Oxazine dyes and their use in nucleic acid amplification reactions - Patents.com

Abstract

Disclosed herein are functionalized oxazine dye compounds, compositions containing the compounds, and methods of using the compounds (e.g., in nucleic acid amplification reactions). Also disclosed herein are labeled oligonucleotides and labeled nucleotide triphosphate compounds.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 310,796, filed February 16, 2022, which is incorporated by reference in its entirety.

[0002] Disclosed herein are functionalized oxazine dye compounds, compositions containing the compounds, and methods of using the compounds (e.g., in nucleic acid amplification reactions). Also disclosed herein are labeled oligonucleotides and labeled nucleotide triphosphate compounds. [Background technology]

[0003] Fluorescent dyes are widely used in biological research and medical diagnostics. The availability of a wide variety of fluorescent dyes with a range of distinguishable colors makes it more practical to perform multiplex assays that can simultaneously detect multiple biological targets. For certain applications, such as those involving the polymerase chain reaction (PCR), the dyes must be compatible with the reaction conditions, including the high temperatures used in the denaturation step. There is a need for PCR-compatible dyes, especially those with longer emission wavelengths. Summary of the Invention

[0004] In one aspect, provided herein is a compound of formula (I): [ka] or a tautomer or a salt thereof, wherein: A is a 5-, 6-, or 7-membered heterocyclyl; R 1 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 1 -X, where L 1is alkylene or heteroalkylene, and X is selected from -COOH, -SOH, -POH, -OPOH, and a reactive moiety, or R 1 is R 6 and together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 2 , R 3 , and R 4 is defined as follows: (i)R 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a reactive moiety; or (ii)R 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a reactive moiety; or (iii)R 2 is C1-C4 alkyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl; q is 0, 1, 2, or 3; Each R 5 is independently selected from C1 to C4 alkyl; R 6 is hydrogen or R 6 is R 1 and together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; Where: (a)R 1 -L 1 -X, where X is a reactive moiety selected from an active ester, -N3, -C≡CH, -N=C=O, -N=C=S, maleimide, -C(O)-CH=CH2, optionally substituted 1,2,4,5-tetrazinyl, cycloalkenyl, and cycloalkynyl; or (b)R 4 -L 2 -Z, where Z is a reactive moiety selected from active ester, -N3, -C≡CH, -N=C=O, -N=C=S, maleimide, -C(O)-CH=CH2, optionally substituted 1,2,4,5-tetrazinyl, cycloalkenyl, and cycloalkynyl; The compound does not have two or more reactive moieties; The compound is as follows: [ka] isn't it.

[0005] In some embodiments, A is selected from a pyrrolidine ring, a piperidine ring, and a morpholine ring.

[0006] In some embodiments, R 1 is hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl. 1 -L 1 In some embodiments, L 1 is a C2-C4 alkylene. In some embodiments, X is selected from -COOH, -SO3H, -PO3H2, and a reactive moiety, where the reactive moiety is selected from an active ester, -N3, -C≡CH, -N=C=O, -N=C=S, maleimide, -C(O)-CH=CH2, tetrazinyl, cycloalkenyl, and cycloalkynyl.

[0007] In some embodiments, R 2 is C1-C4 alkyl, R3 is hydrogen and R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a reactive moiety.

[0008] In some embodiments, R 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a reactive moiety.

[0009] In some embodiments, R 4 is hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl. 4 -L 2 In some embodiments, L 2 is a C2-C4 alkylene. In some embodiments, Z is selected from -COOH, -SO3H, and a reactive moiety, where the reactive moiety is selected from an active ester, -N3, -C≡CH, -N=C=O, -N=C=S, maleimide, -C(O)-CH=CH2, optionally substituted 1,2,4,5-tetrazinyl, cycloalkenyl, and cycloalkynyl.

[0010] In some embodiments, R 2 is C1-C4 alkyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl.

[0011] In some embodiments, q is 0.

[0012] In some embodiments, R 6 is hydrogen.

[0013] In some embodiments, A is a pyrrolidine ring; R 1 -L 1 -X, where L 1 is a C2-C4 alkylene and X is an N-succinimidyl ester reactive moiety; R 2 and R 3 together with the atoms to which they are attached form a 5-membered heterocyclyl having one nitrogen atom, R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is a C2-C4 alkylene, Z is -SO3H, q is 0, R 6 is hydrogen.

[0014] In some embodiments, A is a morpholine ring; R 1 -L 1 -X, where L 1 is a C2-C4 alkylene and X is an N-succinimidyl ester reactive moiety; R 2 is C1-C4 alkyl, R 3 is hydrogen or R 2 and R 3 form together with the atoms to which they are attached a six-membered ring containing one nitrogen atom and one oxygen atom, R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is a C2-C4 alkylene, Z is -SO3H, q is 0, R 6 is hydrogen.

[0015] In some embodiments, the compound is: [ka] [ka] or a tautomer thereof, or a salt thereof.

[0016] In another aspect, provided herein is a compound of formula (Ia): [ka] or a tautomer or a salt thereof, wherein A is a 5-, 6-, or 7-membered heterocyclyl; R 1 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 1 -X, where L 1 is alkylene or heteroalkylene, and X is selected from -COOH, -SOH, -POH, -OPOH, and a point of attachment to the oligonucleotide, or R 1 is R 6 and together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 2 , R 3 , and R 4 is defined as follows: (i)R 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a point of attachment to the oligonucleotide; or (ii)R 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a point of attachment to the oligonucleotide; or (iii)R 2 is C1-C4 alkyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl; q is 0, 1, 2, or 3; Each R 5 is independently selected from C1 to C4 alkyl; R 6 is hydrogen or R 6 is R 1 and together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; Where: (a)R 1 -L 1 -X, where X is the point of attachment to the oligonucleotide, or (b)R 4 -L 2 -Z, where Z is the point of attachment to the oligonucleotide; The moiety of formula (Ia) does not have more than one point of attachment to the oligonucleotide.

[0017] In some embodiments, A is selected from a pyrrolidine ring, a piperidine ring, and a morpholine ring.

[0018] In some embodiments, R1 is hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl. 1 -L 1 In some embodiments, L 1 is a C2-C4 alkylene. In some embodiments, X is selected from -COOH, -SO3H, -PO3H2, and a point of attachment to the oligonucleotide.

[0019] In some embodiments, R 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a point of attachment to the oligonucleotide.

[0020] In some embodiments, R 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a point of attachment to the oligonucleotide.

[0021] In some embodiments, R 4 is hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl. 4 -L 2 In some embodiments, L 2is a C2-C4 alkylene. In some embodiments, Z is selected from -COOH, -SO3H, -PO3H2, and a point of attachment to the oligonucleotide.

[0022] In some embodiments, R 2 is C1-C4 alkyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl.

[0023] In some embodiments, q is 0.

[0024] In some embodiments, R 6 is hydrogen.

[0025] In some embodiments, A is a pyrrolidine ring and R 1 -L 1 -X, where L 1 is a C2-C4 alkylene, X is the point of attachment to the oligonucleotide, and R 2 and R 3 together with the atoms to which they are attached form a 5-membered heterocyclyl having one nitrogen atom, R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is a C2-C4 alkylene, Z is -SO3H, q is 0, and R 6 is hydrogen.

[0026] In some embodiments, A is a morpholine ring and R 1 -L 1 -X, where L 1 is a C2-C4 alkylene, X is the point of attachment to the oligonucleotide, and R 2 is C1-C4 alkyl, R 3 is hydrogen or R 2 and R 3form, together with the atoms to which they are attached, a six-membered ring having one nitrogen atom and one oxygen atom, R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is a C2-C4 alkylene, Z is -SO3H, q is 0, and R 6 is hydrogen.

[0027] In some embodiments, the oligonucleotide has the formula: [ka] or a tautomer or a salt thereof, where [ka] represents the point of attachment of the moiety to the oligonucleotide.

[0028] In some embodiments, the moiety of formula (Ia) is bound to oligonucleotide by direct bond.In some embodiments, the moiety of formula (Ia) is bound to oligonucleotide via linker.In some embodiments, the moiety of formula (Ia) is bound to oligonucleotide via linker comprising amide moiety, carbamate moiety, 5-membered heteroaryl ring, alkylene moiety, fused bicyclic heterocycle, or any combination thereof.

[0029] In some embodiments, the oligonucleotide is about 5 to about 50 bases in length. In some embodiments, the oligonucleotide is about 15 to about 35 bases in length.

[0030] In another aspect, the present invention provides a compound of formula (Ib): [ka] or a tautomer or a salt thereof, wherein: A is a 5-, 6-, or 7-membered heterocyclyl; R 1 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 1 -X, where L 1 is alkylene or heteroalkylene, and X is selected from -COOH, -SOH, -POH, -OPOH, and a point of attachment to a nucleotide triphosphate compound, or R 1 is R 6 and together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 2 , R 3 , and R 4 is defined as follows: (i)R 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a point of attachment to a nucleotide triphosphate compound; or (ii)R 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a point of attachment to a nucleotide triphosphate compound; or (iii)R 2 is C1-C4 alkyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl; q is 0, 1, 2, or 3; Each R 5 is independently selected from C1 to C4 alkyl; R 6 is hydrogen or R 6 is R 1 and together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocycle; Where: (a)R 1 -L 1 -X, where X is the point of attachment to a nucleotide triphosphate compound, or (b)R 4 -L 2 -Z, where Z is the point of attachment to the nucleotide triphosphate compound; The moiety of formula (Ib) does not have more than one point of attachment to a nucleotide triphosphate compound.

[0031] In some embodiments, A is selected from a pyrrolidine ring, a piperidine ring, and a morpholine ring.

[0032] In some embodiments, R 1 is hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl. 1 -L 1 In some embodiments, L 1 is a C2-C4 alkylene. In some embodiments, X is selected from -COOH, -SO3H, -PO3H2, and a point of attachment to a nucleotide triphosphate compound.

[0033] In some embodiments, R 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2is alkylene or heteroalkylene, and Z is selected from -COOH, -SOH, -POH, -OPOH, and a point of attachment to a nucleotide triphosphate compound. 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a point of attachment to the nucleotide triphosphate compound.

[0034] In some embodiments, R 4 is hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl. 4 -L 2 In some embodiments, L 2 is a C2-C4 alkylene. In some embodiments, Z is selected from -COOH, -SO3H, -PO3H2, and a point of attachment to a nucleotide triphosphate compound.

[0035] In some embodiments, R 2 is C1-C4 alkyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl.

[0036] In some embodiments, q is 0.

[0037] In some embodiments, R 6 is hydrogen.

[0038] In some embodiments, A is a pyrrolidine ring and R 1 -L 1 -X, where L1 is a C2-C4 alkylene, X is the point of attachment to the nucleotide triphosphate compound, and R 2 and R 3 together with the atoms to which they are attached form a 5-membered heterocyclyl having one nitrogen atom, R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is a C2-C4 alkylene, Z is -SO3H, q is 0, and R 6 is hydrogen.

[0039] In some embodiments, A is a morpholine ring and R 1 -L 1 -X, where L 1 is a C2-C4 alkylene, X is the point of attachment to the nucleotide triphosphate compound, and R 2 is C1-C4 alkyl, R 3 is hydrogen or R 2 and R 3 form, together with the atoms to which they are attached, a six-membered ring having one nitrogen atom and one oxygen atom, R 4 is C1-C6 alkyl and L 2 -Z, where L 2 is a C2-C4 alkylene, Z is -SO3H, q is 0, and R 6 is hydrogen.

[0040] In some embodiments, the nucleotide triphosphate compound has the formula: [ka] or a tautomer or a salt thereof, where [ka] represents the point of attachment of the moiety to the nucleotide triphosphate compound.

[0041] In some embodiments, the moiety of formula (Ib) is linked to the nucleotide triphosphate compound by direct bond.In some embodiments, the moiety of formula (Ib) is linked to the nucleotide triphosphate compound via a linker.In some embodiments, the moiety of formula (Ib) is linked to the nucleotide triphosphate compound via a linker comprising an amide moiety, a carbamate moiety, a 5-membered heteroaryl ring, an alkylene moiety, a fused bicyclic heterocycle, or any combination thereof.

[0042] In some embodiments, the compound is a modified deoxynucleotide triphosphate compound selected from deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate. In some embodiments, the compound is a modified deoxynucleotide triphosphate compound selected from dideoxyadenosine triphosphate, dideoxycytidine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate.

[0043] In another aspect, provided herein is a method for performing a nucleic acid amplification reaction, comprising: (a) adding an oligonucleotide compound disclosed herein (e.g., an oligonucleotide comprising a moiety of Formula (Ia)) to a reaction mixture; and (b) performing an amplification reaction; The method is disclosed, comprising:

[0044] In some embodiments, the nucleic acid amplification reaction is selected from the group consisting of: polymerase chain reaction (PCR), quantitative PCR, real-time PCR, hot start PCR, single cell PCR, nested PCR, in situ colony PCR, digital PCR (dPCR), Droplet Digital™ PCR (ddPCR), emulsion PCR, ligase chain reaction (LCR), transcription-based amplification system (TAS), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), and hyperbranched RCA (HRCA). In some embodiments, the nucleic acid amplification reaction is a multiplex nucleic acid amplification reaction.

[0045] In another aspect, provided herein is a method for performing a chain termination DNA sequencing reaction, comprising: (a) adding a modified dideoxynucleotide triphosphate compound disclosed herein (e.g., a modified dideoxynucleotide triphosphate compound comprising a moiety of formula (Ib)) to a polymerase chain reaction (PCR) mixture and performing PCR; (b) removing unincorporated modified dideoxynucleotide triphosphate compounds from the PCR mixture; and (c) performing sequence analysis; The method is disclosed, comprising:

[0046] In some embodiments, the sequence analysis includes fragment analysis and / or Sanger sequencing analysis.

[0047] In another aspect, provided herein is a method for performing a chain termination DNA sequencing reaction, comprising: (a) adding a modified deoxynucleotide triphosphate compound disclosed herein (e.g., a modified dideoxynucleotide triphosphate compound comprising a moiety of formula (Ib)) to a polymerase chain reaction (PCR) mixture; and (b) performing a PCR; Including, The method is disclosed in which a fluorescent signal from the PCR mixture indicates which dNTP has been added, and the terminator is cleaved to facilitate the addition of the subsequent dNTP.

[0048] In some embodiments, the method further comprises performing fragment analysis and / or next generation sequencing. In some embodiments, the method is multiplexed. [Brief description of the drawings]

[0049] [Figure 1] 1 shows an electropherogram of a sample amplified using oligonucleotide primers labeled with the dye compounds disclosed herein (JC-0025 and JC-0081). [Diagram 2] 1 shows electropherograms of samples amplified using oligonucleotide primers labeled with the dye compounds disclosed herein (CS-1341, CS-1377, and JC-0084). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Disclosed herein are oxazine dye compounds that are compatible with PCR reaction conditions. The dyes contain reactive moieties that can be used to label, for example, oligonucleotide primers and nucleotide triphosphate compounds (dNTPs). The dyes, or compounds labeled with the dyes (e.g., labeled oligonucleotide primers and labeled dNTPs), can be used in a variety of sequencing methods, including multiplex PCR assays.

[0051] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present specification, including definitions, will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0052] Definitions of certain functional groups and chemical terms are set forth in more detail below. For purposes of this disclosure, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. (endpapers), and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry, and specific functional moieties and reactivities are described in Sorrell, Organic Chemistry, 2001, pp. 111-115, 1997. nd edition,University Science Books,Sausalito,2006,Smith,March's Advanced Organic Chemistry:Reactions,Mechanism,and Structure,7 th Edition, John Wiley&Sons, Inc., New York, 2013, Larock, Comprehensive Organic Transformations, 3 rd Edition, John Wiley&Sons, Inc., New York, 2018, and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987, the entire contents of each of which are incorporated herein by reference.

[0053] As used herein, the term "alkyl" refers to a linear or branched saturated hydrocarbon chain. An alkyl group can be, for example, one to sixteen carbon atoms (C1-C 16 Alkyl), 1 to 14 carbon atoms (C1 to C 14 Alkyl), 1 to 12 carbon atoms (C1 to C 12 Alkyl), 1 to 10 carbon atoms (C1 to C 10 alkyl), 1 to 8 carbon atoms (C1 to C8 alkyl), 1 to 6 carbon atoms (C1 to C6 alkyl), 1 to 4 carbon atoms (C1 to C4 alkyl), 6 to 20 carbon atoms (C6 to C 20 alkyl), or 8 to 14 carbon atoms (C8 to C 14 Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0054] As used herein, the term "alkylene" refers to a divalent group derived from a straight or branched saturated hydrocarbon chain. Representative examples of alkylene include, but are not limited to, -CH-, -CHCH-, -CH(CH)-, -CHCHCH-, -CHCH(CH)-, -CHCHCHCH-, -CHCH(CH)-, -CHCHCHCH(CH)-, -CHCHCHCH(CH)-, -CHCHCHCHCH(CH)-, -CHCHCHCHCHCH-, -CHCH(CH)CHCH-, -CH(CH)CHCHCH-, -CHCHCHCHCHCHCH-, -CHCH(CH)CHCHCH-, -CHCH(CH)CHCHCH-, -CHCH(CH)CHCHCH-, -CHCH(CH)CHCHCH-, -CHCH(CH)CHCHCH-, and -CH(CH)CHCHCHCHCH-.

[0055] As used herein, the term "active ester" refers to an ester functional group that is susceptible to nucleophilic attack by functional groups such as, for example, amines or thiols. Examples include, but are not limited to, N-hydroxysuccinimidyl esters, N-hydroxysulfosuccinimidyl esters, pentafluorophenyl esters, and the like.

[0056] As used herein, the term "cycloalkyl" refers to a saturated cyclic carbocyclic system containing 3 to 10 carbon atoms and 0 heteroatoms. Cycloalkyls can be monocyclic, bicyclic, bridged, fused, or spirocyclic. Representative examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl.

[0057] As used herein, the term "cycloalkenyl" refers to a non-aromatic, monocyclic or polycyclic cyclic carbon system containing at least one carbon-carbon double bond. Cycloalkenyl can be a monocyclic cycloalkenyl (e.g., cyclopentenyl), a fused bicyclic cycloalkenyl (e.g., octahydronaphthalenyl), or a bridged cycloalkenyl (e.g., bicyclo[2.2.1]heptenyl) in which two non-adjacent atoms of the ring are connected by an alkylene bridge of 1, 2, 3, or 4 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0058] As used herein, the term "cycloalkynyl" refers to a non-aromatic, monocyclic or polycyclic carbon ring system containing at least one carbon-carbon triple bond. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like.

[0059] As used herein, the term "heteroalkyl" refers to an alkyl group, as defined herein, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatom group, such as -NH-, -O-, -S-, -S(O)-, -S(O)2-, and the like. By way of example, one, two, or three carbon atoms may be independently replaced with the same or different heteroatom groups. Examples of heteroalkyl groups include, but are not limited to, -OCH3, -CHOCH3, -SCH3, -CH2SCH3, -NHCH3, and -CH2NHCH3, in which R is hydrogen, alkyl, aryl, arylalkyl, heteroalkyl, or heteroaryl, each of which may be optionally substituted. Heteroalkyl also includes groups in which an alkyl carbon atom is oxidized (i.e., -C(O)-).

[0060] As used herein, the term "heteroalkylene" refers to an alkylene group, as defined herein, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatom group, such as -NH-, -O-, -S-, -S(O)-, -S(O)2-, and the like. By way of example, one, two, or three carbon atoms may be independently replaced with the same or different heteroatom groups. Heteroalkylene also includes groups in which the alkylene carbon atoms are oxidized (i.e., -C(O)-). Examples of heteroalkylene groups include, but are not limited to, -CH2-O-CH2-, -CH2-S-CH2-, -CH2-NH-CH2-, -CH2-NH-C(O)-CH2-, and the like, as well as polyethylene oxide chains, polypropylene oxide chains, and polyethyleneimine chains.

[0061] As used herein, the term "heterocycle" or "heterocyclic" refers to a saturated or partially unsaturated non-aromatic ring group having one or more ring heteroatoms independently selected from O, N, and S, meaning a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from O, N, and S. A 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from O, N, and S. A 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from O, N, and S. A 6-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from O, N, and S. The 7- and 8-membered rings contain 0, 1, 2, or 3 double bonds and 1, 2, or 3 heteroatoms selected from O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, pipera, These include thienyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl.A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocyclic group, or a bridged monocyclic heterocyclic ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclic heterocycles are exemplified by bicyclic heterocycles fused to a phenyl group, or bicyclic heterocycles fused to a monocyclic cycloalkyl, or bicyclic heterocycles fused to a monocyclic cycloalkenyl, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3,7 ]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 ]decane. The monocyclic, bicyclic, and tricyclic heterocycles are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring.

[0062] compound As used herein, a compound of formula (I): [ka] or a tautomer or a salt thereof, wherein: A is a 5-, 6-, or 7-membered heterocyclyl; R 1 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 1 -X, where L 1 is alkylene or heteroalkylene, and X is selected from -COOH, -SOH, -POH, -OPOH, and a reactive moiety, or R 1 is R 6 and together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 2 , R 3 , and R 4 is defined as follows: (i)R 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a reactive moiety; or (ii)R 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a reactive moiety; or (iii)R 2 is C1-C4 alkyl, R 3 and R 4together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl; q is 0, 1, 2, or 3; Each R 5 is independently selected from C1 to C4 alkyl; R 6 is hydrogen or R 6 is R 1 and together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; Where: (a)R 1 -L 1 -X, where X is a reactive moiety selected from an active ester, -N3, -C≡CH, -N=C=O, -N=C=S, maleimide, -C(O)-CH=CH2, optionally substituted 1,2,4,5-tetrazinyl, cycloalkenyl, and cycloalkynyl; or (b)R 4 -L 2 -Z, where Z is a reactive moiety selected from active ester, -N3, -C≡CH, -N=C=O, -N=C=S, maleimide, -C(O)-CH=CH2, optionally substituted 1,2,4,5-tetrazinyl, cycloalkenyl, and cycloalkynyl; The compound does not have two or more reactive moieties; The compound is as follows: [ka] isn't it.

[0063] In some embodiments, A is selected from a pyrrolidine ring, a piperidine ring, and a morpholine ring. In some embodiments, A is a morpholine ring. In some embodiments, A is a piperidine ring. In some embodiments, A is a pyrrolidine ring.

[0064] In some embodiments, R 1is hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl. 1 is hydrogen or C1-C4 alkyl. In some embodiments, R 1 is hydrogen or ethyl. In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is ethyl.

[0065] In some embodiments, R 1 -L 1 In some embodiments, L 1 is a C2-C4 alkylene, or L 1 is C2-C4 heteroalkylene. 1 is C2-C4 alkylene. 1 is -CH2CH2CH2-. In some embodiments, X is selected from -COOH, -SO3H, -PO3H2, and a reactive moiety, where the reactive moiety is selected from an active ester, -N3, -C≡CH, -N=C=O, -N=C=S, maleimide, -C(O)-CH=CH2, tetrazinyl, cycloalkenyl, and cycloalkynyl. In some embodiments, X is selected from -SO3H, -PO3H2, and an active ester. In some embodiments, X is -SO3H. In some embodiments, X is -PO3H2. In some embodiments, X is an active ester (such as a succinimidyl ester or a pentafluorophenyl ester). In some embodiments, X is a succinimidyl ester (or a salt thereof) that is unsubstituted or substituted with a -SO3H group. In some embodiments, X is the following: [ka] is selected from.

[0066] In some embodiments, R 2 is C1-C4 alkyl, R3 is hydrogen and R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SOH, -POH, -OPOH, and a reactive moiety. 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is alkylene or heteroalkylene and Z is -SOH. In some embodiments, R 2 is C1-C2 alkyl, R 3 is hydrogen and R 4 is C1-C2 alkyl or -(CH2) n -SO3H, where n is 3, 4, or 5. In some embodiments, R 2 is methyl, R 3 is hydrogen and R 4 is methyl, ethyl, or -(CH2)4-SO3H. In some embodiments, R 2 is methyl, R 3 is hydrogen and R 4 is ethyl. In some embodiments, R 2 is methyl, R 3 is hydrogen and R 4 is -(CH2)4-SO3H.

[0067] In some embodiments, R 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2is alkylene or heteroalkylene, and Z is selected from -COOH, -SOH, -POH, -OPOH, and a reactive moiety. 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is selected from hydrogen, C1-C6 alkyl, and C1-C6 heteroalkyl. 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is selected from hydrogen and C1-C2 alkyl. 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 -L 2 In some embodiments, L 2 is C2-C4 alkylene. 2 is -CH2CH2CH2-. In some embodiments, Z is selected from -COOH, -SO3H, and a reactive moiety, where the reactive moiety is selected from an active ester, -N3, -C≡CH, -N=C=O, -N=C=S, maleimide, -C(O)-CH=CH2, optionally substituted 1,2,4,5-tetrazinyl, cycloalkenyl, and cycloalkynyl. In some embodiments, Z is an active ester. In some embodiments, Z is a succinimidyl ester.

[0068] In some embodiments, R 2 and R 3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclyl, R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2is alkylene or heteroalkylene, and Z is selected from -COOH, -SOH, -POH, -OPOH, and a reactive moiety. 2 and R 3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclyl, R 4 is hydrogen and -L 2 -Z, where L 2 is a C2-C4 alkylene or C2-C4 heteroalkylene, and Z is a reactive moiety selected from an active ester, -N3, -C≡CH, -N=C=O, -N=C=S, maleimide, -C(O)-CH=CH2, tetrazinyl, cycloalkenyl, and cycloalkynyl. 2 and R 3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclyl, R 4 is hydrogen and -L 2 -Z, where L 2 is -CH2CH2CH2- and Z is a reactive moiety selected from an active ester (such as a succinimidyl ester). In some embodiments, the group in formula (I) [ka] is the following: [ka] having a structure selected from Here, R 4 is as defined and described above.

[0069] In some embodiments, R 2 is C1-C4 alkyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl. 2 is methyl, R 3 and R 4together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl.

[0070] In some embodiments, q is 0. In some embodiments, q is 1 and R 5 is methyl.

[0071] In some embodiments, R 6 is hydrogen.

[0072] In some embodiments, in the compound of formula (I), A is a pyrrolidine ring; R 1 -L 1 -X, where L 1 is a C2-C4 alkylene and X is an N-succinimidyl ester reactive moiety; R 2 and R 3 together with the atoms to which they are attached form a 5-membered heterocyclyl having one nitrogen atom, R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is a C2-C4 alkylene, Z is -SO3H, q is 0, R 6 is hydrogen.

[0073] In some embodiments, in the compound of formula (I), A is a morpholine ring; R 1 -L 1 -X, where L 1 is a C2-C4 alkylene and X is an N-succinimidyl ester reactive moiety; R 2 is C1-C4 alkyl, R 3 is hydrogen or R 2 and R 3form together with the atoms to which they are attached a six-membered ring containing one nitrogen atom and one oxygen atom, R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is a C2-C4 alkylene, Z is -SO3H, q is 0, R 6 is hydrogen.

[0074] In some embodiments, the compound is: [ka] [ka] and tautomers and salts thereof.

[0075] Further compounds include the following: [ka] [ka] [ka] [ka] Examples include:

[0076] Compounds and intermediates can be isolated and purified by methods well known to those skilled in the art of organic synthesis. Examples of conventional methods for isolating and purifying compounds include, but are not limited to, chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilane groups, optionally pretreated with activated carbon, recrystallization at high or low temperatures, thin layer chromatography, distillation at various pressures, sublimation under vacuum, and trituration, as described, for example, in "Vogel's Textbook of Practical Organic Chemistry" by Furniss, Hannaford, Smith, and Tatchell, 5th edition (1989), pub. Longman Scientific & Technical, Essex CM20 2JE, England.

[0077] The reaction conditions and reaction times for each of the individual steps may vary depending on the particular reactants employed and the substituents present in the reactants used. The reactants may be worked up in a conventional manner (e.g., by removing the solvent from the residue) and may be further purified according to methodologies commonly known in the art, such as, but not limited to, crystallization, distillation, extraction, trituration and chromatography. Unless otherwise stated, starting materials and reagents are either commercially available or may be prepared from commercially available materials by one skilled in the art using methods described in the chemical literature.

[0078] Standard experimentation, including appropriate manipulation of reaction conditions, reagents and sequence of the synthetic route, protection of any chemical functionality that may not be compatible with the reaction conditions, and deprotection at appropriate points in the reaction sequence of the method, is within the scope of this disclosure. Suitable protecting groups and methods for protecting and deprotecting various substituents using such suitable protecting groups are well known to those skilled in the art, and examples thereof can be found in the book by Greene, PGM Wuts and TW Greene, entitled Protective Groups in Organic Synthesis (4 thed.), John Wiley & Sons, NY (2006).

[0079] If an optically active form of a disclosed compound is required, it may be obtained by carrying out one of the procedures described herein using optically active starting materials (prepared, for example, by asymmetric induction of an appropriate reaction step) or by resolving a stereoisomeric mixture of the compound or intermediate using standard procedures (such as chromatographic separation, recrystallization, or enzymatic resolution).

[0080] Similarly, if a pure geometric isomer of a compound is required, it can be obtained by carrying out one of the procedures described herein using a pure geometric isomer as a starting material, or by resolving a geometric isomeric mixture of the compound or intermediate using standard procedures such as chromatographic separation.

[0081] The synthetic schemes and specific examples disclosed herein are illustrative and should not be construed as limiting the scope of the disclosure or the claims. Alternatives, modifications, and equivalents of the synthetic methods and specific examples are contemplated.

[0082] The compounds exemplified above are either in zwitterionic form or have a net charge, in which case the compound will further include ions to balance the net charge. In particular, the compounds may be anionic or may have functional groups that can be anionic (e.g., -COOH is -COO - In the case of cations having an acid salt such as phosphate, phosphate groups, etc., salts may be formed with one or more suitable cations. Examples of suitable inorganic cations include, but are not limited to, alkali metal cations (Li + , Na + , and K + etc.), alkaline earth cations (Ca 2+ , Mg 2+ etc.), and other cations. In particular, sodium salts may be suitable. Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH+ ) and substituted ammonium ions (e.g., NH3R1 + , NH2R2 + , NHR3 + , and NR4 + ). Some examples of suitable substituted ammonium ions include those derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. In some embodiments, the compound is a sodium salt. The compound may be cationic or may have a functional group that can be cationic (e.g., -NH2 is -NH3 + (which may be 0, 1, 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, Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, tetrafluoroboric acid, toluenesulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, and valeric acid. In some embodiments, the compounds disclosed herein are trifluoroacetate salts.

[0083] Also included in the present disclosure are isotopically labeled compounds that are identical to the compounds disclosed herein except for the fact that one or more atoms have been replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes suitable for inclusion in the compounds of the present disclosure include, but are not limited to, 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 31 P, 35 S, 18 F, and 36 The isotopically labeled compounds of formula (I) or (II) can generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described herein, substituting the appropriate isotopically labeled reagent for the non-isotopically labeled reagent.

[0084] Oligonucleotides Also disclosed herein are oligonucleotides labeled with the dye compounds disclosed herein, such as the dye compounds of formula (I). For example, the dye compounds of formula (Ia): [ka] or a tautomer or a salt thereof, wherein A is a 5-, 6-, or 7-membered heterocyclyl; R 1 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 1 -X, where L 1 is alkylene or heteroalkylene, and X is selected from -COOH, -SOH, -POH, -OPOH, and a point of attachment to the oligonucleotide, or R 1 is R 6 and together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 2 , R3 , and R 4 is defined as follows: (i)R 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a point of attachment to the oligonucleotide; or (ii)R 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a point of attachment to the oligonucleotide; or (iii)R 2 is C1-C4 alkyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl; q is 0, 1, 2, or 3; Each R 5 is independently selected from C1 to C4 alkyl; R 6 is hydrogen or R 6 is R 1 and together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; Where: (a)R 1 -L 1 -X, where X is the point of attachment to the oligonucleotide, or (b)R 4 -L 2-Z, where Z is the point of attachment to the oligonucleotide; The moiety of formula (Ia) does not have more than one point of attachment to the oligonucleotide.

[0085] In some embodiments, A is selected from a pyrrolidine ring, a piperidine ring, and a morpholine ring. In some embodiments, A is a morpholine ring. In some embodiments, A is a piperidine ring. In some embodiments, A is a pyrrolidine ring.

[0086] In some embodiments, R 1 is hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl. 1 is hydrogen or C1-C4 alkyl. In some embodiments, R 1 is hydrogen or ethyl. In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is ethyl.

[0087] In some embodiments, R 1 -L 1 In some embodiments, L 1 is a C2-C4 alkylene, or L 1 is C2-C4 heteroalkylene. 1 is C2-C4 alkylene. 1 is -CH2CH2CH2-. In some embodiments, X is selected from -COOH, -SO3H, -PO3H2, and a point of attachment to an oligonucleotide. In some embodiments, X is selected from -SO3H, -PO3H2, and a point of attachment to an oligonucleotide. In some embodiments, X is -SO3H. In some embodiments, X is -PO3H2. In some embodiments, X is a point of attachment to an oligonucleotide.

[0088] In some embodiments, R 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SOH, -POH, -OPOH, and a point of attachment to the oligonucleotide. 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is alkylene or heteroalkylene and Z is -SOH. In some embodiments, R 2 is C1-C2 alkyl, R 3 is hydrogen and R 4 is C1-C2 alkyl or -(CH2) n -SO3H, where n is 3, 4, or 5. In some embodiments, R 2 is methyl, R 3 is hydrogen and R 4 is methyl, ethyl, or -(CH2)4-SO3H. In some embodiments, R 2 is methyl, R 3 is hydrogen and R 4 is ethyl. In some embodiments, R 2 is methyl, R 3 is hydrogen and R 4 is -(CH2)4-SO3H.

[0089] In some embodiments, R 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2-Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SOH, -POH, -OPOH, and a point of attachment to the oligonucleotide. 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is selected from hydrogen, C1-C6 alkyl, and C1-C6 heteroalkyl. 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is selected from hydrogen and C1-C2 alkyl. 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 -L 2 In some embodiments, L 2 is C2-C4 alkylene. 2 is -CH2CH2CH2-. In some embodiments, Z is selected from -COOH, -SO3H, and a point of attachment to the oligonucleotide.

[0090] In some embodiments, R 2 and R 3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclyl, R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SOH, -POH, -OPOH, and a point of attachment to the oligonucleotide. 2 and R 3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclyl, R 4is hydrogen and -L 2 -Z, where L 2 is a C2-C4 alkylene or a C2-C4 heteroalkylene, and Z is the point of attachment to the oligonucleotide. 2 and R 3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclyl, R 4 is hydrogen and -L 2 -Z, where L 2 is -CH2CH2CH2- and Z is the point of attachment to the oligonucleotide.

[0091] In some embodiments, R 2 is C1-C4 alkyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl. 2 is methyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl.

[0092] In some embodiments, q is 0. In some embodiments, q is 1 and R 5 is methyl.

[0093] In some embodiments, R 6 is hydrogen.

[0094] In some embodiments, the moiety of formula (Ia) is A is a pyrrolidine ring; R 1 -L 1 -X, where L 1 is a C2-C4 alkylene and X is the point of attachment to the oligonucleotide; R 2 and R 3together with the atoms to which they are attached form a 5-membered heterocyclyl having one nitrogen atom, R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is a C2-C4 alkylene, Z is -SO3H, q is 0, R 6 is hydrogen.

[0095] In some embodiments, the moiety of formula (Ia) is A is a morpholine ring; R 1 -L 1 -X, where L 1 is a C2-C4 alkylene and X is the point of attachment to the oligonucleotide; R 2 is C1-C4 alkyl, R 3 is hydrogen or R 2 and R 3 form together with the atoms to which they are attached a six-membered ring containing one nitrogen atom and one oxygen atom, R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is a C2-C4 alkylene, Z is -SO3H, q is 0, R 6 is hydrogen.

[0096] In some embodiments, the moiety of formula (Ia) is: [ka] or a tautomer thereof or a salt thereof; where [ka] represents the point of attachment of the moiety to the oligonucleotide.

[0097] Further moieties of formula (Ia) include: [ka] Examples include:

[0098] The moiety of formula (Ia) can be linked to the oligonucleotide by direct bond or through a linker. The linker can include one or more groups independently selected from methylene (-CH2-), ethylene (-CH=CH-), ethynylene (-C≡C-), ether (-O-), amine (-NH-), thioether (-S-), carbonyl (-C(O)-), or sulfonyl (-S(O)2-) moieties, or any combination thereof, such as amide (-C(O)NH-), ester (-C(O)O-), carbamate (-OC(O)NH-), or sulfonamide (-S(O)2NH-) groups, and any combination thereof. The linker can also include one or more cyclic groups, such as arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties. For example, if a compound of formula (I) contains an alkyne or azide group and the compound is attached to an oligonucleotide via click chemistry, a triazole linking group will be formed and the linker will contain a 1,2,3-triazole moiety.Those skilled in the art will appreciate that copper-free click chemistry reactions can also be performed, resulting in other types of linking moieties.

[0099] In some embodiments, the linker comprises one or more -(CH2CHO)-(oxyethylene) groups, for example, 1 to 20 -(CH2CHO)- groups (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 -(CH2CHO)- groups, or any range therebetween). In some embodiments, the linker comprises a -(CH2CHO)-, -(CH2CHO)2-, -(CH2CHO)3-, -(CH2CHO)4-, -(CH2CHO)5-, or -(CH2CHO)6- group.

[0100] In some embodiments, the linker comprises one or more alkylene groups (e.g., -(CH) n -), where n is 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any suitable range therebetween). In some embodiments, the linker comprises one or more branched alkylene groups.

[0101] In some embodiments, the linker comprises at least one amide group (-C(O)NH-). In some embodiments, the linker comprises two amide groups.

[0102] In some embodiments, the moiety of formula (Ia) is attached to the oligonucleotide via an amide moiety (-C(O)NH-). Such a linker may result after reaction of a compound of formula (I) containing a succinimidyl ester group with an amine-modified oligonucleotide.

[0103] Labeled oligonucleotides can be synthesized according to standard methods.For example, modified oligonucleotides containing primary amino groups can be attached to the compound of formula (I), which has a reactive moiety that reacts with primary amines, such as active esters (e.g., succinimidyl esters).Therefore, in one aspect, disclosed herein is a method for synthesizing labeled oligonucleotides, comprising reacting oligonucleotides with the compound of formula (I) disclosed herein to obtain labeled oligonucleotides (e.g., the oligonucleotides that contain the moiety of formula (Ia)).

[0104] The oligonucleotides can be of any suitable length (e.g., suitable for use as primers in sequencing reactions). In some embodiments, the oligonucleotides are about 5 bases to about 50 bases in length, or any range therebetween (such as about 15 bases to about 35 bases in length). In some embodiments, the oligonucleotides are about 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, or 50 bases in length, or any range therebetween.

[0105] The labeled oligonucleotides of the present disclosure may be used in sequencing methods, such as the methods described herein below. For use in such methods, the present disclosure also provides compositions comprising the labeled oligonucleotides. The compositions may further comprise one or more nucleic acid amplification reagents. In some embodiments, the one or more amplification reagents are selected from the group consisting of: deoxynucleotide triphosphates (e.g., unlabeled deoxynucleotide triphosphates), a buffer, a magnesium salt (e.g., MgCl2 or MgSO4), a template nucleic acid, and a DNA polymerase (e.g., a thermostable DNA polymerase (such as Taq, Tca, Tfu, Tbr, Tth, Tih, Tfi, Tli, Tfl, Pfu, Pwo, KOD, Tma, Tne, Bst, Pho, Sac, Sso, or ES4, or any mutant, variant, or derivative thereof)).

[0106] Modified Nucleotide Triphosphate Compounds Also disclosed herein are modified nucleotide triphosphate compounds labeled with the dye compounds disclosed herein (such as the dye compounds of formula (I)). When used in connection with a "nucleotide triphosphate compound," the term "modified" refers to the nucleotide triphosphate compound being covalently linked to the dye compound (such as the moiety of formula (Ib) described below), for example, by a direct bond or via a linker, as described below.

[0107] For example, as used herein, formula (Ib): [ka] or a tautomer or a salt thereof, wherein: A is a 5-, 6-, or 7-membered heterocyclyl; R 1 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 1 -X, where L 1is alkylene or heteroalkylene, and X is selected from -COOH, -SOH, -POH, -OPOH, and a point of attachment to a nucleotide triphosphate compound, or R 1 is R 6 and together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 2 , R 3 , and R 4 is defined as follows: (i)R 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a point of attachment to a nucleotide triphosphate compound; or (ii)R 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene and Z is selected from -COOH, -SO3H, -PO3H2, -OPO3H2, and a point of attachment to a nucleotide triphosphate compound; or (iii)R 2 is C1-C4 alkyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl; q is 0, 1, 2, or 3; Each R 5 is independently selected from C1 to C4 alkyl; R 6 is hydrogen or R 6 is R 1and together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; Where: (c)R 1 -L 1 -X, where X is the point of attachment to a nucleotide triphosphate compound, or (d)R 4 -L 2 -Z, where Z is the point of attachment to the nucleotide triphosphate compound.

[0108] In some embodiments, A is selected from a pyrrolidine ring, a piperidine ring, and a morpholine ring. In some embodiments, A is a morpholine ring. In some embodiments, A is a piperidine ring. In some embodiments, A is a pyrrolidine ring.

[0109] In some embodiments, R 1 is hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl. 1 is hydrogen or C1-C4 alkyl. In some embodiments, R 1 is hydrogen or ethyl. In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is ethyl.

[0110] In some embodiments, R 1 -L 1 In some embodiments, L 1 is a C2-C4 alkylene, or L 1 is C2-C4 heteroalkylene. 1 is C2-C4 alkylene. 1is -CH2CH2CH2-. In some embodiments, X is selected from -COOH, -SO3H, -PO3H2, and a point of attachment to a nucleotide triphosphate compound. In some embodiments, X is selected from -SO3H, -PO3H2, and a point of attachment to a nucleotide triphosphate compound. In some embodiments, X is -SO3H. In some embodiments, X is -PO3H2. In some embodiments, X is a point of attachment to a nucleotide triphosphate compound.

[0111] In some embodiments, R 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SOH, -POH, -OPOH, and a point of attachment to a nucleotide triphosphate compound. 2 is C1-C4 alkyl, R 3 is hydrogen and R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is alkylene or heteroalkylene and Z is -SOH. In some embodiments, R 2 is C1-C2 alkyl, R 3 is hydrogen and R 4 is C1-C2 alkyl or -(CH2) n -SO3H, where n is 3, 4, or 5. In some embodiments, R 2 is methyl, R 3 is hydrogen and R 4 is methyl, ethyl, or -(CH2)4-SO3H. In some embodiments, R 2 is methyl, R 3 is hydrogen and R 4is ethyl. In some embodiments, R 2 is methyl, R 3 is hydrogen and R 4 is -(CH2)4-SO3H.

[0112] In some embodiments, R 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SOH, -POH, -OPOH, and a point of attachment to a nucleotide triphosphate compound. 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is selected from hydrogen, C1-C6 alkyl, and C1-C6 heteroalkyl. 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 is selected from hydrogen and C1-C2 alkyl. 2 and R 3 together with the atoms to which they are attached form a 5-, 6-, or 7-membered heterocyclyl; R 4 -L 2 In some embodiments, L 2 is C2-C4 alkylene. 2 is -CH2CH2CH2-. In some embodiments, Z is selected from -COOH, -SO3H, and a point of attachment to a nucleotide triphosphate compound.

[0113] In some embodiments, R 2 and R 3together with the atoms to which they are attached form a 5- or 6-membered heterocyclyl, R 4 is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, and -L 2 -Z, where L 2 is alkylene or heteroalkylene, and Z is selected from -COOH, -SOH, -POH, -OPOH, and a point of attachment to a nucleotide triphosphate compound. 2 and R 3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclyl, R 4 is hydrogen and -L 2 -Z, where L 2 is a C2-C4 alkylene or a C2-C4 heteroalkylene, and Z is the point of attachment to the nucleotide triphosphate compound. 2 and R 3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclyl, R 4 is hydrogen and -L 2 -Z, where L 2 is -CH2CH2CH2- and Z is the point of attachment to the nucleotide triphosphate compound.

[0114] In some embodiments, R 2 is C1-C4 alkyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl. 2 is methyl, R 3 and R 4 together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocyclyl.

[0115] In some embodiments, q is 0. In some embodiments, q is 1 and R 5 is methyl.

[0116] In some embodiments, R 6 is hydrogen.

[0117] In some embodiments, the moiety of formula (Ib) is A is a pyrrolidine ring; R 1 -L 1 -X, where L 1 is a C2-C4 alkylene and X is the point of attachment to the nucleotide triphosphate compound; R 2 and R 3 together with the atoms to which they are attached form a 5-membered heterocyclyl having one nitrogen atom, R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is a C2-C4 alkylene, Z is -SO3H, q is 0, R 6 is hydrogen.

[0118] In some embodiments, the moiety of formula (Ib) is A is a morpholine ring; R 1 -L 1 -X, where L 1 is a C2-C4 alkylene and X is the point of attachment to the nucleotide triphosphate compound; R 2 is C1-C4 alkyl, R 3 is hydrogen or R 2 and R 3 form together with the atoms to which they are attached a six-membered ring containing one nitrogen atom and one oxygen atom, R 4 is C1-C6 alkyl and -L 2 -Z, where L 2 is a C2-C4 alkylene, Z is -SO3H, q is 0, R 6 is hydrogen.

[0119] In some embodiments, the moiety of formula (Ib) is: [ka] or a tautomer thereof or a salt thereof; where [ka] represents the point of attachment of the moiety to the nucleotide triphosphate compound.

[0120] The moiety of formula (Ib) can be attached to the nucleotide triphosphate compound by a direct bond or through a linker. The linker can include one or more groups independently selected from methylene (-CH2-), ethylene (-CH=CH-), ethynylene (-C≡C-), ether (-O-), amine (-NH-), thioether (-S-), carbonyl (-C(O)-), or sulfonyl (-S(O)2-) moieties, or any combination thereof, such as amide (-C(O)NH-), ester (-C(O)O-), carbamate (-OC(O)NH-), or sulfonamide (-S(O)2NH-) groups, and any combination thereof. The linker can also include one or more cyclic groups, such as arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties. For example, if a compound of formula (I) contains an alkyne or azide group and that compound is attached to a nucleotide triphosphate compound via click chemistry, a triazole linking group will be formed and the linker will contain a 1,2,3-triazole moiety. One of skill in the art will appreciate that copper-free click chemistry reactions can also be performed, resulting in other types of linking moieties.

[0121] In some embodiments, the linker comprises one or more -(CH2CHO)-(oxyethylene) groups, for example, 1 to 20 -(CH2CHO)- groups (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 -(CH2CHO)- groups, or any range therebetween). In some embodiments, the linker comprises a -(CH2CHO)-, -(CH2CHO)2-, -(CH2CHO)3-, -(CH2CHO)4-, -(CH2CHO)5-, or -(CH2CHO)6- group.

[0122] In some embodiments, the linker comprises one or more alkylene groups (e.g., -(CH) n -), where n is 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any suitable range therebetween). In some embodiments, the linker comprises one or more branched alkylene groups.

[0123] In some embodiments, the linker comprises at least one amide group (-C(O)NH-). In some embodiments, the linker comprises two amide groups.

[0124] In some embodiments, the moiety of formula (Ia) is attached to the nucleotide triphosphate compound via an amide moiety (-C(O)NH-). Such linkers can be generated after reaction of a compound of formula (I) containing a succinimidyl ester group with an amine-modified nucleotide triphosphate compound.

[0125] The modified nucleotide triphosphate compound can be a modified deoxynucleotide triphosphate compound or a modified dideoxynucleotide triphosphate compound.For example, in some embodiments, the compound is a modified deoxynucleotide triphosphate compound selected from deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate.In some embodiments, the compound is a modified dideoxynucleotide triphosphate compound selected from dideoxyadenosine triphosphate, dideoxycytidine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate.For example, exemplary modified deoxynucleotides include: [ka]

[0126] Exemplary modified dideoxynucleotides include the following: [ka]

[0127] Modified nucleotide triphosphate compounds can be synthesized according to standard methods.For example, modified nucleotide triphosphate compounds containing primary amino groups can be attached to the compound of formula (I) with reactive moieties that react with primary amines, such as active esters (e.g., succinimidyl esters).Therefore, in one aspect, disclosed herein is a method for synthesizing labeled oligonucleotides, comprising reacting a nucleotide triphosphate compound (e.g., a nucleotide triphosphate compound functionalized with a linker) with the compound of formula (I) disclosed herein to obtain labeled oligonucleotides (e.g., the oligonucleotides that contain the moiety of formula (Ia)).

[0128] The modified nucleotide triphosphate compounds of the present disclosure may be used in sequencing methods, such as the methods described herein below. The modified nucleotide triphosphate compounds of the present disclosure may be used in sequencing methods, such as the methods described herein below. For use in such methods, the present disclosure also provides compositions comprising modified nucleotide triphosphate compounds. The compositions may further comprise one or more nucleic acid amplification reagents. In some embodiments, the one or more amplification reagents are selected from the group consisting of: deoxynucleotide triphosphates (e.g., unlabeled deoxynucleotide triphosphates), buffers, magnesium salts (e.g., MgCl2 or MgSO4), oligonucleotide primers, template nucleic acids, and DNA polymerases (e.g., thermostable DNA polymerases such as Taq, Tca, Tfu, Tbr, Tth, Tih, Tfi, Tli, Tfl, Pfu, Pwo, KOD, Tma, Tne, Bst, Pho, Sac, Sso, or ES4, or any mutant, variant, or derivative thereof).

[0129] How to use The compounds of the present disclosure may be used in any suitable molecular biological or biochemical assay involving detection and / or quantification of labeled nucleotides, oligonucleotides, and / or polynucleotides. As further described herein, the dyes of the present disclosure exhibit many advantageous features, including, but not limited to, stability at high temperatures and longer emission wavelengths. In some embodiments, the dyes of the present disclosure may be used to label individual nucleic acids (e.g., dNTPs) and / or oligonucleotides (e.g., primers / probes), and are useful in any method involving nucleic acid amplification. For example, nucleic acid amplification methods may include, but are not limited to, polymerase chain reaction (PCR), quantitative PCR, real-time PCR, hot start PCR, single cell PCR, nested PCR, in situ colony PCR, digital PCR (dPCR), Droplet Digital™ PCR (ddPCR), emulsion PCR, ligase chain reaction (LCR), transcription-based amplification system (TAS), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), and hyperbranched RCA (HRCA).

[0130] In accordance with these embodiments, RT-PCR generally refers to PCR that first performs a reverse transcription reaction that converts the target RNA into complementary single-stranded DNA, and then amplifies the DNA. Real-time PCR generally refers to PCR in which the amount of reaction product (target amplicon) is monitored as the reaction proceeds. There are many forms of real-time PCR, which differ primarily in the detection chemistry used to monitor the reaction product, such as dyes as further described herein. Nested PCR generally refers to a two-step PCR, where the product of the first PCR amplification is sampled for a second PCR with a new set of primers, at least one of which is located inside the product of the first amplification. Multiplex PCR generally refers to PCR in which multiple target sequences (or a single target sequence and one or more reference sequences) are performed simultaneously in the same reaction mixture. Typically, a different primer set is used for each sequence to be amplified, including primer sets labeled with the dyes of the present disclosure. Quantitative PCR generally refers to PCR designed to measure the abundance of one or more specific target sequences or samples in a sample, which may include the use of the dyes of the present disclosure. Digital PCR generally refers to compartmentalizing a bulk PCR reaction into thousands of nanoliter-scale reactions, with each compartment containing 0, 1, or just a few DNA molecules. Samples can be absolutely quantified by counting positive reactions based on the fluorescence of the probe, including the fluorescent signal generated by the dyes of the present disclosure. dPCR overcomes the common limitations of qPCR, such as the need for a standard curve, poor accuracy when measuring rare targets, and lack of sensitivity under high background conditions.

[0131] In some embodiments, the dyes of the present disclosure, as described above, can be used to label individual nucleic acids (e.g., dNTPs) and / or oligonucleotides (e.g., primers / probes) to detect, track, and / or quantify one or more target nucleic acids during or after amplification. In some embodiments, the target nucleic acid amplification product can be tracked, detected, and / or quantified directly via labeled nucleic acids and / or labeled oligonucleotides (e.g., qPCR, RT-PCR), while in other embodiments, the target nucleic acid amplification product can be tracked, detected, and / or quantified indirectly via labeled nucleic acids and / or labeled oligonucleotides (e.g., Taqman-based assays, strand displacement assays). As would be recognized by one of skill in the art based on the present disclosure, the dyes described herein can be used in any nucleic acid amplification and / or detection assay. In some embodiments, the dyes of the present disclosure can be used to label nucleic acids, oligonucleotide sequences, single-stranded DNA, double-stranded DNA, RNA (e.g., mRNA or miRNA), or DNA-RNA hybrids. In some embodiments, the nucleic acid labeled with the dye of the present disclosure is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides in length. In some embodiments, the dye of the present disclosure is used to label a sequence that is complementary or substantially complementary to a target sequence or another probe sequence. In some embodiments, the dye of the present disclosure can be used with a quencher that anneals to the same target nucleic acid or its complement, thereby allowing detection of the nucleic acid label.

[0132] Embodiments of the present disclosure include the use of the dyes described herein for nucleic acid sequencing applications, including, but not limited to, fragment analysis and next generation sequencing. For example, in some embodiments, fragment analysis involves a series of techniques in which DNA fragments are fluorescently labeled with the dyes of the present disclosure, separated by capillary electrophoresis (CE), and sized by comparison to an internal standard. DNA sequencing by CE is used to determine the specific base sequence of a particular fragment or gene segment, while fragment analysis can size, comparatively quantify, and provide genotyping information for fluorescently labeled DNA fragments obtained by PCR using primers designed against specific DNA targets.

[0133] In some embodiments, the dyes of the present disclosure may be used as part of a method for determining a series of base pairs in DNA and / or RNA molecules (i.e., nucleic acid sequencing), including use in whole genome and region sequencing, transcriptome analysis, metagenomic analysis, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis. For example, in some embodiments, replication of a DNA template strand proceeds in a reaction mixture that includes four standard dNTPs and all four ddNTPs (ddATP, ddCTP, ddGTP, and ddATP), each labeled with a different fluorescent dye, such as those described in this disclosure. Random incorporation of the labeled ddNTPs results in a series of DNA fragments in which the extension of the strand stops at each position in succession, each one being one nucleotide longer than the previous one. Size separation of the fragments results in a sequence ladder as a series of colored bands. In an automated DNA sequencer, the fluorescent dye of each band is activated by a scanning laser as it passes a set point at the bottom of an electrophoretic gel. The color of each successive band is read by a fluorometer (which may be read from bottom to top, like a traditional radiolabeled base sequence ladder), and these are assembled by computer as a gel image. Multiple sequencing reactions on separate templates are run in parallel (each ladder band is read as a separate electropherogram or chromatogram). The dyes of the present disclosure may be used with any currently available nucleic acid sequencing method, including, but not limited to, traditional Sanger sequencing or "next generation sequencing" (NGS), including, but not limited to, sequencing by synthesis, sequencing by ligation, single molecule sequencing, nanopore sequencing, and the like.

[0134] The following examples further illustrate aspects of the present disclosure but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0135] In the examples, the following abbreviations are used: ACN is acetonitrile, AcOH is acetic acid, DCM is dichloromethane, DI is deionized water, DIPEA is N,N-diisopropylethylamine, DMF is dimethylformamide, EtO is diethyl ether, EtOAc is ethyl acetate, HPLC is high performance liquid chromatography, LC-MS is liquid chromatography-mass spectrometry, LRMS is low resolution mass spectrometry, MeOH is methanol, NMR is nuclear magnetic resonance, RT is room temperature, TFA is trifluoroacetic acid, THF is tetrahydrofuran, TMSBr is bromotrimethylsilane, and TSTU is N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate.

[0136] Example 1: Synthesis of Compounds 5-(Ethylamino)-4-methyl-2-nitrosophenol [ka] General procedure 1: 3-Ethylamino-p-cresol (1.0 g, 6.6 mmol, 1.0 equiv.) was dissolved in 5 mL of ice-cold 6M HCl. To the solution was added NaNO2 (479 mg, 6.9, 1.05 equiv.) in three portions over 1 h by keeping the reaction in an ice bath. The reaction was stirred for another 2 h. The precipitate was then filtered in a separatory funnel, washed with 15-20 mL of 2M HCl, and dried under high vacuum to give 5-(ethylamino)-4-methyl-2-nitrosophenol (1.1 g, 93%). LR-MS [M+H] + 181.2.

[0137] Ethyl 4-(6-methoxy-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)butanoate [ka] General procedure 2: 6-Methoxy-3,4-dihydro-2H-benzo[b][1,4]oxazine (320 mg, 1.9 mmol, 1.0 equiv), NaI (29 mg, 0.19 mmol, 0.1 equiv), ethyl 4-bromobutanoate (809 μL, 5.8 mmol, 3.0 equiv), and DIPEA (3.4 mL, 19.4 mmol, 10 equiv) were suspended in toluene (20 mL) and heated to 120° C. for 20 h. The reaction was then cooled and filtered through Celite under vacuum. The precipitate was washed with Et2O / DCM (1 / 1, 50×2 mL). The filtrate was concentrated in vacuo and the desired product was purified by purification on silica gel. 1 H NMR (400 MHz, chloroform-d) δ 6.68 (dd, J = 9.1, 2.2 Hz, 1H), 6.29 (d, J = 2.9 Hz, 1H), 6.15 (dd, J = 8.7, 2.9 Hz, 1H), 4.15 (ddd, J = 17.0, 6.3, 2.4 Hz, 4H), 3.75 (t, J = 1.6 Hz, 3H), 3.40 - 3.18 (m, 4H), 2.36 (t, J = 7.3 Hz, 2H), 1.94 (p, J = 7.4 Hz, 2H), 1.25 (ddd, J = 8.7, 7.0, 1.9Hz, 3H). LRMS m / z: [M + H] + 280.3.

[0138] Ethyl 4-(7-hydroxy-3,4-dihydroquinolin-1(2H)-yl)butanoate [ka] The target compound was synthesized from 1,2,3,4-tetrahydroquinolin-7-ol according to general procedure 2. 1H NMR (400 MHz, chloroform-d) δ 6.80 (d, J = 7.9 Hz, 1H), 6.22 - 5.96 (m, 2H), 4.56 (s, 1H), 4.26 - 4.04 (m, 2H), 3.27 (t, J = 6.9 Hz, 4H), 2.69 (t, J = 6.4 Hz, 2H), 2.38 (dd, J = 8.1, 6.3 Hz, 2H), 1.94 (dd, J = 10.0, 5.8 Hz, 4H), 1.29 (td, J = 7.2, 1.7 Hz, 3H). LRMS m / z: [M + H] + 264.3.

[0139] Ethyl 4-(6-iodoindolin-1-yl)butanoate [ka] The target compound was synthesized from 6-iodoindoline following general procedure 2. 1 H NMR (400 MHz, chloroform-d) δ 6.95 (d, J = 7.7 Hz, 1H), 6.78 (d, J = 7.6 Hz, 1H), 6.73 (s, 1H), 4.15 (q, J = 6.9 Hz, 3H), 3.38 (t, J = 8.4 Hz, 2H), 3.09 (t, J = 7.1 Hz, 2H), 2.92 (t, J = 8.4 Hz, 2H), 2.42 (t, J = 7.3 Hz, 2H), 1.93 (p, J = 7.2 Hz, 2H), 1.28 (t, J = 7.1 Hz, 4H). LRMS m / z: [M + H] + 360.2.

[0140] 4-(6-Methoxy-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)butanoic acid [ka] General procedure 3: Ethyl 4-(6-methoxy-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)butanoate (1.0 g, 3.6 mmol, 1.0 equiv) was dissolved in THF / MeOH (1 / 1, 10 mL). 5 mL of 2N LiOH was added and the reaction mixture was stirred at RT for 3 h. LC-MS indicated complete conversion. Volatile solvents were removed in vacuo and the pH of the remaining solution was adjusted to pH 4.0 using 2N aq. HCl. The suspension was then extracted with EtOAc (50x3 mL). The combined organic solutions were concentrated in vacuo and the desired product was purified by purification on silica gel. 1 H NMR (400 MHz, chloroform-d) δ 6.69 (dd, J = 8.7, 2.1 Hz, 1H), 6.29 (t, J = 2.6 Hz, 1H), 6.16 (dd, J = 8.5, 3.1 Hz, 1H), 5.30 (t, J = 1.7 Hz, 1H), 4.18 (dt, J = 4.8, 2.7 Hz, 2H), 3.74 (d, J = 1.7 Hz, 3H), 3.37 - 3.25 (m, 4H), 2.44 (td, J = 7.0, 2.1 Hz, 2H), 1.95 (p, J = 7.3 Hz, 2H). LRMS m / z: [M - H] - 250.3.

[0141] (Z)-4-(8-(ethyliminio)-9-methyl-2,3-dihydro-[1,4]oxazino[2,3-b]phenoxazin-4(8H)-yl)butanoate [ka] General procedure 4: 4-(6-Methoxy-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)butanoic acid (86 mg, 0.34 mmol, 1.0 equiv.) and 5-(ethylamino)-4-methyl-2-nitrosophenol (61 mg, 0.34 mmol, 1.0 equiv.) were dissolved in AcOH (4 mL). The suspension was heated to 80° C. and left for 30 min. LC-MS indicated complete consumption of the reactants. The reaction mixture turned deep blue and was concentrated in vacuo. The desired product was purified by purification on silica gel with DCM / MeOH / 1% DIPEA. 1 H NMR (400 MHz, methanol-d4) δ 7.62 (s, 1H), 7.31 (s, 1H), 7.22 (s, 1H), 6.93 (s, 1H), 4.38 (d, J = 5.0 Hz, 2H), 3.95 - 3.69 (m, 4H), 3.60 (d, J = 7.3 Hz, 2H), 2.34 (d, J = 21.6 Hz, 5H), 2.05 (t, J = 8.1 Hz, 2H), 1.39 (t, J = 7.3 Hz, 5H). LRMS m / z: [M + H] + 382.4.

[0142] Compound JC-0025 [ka] General procedure 4: (Z)-4-(8-(ethyliminio)-9-methyl-2,3-dihydro-[1,4]oxazino[2,3-b]phenoxazin-4(8H)-yl)butanoate (38 mg, 1.0 mmol, 1.0 equiv) was mixed with TSTU (60 mg, 2.0 mmol, 2.0 equiv) and DIPEA (870 μL, 5.0 mmol, 5.0 equiv) in dry DMF (2 mL). The reaction was stirred at RT for 30 min. LC-MS indicated complete conversion. The desired product was purified using reverse phase preparative HPLC (ACN / 0.5% TFA in DI H2O). LRMS m / z: [M] + 479.74.

[0143] Additional Compounds Compounds JC-0028, JC-0064, JC-0081, JC-0084, CS-1333, CS-1345 and JC-0068 were synthesized similarly to compound JC-0025 using the appropriate starting materials according to general procedure 4 and general procedure 5. Mass spectrometry data is shown in Table 1 below.

[0144] (E)-4-(6-hydroxy-7-((4-nitrophenyl)diazenyl)-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)butanoic acid [ka] General procedure 6: At 0° C., 4-(6-hydroxy-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)butanoic acid (145 mg, 0.61 mmol, 1 equiv.) was dissolved in 2N HCl (2 mL). To the solution, 4-nitrobenzenediazonium tetrafluoroborate (173 mg, 0.73 mmol, 1.2 equiv.) was added by keeping the reaction in an ice bath. The reaction was stirred for an additional hour. The precipitate was then filtered through a funnel, washed with 2M HCl, and dried under high vacuum to give the product (206 mg, 87%). LRMS m / z: [M+H] + 388.2.

[0145] 4-(6-Hydroxy-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)butane-1-sulfonic acid [ka] General Procedure 7: 3,4-Dihydro-2H-benzo[b][1,4]oxazin-6-ol (200 mg, 1.3 mmol, 1.0 equiv) and 1,4-butanesultone (360 mg, 2.7 mmol, 2.0 equiv) were added into a 25 mL pressure flask equipped with a magnetic stir bar. The mixture was heated at 110° C. After approximately 30 min, the reaction mixture became very viscous and stirring was discontinued. At this point, the flask was cooled to RT and the yellow solid was carefully broken up into larger pieces using a spatula. MeOH (3 mL) was added into the flask and the reaction was allowed to proceed for an additional 5 h at 110° C. The mixture was cooled to RT. The white crystalline product (195 mg, 51%) was filtered, washed with MeOH and dried in vacuum. LRMS [M+H] + 288.54.

[0146] 4-((5-hydroxy-2-methylphenyl)amino)butane-1-sulfonic acid [ka] The desired product was similarly synthesized following general procedure 7. 1 H NMR (400 MHz, chloroform-d) δ 6.94 (t, J = 7.1 Hz, 2H), 6.34 - 6.04 (m, 4H), 3.65 (d, J = 1.9 Hz, 3H), LRMS [M+H] + 274.55.

[0147] Compound CS-1341 [ka] Step 1: A mixture of ethyl 4-(6-hydroxyindolin-1-yl)butanoate (580 mg, 2.5 mmol, 1.0 equiv), 1-ethyl-6-iodoindoline (785 mg, 3.2 mmol, 1.3 equiv), CuI (140 mg, 0.74 mmol, 0.3 equiv), N,N-dimethylglycine (287 mg, 2.8 mmol, 1.1 equiv), and Cs2CO3 (2.4 g, 7.4 mmol, 3.0 equiv) was purged with Ar and suspended in dioxane (6 mL) in a sealed tube. The reaction was then heated at 100 °C for 20 h. The mixture was then cooled and partitioned between EtOAc (100 mL) and DI H2O (50 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (3x50 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The crude material was purified by purification on silica gel using heptane / EtOAc as eluent to give the desired product, methyl 4-(6-((1-ethylindolin-6-yl)oxy)indolin-1-yl)butanoate. 1 H NMR (400 MHz, chloroform-d) δ 6.94 (t, J = 7.1 Hz, 2H), 6.34 - 6.04 (m, 4H), 3.65 (d, J = 1.9 Hz, 3H), 3.48 - 3.26 (m, 4H), 3.20 - 2.99 (m, 4H), 2.92 (t, J = 8.3 Hz, 4H), 2.39 (td, J = 7.4, 2.0 Hz, 2H), 1.91 (p, J = 7.0 Hz, 2H), 1.15 (t, J = 7.4 Hz, 3H). LRMS m / z: [M + H] + 380.2.

[0148] Step 2: To a solution of methyl 4-(6-((1-ethylindolin-6-yl)oxy)indolin-1-yl)butanoate (70 mg, 0.18 mmol, 1.0 equiv) in THF (4 mL) was added LiOH (22 mg, 0.46 mmol, 5 equiv) in 2 mL of DI HO. The solution was stirred at RT for 3 h. LC-MS indicated complete conversion. The volatile solvents were then removed under vacuum and the aqueous solution was diluted with 20 mL of HO. The pH of the aqueous solution was adjusted to pH 4-5. The suspension was then partitioned between EtOAc (100 mL) and DI HO. The aqueous layer was extracted with EtOAc (20x3 mL). The combined organic layers were then washed with HO (30 mL), brine (30 mL), dried over NaSO and concentrated to give crude 4-(6-((1-ethylindolin-6-yl)oxy)indolin-1-yl)butanoic acid, which was used in the next step without further purification.

[0149] Step 3: The desired mixture of diazenyl intermediates was similarly synthesized following general procedure 6. The crude was used in the next step without further purification.

[0150] Step 4: The diazenyl mixture was dissolved in AcOH (0.05 M) and heated at 80° C. for 30 min. The desired product, 4-(1-ethyl-2,3,7,8-tetrahydro-1H-dipyrrolo[3,2-b:2′,3′-i]phenoxazin-9-ium-9-yl)butanoate, was purified by reverse-phase HPLC using ACN / 0.1% TFA in HO as the mobile phase. 1 H NMR (400 MHz, acetonitrile-d3) δ 7.42 (s, 2H), 6.61 (d, J = 6.0 Hz, 2H), 4.01 (q, J = 7.2 Hz, 4H), 3.61 (dt, J = 16.6, 7.7 Hz, 4H), 3.27 (t, J = 7.4 Hz, 4H), 2.45 (m, 4H), 1.31 (t, J = 7.1 Hz, 3H). LRMS m / z: [M + H] + 378.5.

[0151] Step 5: CS-1341 was synthesized from the product of Step 4 according to General Procedure 5. Mass spectrometry data is shown in Table 1 below.

[0152] Compound CS-1377 [ka] Step 1: A mixture of ethyl 4-(6-hydroxyindolin-1-yl)butanoate (84 mg, 0.36 mmol, 1.0 equiv), 6-iodoindoline (131 mg, 0.54 mmol, 1.5 equiv), CuI (20 mg, 0.11 mmol, 0.3 equiv), N,N-dimethylglycine (33 mg, 0.33 mmol, 0.9 equiv), and Cs2CO3 (350 mg, 1.1 mmol, 3.0 equiv) was purged with Ar and suspended in dioxane (4 mL) in a sealed tube. The reaction was then heated at 100 °C for 20 h. The mixture was then cooled and partitioned between EtOAc (100 mL) and DI H2O (50 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (3x50 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The crude material was purified by purification on silica gel with heptane / EtOAc as eluent to give the desired product, methyl 4-(6-(indolin-6-yloxy)indolin-1-yl)butanoate. 1 H NMR (400 MHz, methylene chloride-d2) δ 6.99 (dd, J = 19.4, 7.8 Hz, 2H), 6.29 (d, J = 9.6 Hz, 2H), 6.21 (d, J = 7.9 Hz, 1H), 6.15 (s, 1H), 3.65 (d, J = 1.8 Hz, 3H), 3.58 (t, J = 8.4 Hz, 2H), 3.41 (t, J = 8.2 Hz, 2H), 3.16 - 2.82 (m, 6H), 2.41 (t, J = 7.4 Hz, 2H), 1.92 (p, J = 7.2 Hz, 2H). LRMS m / z: [M + H] + 353.4.

[0153] Step 2: The target intermediate was synthesized according to general procedure 7. The desired product was isolated as a DIPEA salt. 1 H NMR (400 MHz, methanol-d4) δ 6.93 (d, J = 7.8 Hz, 2H), 6.24 - 6.06 (m, 4H), 3.75 (dd, J = 7.1, 5.3 Hz, 1H), 3.61 (d, J = 2.0 Hz, 3H), 3.43 - 3.35 (m, 4H), 3.28 - 3.18 (m, 1H), 3.03 (dt, J = 10.1, 5.1 Hz, 4H), 2.87 (dt, J = 16.3, 8.5 Hz, 7H), 2.41 (td, J = 7.2, 2.0 Hz, 2H), 1.99 - 1.81 (m, 5H), 1.74 (q, J = 7.5 Hz, 2H), 1.48 - 1.20 (m, 11H). LRMS m / z: [M - H] - 487.2.

[0154] Step 3: The target intermediate was similarly synthesized following general procedure 3. 1 H NMR (400 MHz, heavy water) δ 7.44 (d, J = 7.9 Hz, 2H), 7.13 (dd, J = 9.7, 4.1 Hz, 4H), 4.10 - 3.84 (m, 4H), 3.47 (q, J = 6.8 Hz, 4H), 3.25 (q, J = 7.6, 5.4 Hz, 4H), 2.90 - 2.69 (m, 2H), 2.42 (td, J = 7.1, 2.2 Hz, 2H), 2.07 - 1.57 (m, 6H). LRMS m / z: [M - H] - 473.2.

[0155] Step 4: The desired mixture of diazenyl intermediates was similarly synthesized following general procedure 6. The crude was used in the next step without further purification.

[0156] Step 5: The diazenyl mixture was dissolved in AcOH (0.05 M) and heated at 80° C. for 30 min. The desired product, 4-(9-(3-carboxypropyl)-2,3,7,8-tetrahydro-1H-dipyrrolo[3,2-b:2′,3′-i]phenoxazin-9-ium-1-yl)butane-1-sulfonate, was purified by purification on silica gel using DCM / 1% DIPEA in MeOH as eluent. 1 H NMR (400 MHz, methanol-d4) δ 7.32 (s, 2H), 6.67 (d, J = 8.8 Hz, 2H), 3.98 (d, J = 7.4 Hz, 4H), 3.70 (q, J = 6.6 Hz, 1H), 3.59 (d, J = 6.8 Hz, 3H), 3.28 - 3.12 (m, 4H), 2.86 (t, J = 6.8 Hz, 2H), 2.38 (t, J = 6.9 Hz, 2H), 2.13 - 1.70 (m, 7H), 1.36 (d, J = 6.4 Hz, 6H). LRMS m / z: [M - H] - 500.2.

[0157] Step 6: CS-1377 was similarly synthesized following general procedure 5. Mass spectrometry data is shown in Table 1 below.

[0158] Compound CS-1377 [ka] Step 1: The target intermediate was similarly synthesized following general procedure 7 using diethyl (3-bromopropyl)phosphonate as the alkylating reagent. 1H NMR (400 MHz, chloroform-d) δ 6.96 (t, J = 5.9 Hz, 2H), 6.33 - 6.09 (m, 4H), 4.12 (p, J = 7.4 Hz, 5H), 3.67 (s, 3H), 3.46 - 3.32 (m, 4H), 3.07 (q, J = 7.3, 6.9 Hz, 4H), 2.94 (t, J = 8.4 Hz, 4H), 2.42 (t, J = 7.4 Hz, 2H), 1.99 - 1.76 (m, 7H), 1.35 (q, J = 6.9 Hz, 8H). LRMS m / z: [M + H] + 531.6.

[0159] Step 2: To a solution of methyl 4-(6-((1-(3-(diethoxyphosphoryl)propyl)indolin-6-yl)-oxy)indolin-1-yl)butanoate (100 mg, 0.19 mmol, 1.0 equiv) in DCM (2 mL) was added TMSBr (2 mL) dropwise. The solution was stirred at RT for 20 h. LC-MS indicated complete conversion to the corresponding phosphonic acid. The reaction was then concentrated in vacuo and used in the next step without further purification.

[0160] The crude product from the previous step was dissolved in THF / MeOH (1 / 1, 4 mL). 2 mL of 2N LiOH was added and the reaction mixture was stirred at RT for 3 h. LC-MS indicated complete conversion. Volatile solvents were removed in vacuo and the pH of the remaining solution was adjusted to pH 4.0 using 2N aq. HCl. The suspension was then extracted with EtOAc (50x3 mL). The combined organic solutions were concentrated in vacuo and the desired product was purified by purification on silica gel. 1H NMR (400 MHz, methanol-d4) δ 6.92 (d, J = 7.8 Hz, 2H), 6.26 - 6.07 (m, 4H), 3.45 - 3.32 (m, 6H), 3.04 (q, J = 6.3 Hz, 4H), 2.88 (t, J = 8.3 Hz, 4H), 2.38 (t, J = 7.3 Hz, 2H), 1.87 (p, J = 7.6 Hz, 4H), 1.65 (dt, J = 16.7, 7.9 Hz, 3H), 1.37 (d, J = 6.5 Hz, 5H). LRMS m / z: [M + H] + 481.5.

[0161] Step 3: The desired mixture of diazenyl intermediates was similarly synthesized following general procedure 6. The crude was used in the next step without further purification.

[0162] Step 4: The diazenyl mixture was dissolved in AcOH (0.05 M) and heated at 80° C. for 30 min. The desired product, 4-(1-(3-phosphonopropyl)-2,3,7,8-tetrahydro-1H-dipyrrolo[3,2-b:2′,3′-i]phenoxazin-9-ium-9-yl)butanoate, was purified by reverse-phase HPLC using ACN / 0.1% TFA in DI H2O as the mobile phase. 1 H NMR (400 MHz, methanol-d4) δ 7.47 (s, 2H), 6.80 (d, J = 14.8 Hz, 2H), 4.07 (d, J = 9.1 Hz, 4H), 3.70 (dt, J = 19.7, 7.4 Hz, 4H), 2.46 (t, J = 6.8 Hz, 2H), 2.22 - 1.95 (m, 5H), 1.81 (dt, J = 16.6, 7.8 Hz, 2H). LRMS m / z: [M + H] + 472.5.

[0163] Step 5: CS-1480 was similarly synthesized following general procedure 5. Mass spectrometry data is shown in Table 1 below.

[0164] Mass spectrometry data and characterization data including maximum excitation and emission wavelengths are shown in Table 1. [Table 1-1] [Table 1-2]

[0165] Example 2: General Procedure for Using Oxazine Dye N-Hydroxysuccinimidyl Esters to Conjugate Oligonucleotides A. 1 μmole scale. 5'-amino-labeled or internal amino-deoxyuridine oligonucleotides were synthesized on an ABI 394 DNA synthesizer (1 μmole) using 5'Amino modifier C6 TFA amidite from Glen Research or Aminoallyl-dU amidite from PBI. Deprotection in concentrated ammonium hydroxide at 60° C. overnight gave amino-labeled oligonucleotides. The resulting oligonucleotides were evaporated to dryness, redissolved in 1 ml of 2 M NaCl (performed for counterion exchange), and desalted on a NAP-10 size-exclusion cartridge (GE Healthcare). After desalting, the oligonucleotides were evaporated to dryness and subsequently redissolved in 200 μl of 0.5 M sodium carbonate buffer (pH 8.5). Succinimidyl ester dyes (JC-0025, JC-0081, CS-1341, CS-1377, or JC-0084) were dissolved in DMF at a concentration of 20 μl / mg. Two 20 μl aliquots of the dye / DMF solution were added to the dissolved oligonucleotides, separated by 30 min intervals. After the second addition, the reactions were mixed for 1 h. After 1 h, the reactions were diluted to 1 ml with water and desalted on a NAP-10 column (GE Healthcare). The NAP-10 eluate was purified by reversed-phase HPLC on a Phenomonex Jupiter C18 column using an acetonitrile / 0.1 M TEAA buffer system. The HPLC-purified oligonucleotides were evaporated to dryness, redissolved in 0.01 M triethylammonium bicarbonate, and desalted on a NAP-10 column. After the final desalting step, the oligonucleotides were evaporated to dryness.

[0166] B. 100 μmol scale. 5′-amino-labeled or internal amino-deoxyuridine oligonucleotides were synthesized on an AKTA OligoPilot (100 μmol) DNA synthesizer using 5′ Amino modifier C6 TFA amidite from Glen Research or Aminoallyl dU amidite from PBI. Deprotection in concentrated ammonium hydroxide overnight at 60° C. gave 5′-aminohexyl-labeled oligonucleotides. The resulting oligonucleotides were evaporated to dryness, redissolved in 75 ml of 2 M NaCl, and desalted on a 500 ml G-25 column (GE Healthcare). After desalting, the oligonucleotides were evaporated to dryness and subsequently redissolved in 50 ml of 0.5 M sodium carbonate buffer (pH 8.5). Succinimidyl ester dyes (JC-0025, JC-0081, CS-1341, CS-1377, or JC-0084) were dissolved in DMF at a concentration of 20 μl / mg. 2400 μl of the dye / DMF solution was added dropwise to the dissolved oligonucleotide. The reaction was mixed for 1 hour. The dye-conjugated oligonucleotides were neutralized with sodium acetate (pH 5.5) solution and precipitated from 2× volume of ethanol. The precipitated oligonucleotides were centrifuged at 9000 rpm for 60 minutes. The supernatant was decanted and discarded. The resulting solid was dissolved in 70 ml of water and purified by ion exchange chromatography. The oligonucleotides were concentrated and desalted using tangential flow ultrafiltration, followed by evaporation to dryness.

[0167] Example 3: Multiplex PCR of STRs using dyes of the present disclosure Experiments were conducted during development of embodiments of the present disclosure for potential use in 8-dye multiplex PCR of short tandem repeats (STRs) to determine the energy transfer properties of exemplary dyes JC-0025 and JC-0081.

[0168] To derive primer pairs for the D8S1179, FGA, and DYS385a / b loci, oligonucleotides (oligos) were generated as described above using the dyes JC-0025 and JC-0081. The oligos were generated with either a 2-nucleotide spacer or a 4-nucleotide spacer.

[0169] The primer pairs were then used in a triplex mixture to amplify the 2800M DNA. The oligos were then used in the amplification reaction, which was then analyzed on a Spectrum CE instrument.

[0170] The following amplification reaction conditions were used for each 25ul reaction: 5x Master Mix: 5ul Nanopure water: 12.5ul 5x primer pairs (final concentrations: D8S1179: 1.0uM, FGA: 3.20uM, and DYS385a / b: 2.50uM): 5.0ul

[0171] The reaction mixture was vortexed and dispensed into each well of a 96-well plate. 2.5ul of 2800M DNA was then added to each well. The reaction also included quadruplicate 2800M controls and duplicate no template (water) controls. The reaction mixture was stored at 4°C during thermal cycling and then run as "No Amp" on the CE.

[0172] For amplification reactions in a ProFlex thermal cycler, the reactions were carried out as follows: [Table 2]

[0173] Amplified samples were then analyzed on a Spectrum CE Beta 02 instrument. 10ul of ILS (0.25 CCO ILS 2x fragment mix and 9.75 HiDi) was added to each well of a 96-well plate. 1ul of amplification reaction or No Amp mix was then added to the appropriate well. The plate was swirled briefly to remove air bubbles, denatured in a thermal cycler for 3 minutes, and then placed on ice for at least 3 minutes. Samples were injected at 2kV with a 15 second injection in an 8-color spectrum.

[0174] The electropherograms shown in Figure 1 demonstrate that JC-0025, JC-0081, CS-1341, CS-1377, and JC-0084 dyes are tolerable for use in amplification reactions when compared to the 2ET WEN / current control.

[0175] Example 4: Multiplex PCR of STRs using dyes of the present disclosure Experiments were conducted during development of embodiments of the present invention for the potential use in 8-dye multiplex PCR of short tandem repeats (STRs) to determine the energy transfer properties of the exemplary dyes CS-1341, CS-1377, and JC-0084.

[0176] To derive primer pairs for the D8S1179, FGA, and DYS385a / b loci, oligonucleotides (oligos) were generated as described above using the dyes CS-1341, CS-1377, and JC-0084.

[0177] The primer pairs were then used in a triplex mixture to amplify the 2800M DNA. The oligos were then used in the amplification reaction, which was then analyzed on a Spectrum CE instrument.

[0178] The following amplification reaction conditions were used for each 25ul reaction: 5x Master Mix: 5ul Nanopure water: 12.5ul 5x primer pair (concentration: D8S1179: 1.0uM, FGA: 3.20uM, and DYS385a / b: 2.50uM): 5.0ul

[0179] The reaction mixture was vortexed and dispensed into each well of a 96-well plate. 2.5ul of 2800M DNA was then added to each well. The reaction also included quadruplicate 2800M controls and duplicate no template (water) controls. The reaction mixture was stored at 4°C during thermal cycling and then run as "No Amp" on the CE.

[0180] For amplification reactions in a ProFlex thermal cycler, the reactions were carried out as follows: [Table 3]

[0181] Amplified samples were then analyzed on a Spectrum CE Beta 02 instrument. 10ul of ILS (0.25 CCO ILS 2x fragment mix and 9.75 HiDi) was added to each well of a 96-well plate. 1ul of amplification reaction or No Amp mix was then added to the appropriate well. The plate was swirled briefly to remove air bubbles, denatured in a thermal cycler for 3 minutes, and then placed on ice for at least 3 minutes. Samples were injected at 2kV with a 15 second injection in an 8-color spectrum.

[0182] The electropherograms shown in Figure 2 demonstrate that the CS-1341, CS-1377, and JC-0084 dyes are tolerable for use in amplification reactions when compared to the 2ET WEN / current control.

[0183] All references cited in this specification, including publications, patent applications, and patents, are incorporated by reference herein to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0184] In the context of the present invention (particularly in view of the claims that follow), the use of the terms "a," "an," and "the," as well as "at least one" and the like referents should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" following a description of one or more items (e.g., "at least one of A and B") should be construed to mean one item selected from the listed items (A or B), or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise noted. The recitation of ranges of values ​​herein is intended to serve merely as a shorthand way of referring individually to each individual value falling within the range, and each individual value is incorporated herein as if it were individually recited herein, unless otherwise indicated herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein, and unless otherwise clearly contradicted by context. Any examples provided herein, or the use of illustrative language (e.g., "etc.") are intended merely to better clarify the invention, and do not limit the scope of the invention unless otherwise claimed. No language herein should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0185] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will be adopted by those skilled in the art, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. It is to be understood that unless otherwise indicated herein, or otherwise clearly contradicted by context, the present invention encompasses any combination of the above-described elements in all possible variations thereof.

Claims

1. Compounds of formula (I): 【Chemistry 1】 or a tautomer thereof or a salt thereof (wherein A is a pyrrolidine ring; R 1 is -L 1 -X, where L 1 is a C 2 -C 4 alkylene and X is an N-succinimidyl ester reactive moiety; R 2 and R 3 together with the atoms to which they are attached form a 5-membered heterocyclyl having one nitrogen atom, R 4 is C 1 ~C 6 Alkyl and -L 2 -Z, where L 2 is C 2 -C 4 alkylene and Z is —SO 3 H, q is 0, R 6 is hydrogen).

2. The compound is: 【Chemistry 2】 2. The compound of claim 1, wherein the compound is selected from: or a tautomer thereof, or a salt thereof.

3. Formula (Ia): 【Transformation 3】 or a tautomer thereof or a salt thereof, A is a pyrrolidine ring; R 1 is -L 1 -X, where L 1 is C 2 -C 4 alkylene and X is the point of attachment to the oligonucleotide; R 2 and R 3 together with the atoms to which they are attached form a 5-membered heterocyclyl having one nitrogen atom, R 4 is C 1 ~C 6 Alkyl and -L 2 -Z, where L 2 is C 2 -C 4 alkylene and Z is —SO 3 H, q is 0, R 6 is hydrogen).

4. The oligonucleotide has the formula: 【Chemistry 4】 or a tautomer thereof or a salt thereof, where 【Transformation 5】 represents the point of attachment of the moiety to the oligonucleotide.

5. 4. The oligonucleotide of claim 3, wherein the moiety of formula (Ia) is attached to the oligonucleotide by a direct bond.

6. 4. The oligonucleotide of claim 3, wherein the moiety of formula (Ia) is attached to the oligonucleotide via a linker.

7. 7. The oligonucleotide of claim 6, wherein the moiety of Formula (Ia) is attached to the oligonucleotide via a linker comprising an amide moiety, a carbamate moiety, a 5-membered heteroaryl ring, an alkylene moiety, a fused bicyclic heterocycle, or any combination thereof.

8. The oligonucleotide of any one of claims 3 to 7, wherein the oligonucleotide is about 5 to about 50 bases in length.

9. The oligonucleotide of any one of claims 3 to 7, wherein the oligonucleotide is about 15 to about 35 bases in length.

10. Formula (Ib): 【Transformation 6】 or a tautomer or a salt thereof, A is a pyrrolidine ring; R 1 is -L 1 -X, where L 1 is a C 2 -C 4 alkylene and X is the point of attachment to the nucleotide triphosphate compound; R 2 and R 3 together with the atoms to which they are attached form a 5-membered heterocyclyl having one nitrogen atom, R 4 is C 1 ~C 6 Alkyl and -L 2 -Z, where L 2 is C 2 -C 4 alkylene and Z is —SO 3 H, q is 0, R 6 is hydrogen).

11. The nucleotide triphosphate compound has the formula: 【Transformation 7】 or a tautomer thereof or a salt thereof, where 【Transformation 8】 11. The modified nucleotide triphosphate compound of claim 10, wherein represents the point of attachment of the moiety to the nucleotide triphosphate compound.

12. 11. The modified nucleotide triphosphate compound of claim 10, wherein the moiety of Formula (Ib) is attached to the nucleotide triphosphate compound by a direct bond.

13. 11. The modified nucleotide triphosphate compound of claim 10, wherein the moiety of formula (Ib) is attached to the nucleotide triphosphate compound via a linker.

14. 14. The modified nucleotide triphosphate compound of Claim 13, wherein said moiety of Formula (Ib) is attached to said nucleotide triphosphate compound via a linker comprising an amide moiety, a carbamate moiety, a five-membered heteroaryl ring, an alkylene moiety, a fused bicyclic heterocycle, or any combination thereof.

15. The modified nucleotide triphosphate compound according to any one of claims 10 to 14, wherein the compound is a modified deoxynucleotide triphosphate compound selected from deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate.

16. The modified nucleotide triphosphate compound according to any one of claims 10 to 14, wherein the compound is a modified dideoxynucleotide triphosphate compound selected from dideoxyadenosine triphosphate, dideoxycytidine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate.

17. 1. A method for performing a nucleic acid amplification reaction, comprising: (a) adding an oligonucleotide compound according to any one of claims 3 to 7 to a reaction mixture; and (b) performing the amplification reaction; The method comprising:

18. 18. The method of claim 17, wherein the nucleic acid amplification reaction is selected from the group consisting of polymerase chain reaction (PCR), quantitative PCR, real-time PCR, hot-start PCR, single-cell PCR, nested PCR, in situ colony PCR, digital PCR (dPCR), Droplet Digital™ PCR (ddPCR), emulsion PCR, ligase chain reaction (LCR), transcription-based amplification system (TAS), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), and hyperbranched RCA (HRCA).

19. 18. The method of claim 17, wherein the nucleic acid amplification reaction is a multiplex nucleic acid amplification reaction.

20. 1. A method for performing a chain termination DNA sequencing reaction, comprising: (a) adding the modified dideoxynucleotide triphosphate compound of claim 16 to a polymerase chain reaction (PCR) mixture and performing PCR; (b) removing unincorporated modified dideoxynucleotide triphosphate compounds from the PCR mixture; and (c) performing sequence analysis; The method comprising:

21. 21. The method of claim 20, wherein the sequence analysis comprises fragment analysis and / or Sanger sequencing analysis.

22. 1. A method for performing a chain termination DNA sequencing reaction, comprising: (a) adding the modified deoxynucleotide triphosphate compound of claim 15 to a polymerase chain reaction (PCR) mixture; and (b) performing PCR; Including, The method wherein a fluorescent signal from the PCR mixture indicates which dNTP has been added, and the terminator is cleaved to facilitate the addition of subsequent dNTPs.

23. 23. The method of claim 22, further comprising performing fragment analysis and / or next generation sequencing.

24. 21. The method of claim 20, wherein the method is multiplexed.

25. The method of claim 22, wherein the method is multiplexed.

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