co — T3C-7fT3C-7gWSGR Docket No. 60652-721601WSGR Docket No. 60652-7216010 y yH0=P-° ^N' X) T3C-71NCSO=p— 01Xo ® O=P-< X I o- ypj T3C-8aO NCS0=P-0 — / yO=P-CL (A 1) 0- T3C-8b° z\ 0=P-0 — (NCSO=p-O.d- T3C-8c0 NCS 0=P-0 — / °y* O=P-O.d- T3C-8d Y°>° z\ 0=P-0 — (NCSWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-7216010 Xp 'NH X 1\ 7 o 0 >| NH T3C-9gP=O ^NA00^ 1 X— °\l 0 1 O=P-O- 1 °'Xz"\)O=P-CLQ- 3 ^ NCS T3C-10aOO=P-O’ Oy-? NCS O=P-O 1 o- yT3C-10b0O=P-O"X NCSO=?p-O. < 1O- VNHT3C-10coO=p-O’ dy.X NCSO=9p-O. < 16- VN\ T3C-10d0O=P-O- OyWSGR Docket No. 60652-721601o o-O-P=O o o-O-P=OWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601T3C-10iT3C-10jWSGR Docket No. 60652-7216010x A"O N' X)O=P-O.6- 0-O-P=Oo c T3C-10k ozH9 Q T -O-P=O 0N^I\T 'NH26k CJ1 OIZ II / \ / OQ-O —-- 0O oII / X o=p-crOOQ--- io V iVT3C-1010 SCN^^rp ^NH^0 ^N'^'O1O=p-(XT3C-lla010=P-0- 1°x / 0A ^. NCS.. HN jp0 O^NO=p-O. 1T3C-llb d- pjoO=P-O- VWSGR Docket No. 60652-721601WSGR Docket No. 60652-7216019 r^^NCS °=P-°x.O’ V-JT3C-12a0O=P-O- VT3C-12b6OO" U=- iO=P-O. ° o / d’T3C-12c \ 1 / _0 ) oO=P-O’Vz o GO°=P 'Ox _O’ NCS T3C-12d0O=P-O-NCS01 0 O^NO=p-O. 1T3C-13a 6- 0O=P-O’°xO=p-O>|O’T3C-13bOO=P-O"VWSGR Docket No. 60652-7216010 H> HN'Y^^N'^^^'NCSXo c / JO=p-Ck IT3C-13c 0- PJ0O=P-O- Vio. sO’ ^NCS T3C-13d 1 H0O=P-O’OyA 0 A H / NCS X A JI Ho CANO=P-O. IT3C-13e 6- X-oj0O=P-O- Vio. sn- / A JL / NCS T3C-13foHO=P-CrOy*° IX M0 CXN It xX, NCS O=p-O. IT3C-13g d- V°i0O=P-O-6yio. sO’ VA / AA^X\ N >|T3C-13h 1 H II 1o1XX\ / NCSO=p-O’VWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601TVO )> — NCS0 O^hTT3C-14h O=P-O. 1o- yoJoO=P-O"V0NCS > HN ifX J l|0 O^NT3C-14i O=P-O. 1O’ V°-^l0O=P-O- V X NA■ JL / N\ / \ O=P-O. NCS o- kxxT3C-14j0O=P-O- OyNAJ^ / N^> HN^r ( \°Xo (XN^ / °T3C-14k O=p-O. I \0- V°" J / 1 0050=P-0- / °; / O^NCS X N-\,0L / N— \O=p-Oxod- (A / °T3C-141 O=P-O- \Ox / / 7 oO^NCSWSGR Docket No. 60652-721601NCSi oT3C-14mN“ 0O=P-O- V NCSNCS X All N T3C-14n o \ II 1 °=P-Xn0- I^O^I0O=P-O- VOvNCS K7HNz0 O^NT3C-15a O=P-O- 1o- Vo^l0O=p-O’V NCSo HN—Fy HN^ J V 0 O^N ° O=P-O. 1T3C-15b o- oO=P-O’yWSGR Docket No. 60652-7216010 / — NCS x HN'YVoHO=p-O. 1T3C-15c 0- 0O=P-O’O NCS9 HNO=p-o JT3C-15d°’ ¥oO=P-O’NCSO=p-O HN^° \T3C-15e90O=P-O"Oy>O i — NCS x9 HNO=p-O JT3C-15f°' ¥oO=P-O"X* oo H°=P-°> Hd- VNy^ / T > 1 fl ^N' 90\ T3C-15g O=P-O- / oNCSWSGR Docket No. 60652-721601X" oO IIO=P-% Ho- O (1ZN n<N / Y O \ T3C-15h O=P-O’ ) y k o NCS9 oO=P-< X H II1 II H N 9 °O=P-O" \ T3C-15iC) NCS9 oO=p-O. H II9 SHO=P-O" L N / NT3C-15jC) NCS0HN><YXX-O'XX'NCS 0 O^NO=P-O^ |T3C-16a d- 0O=P-O- Vd" k — \T3C-16b | ° NCSoO=P-O- VWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601T3C-17f\ 7\ °z= —) X o o / X W° °’o / Xr o4 OO)=- _ / , 111 / OO< Q-==- o ^ / \ 1| O _J / . O4 / X o o>- — —T3C-17g zoz> < X / p °, _O Z= / -0NCSz o coXo 0O=p-O. 1T3C-17h 6- V-Q-^lo0=P-0- OyX^NCSX0o-p-o— i o TT3C-17i o- Y°k01-0-P 1 =0°xWSGR Docket No. 60652-721601NCSo0=^-0. TT3C-17j d- 0O=P-O- dy> z> < o T3C-17k9 o °O=P-O \ II1 X / OOCL^-- - r i~^o 9Z°y, iOOQ--- / Vx ii “o 0O=P— o 06- T3C-171-O-P=O1OK>4O=P— o 0T3C-17mo Xx^NCS-0-P=01ox>T3C-17nWSGR Docket No. 60652-721601NCSA0 N^ JT3C-17o O=P-O. |l I Jo- o c o 0 z -O-P=O ( z - 1ox>N"7\ \G z-N\ OO JCL— L-- H, >" °IIO=p-O., 1T3C-17p 6-xO OCL-- o X V H0-O-P=O1OK>NCS4>O=p-O.T3C-17q 6- 0-O-p=o1<3T3C-18aWSGR Docket No. 60652-721601WSGR Docket No. 60652-7216010 NCSXOO=p-CLT3C-18f0-O-P=O1oxO NCSHNYN^-Q " 9T3C-18g O=P-O. / o- 0O=P-O"Oy-o o NCSo cr NO=P-CLT3C-18h 6- o■O-P=O1C)0=l?‘0> / N O’ Y' \ _ / / T3C-19aO NCSO=P-O- V / =\O=P-O- - / NO’ NCS T3C-19boO=P-O- yWSGR Docket No. 60652-721601T3C-19c4ncsO=p-O ) O’ K / T3C-19dOoll ■o-p=oOO-D-- 1' 1 C> o p >^ OOXTJ-- / II - 4 — N NCS o=p-o. Q ° ) 0- P-N T3C-19e o O GO-O-P=O1C)>0 NCS x" / — CN=\ <3 NH ' - 0=P-0 [ V _ 'J T3C-19f O’OO=P-O’V40 O NCS O=P-Ck XX N=\ 6- VNHT3C-19g0-O-P=O1c>WSGR Docket No. 60652-721601T3C-19hZ A] lf^ O Cu ZET3C-19i 1 II 1 Ou - o \O. O^CL-- II OOTI-- 1' 1 o PO b0--- 9* ooMZ II “^ pOXTJ-- / II C 1 "7\qo A AN9 OO=P-O. A W.NCST3C-20a o- 1 IJ 00=P-0" 1 dyA AN HN \ _ / X A jlO O^N \ _ / NCSO=p-O. IT3C-20b 0- WoO=P-O’VX9 MO=P-O ^N'' AN0- tpMT3C-20c 0 NCS O=P-O’OyWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601NCST3C-22c o- 0O=P-O- y / N-'NCS>? *^0 X \j / OOT=- 1 O=p-O / T3C-22d O’oOOT>—-- 0) < O O=p-O"dy>IZ / x >°9 o NCS T3C-23a-O-P=O N"iCt z z- >49 N zCS o z <> T3C-23bO-P=o NiOxT3C-23cWSGR Docket No. 60652-721601O zNCS- v YX£ 1 JJ k pf 'O=p-O. ) < / T3C-23d 06- z007 II^ -O-P=O I.OOTJ-- 1z 1? °>H> u 0Z \ xO z= - O -L,11 0OOT--11 K °zzT3C-23e ■^ OOAT>-- zz\o=L zz4n?° 03 < P ZI, 1O OCL-- Zh 11 “ A °0 0z z-^T3C-23fz^^^T z 0 / w coZ Z'YAz 0 co T3C-23g_ / N*NT3C-23h0 NCS O=P-O’1°yWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601,s Y Y\ _ / N-N / \^N% < YI NHT3C-23m O=P-O. 1° 1- 0i-O-P=O1oxV NNx,r \ R F %0=\ VN 1 NH X=S O=P o- A II’O- I 1 OO-U-- T3C-23n A- V~^ rN\ Y 5?—7 NY o \ < OO-U--— V-- / -O-P=O1oxV / \11 Yl N 1 1 / ,N / \-N z zx""z n / / ; H I >s| NHO=P-O- 1 z T3C-23o 1 o °- 1 Z (> 0i-O-P=O1ox>N,N'", 7 Z \ \ ^ 11 1^, N C / s. N^\ / \ Z—N N-- / ' A (J ' Y Q | NH6T3C-23p“v01-O-P=O10>T3C-23qWSGR Docket No. 60652-721601N / / [ \ NCSA o=<I NHO=P-O. 11 > _T3C-23r °- U- —0i-O-p=O10ozo n.II z^OOTl--. L' 1 zzx7k^ oo- T3C-23s L TVA °A • ' '6? ° / o O CL--, 1 IZV ii ■oN" ' KAA I NHO=P-O. 1 ZIT C-2 t ° 13 - 13 Ao0i-O-P=O10Z=\ / — \ L \ \ZZ^ zZ — — T3C-23uWSGR Docket No. 60652-721601NCSO 7^ o oT3C-24aO=P-O.6- 0-O-P=O1°>NCSoj?s ^NY >=CT3C-24b / XO OHI^A JH 0O=P-O.6- 0-O-P=O1O\0SCNXO N'^O A. O=p-Ck\ \ / -O-P=O6T3C-24c(S -O-P=O1°x0O=p-O’10WSGR Docket No. 60652-7216010'Xf|NHSCNI 5o=p-o.O'\ o? M i jj %T3C-24dw °? ri 'O-P=O ^N^ X) 1°x0O=P-O'10NCSuvwwwx o - - '0 / TA Jo=p~o~ [T3C-24e?0W0=P-01L\°NCS / QX° cn u / T3C-24f0 ' — 'O-P=O1OxWSGR Docket No. 60652-721601T3C-24g O C O Q CQ z z o c o z 0 / J0zzZ<'^ z z'ZCI / \z I X-~T3C-24h \z^OJ° O o?ZO-P=O1 / ° QNCS T IZ XoIZo / O V.< 7 II^- \^OOCL-- T / T I3C-24i O< II7, i < \ O O II CL O— 9~V ii 69 OOCL-- Io, i o 9 ■, O O itL-- oV II ■ PO HuCL / “-- oA O? / \^NO=P-O. H f || ''N6- T3C-24j Y^YX AN? °0WO-P=o(!) NCS >T3C-24kWSGR Docket No. 60652-721601T3C-241O O -u^ II -uA z II OOTJ-- T3C-24m OOTJ-- ■ 1 ' 1° d° 6 / / IV / 1 / \ OOT / \ —-- OO-U —-- II / / II / / T o / n oNCS ZI ziHo=°x\ ° / V HN — \YHN' T' II i \ / X 1 Ji0° ° / / < / X'NT3C-25a o O^N N — e iO=P-O. | / z — ^^^ \ \rNd- o zz -n ^xO=p-O" zzxz - X zOy-z>“ NCS z o 0) X n 0,T3C-25b&Y ° Y-h0 / VvNO=p-O"WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-7216010O=P-CL6- 0■O-P=O1T3C-25j SCN \0? °'xl1 X \ o' 'NO O" U•--“NWZ? ( A < / \ -O-P=O N ^O \p O 0.7ijVko o--- XX*H ZI 0O=P-O’10 Jz / / NCSC O \O— - / X Z • II. °A z / 1\ \ oXO. " N N=S T / z z-^^\ T3C-25k°- — K z o o co O=P-O'NCSZ •. Xs1T3C-251°- k'"''? — 7\oO=P-O'VT3C-25mWSGR Docket No. 60652-721601O=P-O. H 0, NCS O’ < T3C-25no s i y~N o=P-o- YY0^Z\ N-s? Az™ jp^ / NH NPNxo cr^rrO=P-(XT3C-26a6- YJ0■O-P=O1OK>oXo, A6- — \ / NH N NT3C-26b0■o-p=o10 Y'NHo=p-o.6- 0N^l H O-P=OVNYNY^ AT3C-26c* ULQ vHzH? ri O-P=O<'N" X) O.0O=P 1-O- VWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601NCSo / j?x x Y k JYNzo cr N;' — 'T3C-29d O=P-O. 1o- oO=P-O- V NCS01 T3C-29eHNAJHXO O^NO=p-O.6- VA0-O-P=O1°>NCSx0T3C-29f O=p-O.o- v0O=P-O’V NCS x ° r O=P-O HN'XX / \|Z yX-N T3C-29g0O = P-O’WSGR Docket No. 60652-721601

[0279] In some embodiments, the modified nucleotide is formed by conversion of a primary amine or a protected primary amine group from a structure selected from Table 3D.Table 3DModifiedStructureNucleotideNH2X, NH2X I J0 Cr NO=P-O.T3D-la o- oO=P-O- V NH2X0 O^N^NH2O=P-O.T3D-lb 0- 0O=P-O"6 / WSGR Docket No. 60652-721601NH2HNvX0 O^N O=p-O. 1 T3D-1C 0- VJ0O=P-O’NH2N<yNH2<0 CXN O=P-O.T3C-ld d- 0O=P-O’ dy-o HN' yZ^NH29 CXN O=P-CL I T3D-2a 0- 0O=P-O"Voo OX\H2O=P-O.T3D-2c d- oO=P-O" dy> H2N^Ox yv 9 O^N O=p-O. 1 T3D-2d o- 00=P-0’ VWSGR Docket No. 60652-7216010 s A ^NH2HN|T O=P-O- 1 T3D-3a o- ypJ0O=P-O’0 HN' jl O O^N^NH2O=P-O.T3D-3b 0- 0O=P-O" Oy>H2N\ O Xo O^N O=p-O. 1 T3D-3c d- o O=P-O- Oy.NH2. H2N —XQJYST3D-4a O=P-O^0- oO=P-O- yH2N NH2XJO=P-O- T3D-4b d- 0O=P-O’yWSGR Docket No. 60652-721601NH2. H2N — Z^ JTI|N]XONN' T3D-4c O=P-CL0- oO=P-O’ yo hH2N —1N^^NHj * 9 'T3D-5a O=p-O.d- Y°>oO=p-O"H2N*Xoh1N NH2T3D-5b O=P-O.d- oO=P-O’OH2N — e"XorJisT^NHz T3D-5c O=p-O.O- OO=P-O’yO=F>-O. NH2d- T3D-6aoO=P-O"yWSGR Docket No. 60652-721601O=P-< X NH2d- T3D-6boO=P-O"Oy>O=P" CLd- Y°>T3D-6co NH2O=P-O"yNH2z—O=p-O >?.-< o0- OO oTl-- T3D-6d 1 / oOOOTl-- —O=P-CT oOy\O -Z= - z. I l\> 4 ®O=P-O^ I d- VoJT3D-7ao NH2O=P-O’T3D-7bWSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601O=P-O.d- T3D-8dO0=P-0 — (NH2Mnb. ° \\ 7\ 1 Ao z=- { r » \ / / > \ _O o J Q--—— T3D-8e OOQ- —-- 1 I 1I*0N^O \___Z0T3D-8f o o i O-P=o1 0 1 O=P-O- 1 °'y 010—< 7 o 0T3D-8gH2N\^X / O-P=O ^NA0°\ 1 0 1 O=P-O- 1 °' / WSGR Docket No. 60652-721601O=P-O. I o- T3D-9a0 NH2O=P —Z0(A) O=p-Ox1, H° o-,< 7K OOz= - i Aj A= / - T3D-9bo y 1 OZ z= / - ' 0=P^ / \ \ / _> Jo oa—-- / O O II NH2OO0— -—V )zx4T3D-9cO=p-O.d- T3D-9d0 NH2O=P — / 0O=p-O.d- yp^T3D-9eO=oI^>^ / NH2c^\WSGR Docket No. 60652-721601T3D-9fA 0 VOOo.^--A v J 7 o zT3D-9g I o ^YN| AHp=0 <0c< 1 0 1 0=P-0‘ i °'Xz<k)O=P-O.d- VNH2T3D-10a0O=P-O"9 NH O=P-O 12°- yT3D-10boO=P-O’X NH29 / O=P-OXI0- VNH T3D-10c0O=P-O- OyWSGR Docket No. 60652-721601T3D-10dT3D-10eT3D-10fWSGR Docket No. 60652-721601o-O-P=Ooo■o-p=oO=P-O-WSGR Docket No. 60652-721601o o-O-P=O o o■O-P=OWSGR Docket No. 60652-7216010x 6:O N^OO=P-O.6- o-O-P=OT3D-10kH 1 / / NNT NH o < JL A -O-P=ONN NH26.0O=p 1 -O’VXoO-P=O6. H zp VN<£yNH2T3D-1010-o-p=o1°y0^0 ^N'^O1O=P-CLT3D-lla < L- Vj010=P-0- 1°~xo^NHJ HN^p220 O^NO=P-O. 1T3D-llb o- oO=P-O-WSGR Docket No. 60652-7216010>HN^NH20 O^NO=P-O. 1T3D-11C o- 0O=P-O- V'1'^'0 T3D-lld44 OO ooi>==-—■ 111o oI I O O / O O o o "0x -u— —— — — - )— / ^NH24T3D-lle■JH2^N''XX— fZ o fx\ IIT3D-llf o- \_ _ _ / 010-P=01'^NH O=p-O [2O’ V'JT3D-12a00=P-0WSGR Docket No. 60652-721601XOO=P-O ro- T3D-12b0O=P-O’O=P-O6- yo^^^NH2T3D-12c0O=P-O- °y^0O=P-O. o0- T3D-12d0O=p-cro po o^rrO=P-O. 1T3D-13a d- 0O=p-O"r<^X / NH2O=p-O [d- V-JT3D-13b0O=P-O’dy., HNV^ N^-^--NH2Xo o< VO=p-OxIT3D-13c d- 0O=P-CTOyWSGR Docket No. 60652-721601^-0,?6 O’- A ^\ / NH2T3D-13d 1 H0o=p-crdy>A A A / NH2y HNX A Jj HO CANO=P-Ck 1T3D-13e o- yp^i0o=p-crX / io. SO' A^AN^NH2T3D-13f 1 H0O=P-O- Oy>0IX M0 CAN \A / NH2O=P-CL 1T3D-13g O’0O=P-Cr6^ / io. £>O’T3D-13h \ N Ti A1 H0 MA / NH2 OP-0- VO=P-CL HO’T3D-13io0^> A\ / NH2 O=P-CTOyWSGR Docket No. 60652-7216010HX Y h ^l 9 0O=P-Ck ^ANNHH2T3D-13j 6- V-0-^oO=p-O’Oy>O ^^NH2HN' JPO O^NO=P-O. |T3D-14a O’oO=p-O"V / NH2°y HN'A^pO O^NO=P-O. |T3D-14b d- oO=P-O"V NH2I 0T3D-14c < Q\H°=P-°-,nr,' N^' NH2O’ ^0-^0O=p-O’VWSGR Docket No. 60652-721601NH2W NH2T3D-14d° 6-°VyN00=P-0*V NH21 1o=p-oT3D-14e O’ V-J0O=p-O"0. HNN^H<3 O^ITT3D-14f O=P-CX 1O'0O=P-O'OyNN— n— NH2o / o o^rrT3D-14g O=P-O. 1d- 0O=P-O'VWSGR Docket No. 60652-721601o \ XNH20 O^NT3D-14h O=p-O. 1o- 00=P-0- V0NH2 > HN if.x J y0 CXNT3D-14i O=p-Ck IO’0O=p-O’dy-X■ X=^ / N^\x\ O=P-CX NH2d ^\XT3D-14j0OP-0- VX / N^HN^r < \°Xo 0< V / °T3D-14k OP-Ck 1 \0- V°" J / 1 - 0050=P-0- / O^NHz X N-\,0L / N— \O=P-CX o6- 0 / °T3D-141 OP-O- \V o>O'X^NH2WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601o °Vo^NH2oHO=P-O. 1T3D-15c 0- 0O=P-O’VoKNH2x V9 HN°T°¥T3D-15doO=P-O-'Z'b NH20=P-0 HN^°"T3D-15eO0O=P-O- Vo ^NH2X r°9 HNO=P-O JT3D-15f°' ¥oO=P-O’X oO IIO=p-O H uWX / \^N o- Y *NI n9 ° \ T3D-15g O=P-O- / oNH2WSGR Docket No. 60652-721601Xo JO=P-O. HO’ T "n9 S < O=P-O’ ) T3D-15ho NH2X9 oO=p-O. H IIo- VNY^NA^ NX1 II H N 90O=P-O" \ T3D-15i dy> >9w NH2O=p-O H j|o- o1n kJHC ‘'Xki / NO=P-O"NT3D-15jw NH20zf I^O^NHJ ' 9O=P-O. 1T3D-16a o- 0O=P-O’Oy*z<'oO=P-O>p’ V"\ -'-'XT3D-16b 1 ° NH2oO=p-O"WSGR Docket No. 60652-721601i l l oO=P-O. 1T3D-17a d- 0O=P-O" M CA 1 1x T0 CT N Xz"O=P- \ 7\ ° - T3D-17b O. 10- Y°1 ) o o X\ ii A '\ / o O Q_— — —A o 0O o=P-O’1,OO0-=- Oyo nX HN jpx / ^NH2o o^rXO=p-O. IT3D-17c d- y°ioO=P-O"dyo0 O^lXO=P-O- 1T3D-17do- Y°10O=P-O- OyT3D-17eWSGR Docket No. 60652-721601T3D-17f\ 7\ °z= - ) \ o o / / \, ii / A / / / OOQ.-— / Ior o4 o o>=- / _, i11 / OO< CL ==- o \ / iy O / / * 1 SV \ ( O o " U— — —T3D-17g M C z II / k / 1.I MXo0NH2z IO=P-Ck IT3D-17h 6- u-j0O=p-O’Oy / ^NH2X0o-p-o— | o TT3D-17i 0- 01■O-P=OWSGR Docket No. 60652-721601NH2X AoO=p-O TT3D-17j d" V-P0O=p-O" X zJ O Xz 0IZC^T3D-17k\ Z IIX'' °Z"Z\O) OCLV. / o- —- “\O O C —- I II\ / L- 1 o POC O P, iOL-- V ii, 1 •O OCL-- IIV. / “ oo 4OtT3D-171-O-P=O1Ct4O=P— o 0T3D-17m1VaAa.-O-P=O1ox>T3D-17nWSGR Docket No. 60652-721601T3D-17oI ZN^N / O V.\ II\ / O O CL —-- O=p-O. o P', iT3D-17p 6- O OQ--- IIu / “o0-O-P=O1OKNH2O=p-O.T3D-17q 6- y°^r0-O-P=O1°>? jL X 0 cr i\rO=p-O.T3D-18a0O=P-O’1°y’WSGR Docket No. 60652-721601o / =\9 NH O=P-O.2T3D-18b o- x°X0O=P-O- V(? XI IPxo cXHNx>O=P-O.T3D-18c6- 0O=P-O’1°yNH2c ) I # Z-N \\ _ / Jxo Jo=p-o.T3D-18d 6- 0■O-P 1=OOK>o / =\ZNH20 O^N \ / O=p-O. 1T3D-18e o- ypyloO=p-O"yWSGR Docket No. 60652-7216010 NH2xo cr 'rrO=P-CLT3D-18f 6- 0-O-P 1 =Ooxc> NH2x<?T3D-18g O=P-O. / d- 0O=P-O- Vo qxNH2q cr NO=P-CLT3D-18h 6- o■O-P 1 =Oc>>Z<'0 z=x0=P-°x, N O’ Y' _ / J T3D-19a0 NH2O=P-O’V^9 / — \O=P-< N, — 6 NT3D-19b ° iNH2oO=P-O’yWSGR Docket No. 60652-721601T3D-19cl Cs ZEx Z -j i r>v °T3D-19d1I II\^OOD--- O 1o llll OO-D--?b On--- OOTJ ' 1--9' 1O o oo oXX oo^X OOT-- 1 °Q °Qz — T3D-19e z ZE — K ZE>K>0 NH2x* / — CN=\ O=p.-O / |NH' — V 7 / ) T3D-19f O’ 1X0O=P-O"VX.0 0 NH2O=p-Ck XX N=\ 6- VNHT3D-19g0■O-P=O1OK>WSGR Docket No. 60652-721601T3D-19h4O Q NH2T3D-19io 0 N-1II^ z i / OOT-- -O-P 1 =O' lC)o o1 ii1 o O zN.AQ Z, N ANyHNj]”Tl \ / 9 O^N \HO=p-O. I T3D-20a d- 1 ho 0 [H O=p-O"OyO N.A N ANX NH2O=P-O. 1T3D-20b 6- oO=P-O’VX9 MO=P-O AN °- A \ — / T3D-20co NH2O=p-O’OyWSGR Docket No. 60652-721601O=P-O >^N''°’ NH T3D-20d2oO=P-O’NH2oJ.T3D-21aO=P-O. 1o- oO=P-O- V NH2T3D-21bX9 XJO=p-O 1O’oO=P-O’xx T o o^i\rO=P-O. 1T3D-22a d- Vo-j0O=P-O- V0S Q O^fTH0=P-0- IT3D-22b o- yp-j0O=P-CrOyWSGR Docket No. 60652-721601NH2XoO=p-O^T3D-22c 0- 00=P-0- VT3D-22d0O=P-O- Oy>49 / NH2T3D-23aO-P=o N'i> / x9 ZNH2T3D-23b'7:'-O-P=O NiC)>■^o-0-P-Ck O _ / NH2 T3D-23c■ ■.. ■ O=P-O- - - iN1 N°yWSGR Docket No. 60652-721601ONH2O O^N^O=p-O.T3D-23d 6- TM CZ0■O-P=O1ox>o Z-VNH2,..o0=P-0. K °T3D-23e ZI6- o0-0-P=0 O VO^n.-- 1P, 1> O O0.-- / h II “o014 0 O X^N sHY \ k-M / XyANH20=^-0 "—VT3D-23f O’0-O-P=O10T3D-23gJ tk.T3D-23h0 NH2O=P-O- 1°yWSGR Docket No. 60652-721601WSGR Docket No. 60652-7216011 NH / vX XI NHO=P-O. 1T3D-23o 1 1o- L- — -J0i■O-P=o I 1 z — 0>T3D-23pii V...4^0oO. U-- o P, 1OOCL-- II7^ ■o / % V^ XN *NI NHO=P-O. 11 1T3D-23q °- - -d01-O-P=o1N\ 7 \,NH2% °KI NHO=P-O. 11 1 ^.T3D-23r °- 0i-O-P=o1Z\ / NH2 / \ ^N / / 4 X >| NHO = P-CX 11 1T3D-23s °- 01-O-P=O10>WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601T3D-24gTM C Z J OT^9 Z / \^z- 4 O -u A COJ L IT3D 2 h A iiOOT-- O-? P=O ° jQo1i o / v° d / ^-A) IA / 1 NH2 / \ OOT —-- \z=° II / / > o IZoo / u O - < II ZI\ZOO^CL-- IT3D-24i, < II P / O^ 11O O0.-- o PA II “, 1 1o IIO O0.-- z —^^^V ■4 QZZ" / z°=P-A H 16- Y^3 -24NX AN T D jo J XX z I0-P=0 N> 0 NH2>T3D-24kWSGR Docket No. 60652-721601T3D-241z.zzLJ-<xoiiT3D-24m OOTJ-- ■ i° d / 1T / / \ OO-0 —-- o \=> Z II T. / oNH2ZI\ / O OCL --- < Y II'n °\ ° i X-S 16? Y HN \ — \. 1 HN' Y II i \ / X II x JJ00O ° / k / / NT3D-25a o N > - (ZKI ^N"oO=P-O’ X / zyY) NH2z z X H 0. jCx oXo (T3D-25b 6- V o 1 kx9 WO=p-O”WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601WSGR Docket No. 60652-7216010XoO=P-CL6- 0■O-P=OT3D-25jX o? AJ? If X NxO OTJ^--11NZ / A -O-P=O N ^O0.AO OTI--- 1^0-^ZI 0O=P-O’l0 4z / / NH2co \o= — x.Sn \XX II °\\ ANzO x ■AzO-to HN^N=S I / 1 w " Z Z-N^^ T3D-25kd- z I o to O=P-O'NH2T3D-251 ' 1 A O \ / IM"°- XX — KoO=P-O'VT3D-25mWSGR Docket No. 60652-721601O=P-O. H o. NH2d- Y^^NYN^SI^J _ < T3D-25no s r / / " N OP-O- Oy7\ N"T3D-26aO* II^ ZOOTJ-- o 1 ' IIo OO-U o-- ' 1o O fO-U\--— on o o^ OONTJ-- 1 / II yT -o zo= —°0T3D-26b 0 zZE N> z I N>oXO N^OO=P-O.6- o-O-P=OT3D-26cH2Nw.x PH? ri■O-P i =O<'N' X) °oO=p-O"1°yWSGR Docket No. 60652-721601T3D-27aM C z Iz 00o7\ 1 II\ °z- O OCL-- L H 1T3D-27b \O J OQ---— > < °? r 1-^Po PO OQ--- hZ ii “ZO O0- o-- IIoxO Y A'AX)O=P-O^6- 0O-P=OT3D-27c&■O-P l=O ^N^O 00O=P-O- 1°yN=NI JXo O^IMT3D-28a O=p-O- 6- 0O-P=O1WSGR Docket No. 60652-7216010X X1)0 CT 'YO=P-O.T3D-28b 6- 0 NH2■o— p=o1°yT3D-29a NH2oX x Y k Xs" <3 o^X / O=P-Ck 1d- oO=P-O"T3D-29b NH2jQj x09 Y^NO=p-O- 11o- pj0O=p-O"dy-T3D-29c NH2JQJX °9 Y^NO=P-O. 1 I o- 0O=p-O"°xWSGR Docket No. 60652-721601T3D-29d NH20( j? VX x lx k AA O= dP--O. 1 M C 0 \ z ZZ —XX-? O=p-O" \ _ / / \\ VT3D-29e°z=ZIH S?o p ~, iOOCL-- IIu / “oT3D-29f NH2XAX °O=p-Ox\X> N6- v0O=P-O- VT3D-29g NH2X ° ( O=p-Oxo- voO=P-O"AWSGR Docket No. 60652-721601

[0280] In some embodiments, a polynucleotide composition further comprises a second modified nucleotide. In some embodiments, the second modified nucleotide comprises a derivative of ethylenediaminetetraacetic acid (EDTA). In some embodiments, the secondmodified nucleotide comprises a structureof embodiments, the second modified nucleotide dendritic polymer comprises a dendritic polymer.In some embodiments, the second modified nucleotide comprises a structureof 'vuu-. In some embodiments, the second modified nucleotide comprises a structure ofIn some embodiments, the second modified nucleotide is configured to facilitate a proximity-based cleavage. In some embodiments, EDTA, a dendritic polymer, or aWSGR Docket No. 60652-721601derivative of EDTA or of the dendritic polymer is added to the reaction mixture to facilitate proximity-based cleavage. In some embodiments, EDTA, a dendritic polymer, or a derivative of EDTA or of the dendritic polymer is introduced at a specific step (e.g., during or immediately prior to the cleavage step), or is present throughout the protein sequencing process.

[0281] In some embodiments, a polynucleotide composition further comprises one or more abasic nucleotides. In some embodiments, the abasic nucleotide does not comprise adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), or any abasic derivative thereof. In some embodiments, at least one of the one or more abasic nucleotides is disposed 5’ of the modified nucleotide or 3’ of the modified nucleotide. In some embodiments, at least one of the one or more abasic nucleotides is less than 10 nucleotides away from the modified nucleotide. In some embodiments, at least one of the one or more abasic nucleotides is immediately adjacent to the modified nucleotide. In some embodiments, at least one of the one or more abasic nucleotides is immediately adjacent to 5’ end of the modified nucleotide. In some embodiments, at least one of the one or more abasic nucleotides is immediately adjacent to 3’ end of the modified nucleotide

[0282] In some embodiments, the -L-B is represented by the structure of Formula (V):LGR,-A I -R2L.,Formula (V),wherein,L is a linker, wherein the linker comprises one or more electron-withdrawing or electron-donating groups;LG is a leaving group; andR1and R2are each independently hydrogen, 5- or 6- membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl, wherein the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2,WSGR Docket No. 60652-721601CN, COOR”, and CON(R”)2, wherein each R” is independently H or Ci- C3 alkyl.

[0283] In some embodiments, L is as described elsewhere herein.

[0284] In some embodiments, LG is a leaving group, such as a leaving group described elsewhere herein.

[0285] In some embodiments, R1and R2are each independently hydrogen, 5- or 6-membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl, wherein the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or Ci-C3 alkyl.

[0286] In some embodiments, R1is hydrogen, 5- or 6- membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl, wherein the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0287] In some embodiments, R1is hydrogen, 5- or 6- membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl. In some embodiments, R1is hydrogen. In some embodiments, R1is 5- or 6-membered aryl. In some embodiments, R1is 5- or 6-membered heteroaryl. In some embodiments, R1is 5- or 6-membered cycloalkyl. In some embodiments, R1is 5- or 6-membered heterocycloalkyl. In some embodiments, R1is Ci-Ce alkyl. In some embodiments, R1is Ci-Ce alkoxy. In some embodiments, R1is Ci-Ce haloalkyl. In some embodiments, R1is Ci-Ce heteroalkyl.

[0288] In some embodiments, R1is hydrogen, 5- or 6- membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-WSGR Docket No. 60652-721601membered heteroaryl, and 5 or 6-membered heteroaryl. In some embodiments, the 5- or 6-membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl is optionally substituted with oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl. In some embodiments, the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0289] In some embodiments, R2is hydrogen, 5- or 6- membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl, wherein the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0290] In some embodiments, R2is hydrogen, 5- or 6- membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl. In some embodiments, R2is hydrogen. In some embodiments, R2is 5- or 6-membered aryl. In some embodiments, R2is 5- or 6-membered heteroaryl. In some embodiments, R2is 5- or 6-membered cycloalkyl. In some embodiments, R2is 5- or 6-membered heterocycloalkyl. In some embodiments, R2is Ci-Ce alkyl. In some embodiments, R2is Ci-Ce alkoxy. In some embodiments, R2is Ci-Ce haloalkyl. In some embodiments, R2is Ci-Ce heteroalkyl.

[0291] In some embodiments, R2is hydrogen, 5- or 6- membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl. In some embodiments, the 5- or 6-membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl is optionally substituted with oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl. In some embodiments, the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or twoWSGR Docket No. 60652-721601members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0292] In some embodiments, the -L-B is represented by the structure of Formula (VI):LGFormula (VI)wherein,L is a linker, wherein the linker comprises one or more electron-withdrawing or electron-donating groups;LG is a leaving group;R1is hydrogen, 5- or 6- membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl, wherein the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl; andn is an integer from 0 to 2.

[0293] In some embodiments, L, LG, and R1are as described elsewhere herein.

[0294] In some embodiments, n is an integer from 0 to 2. In some embodiments, n is an integer from 0 to 1. In some embodiments, n is an integer from 1 to 2. In some embodiments, n is 1 or 2. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 0.

[0295] In some embodiments, the -L-B is represented by the structure of any one of Formula (VILA), (VII-B), or (VILC):LG LGWSGR Docket No. 60652-721601Formula (VII-A) Formula (VII-B) Formula (VII-C)wherein,LG is a leaving group;L3 and L4 are each individually linkers; or— represents L3 and L4 being taken together to form Cs-Cs heterocycle; andR1is hydrogen, or 5- or 6-membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl, wherein the phenyl, 5 or 6- membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0296] In some embodiments, LG and R1are as described elsewhere herein.

[0297] In some embodiments, L3 and L4 are each individually linkers, such as linkers described elsewhere herein. In some embodiments, L3 is a linker, such as a linker described elsewhere herein. In some embodiments, L4 is a linker, such as a linker described elsewhere herein.

[0298] In some embodiments, — represents L3 and L4 being taken together to form Cs-Cs heterocycle. In some embodiments, — represents L3 and L4 being taken together to form Ce-Cs heterocycle. In some embodiments, — represents L3 and L4 being taken together to form Cs-Ce heterocycle. In some embodiments, — represents L3 and L4 being taken together to form a C5 heterocycle. In some embodiments, — represents L3 and L4 being taken together to form a Ce heterocycle.

[0299] In some embodiments, the leaving group (LG) is electron-withdrawing. In some embodiments, the leaving group is C4-C12 heteroaryl or C4-C12 heterocycle each optionally substituted with one or more electron-withdrawing groups. In some embodiments, the leaving group is C4-C12 heteroaryl optionally substituted with one or more electron-withdrawing groups. In some embodiments, the leaving group is C4-C12 heterocycle optionally substituted with one or more electron-withdrawing groups. In some embodiments, the leaving group is an azole orWSGR Docket No. 60652-721601azine, optionally substituted with one or more electron- withdrawing groups. In some embodiments, the leaving group is an azole. In some embodiments, the leaving group is an azine. In some embodiments, the leaving group is an azole substituted with one or more electron-withdrawing groups. In some embodiments, the leaving group is an azine substituted with one or more electron-withdrawing groups.

[0300] In some embodiments, the leaving group is triazole fused with aryl or heteroaryl. In some embodiments, the leaving group is triazole. In some embodiments, the leaving group is triazole fused with aryl. In some embodiments, the leaving group is triazole fused with heteroaryl. In some embodiments, the triazole fused with aryl or heteroaryl is substituted with one or more electron-withdrawing groups.

[0301] In some embodiments, the leaving group is represented by the structure:X3-X4 x3-x4 / / w u / ,X2^Xs X2v 'xX5-YT4'X! *1orwherein,each of X1-X5 is independently selected from N, CH, or C-EWG; each of Y1-Y4 is independently selected from N, CH, or C-EWG; and EWG is an electron-withdrawing group;wherein at least one of X1-X5 is N.X3-X4 / / X2 xX5Xi

[0302] In some embodiments, the leaving group is. In other embodiments, the leaving ZY1--Y2v'V^yi X Xgroup is—J—. In further embodiments, the leaving group is0

[0303] In some embodiments, each of X1-X5 is independently selected from N, CH, or C- EWG (e.g., C substituted with an electron withdrawing group provided elsewhere herein). In some embodiments, at least one of X1-X5 is N. In some embodiments, at least one of X1-X5 is CH. In some embodiments, at least one of X1-X5 is C-EWG.

[0304] In some embodiments, each of Y1-Y4 is independently selected from N, CH, or C-EWG. In some embodiments, at least one of Yi-Y4is N. In some embodiments, at least one of Yi-Y4is CH. In some embodiments, at least one of Yi-Y4is C-EWG.

[0305] In some embodiments, EWG is an electron-withdrawing group. In some embodiments, each electron-withdrawing group is independently selected from halogen,WSGR Docket No. 60652-721601haloalkyl, NO2, sulfonate, amino, alkylamino, CN, or a carbonyl. In some embodiments, the electron-withdrawing group is halogen. In some embodiments, the electron-withdrawing group is haloalkyl. In some embodiments, the electron-withdrawing group is NO2. In some embodiments, the electron-withdrawing group is sulfonate. In some embodiments, the electron-withdrawing group is amino. In some embodiments, the electron- withdraw! ng group is alkylamino. In some embodiments, the electron-withdrawing group is CN.

[0306] In some embodiments, the electron-withdrawing group is carbonyl. In some embodiments, the carbonyl comprises an acetyl group or a derivative thereof, a carboxylic acid, or an aldehyde.

[0307] In some embodiments, at least one electron-withdrawing group is Ci-Ce haloalkyl. In some embodiments, at least one electron-withdrawing group is NO2. In some embodiments, at least one electron-withdrawing group is halogen. In some embodiments, the halogen is F or Cl.

[0308] In some embodiments, the leaving group is:

[0309] In some embodiments, the reactive group of Formula (V), (VI), (VII- A), (VII-B), or (VII-C) has a structure selected from Table 4 A.Table 4AReactive Group StructureWSGR Docket No. 60652-721601NHT4A-la AeA xNN N AH \ _ / NH A AT4A-lbeN NH \ _ / NHT4A-1C A?AN N AH \ _ / NH / J].T4A-ld H \ / N=<\CF3O NHT4A-le N N AH \ / 0 NHT4A-lf AoA'NAKNN, XH\=7 NHT4A-lgAA,H NHT4A-2a H \ HNXN NHT4A-2b ^N HA r^-\ / A \ HN A. < ZXN^NNHT4A-2c A AHNSXA / Z ^N°2 N NHT4A-2d H ' A \HNSN NHT4A-2e A N A N A,H ' / A N=Z 1WSGR Docket No. 60652-721601T4A-3aO NHT4A-3b A A. NN Ni \=7O NHT4A-3c A A A zNi \=7NH 0T4A-3d z — 1 \HN\N O ACFT4A-4A A N NN= /

[0310] In some embodiments, -L-B of Formula (V), (VI), (VII-A), (VII-B), or (VII-C), as described herein has a structure selected from Table 4B.Table 4BL-B (Linker- Reactive StructureGroup)NHT4B-la A A zN,H \ _ / NHT4B-lb A?AN N ANH / J].T4B-1C NH \ / CF3O NHT4B-ld %A A zNH\=7WSGR Docket No. 60652-721601T4B-leNH A AT4B-lfHHNM AxN NH A AT4B-lgH HN ASzk / l' xO2N NHT4B-2NA N A AH\=JNHT4B-3 S? _ —A <zA y—- N A N A§\= IZ / H\=JNHT4B-4VTCH t 7ov= / NHT4B-5 % / N A A\ If N N A!!H\=1NHT4B-6a / o / — \Y^NAX— AA- N^N'A A X L"N H^= / H \ 'NHA °T4B-6b / \^NA^ / / XXN'XANXN< S A A MN H\=I H X- —7NHT4B-7a / o / — \ / NA^ — N^N'A A A L"N H\=J H - - 'NHT4B-7b / o / A^A^^N'AN'A A A L"N H^=J Hx-7WSGR Docket No. 60652-721601

[0311] In some embodiments, the modified nucleotide comprising Formula (V), (VI), (VII-A), (VII-B), or (VII-C) is selected from Table 4C.Table 4CModifiedNucleotid StructureeNH2H / ^X* 0 CT N NHO=P-CLT4C-la 0- VoJ0O=P-O"V0„ HN^ NHx” T IIo O^N\AN, N.O=p-O. H \ / T4C-lb o- 0O=P-o- VWSGR Docket No. 60652-721601O H / \ x 1T YO O^N NHNO=p-O. 1T4C-1C o- 0O=P-O"yNH2HN= / NX^..HN^ TJ T4C-ld O=P-O.o- 0O=P-O"VN0 °HNY Z,NTNH HN — || 1 XoN N'X^'NH2T4C-le O=P-O.d- oO=p-O"Y),=N0=YnNnX. °- yXN CF3T4C-lf0NHO=P-CrT0< YAC^p-Ck N ^0 T4C-lg d- Vj _0 HN N x, N o Opy-o- nNHWSGR Docket No. 60652-721601Xo ®O=p-O. 1 d- T4C-lho HN NQ O=P-O-NOy- NH 0zo 6:O=p-(X N 0 T4C-li O’ V°®l0 HN.. N. OP-0’ fl Oy- NH Y> (A)O=P-OXId- VMT4C-lj 1 - ' H / \o,i..o^ TNOyNH4 ®O=p-O. I d- T4C-lko=? YNNHynhY Y. N ' HN N x-> O=P-O. | \ / d- YJT4C-11OO=P-O- dy>NH? HN'^'N^Y °Y°y / w T4C-lmoO=P-O’WSGR Docket No. 60652-721601o 1 / \ A _N. X. N 0 NH O=p-O. 1T4C-2a 0- Vo^loO=p-O"Y / NVG o=p-o r nO- NHT4C-2boO=P-O- Oy- / CF3N=< o H ' 0 O^trNHT4C-3a O=p-O. Io- ypJ0O=P-O’V / CF3N=< X N, N' JN 9 < YO=P-% J NHT4C-3b O’ IX0O=P-O-0 N ACCFF3 JL9 HN N^N X I 'N= / T4C-4a <3O=P-O- 1o- YY0O=P-O"VWSGR Docket No. 60652-721601oN ACCFF3> J' O.. NH^N^'N T4C-4b O=P-°x | N=- / o- V-JOO=P-O’NH? HAN= / ^\ xHn '~z"<0 O^N NT4C-5a O=p-O. 1o- oO=P-O"V NHX k9 <NH / N=A \0=l?“0> J HN X / ^NOJ T4C-5b O’ N00=P-0"°yNHO f / 'k JL ^N A y - V 7 — N N HN^p \ _ / H \ _ / O O^N^O=p-(X IT4C-6a d- oO=P-O"yNH X XA -A, N 9N\ O=p-O ' - 'T4C-6b d- o0=P-0’VWSGR Docket No. 60652-721601NHx0 O^N T0CBN> -_7 'O=p-O. 1T4C-7a d- oO=P-O-j NHo=p-o r\ O d- Y o YN\v\=JT4C-7b O OT-- Y O 0\fo o-u--- „ O=P-O’A V Xi° 0Y ZIT4C-8a Y °oIZy NHx H IICzO=P-% / Y HN\ 7O’ oT4C-8boO=P-O’Gy*NH9o / — \^NY —.z K z\ L"N H\=J T4C-9a O=P-O 1Hd- y°YoO=P-O‘Oy>NH°A°'l oT4C-9b 1 A L '"H\= / o - "‘kl / ~-NO=P-O"HOy"WSGR Docket No. 60652-721601

[0312] In some embodiments, the use of the modified nucleotides of e.g., Formula (VII-A), (VII-B), or (VII-C) is shown in FIG. 24.

[0313] In some embodiments, the -L-B is represented by the structure of Formula (VIII):SFormula (VIII)wherein,L is a linker, wherein the linker comprises one or more electron-withdrawing or electron-donating groups; andR3is hydrogen or R’, wherein R’ is 5 or 6-membered aryl, 5 or 6- membered heteroaryl, or Ci-Ce alkyl optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6- membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0314] In some embodiments, L is as described elsewhere herein.

[0315] In some embodiments, R3is hydrogen or R’, wherein R’ is 5 or 6-membered aryl, 5 or 6-membered heteroaryl, or Ci-Ce alkyl optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0316] In some embodiments, R3is hydrogen.

[0317] In some embodiments, R3is R’. In some embodiments, R’ is 5 or 6-membered aryl, 5 or 6-membered heteroaryl, or Ci-Ce alkyl optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from oxo,WSGR Docket No. 60652-721601halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl. In some embodiments, R’ is 5 or 6-membered aryl, 5 or 6-membered heteroaryl, or Ci-Ce alkyl. In some embodiments, the 5 or 6-membered aryl, 5 or 6-membered heteroaryl, or Ci-Ce alkyl is optionally substituted with oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl. In some embodiments, the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2. In some embodiments, R” is H or C1-C3 alkyl. In some embodiments, R” is H. In some embodiments, R” is C1-C3 alkyl.

[0318] In some embodiments, the -L-B is represented by the structure of Formula (IX):S-R4'OAS'R5Formula (IX)wherein,L is a linker, wherein the linker comprises one or more electron-withdrawing or electron-donating groups; andR4is Ci-Ce alkylene, 5- or 6-membered cycloalkylene, or fused polycyloalkylene;R5is hydrogen or R’, wherein R’ is 5 or 6-membered aryl, 5 or 6- membered heteroaryl, or Ci-Ce alkyl optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6- membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0319] In some embodiments, L is as described elsewhere herein.

[0320] In some embodiments, R4is Ci-Ce alkylene, 5- or 6-membered cycloalkylene, or fused polycyloalkylene. In some embodiments, R4is alkylene, cycloalkylene, or fused polycycloalkylene. In some embodiments, R4is Ci-Ce alkylene. In some embodiments, R4is 5-or 6-membered cycloalkylene. In some embodiments, R4is fused polycycloalkylene. In some embodiments, R4is 8-12 membered fused polycycloalkylene. In some embodiments, the (e.g.,WSGR Docket No. 60652-721601Ci-Ce) alkylene, (e.g., 5- or 6-membered) cycloalkylene, or (e.g., 8-12 membered) fused polycycloalkylene, is optionally substituted with oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2, wherein each R” is independently H or C1-C3 alkyl. In some embodiments, the (e.g., Ci-Ce) alkylene, (e.g., 5- or 6-membered) cycloalkylene, or (e.g., 8-12 membered) fused polycycloalkylene, is optionally substituted with oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl. In some embodiments, the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2. In some embodiments, each R” is independently H or C1-C3 alkyl. In some embodiments, R” is H. In some embodiments, R” is C1-C3 alkyl.

[0321] In some embodiments, R5is hydrogen or R’, wherein R’ is 5 or 6-membered aryl, 5 or 6-membered heteroaryl, or Ci-Ce alkyl optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

[0322] In some embodiments, R5is hydrogen. In some embodiments, R’ is 5 or 6-membered aryl, 5 or 6-membered heteroaryl, or Ci-Ce alkyl optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl. In some embodiments, R’ is 5 or 6-membered aryl, 5 or 6-membered heteroaryl, or Ci-Ce alkyl. In some embodiments, the 5 or 6-membered aryl, 5 or 6-membered heteroaryl, or Ci-Ce alkyl is optionally substituted with oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl. In some embodiments, the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy,WSGR Docket No. 60652-721601C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2. In some embodiments, R” is H or C1-C3 alkyl. In some embodiments, R” is H. In some embodiments, R” is C1-C3 alkyl.

[0323] In some embodiments, the reactive group of Formula (VIII) or (IX) has a structure selected from Table 5A.Table 5AReactive Group StructureT5A-la s\)00T5A-lb s0 ^ 3- / VW*T5A-1C s00T5A-ld sc / >^ 0=T5A-les?0T5A-2a sT5A-2bT5A-2cT5A-2d s sH: £ ji^ oAszT5A-2eT5A-3 sOWSGR Docket No. 60652-721601

[0324] In some embodiments, -L-B of Formula (VIII) or (IX), as described herein has a structure selected from Table 5B.Table 5BL-B (Linker- Reactive StructureGroup)s ^T5B-laoosT5B-lbsT5B-1CsT5B-ldQsT5B-lez=\ / fT5B-lfs—T5B-lgT5B-lhT5B-liT5B-ljT5B-lkWSGR Docket No. 60652-721601\o=T5B-11ZZ- Z. - T5B-lms0, k / T5B-2 V _ / so^-NHIZZST5B-3 / VW' |-NH0 A / W’o S A ZI H H oT5B-4a N X XT" U > x 0^ H II V \ _c / >V0\o > ^ o= N"NC / )\T5B-4b

[0325] In some embodiments, the modified nucleotide comprising Formula (VIII) or (IX) is selected from Table 5C.Table 5CModifiedStructureNucleotideetHzl,X VT'0 O NSO=p-O.T5C-la 0- 0O=P-O"VWSGR Docket No. 60652-721601x s O=P-O. H T5C-lb d- Vys0O=P-O’y<oO=P-O. 1T5C-1C d- oO=P-O- Oy<NH2Xx \ANI5 oNT5C-ld O=P-O.0- VOxJ0O=p-O’Vo S N. Xr> X'ONN NH2T5C-le O=P-O.d- oO=P-O"dy-Xo ® °t°vojT5C-lf9 1 O=P-O- sKyWSGR Docket No. 60652-721601Xo ®O=p-O. 1o- v°JT5C-lg 1 —s0 JI O=P-O —yO=P-CL Id- T5C-lh 1 — 'so 11 O=P — s dO=p-O. 1o- T5C-li00=P-0’ Vz<k)O=P-O.6- T5C-ljo S O=P-O’z -roH O O^NXO=P-O- |T5C-2a o- YboO=p-O"Oy-Xsw / 9 / sO=P-O / XAO’ V7 / \ T5C-2boO=P-O"VWSGR Docket No. 60652-721601s011 O O^hTO=p-O. 1T5C-3a 0- 0O=P-O’xs0 II O=p-OO’T5C-3b0O=P-O- V0> HN A^p \ _ / f=\ \ _ Z,'s 0 O^N V — 7 'sO=P-O. 1T5C-4a 0- 0O=P-O- dy>sT5C-4b °T°yoO=P-O- VA oi,'Y° O=p-O. IT5C-5a o- 0O=P-O- VWSGR Docket No. 60652-721601sT5C-5b °T°U0O=P-O- VXHXYXOAS / o cr hrO=P-CL IT5C-6a d- oO=p-O"Vzso II O=p-O>O’T5C-6boO=P-O"s o IL / 0 'X _ / Xx JI HO O^NT5C-7a O=P-O- 1d- 0O=p-O"s 4 1^- O=P-CL / ~^NHT5C-7b o- OO=P-O’VWSGR Docket No. 60652-721601T5C-8a\ oXl l zVXO OT3-- o ^NHO=p-O T5C 8b>|- J oO’ \ r0 0-0-—- IXO A X / ' TX O o0O=P-O’°y*TO=p-O^ 1HT5C-9a:xo-M / oO=p-O"zS T5C-9b ° O A J L AN"O=P-O-HVr. — S? 0 / \ / xHi A A iL / / NO=P-O^ 1HT5C-10a d- Y°J0O=P-O-WSGR Docket No. 60652-721601

[0326] In some embodiments, the -L-B is represented by the structure of Formula (XI):ZX6-R9XXN=CA=NL-R8Formula (XI)wherein,CAis carbon;L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups;Xe is O, S, Se, or NR10; andR8is a bond, alkylene, heteroalkylene, alkenylene, heteroalkenyl ene, alkynylene, heteroalkynylene, arylene, or heteroarylene, each optionally substituted with one or more electron-donating or electron-withdrawing groups;R9is a protecting group configured for cleavage from the Formula (XI); and R10is independently selected from a group comprising hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, aryl, or heteroaryl, each optionally substituted with one or more electron-donating or electron-withdrawing groups.

[0327] In some embodiments, L is as described elsewhere herein.

[0328] In some embodiments, Xe is O, S, Se, or NR10. In some embodiments, Xe is O or S. In some embodiments, Xe is O. In some embodiments, Xe is S. In some embodiments, Xe is Se. In some embodiments, Xe is NR10.

[0329] In some embodiments, R8is a bond, alkylene, heteroalkylene, alkenylene, heteroalkenyl ene, alkynylene, heteroalkynylene, arylene, or heteroarylene, each optionally substituted with one or more electron-donating or electron-withdrawing groups. In some embodiments, R8is a bond. In some embodiments, R8comprises an optionally substituted alkylene group, optionally substituted heteroalkylene group, optionally substituted alkenylene group, optionally substituted heteroalkenylene group, optionally substituted alkynylene group,WSGR Docket No. 60652-721601optionally substituted heteroalkynylene group, optionally substituted arylene group, or optionally substituted heteroarylene group. In some embodiments, R8comprises an alkylene group. In some embodiments, R8comprises a substituted alkylene group. In some embodiments, R8comprises an arylene group. In some embodiments, R8comprises a substituted arylene group. In some embodiments, R8comprises a heteroarylene group. In some embodiments, R8comprises a substituted heteroarylene group. In some embodiments, R8is substituted phenylene. In some embodiments, R8is nitrophenylene. In some embodiments, R8is an electron donating group or an electron withdrawing group. In some embodiments, R8is an electron donating group. In some embodiments, R8is an electron withdrawing group.

[0330] In some embodiments, R9is a protecting group. In some embodiments, R9is a protecting group configured for cleavage from the Formula (XI). In some embodiments, R9comprises an acetyl group, a benzoyl group, a benzyl group, a tosyl group, a triphenylmethane group, a methylthiomethyl ether group, a carbobenzyl oxy group, a p-methoxybenzyl ether (PMB) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a pivaloyl group, a tetrahydropyranyl (THP) group, a silyl group, a methyl ether, an ethoxy ethyl, a sulfonamide group, or any combination thereof. In some embodiments, R9comprises a silyl group. In some embodiments, the silyl group comprises trimethyl silyl (TMS), triethylsilyl (TES), tertbutyldimethylsilyl (TBDMS), tert-Butyldiphenylsilyl (TBDPS), triisopropyl silyl (TIPS), triisopropylsilyloxymethyl (TOM), or any combination thereof. In some embodiments, the silyl group comprises trimethyl silyl (TMS). In some embodiments, R9is trimethyl silyl (TMS). In some embodiments, R9is tert-butyloxycarbonyl (BOC). In some embodiments, R9is fluorenylmethoxycarbonyl (Fmoc). In some embodiments, R9is triphenylmethyl (Tr). In some embodiments, R9is benzoyl (Bz). In some embodiments, R9is p-methoxybenzyl (PMB). In some embodiments, R9is dimethoxytrityl (DMT). In some embodiments, R9is t-butyldimethylsilyl (TBDMS). In some embodiments, R9is t-butyldiphenylsilyl (TBDPS). In some embodiments, R9is acetyl (Ac). In some embodiments, R9is methoxyacetyl (MAc). In some embodiments, R9is benzyl (Bn). In some embodiments, R9is 9-fluorenylmethyl (Fm).

[0331] In some embodiments, R10is independently selected from a group comprising hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, aryl, or heteroaryl, each optionally substituted with one or more electron-donating or electron-withdrawing groups. In some embodiments, R10is hydrogen. In some embodiments, R10comprises optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, R10WSGR Docket No. 60652-721601comprises optionally substituted C1-C9 alkyl, optionally substituted C1-C9 alkenyl, or optionally substituted C1-C9 alkynyl. In some embodiments, R10comprises C1-C9 alkyl. In some embodiments, R10comprises a substituted C1-C9 alkyl. In some embodiments, R10comprises Ci-C9 alkenyl. In some embodiments, R10comprises a substituted C1-C9 alkenyl. In some embodiments, R10comprises C1-C9 alkynyl. In some embodiments, R10comprises a substituted C1-C9 alkynyl. In some embodiments, R10is an electron donating group or an electron withdrawing group. In some embodiments, R10is an electron donating group. In some embodiments, R10is an electron withdrawing group.

[0332] In some embodiments, CAof the Formula (XI) is configured to couple an N-terminal amine of a peptide prior to R9 cleavage. In some embodiments, CAof the Formula (XI) is configured to not couple to an N-terminal amine of a peptide subsequent to R9cleavage. In some embodiments, R9is configured for cleavage by a base. In some embodiments, the base is a halide. In some embodiments, the halide is fluoride. In some embodiments, the cleavage requires temperatures above 25 °C. In some embodiments, the cleavage may be performed in neutral or alkaline organic media. In some embodiments, the cleavage may be performed in alkaline organic media.

[0333] In some embodiments, subsequent to the cleavage, the Formula (XI) comprises an oxime or an oximate. In some embodiments, the oxime or the oximate is configured to couple to an internal amino acid of the peptide. In some embodiments, the Formula (XI) is configured to cleave an N-terminal amino acid from the peptide upon or subsequent to the coupling to the internal amino acid of the peptide. In some embodiments, the cleaving the N-terminal amino acid from the peptide generates a 3-iminol,2,4-oxadiazinanone.

[0334] In some embodiments, the -L-B is represented by the structure of Formula (XII):Formula (XII)wherein,CAis carbon;L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups;WSGR Docket No. 60652-721601X7is O, S, SO, SR14, Se, SeO, SeR14, or NR14;X8is O, S, SR14, SOR14, SO2R14, Se, SeR14, SeOR14, SeO2R14or NR14;X9is O, S, Se, or NR14;R11is independently selected from a group comprising hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with one or more electron-donating or electron-withdrawing groups; andR12is a protecting group configured for cleavage from the Formula (XII);R13is a bond, alkylene, heteroalkylene, alkenylene, heteroalkenyl ene, alkynylene, heteroalkynylene, arylene, or heteroarylene, each optionally substituted with one or more electron-donating or electron-withdrawing groups; andR14is independently selected from a group comprising hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, aryl, or heteroaryl, each optionally substituted with one or more electron-donating or electron-withdrawing groups.

[0335] In some embodiments, L is as described elsewhere herein.

[0336] In some embodiments, X7 is O, S, SO, SR14, Se, SeO, SeR14, or NR14. In some embodiments, X7 is O. In some embodiments, X7 is S. In some embodiments, X7 is SO. In some embodiments, X7 is SR14. In some embodiments, X7 is Se. In some embodiments, X7 is SeO. In some embodiments, X7 is SeR14. In some embodiments, X7 is NR14.

[0337] In some embodiments, X8is O, S, SR14, SOR14, SO2R14, Se, SeR14, SeOR14, SeO2R14or NR14. In some embodiments, X8is O. In some embodiments, X8is S. In some embodiments, X8is SR14. In some embodiments, X8is SOR14. In some embodiments, X8is SO2R14. In some embodiments, X8is Se. In some embodiments, X8is SeR14. In some embodiments, X8is SeOR14. In some embodiments, X8is SeO2R14. In some embodiments, X8is NR14.

[0338] In some embodiments, X9 is O, S, Se, or NR14. In some embodiments, X9 is O. In some embodiments, X9 is S. In some embodiments, X9 is Se. In some embodiments, X9 is NR14.

[0339] In some embodiments, R11is independently selected from a group comprising hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with one or more electrondonating or electron-withdrawing groups. In some embodiments, R11is hydrogen. In some embodiments, R11is independently selected from a group comprising alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In someWSGR Docket No. 60652-721601embodiments, R11is independently selected from a group comprising alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each substituted with one or more electron-donating or electron-withdrawing groups. In some embodiments, R11is heterocycloalkyl. In some embodiments, R11is nitrobenzene. In some embodiments, R11is pyrrolidine-2, 5-dione.

[0340] In some embodiments, R12is a protecting group configured for cleavage from the Formula (XI). In some embodiments, R12comprises an acetyl group, a benzoyl group, a benzyl group, a tosyl group, a triphenylmethane group, a methylthiomethyl ether group, a carbobenzyl oxy group, a p-methoxybenzyl ether (PMB) group, a 9-fluorenylmethyloxycarbonyl (FMOC) group, a pivaloyl group, a tetrahydropyranyl (THP) group, a silyl group, a methyl ether, an ethoxy ethyl, a sulfonamide group, or any combination thereof. In some embodiments, R12comprises a silyl group. In some embodiments, the silyl group comprises trimethyl silyl (TMS), triethylsilyl (TES), tert-butyldimethylsilyl (TBDMS), tert-Butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), triisopropylsilyloxymethyl (TOM), or any combination thereof. In some embodiments, the silyl group comprises trimethyl silyl (TMS). In some embodiments, R12is trimethyl silyl (TMS). In some embodiments, R12is tert-butyloxycarbonyl (BOC). In some embodiments, R12is fluorenylmethoxycarbonyl (Fmoc). In some embodiments, R12is triphenylmethyl (Tr). In some embodiments, R12is benzoyl (Bz). In some embodiments, R12is p-methoxybenzyl (PMB). In some embodiments, R12is dimethoxytrityl (DMT). In some embodiments, R12is t-butyldimethylsilyl (TBDMS). In some embodiments, R12is t-butyldiphenylsilyl (TBDPS). In some embodiments, R12is acetyl (Ac). In some embodiments, R12is methoxyacetyl (MAc). In some embodiments, R12is benzyl (Bn). In some embodiments, R12is 12-fluorenylmethyl (Fm).

[0341] In some embodiments, R13is a bond. In some embodiments, R13is independently selected from a group comprising alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, or heteroarylene, each optionally substituted with one or more electron-donating or electron-withdrawing groups. In some embodiments, R13comprises optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted alkenylene, optionally substituted heteroalkenylene, optionally substituted alkynylene, optionally substituted heteroalkynylene, optionally substituted arylene, or optionally substituted heteroarylene. In some embodiments, R13comprises optionally substituted C1-C9 alkylene, optionally substituted C1-C9 alkenylene, or optionally substituted C1-C9 alkynylene. In some embodiments, R13comprises C1-C9 alkylene. In some embodiments, R13comprises a substituted C1-C9 alkylene. In some embodiments, R13comprises C1-C9 alkenylene. In some embodiments,WSGR Docket No. 60652-721601R13comprises a substituted C1-C9 alkenylene. In some embodiments, R13comprises C1-C9 alkynylene. In some embodiments, R13comprises a substituted C1-C9 alkynylene. In some embodiments, R13is an electron donating group or an electron withdrawing group. In some embodiments, R13is an electron donating group. In some embodiments, R13is an electron withdrawing group.

[0342] In some embodiments, R14is independently selected from a group comprising hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, aryl, or heteroaryl, each optionally substituted with one or more electron-donating or electron-withdrawing groups. In some embodiments, R14is hydrogen. In some embodiments, R14comprises optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, R14comprises optionally substituted C1-C9 alkyl, optionally substituted C1-C9 alkenyl, or optionally substituted C1-C9 alkynyl. In some embodiments, R14comprises C1-C9 alkyl. In some embodiments, R14comprises a substituted C1-C9 alkyl. In some embodiments, R14comprises Ci-C9 alkenyl. In some embodiments, R14comprises a substituted C1-C9 alkenyl. In some embodiments, R14comprises C1-C9 alkynyl. In some embodiments, R14comprises a substituted C1-C9 alkynyl. In some embodiments, R14is an electron donating group or an electron withdrawing group. In some embodiments, R14is an electron donating group. In some embodiments, R14is an electron withdrawing group.

[0343] In some embodiments, Xs and X9 are each independently selected from O and NR14. In some embodiments, Xs is NR14and X9 is O. In some embodiments, X9 is O. In some embodiments, R13is hydrogen.

[0344] In some embodiments, CAof Formula (XII) is configured to couple to an N-terminal amine of a peptide prior to R12cleavage. In some embodiments, CAof Formula (XII) is configured to not couple to an N-terminal amine of the peptide subsequent to R12cleavage. In some embodiments, nucleophilic substitution at CAfavors a loss of (NR13)-O-R12.

[0345] In some embodiments, R12is configured for cleavage by a base. In some embodiments, the base is a halide. In some embodiments, the halide is fluoride.

[0346] In some embodiments, the cleavage requires temperatures above 25 °C.

[0347] In some embodiments, the cleavage may be performed in neutral or alkaline organic solution. In some embodiments, the cleavage may be performed in alkaline organic solution. In some embodiments, subsequent to the cleavage, the Formula (XII) comprises an oxime or an oximate. In some embodiments, the oxime or the oximate is configured to couple to an internalWSGR Docket No. 60652-721601amino acid of a peptide. In some embodiments, the Formula (XII) is configured to couple to an internal atom of a peptide upon or subsequent to the cleavage.

[0348] In some embodiments, the Formula (XII) is configured to cleave an N-terminal amino acid from a peptide upon or subsequent to coupling to an internal atom of the peptide. In some embodiments, the internal atom is a carbonyl carbon of the peptide. In some embodiments, a polynucleotide composition comprises a dimethylsulfoxide (DMSO) solution comprising a structure of Formula (XII), or a salt, a solvate, or a derivative thereof. In some embodiments, the Formula (XII) comprises a DMSO solubility of about 10 mg / mL to about 1 pg / mL. In some embodiments, the Formula (XII) comprises a half-life of about 1 day to 10 years when stored in dry form, in dry conditions, at 25 °C, and in the absence of light. In some embodiments, the Formula (XII) comprises a half-life of about 1 day to 10 years when stored in dimethylsulfoxide (DMSO), at 25 °C, and in the absence of light.

[0349] In some embodiments, the reactive group of Formula (XI) or (XII) has a structure selected from Table 6A.Table 6AReactive Group Structure^O— TMST6A-la N=C=N0— TMSN=C=NT6A-lbi-0lN — BocT6A-2a / N=C=NN— BocN=C=NT6A-2b 1 il J5 |i AS-TrT6A-3a / N=C=Ns-TrN=C=NT6A-3b1-01WSGR Docket No. 60652-721601T6A-4aT6A-4b 9 9o o U 0 0 E \0 / ZI- — / / \\ / V 7 Z -00=VQ WVV / T6A-4cN <z ^o o toz^A z —T6A-4d 0 \ o / O - / VW' 00= / O 00= / VW* / = =% / \ ' Z / \ - 00> d <= 2 / = r\ —\ / / \ \d ( Z> - / \ / / / x' O 0)0) T6A-5a x ' N. -N. _Fmoc 0)N^ / cIINHOT6A-5b\ N. _Fmoc N N=— 7 / CNNHOT6A-5c. ' 9\\ ^,0^. N.. Fmoc Z^N C N( | II H \^k oXOT6A-5dWSGR Docket No. 60652-721601T6A-6aT6A-6bO QE / r. / / zi — r, A \ —vz\ / / z / A / 7 —T6A-7a V. \ / o \o= V = / N / x—2 v / VW 00= > - ' / TMSU x / VW / c 0€1 sT6A-7b\ N- -N- / TMSN N= -= / !C|| °13 o Z / wv' S / / 000 0 ==K) ' Z Z - - / / / / / ( ' ' (JiJi - T6A-8a x ' N.. N.. \FHmocN= / C N-JII H ST6A-8bT6A-9a V N.. N. / Trc SsT6A-9bwherein:TMS is trimethylsilyl; Boc is tert-butyloxycarbonyl; and Tr is triphenylmethyl.

[0350] In some embodiments, -L-B of Formula (XI) or (XII), as described herein has a structure selected from Table 6B.WSGR Docket No. 60652-721601Table 6BL-B(Linker- StructureReactiveGroup)^O— TMST6B-la N=C=N0— TMST6B-lb •y^\ / 'N=C=N / ^0— TMSN=C=NT6B-1CC " -NxT6B-ld XJ _ ( / \\XN — <\TMS0— TMS0 _ ^N=C=NZT6B-leNH O O— TMST6B-lf O'X~X'N=C=Nf— NH / O-TMS o / ^ / N=C=NT6B-lgv ° VT6B-2a \ N., N., FmocN N=— / / CNNHOT6B-2b \ N. _N.. FmocN N=^yZcNNHOWSGR Docket No. 60652-721601T6B-2c / Q\ N. -FmocN N=^ / / CHNH0o oET6B o \ o-2d / E ZI\ > z — / ZI \0 / 0=J z / —o5o o= ^zQVIT6B-2eM C zo. oH 0" NT6B-2f 9T c \\ / / 00=\ N. \ -N.. FmocN ' Z -N— / CI / N— ' I0 IZ H\3o Fmoc o NH O —NT6B-2g^" VNA° ]T6B-2h yl -0-, N. - Fmoc / N C N< I II H\ _ k OxoT6B-2iT6B-2jyl. N. -Fmoc / K" N C N< I II HV— k oxoWSGR Docket No. 60652-721601

[0351] In some embodiments, the modified nucleotide comprising Formula (XI) or Formula (XII) is selected from Table 6C.Table 6CModifiedStructureNucleotideNH2 / °-™SX, N=C = NI JCr NO=p-O.T6C-la 0- oO=p-O’oX* O-TMS90N^N=C=NO=P-O.T6C-lb O- oO=P-O’o— TMSo / u N=C=NX J0 O^NO=p-O. 1T6C-1C o- oO=p-O’VWSGR Docket No. 60652-721601TMSO1 NH2N 1 |\| / xs \\ / / 7i y-N~< JI J * 9 N^-N^ T6C-ldO=P-O.0- Y°J0O=P-O-TMS01 flN\ 11 X NHzN\ Ji J * 0NN'^NHj T6C-leO=P-O.0- VQJoO=P-O- yXo ®O=p-O. 1d- V°JT6C-lf \| - ^O— TMS O N=C=N O=P-O’VXi (A)O=P-O. I0- V' _°' _J / o— TMS T6C-lgQ N=C=N 0 = P-0 — / VXo ®O=p-O. 10- V X| _°J / / O-TMS T6C-lh0 N=C=N 0=P — / 0WSGR Docket No. 60652-721601 / TMS 0y / XoxC*NO=P-CLT6C-li o- 0O=P-O"Xt)O-TMS O’ b-"\ zT6C-lj \ N=C=NOO=P-O’Oy-0 / O-TMS A, N=C=NHN ^pO O^NO=P-O. 1T6C-2a o- oO=P-O’yZ<'o o— TMS 0=^-0. N=C=NoT6C-2boO=P-O’dy-o— TMS o N=C=N HN'JSX^N== / O O^N / )O=p-O. I ' — 'T6C-3a d- oO=F>-O’WSGR Docket No. 60652-7216010o o^rr 4 / ) —O=P-O 1 ' — 'co..oT6C-3b A- 9,oU° \ / NTMS0O=P-O-0oXo. / -TMS T6C-3c o- N=C=N0O=P-O- / TMS o 1 o ML xN0 O^NT6C-3d O=p-O. Id- 0O=p-O"VO— TMS 9 N=C=NT6C-3eEY XO=P-O'°=P-°> / XO’ V"7T6C-3f 1 \\ / ) -NX0r, 0u=P 7-0 °" N 0^ TMS °yWSGR Docket No. 60652-721601O=P-OXT6C-3g9 - \ O— TMSO=?‘°’ N=C=N°x / O— TMS 0 N=C=N0 VyzHrif l nA i_i70 O^NT6C-4a O=p-O. 1o- 0O=P-O’o— TMS S? O N=C=No 'X _ / O=p-O. / \ / A-NT6C-4bQ\ HoO=P-O’yO °W z°-™sA Z- 0 N=C=N. HN'Y^N7O O^KTHO=P-O. 1T6C-5a d- VoJoO=P-O’X* °\\ o— TMS 9 / — O^XN=C=N / o=P-<\ / ^NO’ HT6C-5boO=P-O"WSGR Docket No. 60652-721601O-TMS s ^YN=C=NT6C-6a± ° Yo- Y°>0O=P-O-O— TMSoN=C=NT6C-6bi NO=P-O"Oy-^O— TMS ° _ZN=C=NT6C-7a± ° Yd- oO=P-O"yQ— TMS X H Z / N=C=NT6C-7b9 N"'O=p-O"FmocMk NHZX I Jo CT N C-0O=p-OxT6C-8ao- ^l U0O=P-O-yWSGR Docket No. 60652-721601Q Fmocuu\z NHX A 1 / N 9 oXX / 'c=0O=P-O. / T6C-8b 0- Vo^l zN\ 0O=P-O’VFmoc 9 NHx? 8 / zX A j 'X c=o O=P-CL 1NZT6C-8c“■ Vj o1 X 9 o0 \ OO JQ-—-- V^N O 9= °P-O’\ IIZ 1 obd--- X o Vo HN^\ z1C-N / N"Fm°CX N \ 12V / X J T6C-8d O=P-O.0- Yx0O=P-O- yC-N^oHN— N II Fmoc \ AN N H-^QN'^XN<>^NH2 T6C-8eO=P-O.o- pjoO=P-O- yT6C-8fWSGR Docket No. 60652-721601Xo ®O=p-O. I0- 0 Fmoc T6C-8gO=P-O— \ NH Oy N X’o £ / >Xo ®O=p-O. I0- V0®)k - ' Fmoc T6C-8h 9 / x.. NH O=P — N X ^ o. N0AFmoc-NH O=P-OX / 9 O- podT6C-8i i —7LN9O=P-O"9 FmocO=P-O NH O’T6C-8j V V9 ' O=P-O",N^\6y ® " Fmoc NHO / A ^N X A c=o O O^NNZT6C-9aoO=P-O’VWSGR Docket No. 60652-721601Fmoc<3 NH O=P-O NO’ C=O T6C-9boO=P-O- V. »O Fmoc A ' o o. HN |p y=\ZNOz u-H- E I o o^rr / ) — ZN / x\ / O- O=P-O. 1 ' — ' c=o T6C-10a d- N'9 Cii O=P-O" XXV< 7\ °z= - VOOCL--- L °? °> / ii x OO0.-- X oT6C-10bFmoc— NH O ' / / N-C 7 o fjT6C-10c 0O=p-O. 1o- oO=P-O’Vi Fmoc O=P-O. / \ \ d- V / =\ 'NHT6C-10d 1 X\.N0 — J C=O o=p-o-Nz°y < / ?WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601Fmoc NHOvN A / x C=O O oYH< T? T6C-12a O=p-O.d- OO=P-O’VFmocNHX 0 | / 9 X / \ O=P-O. / \ / p-° T6C-12b 6- y nZN1 OO=P-O’Oy-Frnoc^ O^ NH A / X V-,HY if v C=o C) O^N'JHNzT6C-13a°t°\^ u 0O=P-O'Fmoc X. °\ zNH0=P’°> / ^N C=O T6C-13b O’ VHN'0 uO=p-O’VFmoc O' NH A / x y~o^N AH" Y^N 'C=OzO O^N^H0 / P T6C-13co* O’°'ll^ 0o-^ 1l T \O=P-O"Oy>WSGR Docket No. 60652-721601T6C-13dFmocC) NH A / K Vo^NZHJ IT^N 'C=O F o^N'JHoT6C-13e O=P-O. 'V-'x d- [T 7 X oX n / xO OTl-- 9O=P NO J-2oO’' II — / Vz\O OTl —-- V X OyO IZ,^ FmocX* ° / NHo°'9-0> / ^NC ) I c=oT6 -13f o - yh0 / O C Z< o WV / i O z9? IIT / 0=P-°’ X-s IX ° \ °y‘ NO2zt-BDMS °v 0A XH? if^N C=o * 9 O^N9 Ho7O=p-O. -X T6C-13g d- 79O=P-O- NO2yt-BDMS X °\ / °" 9 'XO'^N70=P’°> X'N C=O T6C-13h °- yhozo=9_FF p-° \-XNO2WSGR Docket No. 60652-721601Fmoc.NH1T6C-14a± °Y M:*d- oO=P-O- VFmoc^ NH1 O / N'' 0° T6C-14bO=P-O"VFmoc^ NH1 O / N^ <°T6C-14cO=P-O Id- oO=P-O’Fmoc^NH1 OT6C-14d •i y~>9 N'"O=P-O’WSGR Docket No. 60652-721601Fmoc^ NH1T6C-14eO’0O=p-O"Fmoc^NH1O N^ T6C-14f■O=P-O’Vt-BDMS / 0 1 O / N^ o°T6C-14gO’ V°^l0O=P-O"t-BDMS O1 o / N^ < O T6C-14hi N''O=P-O’WSGR Docket No. 60652-721601WSGR Docket No. 60652-721601T6C-15e Fmoc.NH0s HN-'SA^HVX 1 0 \ NAMA O=P-O VNd- VoJ0O=P-O’T6C-15f Fmoc- NH1oXo- Xa^oZV ^ Y1\A< NO29 'N’O=p-O"NT6C-15g t-BDMSOo xX' / XA — X C / °NAX O O A^N JI \ \ < / / n1N°2 O=P-O. 16- 0O=P-O"T6C-15h t-BDMS01zO=9p-O H V \ / 0Y A...° \ \ I A1 - - J.N°2 O=P-O’

[0352] In some embodiments, the use of a modified nucleotide of Formula (XI) is shown in FIG. 25B

[0353] In some embodiments, the use of modified nucleotides of Formula (XII) are shown in FIG. 26B and FIG. 27BWSGR Docket No. 60652-721601

[0354] The linker provided herein, may be a bond, a cleavable linker, or a non-cleavable linker. The cleavable linker may be chemically or enzymatically cleavable. In some embodiments, the cleavable linker is chemically cleavable. In some embodiments, the linker is cleavable by acid, base, a reducing agent (e.g., disulfide bonds that are cleavable using TCEP, DTT), or a catalyst (e.g., Pd). In some embodiments, the linker is cleavable by an acid. The acid may be a strong acid or a weak acid. The acid may be hydrochloric acid, hydrobromic acid, perchloric acid, iodic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, nitric acid, or any other suitable acid. The base may be a strong base or a weak base. The base may be sodium hydroxide, potassium hydroxide, lithium hydroxide, barium hydroxide, or any other suitable base. In some embodiments, the linker is cleavable by a reducing agent. In some embodiments, for instance, the linker comprises disulfide bond cleavable using TCEP or DTT. In some embodiments, the linker is cleavable by a catalyst. The catalyst may be an inorganic catalyst, such as platinum, palladium, or nickel.

[0355] In some embodiments, the cleavable linker comprises a disulfide bond, a hydrazone, a PEG linker, a DNA molecule comprising a cleavage site, a peptide that is cleavable, an ester, or a de-click chemistry moiety. In some embodiments, the peptide is cleavable by an enzyme. In some embodiments, the cleavable linker comprises a hydrazone. In some embodiments, the cleavable linker comprises an o-amino benzyl hydrazone.

[0356] In some embodiments, the linker is a polymer, such as a polyethylene glycol (PEG), polyethylene, polypropylene, polyvinyl chloride, polystyrene or other organic or inorganic polymer.

[0357] The linker may be, for example, a linear or branched polyalkylene glycol (PAG). For example, the linker may be a branched PEG linker. The branched PEG linker may have 3 arms or 4 arms. In some embodiments, the linker has a general structure of PEG CCO-CCO-CCO or PPG CCCO-CCCO-CCCO. In some embodiments, the linker is a Polypropylene oxide) linker, such as polypropylene glycol) (PPG), having a structure H[OCH(CH3)CH2]nOH, where n is an integer equal to or greater than 1. The linker may comprise a combination of poly alkylene oxides, such as a poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) diacrylate. For example, the linker may have the following structure:WSGR Docket No. 60652-721601z o, where at least one of x, y, and z is an integer greater than 0.

[0358] The polymer can be a synthetic polymer or naturally-occurring polymer. Nonlimiting examples of polymers include such as a polyalkylene glycol (PAG) (e.g., polyethylene glycol (PEG) or polypropylene glycol (PPG), poly-L-lysine (PLL), poly (DL-lactic acid) (PLA), poly (DL-lactide-co-glycoside) (PLGA), polyomithine, polyarginine, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), peptides, peptoids, etc. In some embodiments, the polymer comprises (PEG). In some embodiments, the polymer comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, the polymer comprises deoxyribonucleic acid (DNA). In some embodiments, the polymer comprises ribonucleic acid (RNA).

[0359] In some embodiments, the linker comprises a moiety that is derived (e.g., resulting from the reaction of the click chemistry moiety with a complementary click chemistry moiety) from a click chemistry moiety. In some embodiments, the linker comprises a moiety that is derived from bicyclononyne (BCN), an alkyne, dibenzocyclooctyne (DBCO), tetrazine, or transcyclooctene (TCO). In some embodiments, the linker comprises a moiety that is derived from BCN. In some embodiments, the linker comprises a moiety that is derived from an alkyne. In some embodiments, the linker comprises a moiety that is derived from DBCO. In some embodiments, the linker comprises a moiety that is derived from TCO. In some embodiments, the linker is tetrazine.

[0360] In some embodiments, the linker comprises a moiety that is derived from a structure of Formula (X):Formula (X)whereinR6is coupling moiety;R7is Ci-Ce alkylene or Ci-Ce heteroalkylene optionally substituted with oxo or absent;WSGR Docket No. 60652-721601X is CH or N; andeach m is independently an integer from 1 to 5.

[0361] In some embodiments, R6is a coupling moiety. In some embodiments, the coupling moiety is N-hydroxysuccinimidyl ester, tetrafluorophenyl ester, maleimide, phoshoramidite, or hydrazide. In some embodiments, the coupling moiety is N-hydroxysuccinimidyl ester. In some embodiments, the coupling moiety is tetrafluorophenyl ester. In some embodiments, the coupling moiety is maleimide. In some embodiments, the coupling moiety is phosphoramidite. In some embodiments, the coupling moiety is hydrazide.

[0362] In some embodiments, R7is Ci-Ce alkylene or Ci-Ce heteroalkylene optionally substituted with oxo or absent.

[0363] In some embodiments, R7is Ci-Ce alkylene optionally substituted with oxo, or absent. In some embodiments, R7is Ci-Ce alkylene. In some embodiments, R7is Ci-Ce alkylene substituted with oxo. In some embodiments, R7is absent.

[0364] In some embodiments, R7is Ci-Ce heteroalkylene optionally substituted with oxo, or absent. In some embodiments, R7is Ci-Ce heteroalkylene. In some embodiments, R7is Ci-Ce heteroalkylene substituted with oxo. In some embodiments, R7is absent.

[0365] In some embodiments, X is CH or N. In some embodiments, X is CH. In some embodiments, X is N.

[0366] In some embodiments, each m is independently an integer from 1 to 5. In some embodiments, each m is independently 1, 2, 3, 4, or 5. In some embodiments, one m is 1 and the other is 2. In some embodiments, both m are 2. In some embodiments, both m are 1.

[0367] In some embodiments, the compound of Formula (X) is represented by the structure of Formula (X-A):Formula (X-A)whereinR7is Ci-Ce alkylene optionally substituted with oxo or absent;X is CH or N;p is an integer from 0 to 6; andWSGR Docket No. 60652-721601each m is independently an integer from 1 to 5.

[0368] In some embodiments, R7is Ci-Ce alkylene optionally substituted with oxo, or absent. In some embodiments, R7is Ci-Ce alkylene. In some embodiments, R7is Ci-Ce alkylene substituted with oxo. In some embodiments, R7is absent.

[0369] In some embodiments, X is CH or N. In some embodiments, X is CH. In some embodiments, X is N.

[0370] In some embodiments, p is an integer from 0 to 6. In some embodiments, p is an integer from 0 to 5. In some embodiments, p is an integer from 0 to 4. In some embodiments, p is an integer from 1 to 5. In some embodiments, p is an integer from 1 to 4. In some embodiments, p is an integer from 1 to 6. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6. In some embodiments, p is 0.

[0371] In some embodiments, each m is independently an integer from 1 to 5. In some embodiments, each m is independently 1, 2, 3, 4, or 5. In some embodiments, one m is 1 and the other is 2. In some embodiments, both m are 2. In some embodiments, both m are 1.

[0372] In some embodiments, the compound of Formula (X-A) is represented by thestructureof. In some embodiments, the compound of Formula (X-A) is P Q.represented by the structureof °

[0373] In some embodiments, the linker is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted heteroalkenylene, substituted or unsubstituted heteroalkynylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. In some embodiments, the linker is a bond, substituted or unsubstituted C1-C12 alkylene, substituted or unsubstituted C1-C12 alkenylene, substituted or unsubstituted C1-C12 alkynylene, substituted or unsubstituted C1-C12 heteroalkylene, substituted or unsubstituted C1-C12 heteroalkenylene, substituted or unsubstituted C1-C12 heteroalkynylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. In someWSGR Docket No. 60652-721601embodiments, the linker is substituted or unsubstituted C1-C12 alkylene, substituted or unsubstituted C1-C12 alkenylene, substituted or unsubstituted C1-C12 alkynylene, substituted or unsubstituted C1-C12 heteroalkylene, substituted or unsubstituted C1-C12 heteroalkenylene, substituted or unsubstituted C1-C12 heteroalkynylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, each substituted with one or more electron-withdrawing or electron-donating groups. In some embodiments, each electron- withdrawing group is independently selected from halogen, haloalkyl, NO2, sulfonate, amino, alkylamino, CN, or a carbonyl. In some embodiments, each electron-donating group is independently selected from alkyl, aryl, alkoxy, aryloxy, hydroxy, amino, alkylamino, thiol, or thioether group. In some embodiments, the linker is a bond, substituted or unsubstituted C1-C12 alkylene, or substituted or unsubstituted C1-C12 heteroalkylene. In some embodiments, the linker is a bond. In some embodiments, the linker is substituted or unsubstituted C1-C12 alkylene. In some embodiments, the linker is substituted or unsubstituted C1-C12 heteroalkylene. In some embodiments, the linker is substituted or unsubstituted C1-C12 alkenylene. In some embodiments, the linker is substituted or unsubstituted C1-C12 alkynylene. In some embodiments, the linker is substituted or unsubstituted C1-C12 heteroalkenylene. In some embodiments, the linker is substituted or unsubstituted C1-C12 heteroalkynylene. In some embodiments, the linker is substituted or unsubstituted arylene. In some embodiments, the linker is substituted or unsubstituted heteroarylene. In some embodiments, the C1-C12 heteroalkylene, C1-C12 heteroalkenylene, C1-C12 heteroalkynylene are substituted with one or more of oxo, fused cycloalkyl, or fused heterocycloalkyl. In some embodiments, the fused cycloalkyl or fused heterocycloalkyl are further substituted with one or more Ci-Ce alkyl (e.g., which is further substituted with the reactive group provided elsewhere herein).

[0374] In some embodiments, the linker is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, the linker is substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C1-C12 alkenyl, substituted or unsubstituted C1-C12 alkynyl, substituted or unsubstituted C1-C12 heteroalkyl, substituted or unsubstituted C1-C12 heteroalkenyl, substituted or unsubstituted C1-C12 heteroalkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, the linker is substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C1-C12 alkenyl, substituted or unsubstituted C1-C12 alkynyl, substituted or un substituted C1-C12 heteroalkyl, substituted or unsubstituted C1-C12WSGR Docket No. 60652-721601heteroalkenyl, substituted or unsubstituted C1-C12 heteroalkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, each substituted with one or more electron-withdrawing or electron-donating groups. In some embodiments, each electron- withdrawing group is independently selected from halogen, haloalkyl, NO2, sulfonate, amino, alkylamino, CN, or a carbonyl. In some embodiments, each electron-donating group is independently selected from alkyl, aryl, alkoxy, aryloxy, hydroxy, amino, alkylamino, thiol, or thioether group. In some embodiments, the linker is substituted or unsubstituted C1-C12 alkyl, or substituted or unsubstituted C1-C12 heteroalkyl. In some embodiments, the linker is substituted or unsubstituted C1-C12 alkyl. In some embodiments, the linker is substituted or unsubstituted C1-C12 heteroalkyl. In some embodiments, the linker is substituted or unsubstituted C1-C12 alkenyl. In some embodiments, the linker is substituted or unsubstituted C1-C12 alkynyl. In some embodiments, the linker is substituted or unsubstituted C1-C12 heteroalkenyl. In some embodiments, the linker is substituted or unsubstituted C1-C12 heteroalkynyl. In some embodiments, the linker is substituted or unsubstituted aryl. In some embodiments, the linker is substituted or unsubstituted heteroaryl. In some embodiments, the C1-C12 heteroalkyl, C1-C12 heteroalkenyl, C1-C12 heteroalkynyl are substituted with one or more of oxo, fused cycloalkyl, or fused heterocycloalkyl. In some embodiments, the fused cycloalkyl or fused heterocycloalkyl are further substituted with one or more Ci-Ce alkyl (e.g., which is further substituted with the reactive group provided elsewhere herein).

[0375] In some embodiments, the linker is a bond.

[0376] In some embodiments, the linker comprises alkylene, alkenylene, or alkynylene, each optionally substituted with one or more electron- withdrawing or electron-donating groups. In some embodiments, the linker comprises heteroalkylene, heteroalkenylene, heteroalkynylene, each optionally substituted with one or more electron-withdrawing or electron-donating groups. In some embodiments, the linker comprises a structure of-(Ci-Ce alkyl)-(NH(C=O))-, -(Ci-Ce alkyl)-O-(NH(C=O))-(Ci-C6alkyl)-, or-(Ci-C6alkyl)-(NH(C=O))-(Ci-C6alkyl)-. In some embodiments, the linker comprises arylene or heteroarylene, each optionally substituted with one or more electron-withdrawing or electron-donating groups. In some embodiments, the linker comprises one or more structures selected from pyridinediyl, pyrazinediyl, pyrimidinediyl, pyridazinediyl, quinolinediyl, isoquinolinediyl, benzo[h]quinolinediyl, and phenanthridinediyl. In some embodiments, the linker has a structure of-(Ci-Ce alkyl)-(2,5-pyridindiyl)-. In some embodiments, the linker has a structure of-(CH2CH2)-(2,5-pyridindiyl)-. In some embodiments, the 2,5-pyridindiyl in the structure of-(CH2CH2)-(2,5-pyridindiyl)- is substituted with a dimethylamino group. In some embodiments, the linker has a structure of-(Ci-Ce alkyl)-WSGR Docket No. 60652-721601(NH(C=O))- and a structure of-(Ci-Ce alkyl)-(2,5-pyridindiyl)-. In some embodiments, the linker comprises one or more structures selected from imidazolyl, triazolyl, tetrazolyl, indolyl, and carbazolyl. In some embodiments, the linker comprises one or more structures selected from furanyl, benzofuranyl, dibenzofuranyl, chromenyldiyl, xanthenyl, thiophenyl, benzothiophenyl, dibenzothiophenyl, and thioxanthyl.

[0377] In some embodiments, the linker is -(Ci-Ce alkyl)-(NH(C=O))- or -(Ci-Ce alkyl)-(NH(C=O))-(Ci-Ce alkyl)-. In some embodiments, the linker is -(Ci-Ce alkyl)-(NH(C=O))-. In some embodiments, the linker is -(Ci-Ce alkyl)-(NH(C=O))-(Ci-Ce alkyl)-. In some embodiments, the linker is stable throughout the entire Edman degradation process.

[0378] In some embodiments, the linker has a structure selected from Table 7.Table 7Linker StructureT7-1 bondT7-2a -alkylT7-2b -alkylene- T7-3a -alkenylT7-3b -alkenylene- T7-4a -alkynylT7-4b -alkynylene- T7-5a -arylT7-5b -arylene- T7-6a -heteroalkylT7-6b -heteroalkylene- T7-7a -heteroalkenylT7-7b -heteroalkenylene- T7-8a -heteroalkynylT7-8b -heteroalkynylene- T7-9a -heteroarylT7-9b -heteroarylene- T7-10 -(Ci-C6alkyl)-O- T7-11 -(Ci-C6alkyl)-(NH(C=O))- T7-12 -(Ci-C6alkyl)-O-(NH(C=O))-(Ci-C6alkyl)- T7-13 -(Ci-C6alkyl)-(NH(C=O))-(Ci-C6alkyl)-WSGR Docket No. 60652-721601T7-14a — (C i-Ce alkyl)-(2, 5 -pyridindiyl)- T7-14b -(CH2CH2)-(2, 5 -pyridindiyl)- T7-14c -(CH2CH2)-(2, 5 -pyridindiyl)T7-15a -(1,2,3 -tri azol- 1,4 -diyl)—T7-15b -(1,2,3-triazol-l-yl)T7-15c -(l,2,3-triazol-4-yl)T7-16 -(2,9-9H-fluorene)- T7-17 -(1,4-cubane)- T7-18T7-19A — \N J 'OH z z—T7-20, O,koJAJi k A\ _ < / 14VT7-21

[0379] In some embodiments, the nitrogenous base is adenine, thymine, cytosine, guanine, uracil, or a modified version thereof. In some embodiments, the polynucleotide composition further comprises an additional modified nucleotide comprising a catalytic group configured to facilitate proximity-based cleavage.

[0380] In any of the embodiments provided herein, the polynucleotide compositions may be sequencing reagents (or sequencing reagent compositions).

[0381] In some embodiments, the polynucleotide compositions comprise an additional modified nucleotide comprising a catalytic group. The catalytic group may be configured to facilitate proximity-based cleavage. The inclusion of an additional nucleotide comprising a catalytic group configured to facilitate proximity-based cleavage may be beneficial to increaseWSGR Docket No. 60652-721601the rate at which the sequencing described herein can take place. The catalytic group may be a 5-or 6-membered heterocycle or 5- or 6-membered heteroaryl (e.g., a nucleotide substituted with a 5- 6-membered heterocycle or 5- or 6-membered heteroaryl). In some embodiments, the catalytic group is an N-heterocycle or N-heteroaryl (e.g., a nucleotide substituted with N-heterocycle or N-heteroaryl). In some embodiments, the catalytic group is histidine or a histidine side chain. The catalytic group may promote proton exchange. In some embodiments, the catalytic group is comprised on the same polynucleotide as the modified polynucleotides described elsewhere herein (e.g., comprising substitution with -L-B). In some embodiments, when the catalytic group is comprised on the same nucleotide as the modified polynucleotides described elsewhere herein, the two modified polynucleotides are located with 10 nucleotides (e.g., within 8, 6, 5, 4, 3, or 2 nucleotides of each other). In other embodiments, the catalytic group is located on a complementary polynucleotide.Method of Using the Sequencing Reagent

[0382] Provided herein are methods of using any of the sequencing reagents or polynucleotide compositions provided herein. In some embodiments, the method comprises providing the sequencing reagent, wherein the sequencing reagent optionally comprises a catalytic group. In some embodiments, the method comprises contacting the sequencing reagent with a polymeric analyte. In some embodiments, the method comprises cleaving a monomer of the polymeric analyte, thereby providing a sequencing reagent-monomer complex. In some embodiments, the sequencing reagent comprises a structure selected from Tables 3C, 4C, 5C, and 6C.

[0383] In some embodiments, the method comprises providing the sequencing reagent (e.g., any of the polynucleotide compositions provided herein) and a polymeric analyte comprising a plurality of monomers.

[0384] In some embodiments, the sequencing reagent comprises a single-stranded nucleic acid molecule. In some embodiments, the sequencing reagent comprises a double-stranded nucleic acid molecule comprising a first strand comprising the modified nucleotide, and a second strand. In some embodiments, the first strand and the second strand are complementary.

[0385] In some embodiments, the sequencing reagent comprises a catalytic group. In some embodiments, the catalytic group is comprised in the first strand of the double-stranded nucleic acid molecule. In some embodiments, the catalytic group is comprised in the first strand of the double-stranded nucleic acid molecule. In some embodiments, the catalytic group is comprisedWSGR Docket No. 60652-721601in the second strand of the double-stranded nucleic acid molecule. In some embodiments, the catalytic group is configured to facilitate proximity-based cleavage.

[0386] In some embodiments, the polymeric analyte comprises a polypeptide.

[0387] In some embodiments, the method further comprises, prior to providing the sequencing reagent and a polymeric analyte comprising a plurality of monomers, converting a primary amine group or a protected primary amine group to an isothiocyanate, thereby generating the sequencing reagent.

[0388] In some embodiments, the method comprises contacting the sequencing reagent with a monomer of the plurality of monomers, thereby generating a sequencing reagent-monomer complex. In some embodiments, the monomer is an N-terminal amino acid.

[0389] In some embodiments, the method further comprises coupling the sequencing reagent or the sequencing reagent-monomer complex to a capture moiety. In some embodiments, the coupling of the sequencing reagent or sequencing reagent-monomer complex to the capture moiety comprises ligation.

[0390] In some embodiments, the method comprises cleaving a monomer of the polymeric analyte, thereby providing a modified monomer. In some embodiments, the cleaving is performed chemically. In some embodiments, the cleaving is performed chemically in the presence of a base. In some embodiments, the cleaving is performed enzymatically.

[0391] In some embodiments, the method comprises detecting the modified monomer. In some embodiments, the detecting comprises contacting the modified monomer complex with a binding agent. In some embodiments, the binding agent comprises an antibody, nanobody, single chain variable fragment (scFv), or aptamer. In some embodiments, the binding agent comprises a polymerizable molecule. In some embodiments, the polymerizable molecule comprises a nucleic acid molecule. In some embodiments, the detecting is performed using a nanopore sequencer.

[0392] In some embodiments, the method further comprises contacting the polymeric analyte with an additional sequencing reagent, wherein the additional sequencing reagent binds to an additional monomer of the plurality ...

Claims

WSGR Docket No. 60652-721601CLAIMS WHAT IS CLAIMED IS:

1. A composition comprising a polymer substituted with -L-B, wherein:L is a linker, wherein the linker comprises one or more electron-withdrawing or electrondonating groups, andB is a reactive group configured to react with an N-terminus of a peptide.

2. The composition of claim 1, wherein the polymer is a polynucleotide, a polypeptide, a polymeric material, or a combination thereof.

3. The composition of claim 2, wherein the composition is a polynucleotide composition comprising a modified nucleotide substituted with -L-B.

4. A polynucleotide composition comprising a modified nucleotide of Formula (IA):X?°-P-°^ n L^B0\ / OO=p-O‘I°yFormula (I A)wherein,L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups;B is a reactive group configured to react with an N-terminus of a peptide.

5. A polynucleotide composition comprising a modified nucleotide of Formula (IIA):Formula (IIA)wherein,WSGR Docket No. 60652-721601L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups;Ring A is a nitrogenous base or modified version thereof; andB is a reactive group configured to react with an N-terminus of a peptide.

6. A polynucleotide composition, wherein the polynucleotide composition comprises a modified nucleotide of Formula (IB), (IB-a), (IB-b), (IB-c), (IB-d), (IIB), or (IIB-a):Formula (IB) Formula (IB-a) Formula (IB-b)b-p=o°^xea0O=P-O— L-B0Formula (IB-c)O0=P— L-BFormula (IB-d) Formula (IIB)QrFormula (IIB-a)wherein,WSGR Docket No. 60652-721601L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups;B is a reactive group configured to react with an N-terminus of a peptide;Xais O, CH(L-B), or CH2;Xbis O, CH(L-B), or CH2;Xcis absent, CH(L-B), or CH2;Xdis absent, H, O, OH, halogen, CH(L-B), or OMe;Xeis absent, O, CH(L-B), or CH2.

7. A polynucleotide composition, wherein the polynucleotide composition comprises a modified nucleotide of Formula (IVA-A), (IVA-B), (IVA-C), (IVA-D), or (IVA-E):°=P~O— L-B O=p — L-BFormula (IVA- A) Formula (IVA-B) Formula (IVA-C)Formula (I VA-D) Formula (IVA-E)wherein,L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups; andB is a reactive group configured to react with an N-terminus of a peptide;L1and L2are each individually substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene,WSGR Docket No. 60652-721601substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, or a bond; andL3and L4are each individually substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or H.

8. A polynucleotide composition, wherein the polynucleotide composition comprises a modified nucleotide of Formula (IVB-A), (IVB-B), or (IVB-C):Formula (I VB- A) Formula (IVB-B) Formula (IVB-C)wherein,L is a linker, wherein the linker comprises one or more electron- withdrawing or electron-donating groups;B is a reactive group configured to react with an N-terminus of a peptide; andis cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, each optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2.

9. The polynucleotide composition of any one of claims 1-8, wherein B is or comprises isothiocyanate (ITC).

10. The polynucleotide composition of claim 9, wherein the isothiocyanate is formed by conversion of an amine or a protected amine group.

11. The polynucleotide composition of any one of claims 1-8, wherein B is or comprises the structure:WSGR Docket No. 60652-721601N=C=S12. The polynucleotide composition of any one of the preceding claims, wherein B is or comprises phenylisothiocyanate (PITC).

13. The polynucleotide composition of any one of claims 1-8 wherein B is or comprises - NRX(C=S)-LG;wherein,Rxis independently H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2, wherein each R” is independently H or Ci- C3 alkyl; andwherein LG is a leaving group.

14. The polynucleotide composition of claim 13, wherein B is or comprises -NH(C=S)-aryl or - NH(C=S)-heteroaryl.

15. The polynucleotide composition of claim 13, wherein the -NRX(C=S)-LG is -N(CH3)(C=S)- imidazole or -N(cyclopentyl)(C=S)-imidazole.

16. The polynucleotide composition of claim 13 or claim 15, wherein the nitrogen atom of the - NRX(C=S)-LG group is a member of the heterocycloalkyl or the heteroaryl.

17. The polynucleotide composition of claim 13, wherein Rxis five- or six-membered aryl or heteroaryl.

18. The polynucleotide composition of any one of claims 13 or 15-17, wherein Rxis configured to facilitate an interaction with a peptide.

19. The polynucleotide composition of any one of the preceding claims, further comprising a derivative of ethylenediaminetetraacetic acid (EDTA), a dendritic polymer, or any combination thereof.

20. The polynucleotide composition of any one of the preceding claims, further comprising one or more abasic nucleotides.WSGR Docket No. 60652-72160121. The polynucleotide composition of claim 20, wherein at least one of the one or more abasic nucleotides is immediately adjacent to the modified nucleotide.

22. The polynucleotide composition of any one of the preceding claims, wherein B has a structure selected from Table 3A.

23. The polynucleotide composition of any one of the preceding claims, wherein -L-B has a structure selected from Table 3B.

24. The polynucleotide composition of any one of the preceding claims, wherein the modified nucleotide has a structure selected from Table 3C.

25. The polynucleotide composition of any one of claims 1-8, wherein the -L-B is or comprises the structure of Formula (V):LGR!I 'R2LyFormula (V),wherein:LG is a leaving group;R1and R2are each independently hydrogen, 5- or 6- membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6- membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl, wherein the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

26. The polynucleotide composition of any one of claims 1-8, wherein the -L-B is or comprises the structure of Formula (VI):WSGR Docket No. 60652-721601LGFormula (VI)wherein:LG is a leaving group;R1is hydrogen, 5- or 6-membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl, wherein the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and C0N(R”)2, wherein each R” is independently H or C1-C3 alkyl; andn is an integer from 0 to 2.

27. The polynucleotide composition of any one of claims 1-8, wherein the -L-B is or comprises the structure of any one of Formula (VII-A), (VII-B), or (VILC):LG LGFormula (VII-A) Formula (VII-B) Formula (VILC)wherein,LG is a leaving group;L3 and L4 are each individually linkers; or— represents L3 and L4 being taken together to form Cs-Cs heterocycle;andR1is hydrogen, or 5- or 6-membered aryl, 5 or 6-membered heteroaryl, 5 or 6-membered cycloalkyl, 5 or 6-membered heterocycloalkyl, Ci-WSGR Docket No. 60652-721601Ce alkyl, Ci-Ce alkoxy, Ci-Ce haloalkyl, or Ci-Ce heteroalkyl, each optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl, wherein the phenyl, 5 or 6-membered heteroaryl, and 5 or 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, N02, CN, COOR”, and C0N(R”)2, wherein each R” is independently H or C1-C3 alkyl.

28. The polynucleotide composition of any one of claims 25-27, wherein the leaving group is: / YI-Y2X3-X4 x3-x4 / / w n / V3X2, X5x2X5-Y4Xi XIwherein,each of X1-X5 is independently selected from N, CH, or C-EWG; each of Y1-Y4 is independently selected from N, CH, or C-EWG; and EWG is an electron-withdrawing group;wherein at least one of X1-X5 is N.

29. The polynucleotide composition of any one of claims 25-28, wherein the modified polynucleotide has the structure selected from Table 4C.

30. The polynucleotide composition of any one of claims 1-8, wherein the -L-B is or comprises the structure of Formula (VIII):SFormula (VIII)wherein,R3is hydrogen or R’, wherein R’ is 5 or 6-membered aryl, 5 or 6- membered heteroaryl, or Ci-Ce alkyl optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6- membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one orWSGR Docket No. 60652-721601two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

31. The polynucleotide composition of any one of claims 1-8, wherein the -L-B is or comprises the structure of Formula (IX):SFormula (IX)wherein,R4is Ci-Ce alkylene, 5- or 6-membered cycloalkylene, or fused polycyloalkylene;R5is hydrogen or R’, wherein R’ is 5 or 6-membered aryl, 5 or 6- membered heteroaryl, or Ci-Ce alkyl optionally substituted with one or more members selected from oxo, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, phenyl, 5-membered heteroaryl, and 6- membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from oxo, halo, OH, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, NO2, CN, COOR”, and CON(R”)2, wherein each R” is independently H or C1-C3 alkyl.

32. The polynucleotide composition of any one of claims 30-31, wherein the modified polynucleotide has a structure selected from Table 5C.

33. The polynucleotide composition of any one of claims 1-8, wherein the -L-B is or comprises the structure of Formula (XI):Formula (XI),wherein,X6is O, S, Se, or NR10;WSGR Docket No. 60652-721601R8is a bond, alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, or heteroarylene, each optionally substituted with one or more electron-donating or electron-withdrawing groups;R9is a protecting group configured for cleavage from said Formula (XI); andR10is independently selected from a group comprising hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, aryl, or heteroaryl, each optionally substituted with one or more electron-donating or electron-withdrawing groups.

34. The polynucleotide composition of any one of claims 1-8, wherein the -L-B is or comprises the structure of Formula (XII):Formula (XII),wherein,X7is O, S, SO, SR14, Se, SeO, SeR14, or NR14;X8is O, S, SR14, SOR14, SO2R14, Se, SeR14, SeOR14, SeO2R14or NR14; X9is O, S, Se, or NR14;R11is independently selected from a group comprising hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with one or more electron-donating or electron-withdrawing groups;R12is a protecting group configured for cleavage from said Formula (XII);R13is a bond, alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, arylene, or heteroarylene, each optionally substituted with one or more electron-donating or electron-withdrawing groups; andWSGR Docket No. 60652-721601R14is independently selected from a group comprising hydrogen, alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, aryl, or heteroaryl, each optionally substituted with one or more electron-donating or electron-withdrawing groups.

35. The polynucleotide composition of any one of claims 33-34, wherein the modified polynucleotide has a structure selected from Table 6C.

36. The polynucleotide composition of any one of the preceding claims, wherein the linker comprises a moiety that is derived from a click chemistry moiety.

37. The polynucleotide composition of any one of the preceding claims, wherein the linker comprises a moiety that is derived from bicyclononyne (BCN), an alkyne, an azide, dibenzocyclooctyne (DBCO), tetrazine, trans-cyclooctene (TCO), or a structure of Formula (X):Formula (X)whereinR6is coupling moiety;R7is Ci-Ce alkylene or Ci-Ce heteroalkylene optionally substituted with oxo or absent;X is CH or N; andeach m is independently an integer from 1 to 5.

38. The polynucleotide composition of claim 37, wherein the compound of Formula (X) has the structure of Formula (X-A):Formula (X-A)whereinWSGR Docket No. 60652-721601R7is Ci-Ce alkylene optionally substituted with oxo or absent;X is CH or N;p is an integer from 0 to 6; andeach m is independently an integer from 1 to 5.

39. The polynucleotide composition of claim 38, wherein the compound of Formula (X-A) is selected from:

40. The polynucleotide composition of any one of the preceding claims, wherein the linker comprises a bond, alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, arylene, or heteroarylene, each optionally substituted with one or more electron-withdrawing or electron-donating groups.

41. The polynucleotide composition of any one of the preceding claims, wherein the linker comprises one or more structures selected from Table 7.

42. The polynucleotide composition of any one of the preceding claims, wherein the polynucleotide composition is a sequencing reagent.

43. A method of using the sequencing reagent of claim 42, the method comprising:(a) providing the sequencing reagent and a polymeric analyte comprising a plurality of monomers, wherein the sequencing reagent optionally comprises a catalytic group;(b) contacting the sequencing reagent with a monomer of the plurality of monomers, thereby generating a sequencing reagent-monomer complex;(c) cleaving a monomer of the polymeric analyte, thereby providing a modified monomer; and(d) detecting the modified monomer.WSGR Docket No. 60652-72160144. The method of claim 43, further comprising, prior to (a), converting a primary amine group or a protected primary amine group to an isothiocyanate, thereby generating the sequencing reagent.

45. The method of any one of claims 43-44, wherein the polymeric analyte comprises a polypeptide, and wherein the monomer is an N-terminal amino acid.

46. The method of claim 43, wherein the detecting of (d) comprises contacting the modified monomer complex with a binding agent.

47. The method of claim 46, wherein the binding agent comprises a polymerizable molecule, and wherein the polymerizable molecule comprises a nucleic acid molecule.

48. The method of any one of claims 43-47, further comprising coupling the sequencing reagent or the sequencing reagent-monomer complex to a capture moiety.

49. The method of any one of claims 43-48, wherein the detecting is performed using a nanopore sequencer.

50. The method of any one of claims 43-49, further comprising:(h) contacting the polymeric analyte with an additional sequencing reagent, wherein the additional sequencing reagent binds to an additional monomer of the plurality of monomers; wherein binding of the additional sequencing reagent to the additional monomer of the polymeric analyte provides an additional sequencing reagent-monomer complex;(i) coupling the additional sequencing reagent to the modified monomer; and(j) cleaving the additional sequencing reagent-monomer complex from the polymeric analyte, thereby providing a stacked sequencing reagent-monomer complex.

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