Pyrrolo[h]coumarin dye and use thereof in sequencing

Abstract

Provided are a pyrrolo[h]coumarin dye and a use thereof in sequencing. Specifically provided are a compound represented by formula I or a stereoisomer, tautomer, crystal form, salt, ester or solvate thereof. The compound has the advantages of high brightness and high stability. The compound exhibits strong fluorescence under blue light excitation. When used for labeling dNTPs and then applied in gene sequencing, the sequencing signal, resolution and throughput are all significantly improved, and the sequencing quality is significantly improved.

Description

A pyrrolo[h]coumarin dye and its application in sequencing Technical Field

[0001] This invention relates to the field of nucleic acid sequencing, specifically to a pyrrolo[h]coumarin dye and its application in sequencing. Background Technology

[0002] Gene sequencing technology uses fluorescent markers of four different bases to label the DNA with different colors. When DNA polymerase synthesizes the complementary strand, each addition of a base releases a different fluorescence. The captured fluorescence signals are processed by specific computer software to obtain the sequence information of the DNA being sequenced. Currently, commercially available sequencers primarily use red-green light sequencing technology, where dyes labeled with the four bases emit wavelengths in the green and red light regions. According to the Rayleigh criterion, the resolution of the optical system is x = 0.61λ / NA. With red light wavelength approximately 720nm and lens NA of 0.8, the theoretical resolution is approximately 550nm. However, because the lens NA is difficult to improve due to manufacturing processes, it is challenging to achieve significant resolution breakthroughs in red-green light sequencing technology. Therefore, developing new dyes with shorter wavelengths in the blue-green light region for blue-green light sequencing technology would help improve its resolution and throughput, ultimately leading to a comprehensive upgrade of all sequencers. Summary of the Invention

[0003] Current sequencers mainly use red-green light sequencing technology, which uses dyes that label the four bases and emit wavelengths in the green and red light regions. Due to the longer wavelength of red-green dyes, their theoretical resolution is poor. In sequencing, the throughput and resolution of red-green light sequencing technology are not high and it is difficult to make breakthroughs. As a result, its sequencing quality is not as good as that of blue light sequencing technology.

[0004] To address the problems existing in the prior art, the inventors of this application, after in-depth research, designed and synthesized a new type of pyrrolo[h]coumarin dye, which has the advantages of high brightness and high stability. This type of dye exhibits strong fluorescence under blue light excitation. Furthermore, when used to label dNTPs and applied to gene sequencing, the sequencing signal, resolution, and throughput are significantly improved, and the sequencing quality is significantly enhanced.

[0005] Therefore, in a first aspect, the present invention provides a compound of Formula I or a stereoisomer, tautomer, crystal form, salt, ester, or solvate thereof.

[0006] in:

[0007] R 1 Selected from hydrogen, -COOH,

[0008] R 1a R 1b R 1c R 1’a R 1’b R 1’c R 1d R 1e R 1f R 1g R 1h R 1i R 1j R 1k R 1l R 1m R 1n R 1o R 1p R 1q R 1r R 1s R 1t R 1u R 1v R 1w R 1x R 1y Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - ), halogen, halogenated C1-C6 alkyl group, wherein the C1-C6 alkyl group is optionally selected from carboxyl group (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group replaced by )

[0009] R 2 R 3 R 4 Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl;

[0010] R 5a R 5b R 6a R 6b Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl;

[0011] R7 Selected from -COOH, -SO3H;

[0012] n is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4, and more preferably from 3;

[0013] X is selected from O and S, with O being the preferred choice.

[0014] In some implementation schemes, R 1 Selected from hydrogen, -COOH,

[0015] In some implementation schemes, R 1 Selected from hydrogen, -COOH,

[0016] In some implementation schemes, R 1 Selected from hydrogen, -COOH,

[0017] In some implementation schemes, R 1 Selected from hydrogen, -COOH,

[0018] In some implementation schemes, R 1a R 1b R 1c R 1d Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - ), halogen, halogenated C1-C6 alkyl group, wherein the C1-C6 alkyl group is optionally selected from carboxyl group (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group is replaced by )

[0019] In some implementation schemes, R 1a R 1b R 1c R 1d In, any one (such as R) 1b Or R 1c ) or any two (such as R) 1b and R 1c Selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO) -), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - ), halogen, halogenated C1-C6 alkyl, wherein the C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group is replaced by ) and the rest is hydrogen.

[0020] In some implementation schemes, R 1a R 1b R 1c R 1d Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), sulfonic acid (-SO3H), halogen, or halogenated C1-C6 alkyl, wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) or sulfonic acid (-SO3H).

[0021] In some implementation schemes, R 1a R 1b R 1c R 1d In, any one (such as R) 1b Or R 1c ) or any two (such as R) 1b and R 1c The group is selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), sulfonic acid (-SO3H), halogen, and halogenated C1-C6 alkyl, wherein the C1-C6 alkyl is optionally replaced by a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H), and the remainder is hydrogen.

[0022] In some implementation schemes, R 1a R 1b R 1c R 1d Each is independently selected from hydrogen, C1-C6 alkyl, sulfonic acid group (-SO3H), halogen, wherein the C1-C6 alkyl is optionally replaced by sulfonic acid group (-SO3H).

[0023] In some implementation schemes, R 1a R 1b R 1c R 1d In, any one (such as R) 1b Or R 1c ) or any two (such as R) 1b and R 1c The group is selected from hydrogen, C1-C6 alkyl, sulfonic acid group (-SO3H), halogen, and the C1-C6 alkyl is optionally replaced by sulfonic acid group (-SO3H), with the remainder being hydrogen.

[0024] In some implementation schemes, R 1a R 1b R 1c R 1d Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Halogen; p is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4.

[0025] In some implementation schemes, R 1a R 1b R 1c R 1d In, any one (such as R) 1b Or R 1c Selected from hydrogen, sulfonic acid group (-SO3H), Halogen, the rest being hydrogen, or any two (such as R). 1b and R 1c The elements are selected from hydrogen and halogens, with the remainder being hydrogen; where p is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4.

[0026] In some implementation schemes, R 1a R 1b R 1c R 1d Each is independently selected from hydrogen, sulfonic acid group (-SO3H), -F.

[0027] In some implementation schemes, R 1a R 1b R 1c R 1d In, any one (such as R) 1b Or R 1c Selected from hydrogen, sulfonic acid group (-SO3H), -F, the rest are hydrogen, or any two (such as R). 1b and R 1c () is selected from hydrogen and -F, and the rest are hydrogen.

[0028] In some implementation schemes, R 1a R 1b R 1c R 1d Each group is independently selected from hydrogen, sulfonic acid group (-SO3H), and -F.

[0029] In some implementation schemes, R 1a R 1b R 1c R 1d In, any one (such as R) 1b Or R 1cThe group is selected from hydrogen, sulfonic acid group (-SO3H), -F, and the remainder is hydrogen, or any two (such as R). 1b and R 1c () is selected from hydrogen and -F, and the rest are hydrogen.

[0030] In some implementation schemes, R 1e Selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group is replaced by )

[0031] In some implementation schemes, R 1e The group is selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), sulfonic acid (-SO3H), wherein the C1-C6 alkyl is optionally replaced by a group selected from carboxyl (-COOH) or sulfonic acid (-SO3H).

[0032] In some implementation schemes, R 1e Selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, with a preference for 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, with a preference for 1, 2, 3.

[0033] In some implementation schemes, R 1e Selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH),

[0034] In some implementation schemes, R 1e Selected from hydrogen,

[0035] In some implementation schemes, R 1f Selected from C1-C6 alkyl and carboxylate ions (-COO) - ), sulfonate ions (-SO3) - The C1-C6 alkyl group is selected from carboxylate ions (-COO). - ), sulfonate ions (-SO3) - The group is replaced by )

[0036] In some implementation schemes, R 1f Selected from C1-C6 alkyl, sulfonate ions (-SO3) -The C1-C6 alkyl group is surrounded by sulfonate ions (-SO3). - ) replaced.

[0037] In some implementation schemes, R 1f Selected from sulfonate ions (-SO3) - ), Carboxylate ion (-COO) - ), q is selected from 1, 2, 3, 4, 5, 6, with 2, 3, 4 being preferred; t is selected from 1, 2, 3, 4, 5, 6, with 1, 2, 3 being preferred.

[0038] In some implementation schemes, R 1f Selected from sulfonate ions (-SO3) - ), Carboxylate ion (-COO) - ), In some implementation schemes, R 1f Selected from sulfonate ions (-SO3) - ),

[0039] In some implementation schemes, R 1f for

[0040] In some implementation schemes, R 1g R 1h Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group is replaced by )

[0041] In some implementation schemes, R 1g R 1h Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H).

[0042] In some implementation schemes, R 1g R 1h Each is independently selected from hydrogen, C1-C6 alkyl, and sulfonic acid group (-SO3H), wherein the C1-C6 alkyl is optionally replaced by sulfonic acid group (-SO3H).

[0043] In some implementation schemes, R 1g R 1h Each is independently selected from hydrogen, sulfonic acid group (-SO3H), p is selected from 1, 2, 3, 4, 5, 6, with 2, 3, 4 being the preferred selection.

[0044] In some implementation schemes, R 1g R 1h Each is independently selected from hydrogen, sulfonic acid group (-SO3H),

[0045] In some implementation schemes, R 1i Selected from C1-C6 alkyl and carboxylate ions (-COO) - ), sulfonate ions (-SO3) - The C1-C6 alkyl group is selected from carboxylate ions (-COO). - ), sulfonate ions (-SO3) - The group is replaced by )

[0046] In some implementation schemes, R 1i Selected from sulfonate ions (-SO3) - ), Carboxylate ion (-COO) - ), q is selected from 1, 2, 3, 4, 5, 6, with 2, 3, 4 being preferred; t is selected from 1, 2, 3, 4, 5, 6, with 1, 2, 3 being preferred.

[0047] In some implementation schemes, R 1i Selected from sulfonate ions (-SO3) - ), Carboxylate ion (-COO) - ), In some implementation schemes, R 1i for

[0048] In some implementation schemes, R 1j R 1k Each is independently selected from C1-C6 alkyl and carboxylate ions (-COO). - ), sulfonate ions (-SO3) - The C1-C6 alkyl group is selected from carboxylate ions (-COO). - ), sulfonate ions (-SO3) - The group is replaced by )

[0049] In some implementation schemes, R 1j R 1kEach is independently selected from sulfonate ions (-SO3-) - ), Carboxylate ion (-COO) - ), q is selected from 1, 2, 3, 4, 5, 6, with 2, 3, 4 being preferred; t is selected from 1, 2, 3, 4, 5, 6, with 1, 2, 3 being preferred.

[0050] In some implementation schemes, R 1j R 1k Each is independently selected from sulfonate ions (-SO3-) - ), Carboxylate ion (-COO) - ),

[0051] In some implementation schemes, R 1’a R 1’b R 1’c Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group is replaced by )

[0052] In some implementation schemes, R 1’a R 1’b R 1’c Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H).

[0053] In some implementation schemes, R 1’a R 1’b R 1’c Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, with a preference for 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, with a preference for 1, 2, 3.

[0054] In some implementation schemes, R 1’a R 1’b R 1’c Each is independently selected from hydrogen, sulfonic acid group (-SO3H), carboxyl

[0055] In some implementation schemes, R 1’a R 1’b R 1’c Each is independently selected from hydrogen, In some implementation schemes, R 1’a R 1’b R 1’c Both are hydrogen.

[0056] In some implementation schemes, R 1l R 1m Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group is replaced by )

[0057] In some implementation schemes, R 1l R 1m Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H).

[0058] In some implementation schemes, R 1l R 1m Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, with a preference for 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, with a preference for 1, 2, 3.

[0059] In some implementation schemes, R 1l R 1m Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH),

[0060] In some implementation schemes, R 1l R 1m Each is independently selected from hydrogen,

[0061] In some implementation schemes, R 1l R1m Each is independently selected from hydrogen,

[0062] In some implementation schemes, R 1l R 1m In, any one (such as R) 1l Selected from hydrogen, The other is hydrogen.

[0063] In some implementation schemes, R 1n R 1o Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group is replaced by )

[0064] In some implementation schemes, R 1n R 1o Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H).

[0065] In some implementation schemes, R 1n R 1o Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, with a preference for 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, with a preference for 1, 2, 3.

[0066] In some implementation schemes, R 1n R 1o Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH),

[0067] In some implementation schemes, R 1n R 1o Each is independently selected from hydrogen,

[0068] In some implementation schemes, R 1n R 1o Both are hydrogen.

[0069] In some implementation schemes, R 1p R 1q R 1r Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group is replaced by )

[0070] In some implementation schemes, R 1p R 1q R 1r Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H).

[0071] In some implementation schemes, R 1p R 1q R 1r Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, with a preference for 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, with a preference for 1, 2, 3.

[0072] In some implementation schemes, R 1p R 1q R 1r Each is independently selected from hydrogen, sulfonic acid group (-SO3H), carboxyl(-

[0073] In some implementation schemes, R 1p R 1q R 1r Each is independently selected from hydrogen, In some implementation schemes, R 1p R 1q R 1r Both are hydrogen.

[0074] In some implementation schemes, R 1s R 1t R 1u R 1vEach is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group is replaced by )

[0075] In some implementation schemes, R 1s R 1t R 1u R 1v Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H).

[0076] In some implementation schemes, R 1s R 1t R 1u R 1v Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, with a preference for 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, with a preference for 1, 2, 3.

[0077] In some implementation schemes, R 1s R 1t R 1u R 1v Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH),

[0078] In some implementation schemes, R 1s R 1t R 1u R 1v Each is independently selected from hydrogen,

[0079] In some implementation schemes, R 1s R 1t R 1u R 1v Both are hydrogen.

[0080] In some implementation schemes, R 1w R 1x R 1yEach is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group is replaced by )

[0081] In some implementation schemes, R 1w R 1x R 1y Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H).

[0082] In some implementation schemes, R 1w R 1x R 1y Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, with a preference for 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, with a preference for 1, 2, 3.

[0083] In some implementation schemes, R 1w R 1x R 1y Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH),

[0084] In some implementation schemes, R 1w R 1x R 1y Each is independently selected from hydrogen,

[0085] In some implementation schemes, R 1w R 1x R 1y Both are hydrogen.

[0086] In some implementation schemes, R 2 R 3 R 4 Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, and halogenated C1-C6 alkyl.

[0087] In some implementation schemes, R 2 R 3 R 4 Each is independently selected from hydrogen, C1-C6 alkyl, and halogenated C1-C6 alkyl.

[0088] In some implementation schemes, R 2 Selected from hydrogen, C1-C6 alkyl, halo-C1-C6 alkyl, R 3 R 4 It is hydrogen.

[0089] In some implementation schemes, R 2 R 3 R 4 Each is independently selected from hydrogen, methyl, and trifluoromethyl.

[0090] In some implementation schemes, R 2 Selected from hydrogen, methyl, trifluoromethyl, R 3 R 4 It is hydrogen.

[0091] In some implementation schemes, R 2 R 3 R 4 It is hydrogen.

[0092] In some implementation schemes, R 5a R 5b Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl.

[0093] In some implementation schemes, R 5a R 5b Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, and C1-C6 alkyl.

[0094] In some implementation schemes, R 5a R 5b Each is independently selected from hydrogen and C1-C6 alkyl groups.

[0095] In some implementation schemes, R 5a R 5b Each is independently selected from hydrogen and methyl.

[0096] In some implementation schemes, R 5a R 5b It is hydrogen.

[0097] In some implementation schemes, R 6a R 6bEach is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl.

[0098] In some implementation schemes, R 6a R 6b Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, and C1-C6 alkyl.

[0099] In some implementation schemes, R 6a R 6b Each is independently selected from hydrogen and C1-C6 alkyl groups.

[0100] In some implementation schemes, R 6a R 6b Each is independently selected from hydrogen and methyl.

[0101] In some implementation schemes, R 6a R 6b It is hydrogen.

[0102] In some embodiments, the compound has the structural formula shown in formula I-(1).

[0103] in:

[0104] R 1-1 Selected from

[0105] Preferably, R 1-1 Selected from

[0106] More preferably, R 1-1 Selected from

[0107] More preferably, R 1-1 Selected from

[0108] Most preferably, R 1-1 Selected from

[0109] R 1a R 1b R 1c R 1’a R 1’b R1’c R 1d R 1e R 1f R 1g R 1h R 1i R 1j R 1k R 2 R 3 R 4 R 5a R 5b R 6a R 6b R 7 Each of , n, and X is independently described as in any of the aforementioned technical solutions.

[0110] In some embodiments, the compound has the structural formula shown in formula I-(2).

[0111] in:

[0112] R 1-2 Selected from

[0113] Preferably, R 1-2 Selected from

[0114] More preferably, R 1-2 Selected from

[0115] More preferably, R 1-2 Selected from

[0116] Most preferably, R 1-2 Selected from

[0117] R 1e R 1f R 1i R 1j R 1k R 1l R 1m R 1n R 1o R 1p R 1q R 1r R 1s R 1t R1u R 1v R 1w R 1x R 1y R 2 R 3 R 4 R 5a R 5b R 6a R 6b R 7 Each of , n, and X is independently described as in any of the aforementioned technical solutions.

[0118] In some embodiments, the compound is selected from:

[0119] In a second aspect, the present invention provides an intermediate having the structural formula shown in Formula Ii.

[0120] in:

[0121] R 2 R 3 R 4 R 5a R 5b R 6a R 6b Each of and n is independently described as in any of the technical solutions of the first aspect;

[0122] R 8 Selected from -SO3H, -C(=O)-O-C1-C6 alkyl groups, preferably selected from -SO3H,

[0123] In some implementations, the intermediate is selected from:

[0124] In a third aspect of the invention, the invention provides a modified nucleoside, nucleotide, or oligonucleotide comprising a nucleoside, nucleotide, or oligonucleotide, and a detectable label; wherein the nucleoside, nucleotide, or oligonucleotide is linked to the detectable label, and the detectable label is a compound or a stereoisomer, tautomer, crystal form, salt, ester, or solvate described in any of the technical solutions of the first aspect.

[0125] In some embodiments, the nucleoside, nucleotide, or oligonucleotide is linked to the detectable labeled carboxyl group (-COOH) or sulfonic acid group (-SO3H).

[0126] In some embodiments, the nucleoside, nucleotide, or oligonucleotide comprises a base, a denitrogenated base, or a tautomer thereof, wherein the base, denitrogenated base, or tautomer thereof is linked to the detectable labeled carboxyl group (-COOH) or sulfonic acid group (-SO3H); preferably, the base is selected from adenine, thymine, uracil, cytosine, and guanine; preferably, the denitrogenated base is selected from 7-denitroadenine and 7-denitroguanine; preferably, the nucleoside, nucleotide, or oligonucleotide comprises The The first site is linked to the detectable labeled carboxyl group (-COOH) or sulfonic acid group (-SO3H); preferably, the nucleoside, nucleotide, or oligonucleotide further comprises ribose or deoxyribose. The 2nd site is connected to the 1'-carbon atom of the ribose or deoxyribose.

[0127] In some embodiments, the modified nucleoside, nucleotide, or oligonucleotide further comprises a linker, which is linked to the detectable label via the linker. The detectable label is a compound or its stereoisomer, tautomer, crystal form, salt, ester, or solvate as described in any of the first aspects.

[0128] In some embodiments, the nucleoside, nucleotide, or oligonucleotide is linked to the detectable labeled carboxyl group (-COOH) or sulfonic acid group (-SO3H) via a linker.

[0129] In some embodiments, the nucleoside, nucleotide, or oligonucleotide comprises a base, a denitrogenated base, or a tautomer thereof, which is linked to the detectable labeled carboxyl group (-COOH) or sulfonic acid group (-SO3H) via a linker; preferably, the base is selected from adenine, thymine, uracil, cytosine, and guanine; preferably, the denitrogenated base is selected from 7-denitroadenine and 7-denitroguanine; preferably, the denitrogenated base is selected from 7-denitroadenine and 7-denitroguanine; preferably, the nucleoside, nucleotide, or oligonucleotide comprises The The 1st site is connected to the linker; preferably, the nucleoside, nucleotide, or oligonucleotide further comprises ribose or deoxyribose. or The 2nd site is connected to the 1'-carbon atom of the ribose or deoxyribose.

[0130] In some embodiments, the nucleoside, nucleotide, or oligonucleotide further comprises a reversible blocking group; preferably, the reversible blocking group is attached to the 3'-OH of the ribose or deoxyribose contained in the nucleoside, nucleotide, or oligonucleotide.

[0131] In some embodiments, the modified nucleotide has a structure selected from the following:

[0132] Where T stands for -LN, L is a linker, and N is a nucleotide;

[0133] Preferably, the connector is

[0134] Preferably, the nucleotides are selected from: Among them, R 0 It is a reversible blocking group;

[0135] Preferably, the reversible blocking group is

[0136] In some embodiments, the modified nucleotide has a structure selected from the following:

[0137] In a fourth aspect of the invention, the invention provides a kit comprising a first nucleotide linked to a first detectable label, the first detectable label being a compound or a stereoisomer, tautomer, crystal form, salt, ester or solvate thereof as described in any of the technical solutions of the first aspect.

[0138] In some embodiments, the kit further comprises a first nucleotide linked to a second detectable label, the second detectable label being a compound or its stereoisomer, tautomer, crystal form, salt, ester, or solvate that is different from the first detectable label.

[0139] In some embodiments, the kit further comprises a second nucleotide linked to a first detectable label, wherein the first detectable label is a compound or a stereoisomer, tautomer, crystal form, salt, ester or solvate thereof described in any of the first aspects.

[0140] In some embodiments, the kit further comprises a third nucleotide linked to a second detectable label, the second detectable label being a compound or its stereoisomer, tautomer, crystal form, salt, ester, or solvate that is different from the first detectable label.

[0141] In some implementations, the kit also contains a fourth nucleotide not linked to a detectable label.

[0142] In some implementations, a first nucleotide with a first detectable label and a second nucleotide with the first detectable label can be excited by a first light source wavelength, a first nucleotide with a second detectable label and a third nucleotide with the second detectable label can be excited by a second light source wavelength, and a fourth nucleotide without a detectable label cannot be excited by the first light source wavelength and the second light source wavelength.

[0143] In some embodiments, the kit further comprises a polymerase for carrying out nucleotide polymerization reactions and one or more buffer solutions.

[0144] In a fifth aspect, the present invention provides a method for determining the sequence of a target polynucleotide, comprising incorporating a modified nucleotide as described in any of the third aspects into the complementary sequence of the target polynucleotide.

[0145] In some embodiments, the method further includes detecting a detectable marker contained in the modified nucleotide.

[0146] In some embodiments, the method for determining the sequence of the target polynucleotide is performed on an automated sequencing instrument, wherein the automated sequencing instrument includes an excitation source; preferably, the automated sequencing instrument includes two excitation sources operating at different wavelengths.

[0147] In some implementations, the method includes:

[0148] (A) Incorporating at least one modified nucleotide as described in any of the third aspects into the complementary sequence of the target polynucleotide; and

[0149] (B) The type of incorporated nucleotide is determined by detecting the fluorescent signal of the detectable label contained in the modified nucleotide.

[0150] In some implementations, the method includes:

[0151] (a) Provides a variety of different nucleotides, wherein at least one nucleotide is a modified nucleotide as described in any of the technical solutions of the third aspect;

[0152] (b) Incorporating the various different nucleotides into the complementary sequence of the target polynucleotide, wherein the various different nucleotides can be distinguished from each other during detection;

[0153] (c) Detect the nucleotides in (b) to determine the type of incorporated nucleotides;

[0154] (d) Remove detectable markers and reversible blocking groups from the nucleotides in (b); and

[0155] (e) Optionally repeat steps (a)-(d) once or more;

[0156] This allows for the determination of the sequence of the target polynucleotide;

[0157] Preferably, the detectable marker and reversible blocking group are removed using an excision reagent;

[0158] Preferably, the excision reagent for removing the detectable marker and the reagent for removing the reversible blocking group are the same;

[0159] Preferably, the removal of the detectable marker and the removal of the reversible blocking group are performed simultaneously.

[0160] In some implementations, the method includes the following steps:

[0161] (1) Provide a first nucleotide, a second nucleotide, a third nucleotide and a fourth nucleotide, wherein at least one of the four nucleotides is a modified nucleotide as described in any of the technical solutions of the third aspect;

[0162] (2) Contact the four nucleotides with the target polynucleotide; remove the nucleotides not incorporated into the grown nucleic acid chain; detect the nucleotides incorporated into the grown nucleic acid chain; remove the reversible blocking groups and detectable labels contained in the nucleotides incorporated into the grown nucleic acid chain;

[0163] Optionally, it also includes (3): repeating (1)-(2) once or more.

[0164] In some implementations, the method includes the following steps:

[0165] (a') Provides a mixture comprising a duplex, a nucleotide comprising at least one modified nucleotide as described in any of the third aspects, a polymerase, and an excision reagent; said duplex comprises a grown nucleic acid strand and a nucleic acid strand to be sequenced;

[0166] (b') Perform the reaction comprising the steps (i), (ii) and (iii), optionally, repeating it once or more:

[0167] Step (i): Using a polymerase, the modified nucleotide is incorporated into the growing nucleic acid chain to form a nucleic acid intermediate containing a reversible blocking group and a detectable label:

[0168] Step (ii): Detect the nucleic acid intermediate;

[0169] Step (iii): Use an excision reagent to excise the reversible blocking group and detectable label contained in the nucleic acid intermediate;

[0170] Preferably, the excision reagent used for the excision of the reversible blocking group and the excision of the detectable label is the same reagent.

[0171] In some embodiments, the method further has one or more technical features selected from the following (I)-(IX):

[0172] (I) The double chain is fixed to the support;

[0173] (II) The grown nucleic acid strand is a primer; preferably, the primer is annealed to the nucleic acid strand to be sequenced to form the double strand;

[0174] (III) The double strand, the modified nucleotide, and the polymerase together form a reaction system;

[0175] (IV) Using a polymerase, under conditions that allow the polymerase to perform nucleotide polymerization, the modified nucleotide is incorporated into a growing nucleic acid chain to form a nucleic acid intermediate containing a reversible blocking group and a detectable label.

[0176] (V) Before any step of detecting the nucleic acid intermediate, remove the solution phase of the reaction system of the previous step, leaving the duplexes immobilized on the support;

[0177] (VI) The excision reagent is in contact with the duplex or the grown nucleic acid strand in the reaction system;

[0178] (VII) The excision reagent is capable of excising the reversible blocking groups and detectable labels contained in the modified nucleotides incorporated into the growing nucleic acid chain, without affecting the phosphodiester bonds on the duplex backbone.

[0179] (VIII) After any step of removing the reversible blocking group and the detectable label contained in the nucleic acid intermediate, remove the solution phase of the reaction system of this step;

[0180] (IX) Following step (ii), the method further includes: determining the type of nucleotides modified by the nucleic acid strand incorporated into the growing nucleic acid strand in step (i) based on the signal detected in step (ii), and determining the type of nucleotides at the corresponding positions in the nucleic acid strand to be sequenced based on the base complementary pairing principle.

[0181] In a sixth aspect, the present invention provides the use of the compound or its stereoisomers, tautomers, crystal forms, salts, esters or solvates described in any of the technical solutions of the first aspect in the fields of sequencing, expression analysis, hybridization analysis, gene analysis, RNA analysis, protein binding assay, in vitro diagnostics, immunoassay, and molecular markers.

[0182] In some implementations, the molecular markers are used for cell imaging, tissue imaging, or in vivo biological imaging.

[0183] In a seventh aspect of the invention, the invention provides the use of the compounds described in any of the first aspects, or their stereoisomers, tautomers, crystal forms, salts, esters, or solvates, in the fluorescent labeling, quantification, or detection of proteins, enzymes, or nucleic acids.

[0184] In an eighth aspect of the invention, the invention provides the use of nucleosides, nucleotides or oligonucleotides as described in any of the technical solutions of the third aspect or kits as described in any of the technical solutions of the fourth aspect in sequencing. Beneficial effects

[0185] 1. The pyrrolo[h]coumarin dye synthesized in this invention has a maximum excitation wavelength and emission wavelength in the blue and cyan light wavelength regions. At the maximum excitation wavelength of blue light (e.g., 400nm-490nm), it can produce strong fluorescence and can be used to label nucleic acids for gene sequencing.

[0186] 2. The novel dyes developed in this invention, which label dNTPs in the blue or cyan regions, can help improve sequencing resolution and throughput, and enhance sequencing quality in the field of gene sequencing. Attached Figure Description

[0187] Figure 1: The left figure shows the excitation and emission spectra of dye I-1, and the right figure shows the excitation and emission spectra of dye I-2.

[0188] Figure 2: The left figure shows the excitation and emission spectra of dye I-3, and the right figure shows the excitation and emission spectra of dye I-4.

[0189] Figure 3: The left figure shows the excitation and emission spectra of dye I-5, and the right figure shows the excitation and emission spectra of dye I-6.

[0190] Figure 4: The left figure shows the excitation and emission spectra of dye I-7, and the right figure shows the excitation and emission spectra of dye I-8.

[0191] Figure 5: The left figure shows the excitation and emission spectra of dTTP (compound 7) labeled with dye I-1, and the right figure shows the excitation and emission spectra of dCTP (compound 8) labeled with dye I-1.

[0192] Figure 6: Excitation and emission spectra of dTTP (compound 9) labeled with dye I-8, and excitation and emission spectra of dCTP (compound 10) labeled with dye I-8.

[0193] Figure 7: Sequencing signal of dye I-1 labeled dTTP (compound 7).

[0194] Figure 8: Sequencing signal of dye I-1 labeled dCTP (compound 8).

[0195] Figure 9: Sequencing results of dye I-1 labeled dNTPs. Detailed Implementation

[0196] It should be understood that the terminology used herein is intended to describe specific embodiments and is not intended to be limiting. Furthermore, although any methods, apparatus, and materials similar to or equivalent to those described herein may be used to practice or test the invention, preferred methods, apparatus, and materials are described here.

[0197] In this invention, unless otherwise explicitly stated, the descriptive phrase “...each independently selected” used throughout this document can mean either that the specific options expressed by the same or different symbols in different groups do not affect each other, or that the specific options expressed by the same or different symbols in the same group do not affect each other.

[0198] The substituents in the compounds of this invention are disclosed according to the type or range of groups. In particular, this invention includes every independent sub-combination of the members of these types and ranges. For example, the term "C1-C6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl groups.

[0199] The term "C1-C6 alkyl" refers to an alkyl group having 1 to 6 carbon atoms, preferably "C1-C4 alkyl", more preferably "C1-C3 alkyl", and most preferably "C1-C2 alkyl". Examples of "C1-C6 alkyl" include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl). Examples of "C1-C4 alkyl" include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), and butyl (e.g., n-butyl, isobutyl, tert-butyl). Examples of "C1-C3 alkyl" include methyl, ethyl, and propyl (e.g., n-propyl, isopropyl). Examples of "C1-C2 alkyl" include methyl and ethyl.

[0200] The term “C1-C6 alkoxy” refers to any of the above C1-C6 alkyl groups that are attached to the rest of the molecule by an oxygen atom (-O-), examples of which include methoxy, ethoxy, isopropoxy, etc.

[0201] The term "C1-C6 alkoxy" refers to the group obtained by replacing the oxygen atom in any of the above C1-C6 alkoxy groups with a sulfur atom, such as C1-C6 alkoxy, C1-C4 alkoxy, C1-C3 alkoxy, etc.

[0202] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0203] The term "halogenated C1-C6 alkyl" refers to a group formed by replacing one or more (preferably one) hydrogen atoms of any of the above C1-C6 alkyl groups with halogen atoms (preferably fluorine), such as trifluoromethyl (-CF3).

[0204] The term "C3-C6 cycloalkyl" refers to a saturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 6 carbon atoms, and non-limiting examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0205] The term "C2-C6 alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon group with 2 to 6 carbon atoms and containing one or more carbon-carbon double bonds, such as vinyl, allyl, etc.

[0206] The term "C2-C6 ynyl" refers to a straight-chain or branched aliphatic hydrocarbon group with 2 to 6 carbon atoms and containing one or more carbon-carbon triple bonds, such as ethynyl.

[0207] The term "substituted" refers to the selective substitution of any hydrogen atom or group by a specified group, provided that the substitution does not exceed the normal valence state of the specified atom.

[0208] The term "stereoisomer" refers to a compound with the same molecular structure as the compound of the present invention but a different spatial configuration. When the stereoisomers of the compounds described herein are specifically designated by chemical name as (R)- or (S)-isomers, they should be understood as having a predominant configuration of (R)- or (S)-isomer, respectively. Any asymmetric carbon atom may be present in (R)-, (S)-, or (R, S)- configurations, preferably in the (R)- or (S)- configuration.

[0209] The terms "solvent" or "solvent compound" are used interchangeably to refer to a compound existing in combination with a solvent molecule. This combination may include a stoichiometric amount of a solvent, such as a monohydrate or dihydrate, or may include any amount of water; similarly, methanol or ethanol may form an "alcohol," which may be stoichiometric or non-stoichiometric. As used herein, the term "solvent compound" refers to a solid form, that is, a compound in solution of a solvent that, while solvated, is not a solvate compound as used herein.

[0210] The term "tautomer" refers to compounds with the same molecular structure but different electron configurations. For example, ketones and enols are a common tautomer pair.

[0211] The term "salt" refers to (i) the salt formed by an acidic functional group (e.g., -COOH) present in the compounds provided by this invention and a suitable inorganic or organic cation (base), including but not limited to, alkali metal salts such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts such as calcium salts, magnesium salts, etc.; other metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts such as ammonium salts; organic base salts such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenylethylamine salts, piperazine salts, tetramethylamine salts, and tris(hydroxymethyl)aminomethane salts. (ii) The salts formed by the basic functional group (e.g., -NH2) present in the compounds provided by the present invention and suitable inorganic or organic anions (acids), including but not limited to, hydrohalides such as hydrofluoric acid, hydrochloride, hydrobromide, hydroiodide, etc.; inorganic acid salts such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkyl sulfonates such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; aryl sulfonates such as benzenesulfonates, p-benzenesulfonates, etc.; organic acid salts such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts such as glycine salts, trimethylglycine salts, arginine salts, ornithine salts, glutamate salts, aspartate salts, etc.

[0212] The term "ester" refers to an ester formed by the -COOH group present in the compounds provided by this invention and a suitable alcohol, or an ester formed by the -OH group present in the compounds provided by this invention and a suitable acid (e.g., a carboxylic acid or an oxy-containing inorganic acid). Suitable ester groups include, but are not limited to, formate, acetate, propionate, butyrate, acrylate, ethyl succinate, stearate, or palmitate. Esters can undergo hydrolysis in the presence of acid or base to produce the corresponding acid or alcohol.

[0213] The term "crystal form" refers to the crystal structure of a substance. During crystallization, various factors alter the intramolecular or intermolecular bonding patterns, resulting in different arrangements of molecules or atoms in the crystal lattice, thus forming different crystal structures. The compounds of this invention can exist in one crystal structure or multiple crystal structures, i.e., they exhibit "polymorphism." The compounds of this invention can exist in different crystal forms.

[0214] In the method of this invention, the nucleic acid molecule to be sequenced can be any target nucleic acid molecule. In some preferred embodiments, the nucleic acid molecule to be sequenced comprises deoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, or any combination thereof. In the method of this invention, the nucleic acid molecule to be sequenced is not limited by its type. In some preferred embodiments, the nucleic acid molecule to be sequenced is DNA or RNA. In some preferred embodiments, the nucleic acid molecule to be sequenced can be genomic DNA, mitochondrial DNA, chloroplast DNA, mRNA, cDNA, miRNA, or siRNA. In some preferred embodiments, the nucleic acid molecule to be sequenced is linear or circular. In some preferred embodiments, the nucleic acid molecule to be sequenced is double-stranded or single-stranded. For example, the nucleic acid molecule to be sequenced can be single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), or a hybrid of DNA and RNA. In some preferred embodiments, the nucleic acid molecule to be sequenced is single-stranded DNA. In some preferred embodiments, the nucleic acid molecule to be sequenced is double-stranded DNA.

[0215] In the method of this invention, the nucleic acid molecule to be sequenced is not limited by its source. In some preferred embodiments, the nucleic acid molecule to be sequenced can be obtained from any source, such as any cell, tissue, or organism (e.g., virus, bacteria, fungus, plant, and animal). In some preferred embodiments, the nucleic acid molecule to be sequenced is derived from mammals (e.g., humans, non-human primates, rodents, or canines), plants, birds, reptiles, fish, fungi, bacteria, or viruses.

[0216] Methods for extracting or obtaining nucleic acid molecules from cells, tissues, or organisms are well known to those skilled in the art. Suitable methods include, but are not limited to, ethanol precipitation, chloroform extraction, etc. Detailed descriptions of such methods can be found, for example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Laboratory Molecular Biology, 3rd ed., John Wiley & Sons, Inc., 1995. In addition, a variety of commercially available kits can be used to extract nucleic acid molecules from various sources, such as cells, tissues, or organisms.

[0217] In the method of this invention, the length of the nucleic acid molecule to be sequenced is not limited. In some preferred embodiments, the length of the nucleic acid molecule to be sequenced can be at least 10 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1000 bp, or at least 2000 bp. In some preferred embodiments, the length of the nucleic acid molecule to be sequenced can be 10-20 bp, 20-30 bp, 30-40 bp, 40-50 bp, 50-100 bp, 100-2000 bp, 200-300 bp, 300-400 bp, 400-500 bp, 500-1000 bp, 1000-2000 bp, or more than 2000 bp. In some preferred embodiments, the nucleic acid molecule to be sequenced can have a length of 10-1000 bp to facilitate high-throughput sequencing.

[0218] In the sequencing method of the present invention, a suitable polymerase can be used to perform nucleotide polymerization. In some exemplary embodiments, the polymerase is capable of synthesizing new DNA chains using DNA as a template (e.g., DNA polymerase). In some exemplary embodiments, the polymerase is capable of synthesizing new DNA chains using RNA as a template (e.g., reverse transcriptase). In some exemplary embodiments, the polymerase is capable of synthesizing new RNA chains using either DNA or RNA as a template (e.g., RNA polymerase). Therefore, in some preferred embodiments, the polymerase is selected from DNA polymerase, RNA polymerase, and reverse transcriptase. A suitable polymerase can be selected to perform the nucleotide polymerization reaction as needed. In some preferred embodiments, the polymerization reaction is a polymerase chain reaction (PCR). In some preferred embodiments, the polymerization reaction is a reverse transcription reaction.

[0219] In the method of this invention, KOD polymerase or its mutants can be used for nucleotide polymerization. KOD polymerase or its mutants (e.g., KOD POL151, KOD POL157, KOD POL171, KOD POL174, KOD POL376, KOD POL391) have acceptable polymerization efficiency for the modified nucleosides or nucleotides of this invention. KOD POL391 and KOD POL171 have acceptable polymerization efficiency for the modified nucleotides of this invention. In some embodiments, the polymerization efficiency of KOD POL391 or KOD POL171 for the modified nucleotides of this invention is above 70%, for example, 70%-80%, 80%-90%, or 90%-100%.

[0220] In the method for preparing polynucleotides or the sequencing method of the present invention, the polymerization reaction of nucleotides is carried out under suitable conditions. Suitable polymerization conditions include the composition of the solution phase and the concentration of each component, the pH of the solution phase, and the polymerization temperature. Polymerization under suitable conditions is beneficial for obtaining acceptable or even high polymerization efficiency.

[0221] This invention provides the use of labeled nucleotides containing the dye moiety according to the invention in polynucleotide synthesis sequencing reactions. Synthesis sequencing typically involves the sequential addition of one or more nucleotides or oligonucleotides to a growing polynucleotide chain in the 5' to 3' direction using a polymerase or ligase to form an extended polynucleotide chain complementary to the template / target nucleic acid to be sequenced. The identity of the bases present in one or more added nucleotides can be determined in a detection or "imaging" step. The identity of the added bases can be determined after each nucleotide incorporation step. The sequence of the template can then be inferred using the conventional Watson-Crick base pairing rule.

[0222] Furthermore, nucleotides labeled with the dye compounds of this invention can also be used for sequencing templates on single-molecule arrays. As used herein, the term "single-molecule array" or "SMA" refers to a group of polynucleotide molecules distributed (or arranged) on a solid support, wherein the spacing between any individual polynucleotide and all other polynucleotides in the group makes it possible to individually resolve individual polynucleotide molecules. Therefore, in some embodiments, target nucleic acid molecules immobilized to the surface of a solid support can be resolved optically. This means that one or more distinct signals (each signal representing a polynucleotide) will appear within the resolvable region of the specific imaging device used.

[0223] In achieving single-molecule detection, the spacing between adjacent polynucleotide molecules on the array is at least 100 nm, more specifically at least 250 nm, even more specifically at least 300 nm, and still more specifically at least 350 nm. Therefore, each molecule can be individually resolved and detected as a single-molecule fluorescent spot, and the fluorescence from said single-molecule fluorescent spot also exhibits single-step photobleaching.

[0224] The terms “individually resolved” and “single-molecule resolved” are used herein to define the ability, when visualized, to distinguish one molecule on an array from its neighboring molecules. The spacing between individual molecules on the array will be determined in part by the specific techniques used to resolve the individual molecules. The general characteristics of single-molecule arrays will be understood by reference to publications WO 00 / 06770 and WO 01 / 57248, each of which is incorporated herein by reference.

[0225] While one use of the labeled nucleotides of this invention is for synthetic sequencing reactions, the utility of such nucleotides is not limited to such methods. In fact, the labeled nucleotides described herein can be advantageously used in any sequencing method that requires the detection of fluorescently labeled nucleotides attached to polynucleotides.

[0226] Specifically, nucleotides labeled with the dye compounds of this invention can be used in automated fluorescence sequencing protocols, particularly fluorescent dye-terminator cycle sequencing based on the chain termination sequencing method of Sanger and colleagues. Such methods typically use enzymes and cycle sequencing to incorporate fluorescently labeled dideoxynucleotides into primer extension sequencing reactions. The so-called Sanger sequencing method and related protocols (Sanger type) utilize randomized chain termination with labeled dideoxynucleotides.

[0227] Therefore, the present invention also covers nucleotides labeled with dye compounds that are dideoxynucleotides lacking hydroxyl groups at both the 3' and 2' positions, such modified dideoxynucleotides being suitable for use in Sanger sequencing methods, etc.

[0228] It should be understood that nucleotides labeled with the dye compounds of the present invention, incorporating 3'-terminal groups, can also be used in the Sanger method and related schemes, because the same effect can be achieved by using nucleotides with 3'-OH-terminal groups as by using dideoxynucleotides: both prevent subsequent nucleotide incorporation. In cases where the nucleotides according to the present invention and having 3'-terminal groups are to be used in Sanger-type sequencing methods, it should be understood that the dye compound or detectable label attached to the nucleotide does not need to be linked via a cleavable linker, because in each case of nucleotides incorporating the labels of the present invention, subsequent nucleotide incorporation is not required, and therefore, removal of the label from the nucleotide is not necessary.

[0229] The present invention will be further explained and described below with reference to specific embodiments. Unless otherwise specified, the reagents and instruments are those that can be routinely purchased or are suitable for use by those skilled in the art.

[0230] Example 1: Synthesis of Dyes

[0231] 1. Reagents and Instruments

[0232] instrument:

[0233] The system consisted of a DIONEX UltiMate 3000 LC-MS / MS system (degasser: SRD-3400, binary pump: HPG-3400RS, autosampler: WPS-3000TRS, column oven: TCC-3000SD, detector: DAD-3000, mass spectrometer: ISQ EM-Mass spectrometer, Thermo Fisher Scientific) and a DIONEX UltiMate 3000 preparative LC-MS system (binary pump: HPG-3200BX, autosampler: WPS-3000TSL, detector: DAD-3000, fraction collector: fraction collector). F, Thermo Fisher Scientific, KQ-300E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), electronic balance (model: BCA324I-10CN, Sartorius Scientific Instruments (Beijing) Co., Ltd.), Biotage automatic column dryer (Bytaiqi Trading (Shanghai) Co., Ltd.), vacuum freeze dryer (Boyikang (Beijing) Instrument Co., Ltd.).

[0234] Reagents:

[0235] 4-Hydroxyindole (Batch No.: DSX373, 98%, Shanghai Bid Pharmaceutical Technology Co., Ltd.), 4-Fluoro-1,2-phenylenediamine (Batch No.: ELL137, 97%, Shanghai Bid Pharmaceutical Technology Co., Ltd.), 1,2-phenylenediamine (Batch No.: V9DUREAB, 98%, Shanghai Saen Chemical Technology Co., Ltd.), o-aminophenol (Batch No.: C14474998, 98%, Shanghai Maclean Biochemical Technology Co., Ltd.), Cycloisopropylmalonate (Batch No.: CTL663, 98%, Shanghai Bid Pharmaceutical Technology Co., Ltd.), 2-Benzimidazolylacetonitrile (Batch No.: DNX899, 98%, Shanghai Bid Pharmaceutical Technology Co., Ltd.) Pharmaceutical Technology Co., Ltd.), ethyl 4-bromobutyrate (batch number: DPU781, 95%, Shanghai Bid Pharmaceutical Technology Co., Ltd.), N,N,N',N'-tetramethyl-1,8-naphthyldiamine (batch number: C13267051, 98%, Shanghai Maclean Biochemical Technology Co., Ltd.), acetonitrile (batch number: WXBD3817V, chromatographic grade, Sigma-Aldrich reagent), N'N-diisopropylethylamine (DIPEA) (batch number: C12677270, 99%, Shanghai Maclean Biochemical Technology Co., Ltd.), anhydrous N'N-dimethylformamide (DMF) (batch number: C14155136, 99%).8%, Shanghai Maclean Biotechnology Co., Ltd.), Hot dTTP-V1 linker dry powder (batch number: HTA022206002, Wuhan BGI Life Science Research Institute), Hot dATP-V1 linker dry powder (batch number: HAA032206001, Wuhan BGI Life Science Research Institute), Hot dCTP-V1 linker dry powder (batch number: HCA032206001, Wuhan BGI Life Science Research Institute), Hot dGTP-V1 Linker dry powder (batch number: HGA032206001, Wuhan BGI Life Science Research Institute), N,N'-disuccinimidyl carbonate (DSC) (batch number: BCCB6748, 95%, Sigma-Aldrich reagent), 4-dimethylaminopyridine (DMAP) (batch number: C12509524, 99%, Shanghai Maclean Biochemical Technology Co., Ltd.), phosphorus oxychloride (batch number: 90KQRVNR, 99%, Shanghai Saen Chemical Technology Co., Ltd.), ethanol (batch number: 20230824, 95%, Shanghai testing), methanol (batch number: 20220125, 95%, Shanghai testing), tetrahydrofuran (THF) (batch number: C12789231, 99%, Shanghai Maclean Biochemical Technology Co., Ltd.), formic acid (FA) (batch number: C13075900, 98%, Shanghai Maclean Biochemical Technology Co., Ltd.), hot dATP-V1-AF532 (batch number: HAA002209004, Wuhan BGI Genomics Technology Co., Ltd.), hot dTTP-V1-AF532 (batch number: HTA002209004, Wuhan BGI Genomics Technology Co., Ltd.).

[0236] The structural formulas of hot dATP-V1-AF532 and hot dTTP-V1-AF532 are shown below:

[0237] 2. Synthetic route

[0238] (1) Synthesis of compound I-1

[0239] Synthesis of Compound 2

[0240] Weigh 0.8 g (6 mmol) of 4-hydroxyindole into a 100 mL round-bottom flask, add 40 mL of acetic acid, dissolve, and cool to 10-15 °C. Add NaBH4 powder (682 mg, 18 mmol) in six portions. After the addition is complete, remove the cold bath and stir at room temperature for 1 h. After the reaction is complete, quench the reaction mixture with saturated NaHCO3 solution at 0-5 °C, adjust the pH to approximately 3-4, extract with ethyl acetate, and purify by silica gel column chromatography to obtain 0.84 g of compound 2. LCMS: calcd for C8H9NO[MH]- :134.0Found,m / z,[MH] - :134.0.

[0241] Synthesis of Compound 3

[0242] In a 250 mL round-bottom flask, weigh compound 2 (5 g, 37 mmol), ethyl 4-bromobutyrate (10.8 g, 55 mmol), NaI (0.85 g, 5.6 mmol), and proton sponge (12 g, 56 mmol) and dissolve them in 100 mL of acetonitrile. Heat under reflux and stir for 48 h. After the reaction is complete, filter the reaction solution, extract with ethyl acetate (200.0 mL * 3), combine the organic phases, wash with saturated sodium chloride solution (200 mL), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to obtain the crude product. Purify by rapid preparative liquid chromatography (0.1 M FA / acetonitrile), concentrate the preparative solution under reduced pressure, and freeze-dry to obtain compound 3. LCMS: calcd for C 14 H 19 NO3[M+H] + :250.14.Found,m / z,[M+H] + :250.14.

[0243] Synthesis of Compound 4

[0244] Under nitrogen protection, place 1.85 g (21 mmol) of anhydrous DMF in a 250 mL three-necked round-bottom flask and stir at 0 °C. Weigh 3.89 g (25 mmol) of POCl3 and add it dropwise to the reaction flask. Continue stirring for 30 min. Weigh compound 3 (5 g (20 mmol) and dissolve it in 50 mL of anhydrous DMF. Add this solution dropwise to the above reaction mixture. After the addition is complete, allow the mixture to react at room temperature for 30 min, then heat to 100 °C and react for another 30 min. Finally, add a small amount of water and stir for 2 h. After the reaction is complete, filter the reaction mixture. Purify the filtrate using rapid preparative liquid chromatography (0.1 M FA / acetonitrile). Concentrate the solution under reduced pressure and freeze-dry to obtain compound 4. LCMS: calcd for C 15 H 19 NO4[M+H] + :278.1.Found,m / z,[M+H] + :278.1.

[0245] Synthesis of compound I-1

[0246] Take a 100 mL round-bottom flask, weigh compound 4 (1 g, 3.6 mmol), and 2-benzimidazolylacetonitrile (0.85 g, 5.4 mmol). Dissolve them in 50 mL of ethanol, add a few drops of piperidine, heat under reflux and stir for 12 h. Then add 20 mL of 2 M hydrochloric acid solution, heat under reflux and stir for 3 h. After the reaction is complete, filter the reaction solution, purify the filtrate by rapid preparative liquid chromatography (0.1 M FA / acetonitrile), concentrate the preparative solution under reduced pressure, and freeze-dry to obtain dye I-1.

[0247] LCMS:calcd for C 22 H 19 N3O4[M+H] + :390.1.Found,m / z,[M+H] + :390.1.

[0248] 1 H NMR (400MHz, DMSO-d6): δ8.87(s,1H),7.65–7.58(m,2H),7.57–7.53(m,1H),7.18(s,2H),7.14–7.11(m,1H),6.64(s,2H),6.61(d,J=8. 4Hz,1H),5.29(t,J=5.0Hz,1H),3.70(t,J=8.9Hz,1H),3.12(t,J=9.0Hz,2H),2.32–2.24(m,2H),1.81–1.73(m,2H),1.46–1.38(m,2H).

[0249] (2) Synthesis of compound I-2

[0250] Synthesis of compound I-2

[0251] Take a 100 mL round-bottom flask, weigh 0.74 g (4.7 mmol) of 2-benzimidazolylacetonitrile and 0.86 g (7.0 mmol) of 1,3-propanesulfonic acid lactone, dissolve them in 50 mL of ethanol, heat to reflux and stir for 3 h. Then, add compound 4 (1 g (3.13 mmol) and a few drops of piperidine, heat to 140 °C and stir for 12 h. After the reaction is complete, filter the reaction solution, and purify the filtrate by rapid preparative liquid chromatography (0.1 M FA / acetonitrile) to obtain the preparative solutions of compound I-2. Concentrate the preparative solutions under reduced pressure and freeze-dry to obtain compound I-2.

[0252] LCMS:calcd for C 25 H 25 N3O7S[M+H] + :512.14.Found,m / z,[M+H]+ :512.15.

[0253] 1 H NMR (400MHz, DMSO-d6): δ8.20(s,1H),7.65(d,J=7.4Hz,1H),7.61(d,J=8.7H z,1H),7.50(d,J=8.4Hz,1H),7.25(t,J=8.2Hz,1H),7.20(t,J=7.6Hz,1H),6 .58(d,J=8.4Hz,1H),4.27(t,J=7.5Hz,2H),3.68(t,J=8.9Hz,2H),3.29(t,J =7.2Hz,2H),3.11(t,J=8.9Hz,2H),1.92(p,J=7.4Hz,2H),1.83–1.71(m,2H).

[0254] (3) Synthesis of compound I-3

[0255] Synthesis of compound I-3

[0256] Take a 100 mL round-bottom flask, weigh 0.74 g (4.7 mmol) of 2-benzimidazolylacetonitrile and 1.72 g (14.0 mmol) of 1,3-propanesulfonic acid lactone, dissolve them in 50 mL of ethanol, heat to reflux and stir for 3 h. Then, add compound 4 (1 g (3.13 mmol) and a few drops of piperidine, heat to reflux and stir for 12 h. After the reaction is complete, filter the reaction solution, and purify the filtrate by rapid preparative liquid chromatography (0.1 M FA / acetonitrile) to obtain the preparative solution of compound I-3. Concentrate the preparative solution under reduced pressure and freeze-dry to obtain compound I-3.

[0257] LCMS:calcd for C 25 H 31 N3O 10 S2[M+H] + :634.1.Found,m / z,[M+H] + :634.1.

[0258] 1H NMR (400MHz, DMSO-d6): δ8.41(s,1H),8.19(dd,J=6.3,3.2Hz,2H),7.72(dd,J=6.3,3.1Hz,2H),7.51(d,J=8.5Hz,1H),6.65(d,J=8.6Hz, 1H),4.59–4.50(m,4H),3.77(t,J=8.9Hz,2H),3.36(t,J=7.1Hz,4H),3.14(t,J=8.9Hz,2H),2.04(p,J=6.7Hz,4H),1.78(p,J=7.7Hz,2H).

[0259] (4) Synthesis of compound I-4:

[0260] Take a 100 mL round-bottom flask, weigh compound 4 (1 g, 3.13 mmol), 4-fluoro-1,2-phenylenediamine (0.59 g, 4.7 mmol), and ethyl cyanoacetate (0.53 g, 4.7 mmol), and dissolve them in 20 mL of n-butanol. Add a few drops of piperidine and glacial acetic acid, heat to 130 °C, and stir under reflux for 12 h. Then, add 20 mL of hydrochloric acid solution, heat under reflux, and stir for 3 h. After the reaction is complete, filter the reaction solution, purify the filtrate by rapid preparative liquid chromatography (0.1 M FA / acetonitrile), concentrate the preparative solution under reduced pressure, and freeze-dry to obtain dye I-4.

[0261] LCMS:calcd for C 22 H 18 FN3O4[MH] - :406.1.Found,m / z,[MH] - :406.1.

[0262] 1H NMR (400MHz, DMSO-d6): δ8.85(d,J=8.2Hz,1H),7.66–7.58(m,1H),7.55(d,J=10.2Hz,1H),7.40 –7.30(m,1H),7.19(s,2H),7.05–6.95(m,1H),6.65(s,2H),6.61(d,J=8.4Hz,1H),5.29(t,J=4. 9Hz,2H),3.71(t,J=9.0Hz,1H),2.64(d,J=1.9Hz,1H),2.40(q,J=7.2Hz,2H),2.22(t,J=6.7Hz, 2H), 1.75 (t, J = 7.4Hz, 2H), 1.43 (d, J = 7.1Hz, 3H), 1.10 (t, J = 7.2Hz, 2H), 0.90 (t, J = 7.1Hz, 2H).

[0263] (5) Synthesis of compound I-5:

[0264] Take a 100 mL round-bottom flask, weigh compound 4 (1 g, 3.13 mmol), 2-aminothiophenol (0.59 g, 4.7 mmol), and ethyl cyanoacetate (0.53 g, 4.7 mmol), and dissolve them in 20 mL of n-butanol. Add a few drops of piperidine and glacial acetic acid, heat to 130 °C and stir for 12 h. Then, add 20 mL of 2 M hydrochloric acid solution, heat under reflux and stir for 3 h. After the reaction is complete, filter the reaction solution, purify the filtrate by rapid preparative liquid chromatography (0.1 M FA / acetonitrile), concentrate the preparative solution under reduced pressure, and freeze-dry to obtain dye I-5.

[0265] LCMS:calcd for C 25 H 18 N₂O₄S[MH] - :405.1.Found,m / z,[MH] - :405.1.

[0266] 1H NMR (400MHz, DMSO-d6): δ8.94(d,J=1.7Hz,1H),8.07(d,J=7.9Hz,1H),7.94(d,J=8.1Hz,1H),7.70(d,J=8.5Hz,1H),7.49(t,J=7.0Hz,1H),7.37 (t,J=7.6Hz,1H),6.63(d,J=8.6Hz,1H),3.73(t,J=8.8Hz,2H),3.11(t,J=8.8Hz,2H),2.25(t,J=7.2Hz,2H),1.76(p,J=7.2Hz,2H),1.20(s,2H).

[0267] (6) Synthesis of compound I-6:

[0268] Take a 100 mL round-bottom flask, weigh compound 4 (1 g, 3.13 mmol), 2-aminophenol (0.59 g, 4.7 mmol), and ethyl cyanoacetate (0.53 g, 4.7 mmol), and dissolve them in 20 mL of n-butanol. Add a few drops of piperidine and glacial acetic acid, heat to 130 °C and stir for 12 h. Then, add 20 mL of 2 M hydrochloric acid solution, heat under reflux and stir for 3 h. After the reaction is complete, filter the reaction solution, purify the filtrate by rapid preparative liquid chromatography (0.1 MFA / acetonitrile), concentrate the preparative solution under reduced pressure, and freeze-dry to obtain dye I-6.

[0269] LCMS:calcd for C 22 H 18 N2O5[MH] - :389.1.Found,m / z,[MH] - :389.1.

[0270] 1 H NMR (400MHz, DMSO-d6): δ11.02(s,1H),8.70(s,1H),8.35(d,J=7.2Hz,1H),7.64(d,J=8.5Hz,1H),6.86(s,2H),6.83–6.72 (m,1H),6.61(d,J=8.5Hz,1H),3.73(t,J=8.9Hz,2H),3.31(t,J=7.1Hz,2H),2.24(t,J=7.2Hz,2H),1.76(q,J=7.2Hz,2H).

[0271] (7) Synthesis of compound I-7:

[0272] Synthesis of Compound 5

[0273] Take a 250 mL round-bottom flask, weigh compound 2 (5 g, 37 mmol), and dissolve 1,3-propanesulfonic acid lactone (6.77 g, 55.5 mmol) in 100 mL acetonitrile. Heat under reflux and stir overnight. After the reaction is complete, purify by rapid preparative liquid chromatography (0.1 M FA / acetonitrile). Concentrate the prepared solution under reduced pressure and freeze-dry to obtain compound 5. LCMS: calcd for C 11 H 15 NO4S[M+H] + :258.1.Found,m / z,[M+H] + :258.1.

[0274] Synthesis of Compound 6

[0275] Under nitrogen protection, place anhydrous DMF (2.94 g, 23 mmol) in a 250 mL three-necked round-bottom flask and stir at 0 °C. Weigh POCl3 (6.5 g, 42 mmol) and add it dropwise to the reaction flask. Continue stirring for 30 min. Weigh compound 5 (5 g, 19.5 mmol) and dissolve it in 50 mL of anhydrous DMF. Add this solution dropwise to the above reaction mixture. After the addition is complete, allow the mixture to react at room temperature for 30 min, then heat to 100 °C and react for another 30 min. Finally, add a small amount of water and stir for 2 h. After the reaction is complete, filter the reaction mixture. Purify the filtrate using rapid preparative liquid chromatography (0.1 M FA / acetonitrile). Concentrate the solution under reduced pressure and freeze-dry to obtain compound 6. LCMS: calcd for C 12 H 15 NO5S[M+H] + :286.1.Found,m / z,[M+H] + :286.1.

[0276] Synthesis of compound I-7

[0277] Take a 100 mL round-bottom flask and weigh out compound 6 (200 mg, 0.6 mmol), ethyl 3-amino-3-thiopropionate (100 mg, 0.68 mmol), and ethyl 4-bromo-3-oxobutyrate (140 mg, 0.67 mmol). Dissolve them in 50 mL of ethanol and heat under reflux for 3 h. Then, add 4 mL of ethanol and 2 mL of water, followed by 20 mL of 2 M hydrochloric acid solution. Heat under reflux and stir for 3 h. After the reaction is complete, filter the reaction solution. Purify the filtrate by rapid preparative liquid chromatography (0.1 M FA / acetonitrile). Concentrate the preparative solution under reduced pressure and freeze-dry to obtain dye I-7.

[0278] LCMS:calcd for C 19 H 18 N2O7S2[MH]- :449.1.Found,m / z,[MH] - :449.0.

[0279] 1 H NMR (400MHz, DMSO-d6): δ8.66(s,1H),7.63(d,J=8.5Hz,1H),7.39(s,1H),6.64(d,J=8.5Hz,1H),3 .68(s,1H),3.39(t,J=7.2Hz,2H),3.09(t,J=8.9Hz,2H),2.46–2.41(m,2H),1.82(p,J=7.3Hz,2H).

[0280] (8) Synthesis of compound I-8:

[0281] Take a 100 mL round-bottom flask, weigh compound 6 (1 g, 3.5 mmol), cycloisopropyl malonate (1.09 g, 5.25 mmol), and ammonium acetate (1.42 g, 9.0 mmol), and dissolve them in 50 mL of water. React at room temperature for 3 h. Then, add 20 mL of 2 M hydrochloric acid solution, heat under reflux and stir for 3 h. After the reaction is complete, filter the reaction solution, purify the filtrate by rapid preparative liquid chromatography (0.1 M FA / acetonitrile), concentrate the preparative solution under reduced pressure, and freeze-dry to obtain dye I-8.

[0282] LCMS:calcd for C 15 H 15 NO7S[MH] - :352.1.Found,m / z,[MH] - :352.0.

[0283] 1 H NMR (400MHz, DMSO-d6): δ8.35(d,J=22.5Hz,1H),7.52–7.42(m,1H),6.58(t,J=9.6Hz,1H),3.72–3.6 0(m,2H),3.37(q,J=6.9Hz,2H),3.03(d,J=8.9Hz,2H),2.44(t,J=6.2Hz,2H),1.82(p,J=7.3Hz,2H).

[0284] Example 2: Synthesis of dye-labeled dNTPs

[0285] (1) Synthesis of fluorescent dye I-1 labeled dTTP (compound 7)

[0286] Take a 10 mL brown reaction flask, weigh compound I-1 (10.0 mg, 0.026 mmol), dissolve it in anhydrous DMF (2 mL), add DSC (13.5 mg, 0.052 mmol) and DMAP (1.6 mg, 0.012 mmol), and stir magnetically for 2 h at room temperature. Then add hot dTTP Linker (48.1 mg, 0.052 mmol) and NaHCO3 (6.5 mg, 0.078 mmol), and stir magnetically for 12 h at room temperature. Filter the reaction solution, and purify the filtrate by preparative HPLC (0.1 M TEAB / acetonitrile). Concentrate the preparative solution under reduced pressure and freeze-dry to obtain compound 7.

[0287] LCMS:calcd for C 50 H 55 N 14 O 22 P3[MH] - :1295.2.Found,m / z,[MH] - :1295.2.

[0288] 1 H NMR(400MHz, DMSO-d6)δ9.09(t,J=5.3Hz,1H),8.98(t,J=5.7Hz,1H),8.84(s,1H), 8.35(t,J=5.6Hz,1H),8.19(s,1H),7.58(s,2H),7.56(s,1H),7.44(d,J=7.8Hz,1H ),7.31(t,J=7.9Hz,1H),7.13(dd,J=6.1,3.1Hz,3H),7.08–7.01(m,1H),6.57(d,J =8.5Hz,1H),6.09(t,J=6.9Hz,1H),5.17(q,J=4.4Hz,1H),4.92–4.77(m,2H),4.47( s,1H),4.22(qd,J=10.9,4.5Hz,2H),4.07(dd,J=7.2,2.9Hz,1H),4.03(d,J=5.8Hz ,2H),3.96(d,J=4.5Hz,2H),3.92–3.85(m,1H),3.85–3.79(m,1H),3.68(s,0H),3. 64(d,J=8.8Hz,3H),3.32(q,J=6.2Hz,2H),3.23(p,J=7.0Hz,3H),3.08(t,J=8.9Hz ,2H),2.38–2.23(m,1H),2.15(t,J=7.2Hz,2H),1.76(q,J=7.9Hz,2H),1.20(s,1H).

[0289] 31 P NMR (162MHz, DMSO-d6) δ -11.10 (d, J = 22.8Hz), -12.27 (d, J = 22.8Hz), -23.49 (t, J = 23.1Hz).

[0290] (2) Synthesis of fluorescent dye I-1 labeled dCTP (compound 8)

[0291] Take a 10 mL brown reaction flask, weigh compound I-1 (10.0 mg, 0.026 mmol), dissolve it in anhydrous DMF (2 mL), add DSC (13.5 mg, 0.052 mmol) and DMAP (1.6 mg, 0.012 mmol), and stir magnetically for 2 h at room temperature. Then add hot dCTP Linker (48.0 mg, 0.052 mmol) and NaHCO3 (6.5 mg, 0.078 mmol), and stir magnetically for 12 h at room temperature. Filter the reaction solution, and purify the filtrate by preparative HPLC (0.1 M TEAB / acetonitrile). Concentrate the preparative solution under reduced pressure and freeze-dry to obtain compound 8.

[0292] LCMS:calcd for C 50 H 56 N 15 O 21 P3[MH] - :1294.3.Found,m / z,[MH] - :1294.2.

[0293] 1H NMR (400MHz, DMSO-d6) δ8.87(q,J=5.4Hz,2H),8.83(s,1H),8.30(t,J=5.5Hz,1H),8.04(s,1H),7.76(s,0H),7.61–7.54(m,2H),7.5 2(s,1H),7.46(d,J=7.7Hz,1H),7.32(t,J=7.9Hz,1H),7.13(dd,J=6.1,3.1Hz,3H),7.05(dd,J=8.1,2.7Hz,1H),6.91(s,1H),6.56(d ,J=8.5Hz,1H),6.10(t,J=6.8Hz,1H),4.92–4.75(m,3H),4.41(d,J=3.1Hz,0H),4.27–4.12(m,2H),4.09(d,J=5.1Hz,2H),4.01(d,J =6.8Hz,1H),3.96(s,2H),3.93–3.78(m,2H),3.70–3.58(m,2H),3.30(t,J=5.8Hz,2H),3.23(q,J=6.5Hz,3H),3.08(t,J=8.9Hz,2H).

[0294] 31 P NMR (162MHz, DMSO-d6) δ -11.19 (d, J = 23.4Hz), -12.04 (d, J = 22.8Hz), -23.62 (t, J = 22.8Hz).

[0295] (3) Synthesis of fluorescent dye I-8 labeled dTTP (compound 9)

[0296] Take a 10 mL brown reaction flask, weigh compound I-8 (10.0 mg, 0.028 mmol), dissolve it in anhydrous DMF (2 mL), add DSC (14.5 mg, 0.056 mmol) and DMAP (1.72 mg, 0.013 mmol), and stir magnetically for 2 h at room temperature. Then add hot dTTP Linker (51.9 mg, 0.056 mmol) and NaHCO3 (7.0 mg, 0.084 mmol), and stir magnetically for 12 h at room temperature. Filter the reaction solution, and purify the filtrate by preparative HPLC (0.1 M TEAB / acetonitrile). Concentrate the preparative solution under reduced pressure and freeze-dry to obtain compound 9.

[0297] LCMS:calcd for C 46 H 57 N 12 O 25 P3S[MH]- :1259.2.Found,m / z,[MH] - :1259.2.

[0298] 1 H NMR (400MHz, DMSO-d6) δ8.75(t,J=5.8Hz,1H),8.71(t,J=5.2Hz,1H),8.66(t,J=5.2Hz ,1H),8.59(s,1H),7.99(s,1H),7.74(s,1H),7.58(d,J=8.5Hz,1H),7.46–7.40(m,2H), 7.35(t,J=8.1Hz,1H),7.15–7.07(m,1H),6.89(s,1H),6.63(d,J=8.6Hz,1H),6.09(dd ,J=8.0,5.8Hz,1H),5.14(t,J=4.8Hz,1H),4.88(d,J=8.9Hz,1H),4.79(d,J=8.8Hz,1H) ,4.42–4.36(m,1H),4.21(dd,J=10.5,4.4Hz,1H),4.16–4.12(m,1H),4.09(d,J=5.2Hz ,1H),3.95(s,2H),3.89(s,0H),3.86–3.79(m,1H),3.70(d,J=8.5Hz,1H),3.65(d,J=2. 7Hz,1H),3.06(t,J=9.0Hz,2H),2.46–2.37(m,2H),2.33–2.24(m,1H),2.19–2.09(m,1 H),1.97(q,J=7.0Hz,1H),1.81(p,J=7.2Hz,2H),1.48–1.36(m,1H),0.86–0.78(m,1H).

[0299] 31 P NMR (162MHz, DMSO-d6) δ -10.66 (d, J = 19.7Hz), -12.87 (d, J = 24.0Hz), -23.20 (dd, J = 24.3, 20.0Hz).

[0300] (4) Synthesis of fluorescent dye I-8 labeled dCTP (compound 10)

[0301] Take a 10 mL brown reaction flask, weigh compound I-8 (10.0 mg, 0.028 mmol), dissolve it in anhydrous DMF (2 mL), add DSC (14.5 mg, 0.056 mmol) and DMAP (1.72 mg, 0.013 mmol), and stir magnetically for 2 h at room temperature. Then add hot dCTP Linker (51.9 mg, 0.056 mmol) and NaHCO3 (7.0 mg, 0.084 mmol), and stir magnetically for 12 h at room temperature. Filter the reaction solution, and purify the filtrate by preparative HPLC (0.1 M TEAB / acetonitrile). Concentrate the preparative solution under reduced pressure and freeze-dry to obtain compound 10.

[0302] LCMS:calcd for C 43 H 52 N 13 O 24 P3S[MH] - :1258.2.Found,m / z,[MH] - :1258.2.

[0303] 1 H NMR(400MHz, DMSO-d6)δ8.77(q,J=5.2Hz,2H),8.68(t,J=5.4Hz,1H),8.54(s,2H) ,8.00(s,1H),7.75(s,1H),7.44(s,1H),7.42(s,1H),7.35(s,1H),7.10(dd,J=8.2 ,3.7Hz,1H),6.90(s,1H),6.46(s,2H),6.09(dd,J=8.0,5.7Hz,1H),5.13(t,J=4. 8Hz,1H),4.88(d,J=8.8Hz,1H),4.80(d,J=8.9Hz,1H),4.43–4.38(m,1H),4.22(d, J=4.3Hz,0H),4.19(s,0H),4.14(d,J=5.3Hz,1H),4.09(d,J=5.2Hz,2H),3.92–3. 87(m,1H),3.85–3.78(m,1H),3.66(d,J=6.7Hz,2H),3.47(q,J=6.1Hz,2H),3.42–3 .35(m,3H),3.01(t,J=7.7Hz,2H),2.43(t,J=7.3Hz,2H),2.32–2.25(m,1H),2.19– 2.09(m,1H),1.82(p,J=7.4Hz,2H),1.21(d,J=12.7Hz,1H),0.83(d,J=6.1Hz,1H).

[0304] 31 P NMR(162MHz, DMSO-d6)δ-10.80(d,J=20.3Hz),-12.87(d,J=22.8Hz),-23.05–-23.57(m,J=22.5Hz).

[0305] Test Example 1: Excitation and emission spectra of dyes (such as compounds I-1 to I-26)

[0306] Take an aqueous solution of 1.2 μmol / L of the sample to be tested into a 1 cm quartz cuvette, use DMSO solution as a blank reference, and scan in the wavelength range of 350–750 nm to measure the excitation and emission spectra of the sample. The results are shown in Table 1 and Figures 1-4.

[0307] Table 1: Excitation and emission spectra of dyes

[0308] The above results show that the dyes of the present invention (such as compounds I-1 to I-26) can produce strong fluorescence at the maximum excitation wavelength of blue light.

[0309] Test Example 2: Excitation and emission spectra of dNTPs labeled with dyes (such as compounds I-1 to I-26)

[0310] Take an aqueous solution of 1.2 μmol / L of the sample to be tested into a 1 cm quartz cuvette, use DMSO solution as a blank reference, and scan in the wavelength range of 350–750 nm to measure the excitation and emission spectra of the sample. The results are shown in Table 2 and Figures 5-6.

[0311] Table 2: Excitation and emission spectra of dNTPs labeled with dyes

[0312] The above results show that the dyes of the present invention (such as compounds I-1 to I-26) can produce strong fluorescence at the maximum excitation wavelength of blue light.

[0313] Test Example 3: Sequencing experiments of dNTPs labeled with dyes (such as compounds I-1 to I-26)

[0314] The inventors conducted sequencing experiments using dNTPs labeled with dye I-1 as an example. The other dyes of this invention showed similar excellent sequencing results, indicating that the dyes synthesized in this invention can be used for sequencing and can obtain good sequencing quality, thus having significant application value.

[0315] Using the E.coil v3.0 library, 80 fmol was added. DNB was prepared according to the DNBSEQ-200RS DNB Sample Preparation and Loading Kit and its instructions. The RCA preparation time was 25 min. However, the Hot dNTP mix reagent in the kit was replaced with a reagent prepared using the synthetic dye of this invention. Specifically, dNTPs of dye I-1 were selected, and a 2-color Hot dNTP mix was prepared according to the following double-labeled T (compound 7 & hot dTTP-V1-AF532), hot dATP-V1-AF532, and compound 8, G non-luminescent form. The instrument used was a modified DNBSEQ-200RS sequencer (i.e., the instrument laser was changed to 465nm and 532nm laser excitation) for PE100+10 testing.

[0316] The results are shown in Figures 7-9 and Table 3. When the Hot dNTP mix reagent was prepared using the dyes 7 and 8 synthesized in this invention, it could still be sequenced normally on the modified DNBSEQ-200RS sequencer, and good sequencing quality could be obtained. On this instrument, compounds 7 and 8 could be excited to produce strong signals, with a Q30 of 95.9%, a sequencing read length of 659.2M, and a low error rate of only 0.14%. This indicates that the dyes synthesized in this invention can be used for sequencing and can obtain good sequencing quality, and have great application value.

[0317] Table 3 Sequencing of dNTPs labeled with dye I-1

Claims

1. The compound represented by Formula I or its stereoisomers, tautomers, crystal forms, salts, esters, or solvates, in: R 1 Selected from hydrogen, -COOH, R 1a R 1b R 1c R 1’a R 1’b R 1’c R 1d R 1e R 1f R 1g R 1h R 1i R 1j R 1k R 1l R 1m R 1n R 1o R 1p R 1q R 1r R 1s R 1t R 1u R 1v R 1w R 1x R 1y Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - ), halogen, halogenated C1-C6 alkyl group, wherein the C1-C6 alkyl group is optionally selected from carboxyl group (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group replaced by ) R 2 R 3 R 4 Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl; R 5a R 5b R 6a R 6b Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl; R 7 Selected from -COOH, -SO3H; n is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4, and more preferably from 3; X is selected from O and S, with O being the preferred choice.

2. The compound of claim 1 or its stereoisomers, tautomers, crystal forms, salts, esters, or solvates, wherein, R 1 Selected from hydrogen, -COOH, Preferably, R 1 Selected from hydrogen, -COOH, More preferably, R 1 Selected from hydrogen, -COOH, Most preferably, R 1 Selected from hydrogen, -COOH, 3. The compound or its stereoisomers, tautomers, crystal forms, salts, esters or solvates according to any one of claims 1-2, wherein, R 1a R 1b R 1c R 1d Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - ), halogen, halogenated C1-C6 alkyl group, wherein the C1-C6 alkyl group is optionally selected from carboxyl group (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group replaced by ) Or, R 1a R 1b R 1c R 1d In, any one (such as R) 1b Or R 1c ) or any two (such as R) 1b and R 1c Selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - ), halogen, halogenated C1-C6 alkyl, wherein the C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group is replaced by hydrogen; Preferably, R 1a R 1b R 1c R 1d Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), sulfonic acid (-SO3H), halogen, or halo-C1-C6 alkyl, wherein the C1-C6 alkyl group is optionally substituted by a group selected from carboxyl (-COOH) or sulfonic acid (-SO3H); Or preferably, R 1a R 1b R 1c R 1d In, any one (such as R) 1b Or R 1c ) or any two (such as R) 1b and R 1c The group is selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), sulfonic acid (-SO3H), halogen, and halogenated C1-C6 alkyl, wherein the C1-C6 alkyl is optionally replaced by a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H), and the remainder is hydrogen; More preferably, R 1a R 1b R 1c R 1d Each is independently selected from hydrogen, C1-C6 alkyl, sulfonic acid group (-SO3H), halogen, wherein the C1-C6 alkyl is optionally replaced by sulfonic acid group (-SO3H); Or more preferably, R 1a R 1b R 1c R 1d In, any one (such as R) 1b Or R 1c ) or any two (such as R) 1b and R 1c The group is selected from hydrogen, C1-C6 alkyl, sulfonic acid group (-SO3H), halogen, and the C1-C6 alkyl is optionally replaced by sulfonic acid group (-SO3H), and the remainder is hydrogen; More preferably, R 1a R 1b R 1c R 1d Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Halogen; p is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4; Or more preferably, R 1a R 1b R 1c R 1d In, any one (such as R) 1b Or R 1c Selected from hydrogen, sulfonic acid group (-SO3H), Halogen, the rest being hydrogen, or any two (such as R). 1b and R 1c The components are selected from hydrogen and halogens, with the remainder being hydrogen; where p is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4; More preferably, R 1a R 1b R 1c R 1d Each is independently selected from hydrogen, sulfonic acid group (-SO3H), -F； Or more preferably, R 1a R 1b R 1c R 1d In, any one (such as R) 1b Or R 1c Selected from hydrogen, sulfonic acid group (-SO3H), -F, the rest are hydrogen, or any two (such as R). 1b and R 1c The group is selected from hydrogen and -F, and the rest are hydrogen. Most preferably, R 1a R 1b R 1c R 1d Each is independently selected from hydrogen, sulfonic acid group (-SO3H), and -F; Or, most preferably, R 1a R 1b R 1c R 1d In, any one (such as R) 1b Or R 1c The group is selected from hydrogen, sulfonic acid group (-SO3H), -F, and the remainder is hydrogen, or any two (such as R). 1b and R 1c The group is selected from hydrogen and -F, and the rest are hydrogen. R 1e Selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group replaced by ) Preferably, R 1e The group is selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), sulfonic acid (-SO3H), wherein the C1-C6 alkyl is optionally replaced by a group selected from carboxyl (-COOH) or sulfonic acid (-SO3H); More preferably, R 1e Selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, preferably from 1, 2, 3; More preferably, R 1e Selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), Most preferably, R 1e Selected from hydrogen, R 1f Selected from C1-C6 alkyl and carboxylate ions (-COO) - ), sulfonate ions (-SO3) - The C1-C6 alkyl group is selected from carboxylate ions (-COO). - ), sulfonate ions (-SO3) - The group replaced by ) Preferably, R 1f Selected from C1-C6 alkyl, sulfonate ions (-SO3) - The C1-C6 alkyl group is surrounded by sulfonate ions (-SO3). - Replaced by; Or preferably, R 1f Selected from sulfonate ions (-SO3) - ), Carboxylate ion (-COO) - ), q is selected from 1, 2, 3, 4, 5, 6, with a preference for 2, 3, 4; t is selected from 1, 2, 3, 4, 5, 6, with a preference for 1, 2, 3; More preferably, R 1f Selected from sulfonate ions (-SO3) - ), Carboxylate ion (-COO) - ), More preferably, R 1f Selected from sulfonate ions (-SO3) - ), Most preferably, R 1f for R 1g R 1h Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group replaced by ) Preferably, R 1g R 1h Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H); More preferably, R 1g R 1h Each is independently selected from hydrogen, C1-C6 alkyl, and sulfonic acid group (-SO3H), wherein the C1-C6 alkyl is optionally replaced by sulfonic acid group (-SO3H); More preferably, R 1g R 1h Each is independently selected from hydrogen, sulfonic acid group (-SO3H), p is selected from 1, 2, 3, 4, 5, 6, with 2, 3, 4 being the preferred selection; Most preferably, R 1g R 1h Each is independently selected from hydrogen, sulfonic acid group (-SO3H), R 1i Selected from C1-C6 alkyl and carboxylate ions (-COO) - ), sulfonate ions (-SO3) - The C1-C6 alkyl group is selected from carboxylate ions (-COO). - ), sulfonate ions (-SO3) - The group replaced by ) Preferably, R 1i Selected from sulfonate ions (-SO3) - ), Carboxylate ion (-COO) - ), q is selected from 1, 2, 3, 4, 5, 6, with a preference for 2, 3, 4; t is selected from 1, 2, 3, 4, 5, 6, with a preference for 1, 2, 3; More preferably, R 1i Selected from sulfonate ions (-SO3) - ), Carboxylate ion (-COO) - ), Most preferably, R 1i for R 1j R 1k Each is independently selected from C1-C6 alkyl and carboxylate ions (-COO). - ), sulfonate ions (-SO3) - The C1-C6 alkyl group is selected from carboxylate ions (-COO). - ), sulfonate ions (-SO3) - The group replaced by ) Preferably, R 1j R 1k Each is independently selected from sulfonate ions (-SO3-) - ), Carboxylate ion (-COO) - ), q is selected from 1, 2, 3, 4, 5, 6, with a preference for 2, 3, 4; t is selected from 1, 2, 3, 4, 5, 6, with a preference for 1, 2, 3; Most preferably, R 1j R 1k Each is independently selected from sulfonate ions (-SO3-) - ), Carboxylate ion (-COO) - ), R 1’a R 1’b R 1’c Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group replaced by ) Preferably, R 1’a R 1’b R 1’c Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H); More preferably, R 1’a R 1’b R 1’c Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, preferably from 1, 2, 3; More preferably, R 1’a R 1’b R 1’c Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), More preferably, R 1’a R 1’b R 1’c Each is independently selected from hydrogen, Most preferably, R 1’a R 1’b R 1’c All are hydrogen; R 1l R 1m Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group replaced by ) Preferably, R 1l R 1m Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H); More preferably, R 1l R 1m Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, preferably from 1, 2, 3; More preferably, R 1l R 1m Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), More preferably, R 1l R 1m Each is independently selected from hydrogen, More preferably, R 1l R 1m Each is independently selected from hydrogen, Most preferably, R 1l R 1m In, any one (such as R) 1l Selected from hydrogen, The other is hydrogen; R 1n R 1o Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group replaced by ) Preferably, R 1n R 1o Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H); More preferably, R 1n R 1o Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, preferably from 1, 2, 3; More preferably, R 1n R 1o Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), More preferably, R 1n R 1o Each is independently selected from hydrogen, Most preferably, R 1n R 1o All are hydrogen; R 1p R 1q R 1r Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group replaced by ) Preferably, R 1p R 1q R 1r Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H); More preferably, R 1p R 1q R 1r Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, preferably from 1, 2, 3; More preferably, R 1p R 1q R 1r Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), More preferably, R 1p R 1q R 1r Each is independently selected from hydrogen, Most preferably, R 1p R 1q R 1r All are hydrogen; R 1s R 1t R 1u R 1v Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group replaced by ) Preferably, R 1s R 1t R 1u R 1v Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H); More preferably, R 1s R 1t R 1u R 1v Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, preferably from 1, 2, 3; More preferably, R 1s R 1t R 1u R 1v Each is independently selected from hydrogen, sulfonic acid group (-SO3H), carboxyl(- More preferably, R 1s R 1t R 1u R 1v Each is independently selected from hydrogen, Most preferably, R 1s R 1t R 1u R 1v All are hydrogen; R 1w R 1x R 1y Each is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and carboxylate ion (-COO). - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The C1-C6 alkyl group is optionally selected from carboxyl (-COOH), carboxylate ion (-COO) - ), sulfonic acid group (-SO3H), sulfonate ion (-SO3) - The group replaced by ) Preferably, R 1w R 1x R 1y Each group is independently selected from hydrogen, C1-C6 alkyl, carboxyl (-COOH), and sulfonic acid (-SO3H), wherein the C1-C6 alkyl group is optionally substituted with a group selected from carboxyl (-COOH) and sulfonic acid (-SO3H); More preferably, R 1w R 1x R 1y Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), p is selected from 1, 2, 3, 4, 5, 6, preferably from 2, 3, 4; r is selected from 1, 2, 3, 4, 5, 6, preferably from 1, 2, 3; More preferably, R 1w R 1x R 1y Each is independently selected from hydrogen, sulfonic acid group (-SO3H), Carboxyl group (-COOH), More preferably, R 1w R 1x R 1y Each is independently selected from hydrogen, Most preferably, R 1w R 1x R 1y Both are hydrogen.

4. The compound or its stereoisomers, tautomers, crystal forms, salts, esters or solvates according to any one of claims 1-3, wherein, R 2 R 3 R 4 Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, and halogenated C1-C6 alkyl; Preferably, R 2 R 3 R 4 Each is independently selected from hydrogen, C1-C6 alkyl, and halo-C1-C6 alkyl; Or preferably, R 2 Selected from hydrogen, C1-C6 alkyl, halo-C1-C6 alkyl, R 3 R 4 It is hydrogen; More preferably, R 2 R 3 R 4 Each is independently selected from hydrogen, methyl, and trifluoromethyl; More preferably, R 2 Selected from hydrogen, methyl, trifluoromethyl, R 3 R 4 It is hydrogen; Most preferably, R 2 R 3 R 4 It is hydrogen.

5. The compound according to any one of claims 1-4, or its stereoisomers, tautomers, crystal forms, salts, esters, or solvates, wherein, R 5a R 5b Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl; Preferably, R 5a R 5b Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, and C1-C6 alkyl groups; More preferably, R 5a R 5b Each is independently selected from hydrogen and C1-C6 alkyl groups; More preferably, R 5a R 5b Each is independently selected from hydrogen and methyl; Most preferably, R 5a R 5b It is hydrogen; R 6a R 6b Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C3-C6 cycloalkyl; Preferably, R 6a R 6b Each is independently selected from hydrogen, hydroxyl, cyano, nitro, carboxyl, mercapto, halogen, and C1-C6 alkyl groups; More preferably, R 6a R 6b Each is independently selected from hydrogen and C1-C6 alkyl groups; More preferably, R 6a R 6b Each is independently selected from hydrogen and methyl; Most preferably, R 6a R 6b It is hydrogen.

6. The compound or its stereoisomers, tautomers, crystal forms, salts, esters or solvates according to any one of claims 1-5, wherein, The compound has the structural formula shown in formula I-(1). in: R 1-1 Selected from Preferably, R 1-1 Selected from More preferably, R 1-1 Selected from More preferably, R 1-1 Selected from Most preferably, R 1-1 Selected from R 1a R 1b R 1c R 1’a R 1’b R 1’c R 1d R 1e R 1f R 1g R 1h R 1i R 1j R 1k R 2 R 3 R 4 R 5a R 5b R 6a R 6b R 7 Each of the following is defined independently as described in any one of claims 1-5.

7. The compound or its stereoisomers, tautomers, crystal forms, salts, esters or solvates according to any one of claims 1-5, wherein, The compound has the structural formula shown in formula I-(2). in: R 1-2 Selected from Preferably, R 1-2 Selected from More preferably, R 1-2 Selected from More preferably, R 1-2 Selected from Most preferably, R 1-2 Selected from R 1e R 1f R 1i R 1j R 1k R 1l R 1m R 1n R 1o R 1p R 1q R 1r R 1s R 1t R 1u R 1v R 1w R 1x R 1y R 2 R 3 R 4 R 5a R 5b R 6a R 6b R 7 Each of the following is defined independently as described in any one of claims 1-5.

8. The compound or its stereoisomers, tautomers, crystal forms, salts, esters or solvates according to any one of claims 1-7, wherein, The compound is selected from:

9. An intermediate having the structural formula shown in formula Ii, in: R 2 R 3 R 4 R 5a R 5b R 6a R 6b Each of the two, n, is independently defined as described in any one of claims 1-5; R 8 Selected from -SO3H, -C(=O)-O-C1-C6 alkyl groups, preferably selected from -SO3H, 10. The intermediate of claim 9, wherein the intermediate is selected from:

11. A modified nucleoside, nucleotide, or oligonucleotide comprising a nucleoside, nucleotide, or oligonucleotide, and a detectable label; wherein the nucleoside, nucleotide, or oligonucleotide is linked to the detectable label, and the detectable label is a compound of any one of claims 1-8 or a stereoisomer, tautomer, crystal form, salt, ester, or solvate thereof. Preferably, the nucleoside, nucleotide, or oligonucleotide is linked to the detectable labeled carboxyl group (-COOH) or sulfonic acid group (-SO3H); Preferably, the nucleoside, nucleotide, or oligonucleotide comprises a base, a denitrogenated base, or a tautomer thereof, wherein the base, denitrogenated base, or tautomer thereof is linked to the detectable labeled carboxyl group (-COOH) or sulfonic acid group (-SO3H); preferably, the base is selected from adenine, thymine, uracil, cytosine, and guanine; preferably, the denitrogenated base is selected from 7-denitroadenine and 7-denitroguanine; preferably, the nucleoside, nucleotide, or oligonucleotide comprises The The first site is linked to the detectable labeled carboxyl group (-COOH) or sulfonic acid group (-SO3H); preferably, the nucleoside, nucleotide, or oligonucleotide further comprises ribose or deoxyribose. The 2nd site is connected to the 1'-carbon atom of the ribose or deoxyribose; Preferably, the modified nucleoside, nucleotide, or oligonucleotide further comprises a linker, wherein the nucleoside, nucleotide, or oligonucleotide is linked to the detectable label via the linker, wherein the detectable label is a compound of any one of claims 1-8 or its stereoisomer, tautomer, crystal form, salt, ester, or solvate. Preferably, the nucleoside, nucleotide, or oligonucleotide is linked to the detectable labeled carboxyl group (-COOH) or sulfonic acid group (-SO3H) via a linker; Preferably, the nucleoside, nucleotide, or oligonucleotide comprises a base, a denitrogenated base, or a tautomer thereof, wherein the base, denitrogenated base, or tautomer thereof is linked to the detectable labeled carboxyl group (-COOH) or sulfonic acid group (-SO3H) via a linker; preferably, the base is selected from adenine, thymine, uracil, cytosine, and guanine; preferably, the denitrogenated base is selected from 7-denitroadenine and 7-denitroguanine; preferably, the denitrogenated base is selected from 7-denitroadenine and 7-denitroguanine; preferably, the nucleoside, nucleotide, or oligonucleotide comprises The The 1st site is connected to the linker; preferably, the nucleoside, nucleotide, or oligonucleotide further comprises ribose or deoxyribose. The 2nd site is connected to the 1'-carbon atom of the ribose or deoxyribose; Preferably, the nucleoside, nucleotide, or oligonucleotide further comprises a reversible blocking group; preferably, the reversible blocking group is attached to the 3'-OH of the ribose or deoxyribose contained in the nucleoside, nucleotide, or oligonucleotide.

12. The modified nucleoside, nucleotide, or oligonucleotide of claim 11, wherein, The modified nucleotide has a structure selected from the following: Where T stands for -LN, L is a linker, and N is a nucleotide; Preferably, the connector is Preferably, the nucleotides are selected from: Among them, R 0 It is a reversible blocking group; Preferably, the reversible blocking group is 13. The modified nucleoside, nucleotide, or oligonucleotide according to claim 11 or 12, wherein, The modified nucleotide has a structure selected from the following:

14. A kit comprising a first nucleotide linked to a first detectable label, wherein the first detectable label is a compound of any one of claims 1-8 or a stereoisomer, tautomer, crystal form, salt, ester or solvate thereof; Preferably, the kit further comprises a first nucleotide linked to a second detectable label, wherein the second detectable label is a compound or its stereoisomer, tautomer, crystal form, salt, ester or solvate that is different from the first detectable label; Preferably, the kit further comprises a second nucleotide linked to a first detectable label, wherein the first detectable label is a compound of any one of claims 1-8 or a stereoisomer, tautomer, crystal form, salt, ester or solvate thereof; Preferably, the kit further comprises a third nucleotide linked to a second detectable label, wherein the second detectable label is a compound or its stereoisomer, tautomer, crystal form, salt, ester or solvate that is different from the first detectable label. Preferably, the kit further comprises a fourth nucleotide not linked to a detectable label; Preferably, the first nucleotide with the first detectable label and the second nucleotide with the first detectable label can be excited by the first light source wavelength, the first nucleotide with the second detectable label and the third nucleotide with the second detectable label can be excited by the second light source wavelength, and the fourth nucleotide without the detectable label cannot be excited by the first light source wavelength and the second light source wavelength. Preferably, the kit further comprises: a polymerase for carrying out nucleotide polymerization and one or more buffer solutions.

15. A method for determining the sequence of a target polynucleotide, comprising incorporating the modified nucleotide of any one of claims 11-13 into the complementary sequence of the target polynucleotide; Preferably, the method further includes detecting a detectable marker contained in the modified nucleotide; Preferably, the method for determining the sequence of the target polynucleotide is performed on an automated sequencing instrument, wherein the automated sequencing instrument includes an excitation light source; preferably, the automated sequencing instrument includes two excitation light sources operating at different wavelengths.

16. The method of claim 15, wherein, The method includes: (A) Incorporating at least one modified nucleotide as described in any one of claims 11-13 into the complementary sequence of the target polynucleotide; and (B) The type of incorporated nucleotide is determined by detecting the fluorescent signal of the detectable label contained in the modified nucleotide.

17. The method of claim 15 or 16, comprising: (a) Provides a variety of different nucleotides, wherein at least one nucleotide is a modified nucleotide as described in any one of claims 11-13; (b) Incorporating the various different nucleotides into the complementary sequence of the target polynucleotide, wherein the various different nucleotides can be distinguished from each other during detection; (c) Detect the nucleotides in (b) to determine the type of incorporated nucleotides; (d) Remove detectable markers and reversible blocking groups from the nucleotides in (b); and (e) Optionally repeat steps (a)-(d) once or more; This allows for the determination of the sequence of the target polynucleotide; Preferably, the detectable marker and reversible blocking group are removed using an excision reagent; Preferably, the excision reagent for removing the detectable marker and the reagent for removing the reversible blocking group are the same; Preferably, the removal of the detectable marker and the removal of the reversible blocking group are performed simultaneously.

18. The method according to any one of claims 15-17, comprising the following steps: (1) Provide a first nucleotide, a second nucleotide, a third nucleotide and a fourth nucleotide, wherein at least one of the four nucleotides is a modified nucleotide as described in any one of claims 11-13; (2) Contact the four nucleotides with the target polynucleotide; remove the nucleotides not incorporated into the grown nucleic acid chain; detect the nucleotides incorporated into the grown nucleic acid chain; remove the reversible blocking groups and detectable labels contained in the nucleotides incorporated into the grown nucleic acid chain; Optionally, it also includes (3): repeating (1)-(2) once or more.

19. The method according to any one of claims 15-18, comprising the following steps: (a') Provides a mixture comprising a duplex, a nucleotide comprising at least one of the modifications as described in any one of claims 11-13, a polymerase, and an excision reagent; said duplex comprising a grown nucleic acid strand and a nucleic acid strand to be sequenced; (b') Perform the reaction comprising the steps (i), (ii) and (iii), optionally, repeating it once or more: Step (i): Using a polymerase, the modified nucleotide is incorporated into the growing nucleic acid chain to form a nucleic acid intermediate containing a reversible blocking group and a detectable label: Step (ii): Detect the nucleic acid intermediate; Step (iii): Use an excision reagent to excise the reversible blocking group and detectable label contained in the nucleic acid intermediate; Preferably, the excision reagent used for the excision of the reversible blocking group and the excision of the detectable label is the same reagent.

20. The method according to claim 19, wherein, The method further comprises one or more of the following technical features (I)-(IX): (I) The double chain is fixed to the support; (II) The grown nucleic acid strand is a primer; preferably, the primer is annealed to the nucleic acid strand to be sequenced to form the double strand; (III) The double strand, the modified nucleotide, and the polymerase together form a reaction system; (IV) Using a polymerase, under conditions that allow the polymerase to perform nucleotide polymerization, the modified nucleotide is incorporated into a growing nucleic acid chain to form a nucleic acid intermediate containing a reversible blocking group and a detectable label. (V) Before any step of detecting the nucleic acid intermediate, remove the solution phase of the reaction system of the previous step, leaving the duplexes immobilized on the support; (VI) The excision reagent is in contact with the duplex or the grown nucleic acid strand in the reaction system; (VII) The excision reagent is capable of excising the reversible blocking groups and detectable labels contained in the modified nucleotides incorporated into the growing nucleic acid chain, without affecting the phosphodiester bonds on the duplex backbone. (VIII) After any step of removing the reversible blocking group and the detectable label contained in the nucleic acid intermediate, remove the solution phase of the reaction system of this step; (IX) Following step (ii), the method further includes: determining the type of nucleotides modified by the nucleic acid strand incorporated into the growing nucleic acid strand in step (i) based on the signal detected in step (ii), and determining the type of nucleotides at the corresponding positions in the nucleic acid strand to be sequenced based on the base complementary pairing principle.

21. Use of the compound of any one of claims 1-8 or its stereoisomers, tautomers, crystal forms, salts, esters or solvates in the fields of sequencing, expression analysis, hybridization analysis, gene analysis, RNA analysis, protein binding assay, in vitro diagnostics, immunoassay, and molecular markers; Preferably, the molecular marker is used for cell imaging, tissue imaging, or in vivo biological imaging.

22. Use of the compound of any one of claims 1-8 or its stereoisomers, tautomers, crystal forms, salts, esters or solvates in the fluorescent labeling, quantification or detection of proteins, enzymes or nucleic acids.

23. Use of the nucleoside, nucleotide, or oligonucleotide of any one of claims 11-13 or the kit of claim 14 in sequencing.

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