Fluorescent dye containing fused tetracyclic diboron heterocycle and application of fluorescent dye in sequencing

Abstract

The present application relates to dyes containing fused tetracyclic diboron-containing heterocycles and their use as fluorescent labels. These dyes can be used as fluorescent labeling of nucleotides in nucleic acid sequencing applications.

Description

Technical Field

[0001] The present application relates to dyes containing fused tetracyclic biboron heterocycles and their use as fluorescent labels in nucleic acid sequencing applications. Background Art

[0002] Nonradioactive detection of fluorescently labeled nucleic acids is an important technique in molecular biology. Many procedures employed in recombinant DNA technology have previously relied on the use of radioactively labeled nucleotides or polynucleotides, e.g. 32 P. Radioactive compounds allow sensitive detection of nucleic acids and other molecules of interest. However, there are serious limitations in the use of radioisotopes, such as their expense, limited shelf life, insufficient sensitivity, and more importantly, safety considerations. Eliminating the need for radiolabeling reduces both safety risks and environmental impacts and costs associated with, for example, reagent handling. Methods suitable for non-radioactive fluorescence detection include, by way of non-limiting example, automated DNA sequencing, hybridization methods, real-time detection of polymerase chain reaction products, and immunoassays.

[0003] For many applications, it is desirable to employ multiple spectrally distinguishable fluorescent labels in order to achieve independent detection of multiple spatially overlapping analytes. In such multiplex methods, the number of reaction vessels can be reduced, thereby simplifying the experimental protocol and facilitating the production of specialized kits. For example, in multicolor automated DNA sequencing systems, multiplex fluorescence detection allows the analysis of multiple nucleotide bases in a single electrophoretic channel, thereby increasing throughput over a monochromatic approach and reducing the uncertainty associated with inter-channel electrophoretic mobility variations.

[0004] However, multiple fluorescence detection may be problematic, and many important factors present can limit the selection of appropriate fluorescent labels. First, it may be difficult to find dye compounds with substantially resolved absorption and emission spectra in a given application. In addition, when several fluorescent dyes are used together, it may be complicated to produce fluorescent signals in distinguishable spectral regions by simultaneously exciting, because the absorption bands of these dyes are usually very separate, so even two dyes are also difficult to reach a suitable fluorescence excitation efficiency. Many excitation methods use high-power light sources, such as lasers, so dyes must have enough light stability to withstand such excitations. The last consideration that is particularly important to molecular biology methods is the degree to which fluorescent dyes must be compatible with reagent chemistry, such as DNA synthesis solvents and reagents, buffer, polymerase and ligase.

[0005] As sequencing technology progresses, there is a need to develop additional fluorescent dye compounds, nucleic acid conjugates thereof, and multiple dye sets that meet all of the above limitations and are particularly suitable for high-throughput molecular methods (such as solid-phase sequencing, etc.).

[0006] Fluorescent dye molecules with improved fluorescence properties (such as the wavelength maximum of suitable fluorescence intensity, shape and fluorescence band) can improve the speed and accuracy of nucleic acid sequencing. When measured in water-based biological buffer and at higher temperatures, strong fluorescent signals are particularly important because the fluorescence intensity of most organic dyes is significantly lower under such conditions. In addition, the property of the base to which the dye is attached also affects other spectral characteristics of fluorescence maximum, fluorescence intensity and dye. The sequence-specific interaction between core base and fluorescent dye can be customized by the specific design of fluorescent dye. The optimization of fluorescent dye structure can improve the efficiency of nucleotide incorporation, reduce the level of sequencing errors, and reduce the use of reagents in nucleic acid sequencing, thereby reducing the cost of nucleic acid sequencing.

[0007] Some optical and technological developments have led to great improvements in image quality, but are ultimately limited by poor optical resolution. In general, the optical resolution of optical microscopy is limited to objects spaced at approximately half the wavelength of the light used. In practice, only objects that are quite far apart (at least 200nm to 350nm) can be resolved by optical microscopy. One way to improve image resolution and increase the number of resolvable objects per unit surface area is to use excitation light of a shorter wavelength. For example, if the wavelength of light is shortened by Δλ by about 100nm with the same optics, the resolution will be better (about Δ50nm / (about 15%)), less distorted images will be recorded, and the density of objects on the identifiable area will increase by about 35%.

[0008] Some nucleic acid sequencing methods use lasers to excite dye-labeled nucleotides and detect them. These instruments use longer wavelength light, such as red lasers, together with appropriate dyes that can be excited at 660nm. In order to detect more densely packed nucleic acid sequencing clusters while maintaining available resolution, a shorter wavelength blue light source (450nm to 460nm) can be used. In this case, the optical resolution will not be limited by the emission wavelength of the longer wavelength red fluorescent dye, but by the emission of the dye that can be excited by the secondary long wavelength light source (for example, by the "green laser" at 532nm). Therefore, it is necessary to use blue dye labels for fluorescence detection in sequencing applications.

[0009] Although blue dye chemistry and related laser technology have improved, suitable commercially available blue dyes with strong fluorescence for nucleotide labeling are still quite rare. However, some dyes, especially coumarin dyes, are unstable in aqueous environments over extended periods of time. For example, under alkaline conditions, some coumarin dyes may be easily attacked by nucleophiles, resulting in interference or degradation of the dye. Boron-containing fluorescent dyes such as BODIPY, BOPHY, BOPPY, BOPYPY, BOAHY, and BOPAHY have been described in many scientific literatures and patent publications, such as J Am Chem Soc 2014, 136(15):5623-5626, Organic Letters 2014, 16(11):3048-3051, Organic Letters 2018, 20(15):4462-4466, Chinese Chemical Letters 2019, 30:2271-2273, Organic Letters 2020, 22(12):4588-4592, Chem Communications 2020, 56(43):5791-5794, Chemistry A Eur J 2020, 26(4):863-872, WO 201805044. 2015 / 77427 and CN108516985A. However, designing new boron-containing dyes with appropriate adsorption, good chemical stability and tailored Stokes shift as nucleic acid labels for sequencing applications remains challenging. Summary of the invention

[0010] A new class of dyes containing fused tetracyclic biboron-containing heterocycles are described herein, which have improved chemical stability and strong fluorescence under blue light excitation (e.g., a blue LED or laser, e.g., at about 450 nm to about 460 nm). These dyes also have highly tunable absorption and emission properties suitable for nucleic acid labeling.

[0011] One aspect of the present disclosure relates to compounds of formula (I), salts or meso forms thereof:

[0012]

[0013] Where R 1 , R 2 , R 3 , R 6 , R 7 and R 8Each of the above is independently H, unsubstituted or substituted C1-C6 alkyl, C1-C6 alkoxy, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, (C1-C6 alkoxy)(C1-C6 alkyl), unsubstituted or substituted amino, halo, cyano, carboxyl, hydroxyl, nitro, sulfonyl, sulfinyl, sulfonyl, sulfonate, S-sulfonamido, N-sulfonamido, -C(=O)NR 9 R 10 、-XR 11 , unsubstituted or substituted C3-C 10 Carbocyclic, unsubstituted or substituted C6-C 10 aryl, unsubstituted or substituted 5- to 10-membered heteroaryl, or unsubstituted or substituted 3- to 10-membered heterocyclyl;

[0014] R 4 and R 5 Each of the following is independently H, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C3-C 10 Carbocyclic, unsubstituted or substituted C6-C 10 aryl, unsubstituted or substituted 5- to 10-membered heteroaryl, or unsubstituted or substituted 3- to 10-membered heterocyclyl;

[0015] R 9 and R 10 Each of is independently H, or unsubstituted or substituted C1-C6 alkyl;

[0016] X is -O-, -S-, -CH=CH-, or -C≡C-;

[0017] R 11 Each of which is independently unsubstituted or substituted C3-C 10 Carbocyclic, unsubstituted or substituted C6-C 10 aryl, unsubstituted or substituted 5- to 10-membered heteroaryl, or unsubstituted or substituted 3- to 10-membered heterocyclyl;

[0018] R a , R b , R c and R d Each of the above is independently a halogen group, a cyano group, an unsubstituted C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, a C1-C6 haloalkoxy group, an unsubstituted or substituted C6-C 10 Aryl, unsubstituted or substituted C6-C 10 Aryloxy or -OC(=O)R 12 ;and

[0019] R 12 is an unsubstituted or substituted C1-C6 alkyl, an unsubstituted or substituted C2-C6 alkenyl, an unsubstituted or substituted C6-C 10 aryl or unsubstituted or substituted 5- to 10-membered heteroaryl;

[0020] Alternatively, when R a and R b All are -OC(=O)R 12 When two R 12 Together with the atoms to which they are attached, they form an unsubstituted or substituted 6- to 10-membered heterocyclyl;

[0021] Alternatively, when R c and R d All are -OC(=O)R 12 When two R 12 Together with the atoms to which they are attached, they form an unsubstituted or substituted 6- to 10-membered heterocyclyl;

[0022] The prerequisite is R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 At least one of the comprises a carboxyl group.

[0023] In some aspects, the compounds of the present disclosure are labeled with or conjugated to substrate moieties such as nucleosides, nucleotides, polynucleotides, polypeptides, carbohydrates, ligands, particles, cells, semisolid surfaces (e.g., gels), or solid surfaces. Labeling or conjugation can be via a carboxyl group, which can react with an amino or hydroxyl group on a moiety (such as a nucleotide) or a linker bound thereto to form an amide or ester using methods known in the art.

[0024] Another aspect of the present disclosure relates to dye compounds comprising a linker group to achieve, for example, covalent attachment to a substrate moiety (such as a nucleotide). The attachment can be made at any position of the dye. In some embodiments, the attachment can be via R of formula (I). 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 One of them is carried out.

[0025] Another aspect of the present disclosure provides a labeled nucleoside or nucleotide compound defined by the following formula:

[0026] NL-Dye

[0027] wherein N is a nucleoside or a nucleotide;

[0028] L is an optional linker moiety; and

[0029] The dye is a biboronic heterocycle containing moiety of formula (I) according to the present disclosure, wherein a functional group (e.g., a carboxyl group) of the compound of formula (I) reacts with an amino or hydroxyl group of a linker moiety or a nucleoside / nucleotide to form a covalent bond.

[0030] Some additional aspects of the disclosure relate to oligonucleotides or polynucleotides comprising incorporated therein nucleotides labeled with a compound of formula (I).

[0031] Some other aspects of the present disclosure relate to a test kit comprising dye compounds (free or labeled form) that can be used for various immunological assays, oligonucleotides or nucleic acid markers or for DNA synthesis sequencing. In yet another aspect, the present disclosure provides a test kit comprising a dye "set" that is particularly suitable for synthesis sequencing cycles on an automated instrument platform. In some aspects, a test kit comprising one or more nucleotides, wherein at least one nucleotide is a labeled nucleotide as described herein.

[0032] Another aspect of the present disclosure relates to a method of determining the sequence of a plurality of target polynucleotides, the method comprising:

[0033] (a) contacting a solid support with a solution containing sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;

[0034] (b) contacting the solid support with an aqueous solution comprising a DNA polymerase and one or more of four different types of nucleotides (e.g., comprising dATP, dCTP, dGTP, and dTTP or dUTP, or non-natural nucleotide analogs thereof) under conditions suitable for DNA polymerase-mediated primer extension, wherein at least one type of nucleotide is a labeled nucleotide as described herein, and wherein each of the one or more of the four different types of nucleotides comprises a 3' blocking group;

[0035] (c) incorporating one type of nucleotide into the sequencing primer to produce an extended copy polynucleotide; and

[0036] (d) performing one or more fluorescence measurements on the extended copy polynucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1A and Figure 1B In Illumina with 1Ex-2Ch mode Scatter plot obtained on the instrument at cycle 26 using a spike-in mixture including ffT labeled with the known blue coumarin dye C versus a spike-in mixture including ffT labeled with dye 1-2.

[0038] Figure 2A and Figure 2B In Illumina with 1Ex-2Ch mode Scatter plot obtained on the instrument at cycle 26 using a spike-in mixture including ffT labeled with the known blue coumarin dye C versus a spike-in mixture including ffT labeled with dye I-3.

[0039] Figure 3A and Figure 3B In Illumina with 1Ex-2Ch mode Scatter plot obtained on the instrument at cycle 26 using a spike-in mixture including ffT labeled with the known blue coumarin dye C versus a spike-in mixture including ffT labeled with dye I-8.

[0040] Figure 4A The Illumina with 1Ex-2Ch mode is shown Line graph of the percentage of signal attenuation on the instrument as a function of blue laser dose, comparing the spiked mixture with fft labeled with the known blue coumarin dye C to the spiked mixture with fft labeled with dye I-2. Figure 4B is a line graph showing the percent error rate as a function of blue laser dose for the same sequencing run, comparing fft labeled with coumarin dye A to fft labeled with dye I-2. Figure 4C is a line graph showing the percentage of phasing as a function of blue laser dose for the same sequencing run, comparing fft labeled with coumarin dye A to fft labeled with dye I-2.

[0041] Figure 5A The Illumina with 1Ex-2Ch mode is shown Line graph of the percentage of signal attenuation on the instrument as a function of blue laser dose, comparing a spiked mixture with fft labeled with the known blue coumarin dye C to a spiked mixture with fft labeled with dye I-8. Figure 5B is a line graph showing the percent error rate as a function of blue laser dose for the same sequencing run, comparing fft labeled with coumarin dye A to fft labeled with dye I-8. Figure 5Cis a line graph showing the percentage of phasing as a function of blue laser dose for the same sequencing run, comparing fft labeled with coumarin dye A to fft labeled with dye I-8.

[0042] Fig. 6A The Illumina with 1Ex-2Ch mode is shown Line graph of the percentage of signal attenuation on the instrument as a function of blue laser dose, comparing a spiked mixture with fft labeled with the known blue coumarin dye C to a spiked mixture with fft labeled with dye I-9. Figure 6B is a line graph showing the percent error rate as a function of blue laser dose for the same sequencing run, comparing fft labeled with coumarin dye A to fft labeled with dye I-9. Figure 6C is a line graph showing the percentage of phasing as a function of blue laser dose for the same sequencing run, comparing fft labeled with coumarin dye A to fft labeled with dye I-9.

[0043] Figure 7 is a line graph showing the chemical stability of methylated BOPHY in various culture media. DETAILED DESCRIPTION

[0044] Embodiments of the present disclosure relate to dyes containing fused tetracyclic biboron-containing heterocycles having enhanced fluorescence intensity, tunable Stokes shift, and improved chemical stability. In some embodiments, the Stokes shift of the dyes described herein is in the range of about 25 nm to 60 nm, or 40 nm to about 50 nm. The biboron-containing dyes described herein can be used in Illumina sequencing platforms, for example, MiSeq with dual-channel detection and one blue light excitation. TM .

[0045] definition

[0046] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0047] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless expressly and unequivocally limited to one referent. It will be apparent to those skilled in the art that various modifications and variations may be made to the various embodiments described herein without departing from the spirit or scope of the present teachings. Therefore, the various embodiments described herein are intended to cover other modifications and variations within the scope of the appended claims and their equivalents.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The use of the terms "include" and "comprising" is not restrictive. The use of the term "having" is not restrictive. As used in this specification, the terms "include" and "comprising" are to be interpreted as having an open-ended meaning, whether in a transitional phrase or in the body of the claims. That is, the above terms should be interpreted synonymously with the phrases "at least have" or "at least include". For example, when used in the context of a process, the term "comprising" means that the process includes at least the listed steps, but may also include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the listed features or components, but may also include additional features or components.

[0049] As used herein, common organic abbreviations are defined as follows:

[0050] ℃ Temperature in degrees Celsius

[0051] dATP deoxyadenosine triphosphate

[0052] dCTP deoxycytidine triphosphate

[0053] dGTP deoxyguanosine triphosphate

[0054] dTTP deoxythymidine triphosphate

[0055] ddNTP dideoxynucleotide triphosphate

[0056] ffA fully functionalized A nucleotide

[0057] ffC fully functionalized C nucleotide

[0058] ffG fully functionalized G nucleotide

[0059] ffN Fully functionalized nucleotides

[0060] ffT fully functionalized T nucleotide

[0061] h hour

[0062] RT Room temperature

[0063] SBS Sequencing by Synthesis

[0064] USM Universal Scanning Mixture

[0065] As used herein, the term "array" refers to a group of different probe molecules attached to one or more substrates so that these different probe molecules can be distinguished from each other according to relative position. The array may include different probe molecules at different addressable positions located on the substrate. Alternatively or additionally, the array may include a separate substrate with different probe molecules, wherein different probe molecules can be identified according to the position of the substrate on the surface to which the substrate is attached or according to the position of the substrate in the liquid. The exemplary array in which the separate substrate is located on the surface includes but is not limited to those comprising beads in the hole, such as, for example, U.S. Patent No. 6,355,431B1, US2002 / 0102578 and PCT disclose described in WO 00 / 63437. For example, in U.S. Patent No. 6,524,793, it is described that the exemplary form of beads in the liquid array can be used for example using a microfluidic device, such as a fluorescence activated cell sorter (FACS) is distinguished in the present invention. Additional examples of arrays that can be used in the present invention include, but are not limited to, U.S. Pat. Nos. 5,429,807; 5,436,327; 5,561,071; 5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523; 6,136,269; 6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; 6,346,413; 6,416,949; 6,482,591; 6,514,751 and 6,610,482; and WO 93 / 17126; WO 95 / 11995; WO 95 / 35505; EP 742287; and those described in EP 799 897.

[0066] As used herein, the term "covalently attached" or "covalently bonded" refers to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently attached polymer coating refers to a polymer coating that forms a chemical bond with a functionalized surface of a substrate, as opposed to adhering to the surface via other means (e.g., adhesion or electrostatic interactions). It should be understood that a polymer covalently attached to a surface may also be bonded via other means besides covalent attachment.

[0067] As used herein, the term "halogen" or "halo" means any of the radioactive stable atoms of column 7 of the periodic table, for example, fluorine, chlorine, bromine or iodine, with fluorine and chlorine being preferred.

[0068] As used herein, "C" wherein "a" and "b" are integers a To C b" refers to the number of carbon atoms in an alkyl, alkenyl, or alkynyl group, or the number of ring atoms in a cycloalkyl or aryl group. That is, the rings of alkyl, alkenyl, alkynyl, cycloalkyl, and aryl groups can contain from "a" to "b" (inclusive) carbon atoms. For example, a "C1 to C4 alkyl" group refers to all alkyl groups having from 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2 CH2-, CH3CH2CH(CH3)- and (CH3)3C-; C3 to C4 cycloalkyl groups refer to all cycloalkyl groups having 3 to 4 carbon atoms, i.e., cyclopropyl and cyclobutyl. Similarly, a "4 to 6 membered heterocyclyl" group refers to all heterocyclyl groups having 4 to 6 total ring atoms, such as azetidine, oxetane, oxazoline, pyrrolidine, piperidine, piperazine, morpholine, etc. If there is no alkyl, alkenyl, alkynyl, cycloalkyl or aryl group, the alkyl group may be used. If "a" and "b" are specified, the broadest range described in these definitions will be assumed. As used herein, the term "C1-C6" includes C1, C2, C3, C4, C5, and C6, as well as ranges defined by either of these two numbers. For example, C1-C6 alkyl includes C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl, C2-C6 alkyl, C1-C3 alkyl, etc. Similarly, C2-C6 alkenyl includes C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, and C6 alkenyl. C3-C8 cycloalkyl groups include hydrocarbon rings containing 3, 4, 5, 6, 7 and 8 carbon atoms or ranges defined by any two numbers, such as C3-C7 cycloalkyl groups or C5-C6 cycloalkyl groups.

[0069] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., does not contain double bonds and triple bonds). The alkyl group may have 1 to 20 carbon atoms (whenever it appears in this article, a numerical range such as "1 to 20" refers to each integer in the given range; for example, "1 to 20 carbon atoms" means that the alkyl group may be composed of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, but this definition also covers the term "alkyl" in which no numerical range is specified). The alkyl group can also be a medium-sized alkyl group with 1 to 9 carbon atoms. The alkyl group can also be a low alkyl group with 1 to 6 carbon atoms. By way of example only, "C 1-6"Alkyl" or "C1-C6 alkyl" means that there are from one to six carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Typical alkyl groups include, but are by no means limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like.

[0070] As used herein, "alkoxy" refers to a radical of the formula -OR (wherein R is an alkyl radical as defined above), such as "C 1-9 The term "alkoxy" or "C1-C9 alkoxy" includes, but is not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy and tert-butoxy.

[0071] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. An alkenyl group may have from 2 to 20 carbon atoms, but this definition also encompasses the term "alkenyl" where it appears without a specified numerical range. An alkenyl group may also be a medium-sized alkenyl group having from 2 to 9 carbon atoms. An alkenyl group may also be a lower alkenyl group having from 2 to 6 carbon atoms. By way of example only, "C2-C6 alkenyl" or "C 2-6 “Alkenyl” means that there are two to six carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2-dienyl and buta-1,2-dien-4-yl. Typical alkenyl groups include, but are by no means limited to, ethenyl, propenyl, butenyl, pentenyl and hexenyl, etc.

[0072] As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. An alkynyl group may have from 2 to 20 carbon atoms, but this definition also encompasses the term "alkynyl" where no numerical range is specified. An alkynyl group may also be a medium-sized alkynyl group having from 2 to 9 carbon atoms. An alkynyl group may also be a low-sized alkynyl group having from 2 to 6 carbon atoms. By way of example only, "C 2-6 "Alkynyl" or "C2-C6 alkenyl" means that there are two to six carbon atoms in the alkynyl chain, that is, the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl and 2-butynyl. Typical alkynyl groups include, but are by no means limited to, ethynyl, propynyl, butynyl, pentynyl and hexynyl, etc.

[0073] The term "aromatic" refers to a ring or ring system having a conjugated π electron system, and includes both carbocyclic aromatic groups (e.g., phenyl) and heterocyclic aromatic groups (e.g., pyridine). The term includes monocyclic groups or fused-ring polycyclic (i.e., rings that share adjacent pairs of atoms) groups, provided that the entire ring system is aromatic.

[0074] As used herein, "aryl" refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) that contains only carbon in the ring backbone. When aryl is a ring system, each ring in the ring system is aromatic. Aryl groups can have 6 to 18 carbon atoms, but this definition also covers the term "aryl" that appears without a specified numerical range. In some embodiments, aryl groups have 6 to 10 carbon atoms. Aryl groups can be designated as "C6-C 10 Aryl", "C6 or C 10 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.

[0075] "Aralkyl" or "arylalkyl" is an aryl group attached as a substituent through an alkylene group, such as "C 7-14 "Aralkyl" and the like, including but not limited to benzyl, 2-phenylethyl, 3-phenylpropyl and naphthylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., C 1-6 alkylene group).

[0076] As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more condensed rings sharing two adjacent atoms) containing one or more heteroatoms (i.e., elements other than carbon, including but not limited to nitrogen, oxygen and sulfur) in the ring skeleton. When heteroaryl is a ring system, each ring in the ring system is aromatic. The heteroaryl group can have 5 to 18 ring members (i.e., the number of atoms (including carbon atoms and heteroatoms) constituting the ring skeleton), but the present definition also encompasses the term "heteroaryl" in which no numerical range is specified. In some embodiments, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. The heteroaryl group can be named "5 to 7 yuan heteroaryl", "5 to 10 yuan heteroaryl" or similar names. Examples of heteroaryl rings include, but are not limited to, furanyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolyl, isoquinolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothiophenyl.

[0077] "Heteroaralkyl" or "heteroarylalkyl" is a heteroaryl group attached as a substituent via an alkylene group. Examples include, but are not limited to, 2-thienylmethyl, 3-thienylmethyl, furanylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., C 1-6 alkylene group).

[0078] As used herein, "carbocyclyl" means a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system skeleton. When the carbocyclyl is a ring system, two or more rings can be joined together in a fused, bridged or spiro manner. The carbocyclyl can have any degree of saturation, provided that at least one ring in the ring system is not aromatic. Therefore, carbocyclyl includes cycloalkyl, cycloalkenyl and cycloalkynyl. The carbocyclyl group can have 3 to 20 carbon atoms, but this definition also covers the term "carbocyclyl" in which no numerical range is specified. The carbocyclyl group can also be a medium-sized carbocyclyl with 3 to 10 carbon atoms. The carbocyclyl group can also be a carbocyclyl with 3 to 6 carbon atoms. The carbocyclyl group can be named "C 3-6 "C3-C6 carbocyclyl", "C3-C6 carbocyclyl" or similar designations. Examples of carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicyclo[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

[0079] As used herein, "cycloalkyl" means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0080] As used herein, "heterocyclyl" refers to a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. The heterocyclyl group can be joined together in a fused, bridged or spiro manner. The heterocyclyl group can have any degree of saturation, provided that at least one ring in the ring system is not aromatic. The heteroatom can be present in a non-aromatic ring or an aromatic ring in the ring system. The heterocyclyl group can have 3 to 20 ring members (i.e., the number of atoms (including carbon atoms and heteroatoms) constituting the ring backbone), but this definition also covers the term "heterocyclyl" in which no numerical range is specified. The heterocyclyl group can also be a medium-sized heterocyclyl group with 3 to 10 ring members. The heterocyclyl group can also be a heterocyclyl group with 3 to 6 ring members. The heterocyclyl group can be named "3 to 6-membered heterocyclyl" or similar names. In preferred six-membered monocyclic heterocyclyls, the heteroatoms are selected from one to a maximum of three from O, N or S, and in preferred five-membered monocyclic heterocyclyls, the heteroatoms are selected from one or two heteroatoms selected from O, N or S. Examples of heterocyclyl rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidinedionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxinyl, 1,4-dioxinyl, 1,4-dioxinyl, 1,3-oxathianyl, 1,4-oxathianyl, 1,4-oxathianyl, 2 H-1,2-oxazinyl, trioxanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithienyl, 1,3-dithianyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinone, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiomorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl and tetrahydroquinoline.

[0081] As used herein, "alkoxyalkyl" or "(alkoxy)alkyl" refers to an alkoxy group attached through an alkylene group, such as C2-C8alkoxyalkyl or (C1-C6alkoxy)C1-C6alkyl, for example -(CH2) 1-3 -OCH3.

[0082] An "O-carboxyl" group is one in which R is selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl "-OC(=O)R" groups are as defined herein.

[0083] A "C-carboxyl" group is one in which R is selected from the group consisting of hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 The "-C(=O)OR" group of the group consisting of aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl, as defined herein. Non-limiting examples include carboxyl (ie, -C(=O)OH).

[0084] A "sulfonyl" group is a group in which R is selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 The "-SO2R" group of aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclyl are as defined herein.

[0085] A "sulfinyl" group refers to a "-S(=O)OH" group.

[0086] A "sulfo" group refers to a "-S(=O)2OH" or a "-SO3H" group.

[0087] The "sulfonate" group refers to "-SO3 - ” group.

[0088] The "sulfate" group refers to "-SO4 - ” group.

[0089] An "S-sulfonamido" group refers to a group in which R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 "-SO2NR" of aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclic group A R B " group, as defined herein.

[0090] An "N-sulfonamido" group is one in which R A and R b are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C6-10 "-N(R)" of aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclic group A )SO2R B " group, as defined herein.

[0091] A "C-amido" group is one in which R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 "-C(=O)NR" of aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclic group A R B " group, as defined herein.

[0092] An "N-amido" group is one in which R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 "-N(R)" of aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclic group A )C(=O)R B " group, as defined herein.

[0093] An "amino" group is one in which R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 "-NR" of aryl, 5- to 10-membered heteroaryl and 3- to 10-membered heterocyclic group A R B " group, as defined herein. Non-limiting examples include free amino groups (ie, -NH2).

[0094] An "aminoalkyl" group refers to an amino group attached through an alkylene group.

[0095] An "alkoxyalkyl" group refers to an alkoxy group attached through an alkylene group, such as "C2-C8 alkoxyalkyl" and the like.

[0096] As used herein, "-OAc" or "-O-acyl" refers to an acetoxy group having the structure -OC(=O)CH3.

[0097] When a group is described as "optionally substituted", it can be unsubstituted or substituted. Similarly, when a group is described as "substituted", the substituent can be selected from one or more of the indicated substituents. As used herein, the substituted group is derived from an unsubstituted parent group in which there is an exchange of one or more hydrogen atoms with another atom or group. Unless otherwise specified, when a group is considered to be "substituted", it means that the group is substituted with one or more substituents, which are independently selected from: C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C3-C7 carbocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7 carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3 to 10 C1-C6-alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl-C1-C6-alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), halo, -CN, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy(C1-C6)alkyl (i.e., ether), aryloxy, sulfhydryl (mercapto) , halo(C1-C6)alkyl (e.g., -CF3), halo(C1-C6)alkoxy (e.g., -OCF3), C1-C6alkylthio, arylthio, amino, amino(C1-C6)alkyl, nitro, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino, N-acylamino, S-sulfonylamino, N-sulfonylamino, C-carboxyl, O-carboxyl, acyl, cyanate, isocyanate, thiocyanate, isothiocyanate, sulfinyl, sulfonyl, -SO3H, sulfonate, sulfate, sulfinic acid, -OSO2C 1-C4 alkyl and oxo (=O). Wherever a group is described as "optionally substituted", the group may be substituted with the above substituents. In some embodiments, when an alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl or heterocyclyl group is substituted, they are each independently substituted with one or more substituents selected from the group consisting of: halo, -CN, -SO3 - 、-OSO3 - 、-SO3H、-SR A 、-OR A 、-NR B R C , oxo, -CONR B R C 、-SO2NR B R C , -COOH and -COOR B , where R A , R B and R C Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.

[0098] As will be appreciated by those of ordinary skill in the art, the compounds described herein may exist in an ionized form, for example, -CO2 - 、-SO3 - or -O-SO3 - If the compound contains a positively or negatively charged substituent group, e.g., -SO3 - , it may also contain negatively or positively charged counterions so that the compound is neutral as a whole. In other aspects, the compound may exist in the form of a salt, wherein the counterion is provided by a conjugate acid or base.

[0099] It should be understood that, depending on the context, certain group naming conventions may include a monoradical or a diradical. For example, when a substituent requires two points of attachment to the rest of the molecule, it should be understood that the substituent is a diradical. For example, a substituent identified as an alkyl group requiring two points of attachment includes a diradical, such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc. Other group naming conventions clearly indicate that the group is a diradical, such as "alkylene" or "alkenylene".

[0100] When two "adjacent" R groups are said to form a ring "together with the atoms to which they are attached", this means that the collection of atoms, intervening bonds, and the two R groups is the ring. For example, when the following substructure is present:

[0101]

[0102] And R 1 and R 2 is defined as being selected from the group consisting of hydrogen and alkyl, or R 1 and R 2 Together with the atoms to which they are attached, they form an aryl or carbocyclic group, which means that R 1 and R 2 may be selected from hydrogen or alkyl, or alternatively, the substructure has the structure:

[0103]

[0104] wherein A is an aromatic ring or a carbocyclic group containing a double bond as depicted.

[0105] When a substituent is described as a diradical (i.e., having two points of attachment to the rest of the molecule), it is understood that the substituent may be attached in any orientation unless otherwise indicated. Thus, for example, a substituent depicted as -AE- or include substituents oriented so that A is attached at the leftmost point of attachment of the molecule, as well as substituents in which A is attached at the rightmost point of attachment of the molecule. In addition, if a group or substituent is depicted as Then L is defined as an optionally present linker moiety; when L is absent (or missing), such group or substituent is equivalent to

[0106] The compounds described herein can be represented as several meso forms. Where a single structure is drawn, any relevant meso forms are contemplated. The biboron-containing dyes described herein are represented by a single structure, but can likewise be shown as any relevant meso forms. Exemplary meso structures are shown below:

[0107]

[0108] In each case where a single meso form of a compound described herein is shown, the alternative meso form is also contemplated. In addition, for simplicity, the positive charge on the nitrogen atom of the compound (when there are four bonds to the nitrogen atom) and the negative charge on the boron atom (when there are four bonds to the boron atom) may not be shown in certain compound structures.

[0109] As used herein, "nucleotides" include nitrogenous heterocyclic bases, sugars, and one or more phosphate groups. They are monomeric units of nucleic acid sequences. In RNA, the sugar is ribose, and in DNA it is deoxyribose, i.e., a sugar lacking the hydroxyl groups present in ribose. The nitrogenous heterocyclic bases may be purines, deazapurines, or pyrimidine bases. Purine bases include adenine (A) and guanine (G) and their modified derivatives or analogs, such as 7-deazaadenine or 7-deazaguanine. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U) and their modified derivatives or analogs. The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine.

[0110] As used herein, "nucleoside" is structurally similar to a nucleotide, but lacks a phosphate moiety. An example of a nucleoside analog is a nucleoside analog in which a label is attached to a base and no phosphate group is attached to a sugar molecule. The term "nucleoside" is used herein in the conventional sense as understood by those skilled in the art. Examples include, but are not limited to, ribonucleosides comprising a ribose moiety and deoxyribonucleosides comprising a deoxyribose moiety. A modified pentose moiety is a pentose moiety in which an oxygen atom has been replaced by a carbon and / or a carbon has been replaced by a sulfur or oxygen atom. A "nucleoside" is a monomer that may have a substituted base and / or sugar moiety. In addition, nucleosides may be incorporated into larger DNA and / or RNA polymers and oligomers.

[0111] The term "purine base" is used herein with the common meaning understood by those skilled in the art, and includes its tautomers. Similarly, the term "pyrimidine base" is used herein with the common meaning understood by those skilled in the art, and includes its tautomers. The non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, deazapurine, 7-deazaadenine, 7-deazaguanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. The example of pyrimidine bases includes, but is not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine).

[0112] As used herein, when oligonucleotide or polynucleotide are described as "comprising " nucleoside or nucleotide as described herein, this means that nucleoside or nucleotide as described herein forms covalent bond with oligonucleotide or polynucleotide.Similarly, when nucleoside or nucleotide are described as a part of oligonucleotide or polynucleotide, such as "being incorporated into " oligonucleotide or polynucleotide, this means that nucleoside or nucleotide as described herein forms covalent bond with oligonucleotide or polynucleotide.In some such embodiments, covalent bond is formed between 5' phosphate groups of the nucleotide of phosphodiester bond between 3' carbon atom of oligonucleotide or polynucleotide and 5' carbon atom of nucleotide described in this article.

[0113] As used herein, the term "cleavable linker" is not intended to imply that the entire linker needs to be removed. The cleavage site can be located at a position on the linker that ensures that a portion of the linker remains attached to the detectable label and / or nucleoside or nucleotide moiety after cleavage.

[0114] As used herein, "derivative" or "analog" means a synthetic nucleotide or nucleoside derivative with a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are discussed in, for example, Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90: 543-584, 1990. Nucleotide analogs can also include modified phosphodiester bonds, including phosphorothioate bonds, phosphorodithioate bonds, alkylphosphonate bonds, aniline phosphate bonds and phosphoramidate bonds. As used herein, "derivative", "analog" and "modified" can be used interchangeably, and are covered by the terms "nucleotide" and "nucleoside" defined herein.

[0115] As used herein, the term "phosphate" is used with the ordinary meaning understood by those skilled in the art and includes its protonated form (e.g., ). As used herein, the terms "monophosphate," "diphosphate," and "triphosphate" are used with their ordinary meaning as understood by those skilled in the art, and include protonated forms.

[0116] As used herein, the term "phasing" refers to a phenomenon in SBS, which is caused by the incomplete removal of 3' terminators and fluorophores and / or failure to complete the incorporation of a portion of the DNA chain in the cluster by polymerase under a given sequencing cycle. Prephasing is caused by the incorporation of nucleotides that do not have an effective 3' terminator, wherein the incorporation event is advanced by 1 cycle due to termination failure. Phasing and prephasing cause the measured signal intensity of a specific cycle to be composed of the signal from the current cycle and the noise from the previous and next cycles. As the number of cycles increases, the sequence score of each cluster affected by phasing and prephasing increases, hindering the identification of the correct base. Prephasing can be caused by the presence of traces of unprotected or uncapped 3'-OH nucleotides during sequencing by synthesis (SBS). Unprotected 3'-OH nucleotides can be produced during the manufacturing process or may be stored and during the reagent handling process. Therefore, the discovery of nucleotide analogs that reduce the incidence of prephasing is surprising, and relative to existing nucleotide analogs, provides a huge advantage in SBS applications. For example, the provided nucleotide analogs can produce faster SBS cycle times, lower phasing and prephasing values, and longer sequencing read lengths.

[0117] Dyes containing a diboron dye of formula (I)

[0118] Some aspects of the present disclosure relate to diboron-containing dyes of formula (I) and salts and meso forms thereof:

[0119] Its salt or meso form, wherein R 1 , R 2 , R 3 , R 6 , R 7 and R 8 Each of the above is independently H, unsubstituted or substituted C1-C6 alkyl, C1-C6 alkoxy, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, (C1-C6 alkoxy)(C1-C6 alkyl), unsubstituted or substituted amino, halo, cyano, carboxyl, hydroxyl, nitro, sulfonyl, sulfinyl, sulfonyl, sulfonate, S-sulfonamido, N-sulfonamido, -C(=O)NR 9 R 10 、-XR 11 , unsubstituted or substituted C3-C 10 Carbocyclic, unsubstituted or substituted C6-C 10 aryl, unsubstituted or substituted 5- to 10-membered heteroaryl, or unsubstituted or substituted 3- to 10-membered heterocyclyl;

[0120] R 4 and R5 Each of the following is independently H, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C3-C 10 Carbocyclic, unsubstituted or substituted C6-C 10 aryl, unsubstituted or substituted 5- to 10-membered heteroaryl, or unsubstituted or substituted 3- to 10-membered heterocyclyl;

[0121] R 9 and R 10 Each of is independently H, or unsubstituted or substituted C1-C6 alkyl;

[0122] X is -O-, -S-, -CH=CH-, or -C≡C-;

[0123] R 11 Each of which is independently unsubstituted or substituted C3-C 10 Carbocyclic, unsubstituted or substituted C6-C 10 aryl, unsubstituted or substituted 5- to 10-membered heteroaryl, or unsubstituted or substituted 3- to 10-membered heterocyclyl;

[0124] R a , R b , R c and R d Each of the above is independently a halogen group, a cyano group, an unsubstituted C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, a C1-C6 haloalkoxy group, an unsubstituted or substituted C6-C 10 Aryl, unsubstituted or substituted C6-C 10 Aryloxy or -OC(=O)R 12 ;

[0125] R 12 is an unsubstituted or substituted C1-C6 alkyl, an unsubstituted or substituted C2-C6 alkenyl, an unsubstituted or substituted C6-C 10 aryl or unsubstituted or substituted 5- to 10-membered heteroaryl;

[0126] Alternatively, when R a and R b All are -OC(=O)R 12 When two R 12 Together with the atoms to which they are attached, form an unsubstituted or substituted 6- to 10-membered heterocyclyl; and

[0127] Alternatively, when R c and R d All are -OC(=O)R 12 When two R 12Together with the atoms to which they are attached, they form an unsubstituted or substituted 6- to 10-membered heterocyclyl. 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 At least one of comprises a carboxyl group (-C=O)OH). In another embodiment, R 1 , R 2 , R 3 , R 6 , R 7 and R 8 At least one of the comprises a carboxyl group.

[0128] In some embodiments of the compound of Formula (I), R 1 , R 2 and R 3 Each of R is H or unsubstituted C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl or n-hexyl). 1 and R 3 Each of R is methyl and R 2 In other embodiments, wherein R 1 , R 2 and R 3 Both of them are H or unsubstituted C1-C6 alkyl, and R 1 , R 2 and R 3 One of them is halo (eg, fluoro or chloro), carboxyl, -C(=O)NR 9 R 10 , C1-C6 alkyl substituted with carboxyl, carboxylate, sulfonate or sulfonate, unsubstituted or substituted phenyl, unsubstituted or substituted 5- to 6-membered heteroaryl (e.g., 5- or 6-membered heteroaryl having one or more heteroatoms selected from N, O and S, such as pyridyl), or unsubstituted or substituted 5- to 6-membered heterocyclyl (e.g., 5- or 6-membered heterocyclyl having one or more heteroatoms selected from N, O and S, such as piperidinyl). In other embodiments, R 1 , R 2 and R 3 Both of them are H or methyl, and R 1 , R 2 and R 3 One of them is fluorine, chlorine, carboxyl or -C(O)NHR 10 , where R 10is a C1-C6 alkyl group substituted with a carboxyl group, a carboxylate group, a sulfonate group or a sulfonate group. 10 is a C3 alkyl group substituted with a carboxyl group. 1 and R 2 Yes H, R 3 is phenyl optionally substituted by carboxyl, or R 1 and R 3 Yes H, R 2 is phenyl optionally substituted by carboxyl.

[0129] In some embodiments of the compound of Formula (I), R 1 , R 2 and R 3 Both of them are H or unsubstituted C1-C6 alkyl, and R 1 , R 2 and R 3 One of them is -XR 11 , and wherein X is -O-, -S-, or -C≡C-. In another embodiment, R 1 and R 2 are independently H or unsubstituted C1-C6 alkyl, and R 3 Yes - XR 11 , and wherein X is -O-, -S-, or -C≡C-. In another embodiment, R 1 and R 3 are independently H or unsubstituted C1-C6 alkyl, and R 2 Yes - XR 11 , and wherein X is -O-, -S-, or -C≡C-. In some such embodiments, R 11 is phenyl, which is unsubstituted or substituted by one or more substituents selected from the group consisting of halo, unsubstituted C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, carboxyl, carboxylate, sulfonate, sulfonate or -C(O)NR 13 R 14 , and each R 13 and R 14 are independently H, unsubstituted C1-C6 alkyl, or C1-C6 alkyl substituted with carboxyl, carboxylate, sulfonate, or sulfonate. In another embodiment, X is -O- or -C≡C-, and R 11 is unsubstituted phenyl or substituted by halogen, methoxy, carboxyl or -C(O)NHR 14 Substituted phenyl, wherein R 14 is a C1-C6 alkyl group substituted with a carboxyl group, a carboxylate group, a sulfonate group or a sulfonate group. 11is a 5- or 6-membered heterocyclyl or heteroaryl (e.g., a 5- or 6-membered heteroaryl having one or more heteroatoms selected from N, O and S, such as pyridyl), or an unsubstituted or substituted 5- to 6-membered heterocyclyl (e.g., a 5- or 6-membered heterocyclyl having one or more heteroatoms selected from N, O and S, such as piperidinyl), which is unsubstituted or substituted with one or more substituents selected from the group consisting of halo, unsubstituted C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, carboxyl, carboxylate, sulfonate, sulfonate or -C(O)NR 13 R 14 , and each R 13 and R 14 R is independently H, unsubstituted C1-C6 alkyl, or C1-C6 alkyl substituted with carboxyl, carboxylate, sulfonate, or sulfonate. 11 is unsubstituted pyridyl, pyrrolyl, piperidinyl or pyrrolidinyl; or each is independently substituted by halo, methoxy, carboxyl or -C(O)NHR 14 Substituted pyridyl, pyrrolyl, piperidinyl or pyrrolidinyl, wherein R 14 It is a C1-C6 alkyl group substituted by a carboxyl group, a carboxylate group, a sulfonate group or a sulfonate group.

[0130] In some embodiments of the compound of Formula (I), R 6 , R 7 and R 8 Each of R is H or unsubstituted C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl or n-hexyl). 6 and R 8 Each of R is methyl and R 7 In other embodiments, wherein R 6 , R 7 and R 8 Both of them are H or unsubstituted C1-C6 alkyl, and R 6 , R 7 and R 8 One of them is halo (eg, fluoro or chloro), carboxyl, -C(=O)NR 9 R 10 , C1-C6 alkyl substituted with carboxyl, carboxylate, sulfonate or sulfonate, unsubstituted or substituted phenyl, unsubstituted or substituted 5- to 6-membered heteroaryl (e.g., 5- or 6-membered heteroaryl having one or more heteroatoms selected from N, O and S, such as pyridyl), or unsubstituted or substituted 5- to 6-membered heterocyclyl (e.g., 5- or 6-membered heterocyclyl having one or more heteroatoms selected from N, O and S, such as piperidinyl). In other embodiments, R6 , R 7 and R 8 Both of them are H or methyl, and R 6 , R 7 and R 8 One of them is fluorine, chlorine, carboxyl or -C(O)NHR 10 , where R 10 is a C1-C6 alkyl group substituted with a carboxyl group, a carboxylate group, a sulfonate group or a sulfonate group. 10 is a C3 alkyl group substituted with a carboxyl group. 6 and R 7 Yes H, R 8 is phenyl optionally substituted by carboxyl, or R 6 and R 8 Yes H, R 7 is phenyl optionally substituted by carboxyl.

[0131] In some embodiments of the compound of Formula (I), R 6 , R 7 and R 8 Both of them are H or unsubstituted C1-C6 alkyl, and R 6 , R 7 and R 8 One of them is -XR 11 , and wherein X is -O-, -S-, or -C≡C-. In another embodiment, R 6 and R 7 are independently H or unsubstituted C1-C6 alkyl, and R 8 Yes - XR 11 , and wherein X is -O-, -S-, or -C≡C-. In another embodiment, R 6 and R 8 are independently H or unsubstituted C1-C6 alkyl, and R 7 Yes - XR 11 , and wherein X is -O-, -S-, or -C≡C-. In some such embodiments, R 11 is phenyl, which is unsubstituted or substituted by one or more substituents selected from the group consisting of halo, unsubstituted C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, carboxyl, carboxylate, sulfonate, sulfonate or -C(O)NR 13 R 14 , and each R 13 and R 14are independently H, unsubstituted C1-C6 alkyl, or C1-C6 alkyl substituted with carboxyl, carboxylate, sulfonate, or sulfonate. In another embodiment, X is -O- or -C≡C-, and R 11 is unsubstituted phenyl or substituted by halogen, methoxy, carboxyl or -C(O)NHR 14 Substituted phenyl, wherein R 14 is a C1-C6 alkyl group substituted with a carboxyl group, a carboxylate group, a sulfonate group or a sulfonate group. 11 is a 5- or 6-membered heterocyclyl or heteroaryl (e.g., a 5- or 6-membered heteroaryl having one or more heteroatoms selected from N, O and S, such as pyridyl), or an unsubstituted or substituted 5- to 6-membered heterocyclyl (e.g., a 5- or 6-membered heterocyclyl having one or more heteroatoms selected from N, O and S, such as piperidinyl), which is unsubstituted or substituted with one or more substituents selected from the group consisting of halo, unsubstituted C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, carboxyl, carboxylate, sulfonate, sulfonate or -C(O)NR 13 R 14 , and each R 13 and R 14 R is independently H, unsubstituted C1-C6 alkyl, or C1-C6 alkyl substituted with carboxyl, carboxylate, sulfonate, or sulfonate. 11 is unsubstituted pyridyl, pyrrolyl, piperidinyl or pyrrolidinyl; or each is independently substituted by halo, methoxy, carboxyl or -C(O)NHR 14 Substituted pyridyl, pyrrolyl, piperidinyl or pyrrolidinyl, wherein R 14 It is a C1-C6 alkyl group substituted by a carboxyl group, a carboxylate group, a sulfonate group or a sulfonate group.

[0132] In some embodiments of the compound of Formula (I), R 4 is H. In some embodiments, R 5 is H. In another embodiment, R 4 and R 5 Each of is H.

[0133] In some embodiments of the compound of Formula (I), R a and R b Each of the groups is independently fluoro, cyano, methyl, trifluoromethyl, methoxy or -OC(=O)R 12 , where R 12 is unsubstituted C1-C6 alkyl, unsubstituted C2-C6 alkenyl or unsubstituted phenyl. In another embodiment, R a and R bare fluorine, methyl, trifluoromethyl, methoxy, ethoxy, -OAc(-OC(=O)CH3), -OC(=O)CH=CH2 or -OC(=O)Ph. a and R b All are -OC(=O)R 12 , and the two R 12 Together with the atoms to which they are attached, they form structures In some embodiments, R c and R d Each of the groups is independently fluoro, cyano, methyl, trifluoromethyl, methoxy or -OC(=O)R 12 , where R 12 is unsubstituted C1-C6 alkyl, unsubstituted C2-C6 alkenyl or unsubstituted phenyl. In another embodiment, wherein R c and R d are fluorine, methyl, trifluoromethyl, methoxy, ethoxy, -OAc, -OC(=O)CH=CH2 or -OC(=O)Ph. c and R d All are -OC(=O)R 12 , and the two R 12 Together with the atoms to which they are attached, they form structures A 6-membered heterocyclic group.

[0134] Additional embodiments of compounds of Formula (I) include, but are not limited to, those described in Table A below.

[0135] Table A .

[0136]

[0137]

[0138]

[0139]

[0140] As well as their salts and meso forms. Non-limiting examples are the corresponding C1-C6 alkyl carboxylates (such as methyl, ethyl, isopropyl and tert-butyl esters formed from the carboxyl group of the compound).

[0141] In any embodiment of the bis-boron fused heterocyclic compound described herein, the compound can be further modified to introduce a photoprotective moiety covalently bonded thereto, for example, a photoprotective cyclooctatetraene (COT) moiety, as described in U.S. Application No. 2021 / 0155983A1, which is incorporated by reference in its entirety. For example, a bis-boron compound of formula (I) can be covalently attached to the COT moiety via an amide bond, wherein the photoprotective COT moiety comprises a structure of formula (II):

[0142]

[0143] wherein Z is absent, is optionally substituted C 2-6 Alkenylene or optionally substituted C 2-6 Alkynylidene;

[0144] Each R x and R y are independently H, carboxyl, carboxylate, amino, sulfonate, sulfonate, -C(O)OR A or -C(O)NR B R C , or amino, carboxyl, carboxylate, sulfonate, sulfonate, -C(O)OR A or -C(O)NR B R C Substituted C1-C6 alkyl,

[0145] Each R N1 and R N2 are independently H or amino, carboxyl, carboxylate, sulfonate, sulfonate, -C(O)OR A or -C(O)NR B R C Substituted C1-C6 alkyl,

[0146] R A is an optionally substituted C1-C6 alkyl, an optionally substituted C6-C 10 aryl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted C3-C7 cycloalkyl;

[0147] R B and R C Each of the following is independently H, optionally substituted C1-C6 alkyl, optionally substituted C6-C 10 aryl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted C3-C7 cycloalkyl;

[0148] exist Medium R x and R yThe carbon atom to which it is attached is optionally substituted with O, S or N, provided that when the carbon atom is substituted with O or S, then R x and R y When the carbon atom is replaced by N, R y does not exist; and

[0149] m is an integer between 1 and 10;

[0150] wherein the asterisk * indicates the point of attachment of the photoprotective cyclooctatetraene moiety to the carbonyl group of the amide bond formed by the reaction of the amino group of the photoprotective cyclooctatetraene moiety with the carboxyl group of the compound of formula (I) as described herein.

[0151] In some additional embodiments, the photoprotective COT moiety may comprise the following structure:

[0152]

[0153] The COT moiety can be covalently attached to the biboron dye described herein by a reaction between a functional group (e.g., a carboxyl group) of the biboron dye described herein and an amino group of a COT derivative to form an amide bond (wherein the carbonyl group of the amide bond is not shown). Alternatively, the biboron dye described herein can have an amino group (rather than a carboxyl group) that forms an amide bond with the carboxyl group of the COT derivative.

[0154] Labeled nucleotides

[0155] According to one aspect of the present disclosure, dye compound as described herein is applicable to be attached to substrate part, particularly comprises linker group so that it is possible to attach to the dye compound of substrate part.Substrate part can in fact be any molecule or material that dye of the present disclosure can be put together with it, and in the manner of non-limiting example, can include nucleosides, nucleotides, polynucleotides, carbohydrates, ligands, particles, solid surfaces, organic polymers and inorganic polymers, chromosomes, cores, living cells, and their combinations or assemblies.Dye can be put together by various means (including hydrophobic attraction, ion attraction and covalent connection) by optional linker.In some respects, dye is put together to substrate by covalent attachment.More specifically, covalent connection is realized by means of linker group.In some cases, the nucleotide of such labeling is also referred to as " modified nucleotide ".

[0156] Some aspects of the present disclosure relate to nucleotides labeled with a dye of formula (I) as described herein or a salt of its meso form, or a derivative thereof containing a light protection moiety COT as described herein. The labeled nucleotides or oligonucleotides can be attached to the dye compounds disclosed herein to form amides or alkyl-amide bonds via a carboxyl group (-CO2H) or an alkyl-carboxyl group. In some other embodiments, the carboxyl group can be in the form of an activated form of a carboxyl group, for example, an amide or an ester, which can be used to attach to an amino or hydroxyl group of a nucleotide or oligonucleotide. As used herein, the term "activated ester" refers to a carboxyl group derivative that can react under mild conditions, for example, with a compound containing an amino group. Non-limiting examples of activated esters include, but are not limited to, p-nitrophenyl, pentafluorophenyl, and succinimide esters.

[0157] For example, the dye compound of formula (I) can be obtained by 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 In some such embodiments, R of formula (I) 1 , R 2 , R 3 , R 6 , R 7 and R 8 One of the compounds of formula (I) comprises a carboxyl group, and the attachment forms an amide moiety between the carboxyl functional group of the compound of formula (I) and the amino functional group of the nucleotide or nucleotide linker.

[0158] In some embodiments, dye compounds can be covalently attached to nucleotides via nucleotide bases. In some such embodiments, the nucleotides of the labeling can have dyes that are optionally attached to the C5 position of pyrimidine bases or the C7 position of 7-deazapurine bases by a linker moiety. For example, the core base can be 7-deazaadenine, and the dye is optionally attached to 7-deazaadenine at the C7 position by a linker. The core base can be 7-deazaguanine, and the dye is optionally attached to 7-deazaguanine at the C7 position by a linker. The core base can be cytosine, and the dye is optionally attached to cytosine at the C5 position by a linker. As another example, the core base can be thymine or uracil, and the dye is optionally attached to the C5 position of thymine or uracil by a linker.

[0159] 3' blocking group

[0160] The nucleotide of labeling can also have the capping group of the ribose or deoxyribose of nucleotide covalently attached.The capping group can be attached at any position on ribose or deoxyribose.In a specific embodiment, the capping group is located at the 3'OH position of the ribose or deoxyribose of nucleotide.Various 3'OH capping groups are disclosed in WO2004 / 018497 and WO2014 / 139596, and these applications are hereby incorporated by reference.For example, the capping group can be azidomethyl (-CH2N3) or substituted azidomethyl (for example, -CH(CHF2)N3 or CH(CH2F)N3), or the allyl group of the 3' oxygen atom connected to ribose or deoxyribose moiety.In some embodiments, the 3' capping group is azidomethyl, and 3'-OCH2N3 is formed with the 3' carbon of ribose or deoxyribose.

[0161] Additional 3' blocking groups are disclosed in U.S. Publication No. 2020 / 0216891 A1, which is incorporated by reference in its entirety. Non-limiting examples of 3' blocking groups include: (AOM), Each is covalently attached to the 3' carbon of ribose or deoxyribose.

[0162] Deprotection of 3' blocking group

[0163] In some embodiments, the 3'-capping group can be removed or deprotected by a chemical reagent, for example, in the presence of a water-soluble phosphine reagent to produce a free hydroxyl group. Non-limiting examples include tris(hydroxymethyl)phosphine (THMP), tris(hydroxyethyl)phosphine (THEP), or tris(hydroxypropyl)phosphine (THP or THPP). The 3'-acetal capping groups described herein can be removed or cleaved under various chemical conditions. For 3'-acetal capping groups such as Non-limiting cleavage conditions include Pd (II) complexes such as Pd (OAc) 2 or allyl chloride Pd (II) dimers in the presence of a phosphine ligand, for example tris (hydroxymethyl) phosphine (THMP) or tris (hydroxypropyl) phosphine (THP or THPP). For those end-capping groups containing alkynyl groups (e.g., ethynyl), they can also be removed by Pd (II) complexes (e.g., Pd (OAc) 2 or allyl chloride Pd (II) dimers) in the presence of a phosphine ligand (e.g., THP or THMP).

[0164] Palladium cleavage reagent

[0165] In some other embodiments, the 3' blocking group described herein (such as AOM) can be cleaved by a palladium catalyst. In some such embodiments, the Pd catalyst is water-soluble. In some such embodiments, it is a Pd(0) complex (e.g., tris(3,3',3"-phosphinotri(benzenesulfonate)palladium(0) nonahydrate). In some cases, the Pd(0) complex can be generated in situ by reducing the Pd(II) complex with a reagent such as an olefin, an alcohol, an amine, a phosphine, or a metal hydride. Suitable palladium sources include Na2PdCl4, Pd(CH3CN)2Cl2, (PdCl(C3H5))2, [Pd(C3H5)(THP)]Cl, [Pd(C3H5)(THP)2]Cl, Pd(OAc)2, Pd(Ph3)4, Pd(dba)2, Pd (Acac)2, PdCl2(COD) and Pd(TFA)2. In one such embodiment, the Pd(0) complex is generated in situ from Na2PdCl4 or K2PdCl4. In another embodiment, the palladium source is allylpalladium(II) chloride dimer [(PdCl(C3H5))2]. In some embodiments, the Pd(0) complex is generated in aqueous solution by mixing the Pd(II) complex with a phosphine. Suitable phosphines include water-soluble phosphines such as THP, THMP, PTA, TCEP, bis(p-sulfonic acid phenyl)phenylphosphine dihydrate potassium salt or triphenylphosphine-3,3',3"-trisulfonic acid trisodium salt.

[0166] In some embodiments, Pd(0) is prepared by mixing a Pd(II) complex [(PdCl(C3H5))2] with THP in situ. The molar ratio of the Pd(II) complex to THP can be about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some additional embodiments, one or more reducing agents can be added, such as ascorbic acid or a salt thereof (e.g., sodium ascorbate). In some embodiments, the cleavage mixture can contain additional buffer reagents, such as primary amines, secondary amines, tertiary amines, carbonates, phosphates, or borates, or combinations thereof. In some other embodiments, the buffer reagent includes ethanolamine (EA), tris (hydroxymethyl) aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, 2-dimethylethanolamine (DMEA), 2-diethylethanolamine (DEEA), N, N, N', N'-tetramethylethylenediamine (TEMED) or N, N, N', N'-tetraethylethylenediamine (TEEDA) or a combination thereof. In one embodiment, the buffer is DEEA. In another embodiment, the buffer contains one or more inorganic salts, such as carbonate, phosphate or borate, or a combination thereof. In one embodiment, the inorganic salt is a sodium salt.

[0167] Connector

[0168] Dye compounds as disclosed herein can include reactive linker groups at one of the substituent positions for covalently linking the compound to a substrate or another molecule. Reactive linker groups are capable of forming a bond (e.g., covalent or non-covalent bond), particularly a covalent bond. In a specific embodiment, linker groups can be cleavable linkers. The use of the term "cleavable linker" is not intended to imply the need to remove the entire linker. The cleavage site can be located on the linker to ensure that a portion of the linker remains attached to a certain position of the dye and / or substrate portion after cleavage. In a non-limiting example, cleavable linkers can be electrophilic cleavable linkers, nucleophilic cleavable linkers, photocleavable linkers, cleavable under reducing conditions (e.g., linkers containing disulfide or azide), cleavable under oxidative conditions, cleavable by using a secure capture linker, and cleavable by an elimination mechanism. Using a cleavable linker to connect dye compounds to the substrate portion ensures that, if necessary, the label can be removed after detection, thereby avoiding any interfering signal in the downstream step.

[0169] Useful linker groups can be found in PCT Publication WO2004 / 018493 (incorporated herein by reference), examples of which include linkers that can be cleaved using water-soluble phosphines or water-soluble transition metal catalysts formed by transition metals and at least partially water-soluble ligands. In aqueous solution, the latter forms at least partially water-soluble transition metal complexes. Such cleavable linkers can be used to connect the bases of nucleotides to labels, such as dyes described herein.

[0170] Particular linking groups include those disclosed in PCT Publication WO 2004 / 018493 (incorporated herein by reference), such as those comprising a moiety of the formula:

[0171]

[0172] (wherein X is selected from the group consisting of O, S, NH and NQ, wherein Q is a C1-10 substituted or unsubstituted alkyl group, Y is selected from the group consisting of O, S, NH and N (allyl), T is hydrogen or C1-C 10 substituted or unsubstituted alkyl group, and * indicates the position where the moiety is attached to the rest of the nucleotide or nucleoside). In some aspects, the linker connects the base of the nucleotide to a label, such as a dye compound described herein.

[0173] Additional examples of linkers include those disclosed in U.S. Publication No. 2016 / 0040225 (incorporated herein by reference), such as those comprising a moiety of the formula:

[0174]

[0175] (where * indicates the position where the portion is attached to the rest of the nucleotide or nucleoside). The linker moiety presented herein may include all or part of the linker structure between the nucleotide / nucleoside and the label. The linker moiety presented herein may include all or part of the linker structure between the nucleotide / nucleoside and the label.

[0176] Additional examples of linking groups include moieties of the formula:

[0177]

[0178] Wherein B is a nucleobase; Z is -N3 (azido), -O-C1-C6 alkyl, -O-C2-C6 alkenyl or -O-C2-C6 alkynyl; and F1 includes a dye moiety, which may contain an additional linker structure. It is understood by those of ordinary skill in the art that the dye compounds described herein are covalently bound to the linker by reacting the functional groups (e.g., carboxyl) of the dye compound with the functional groups (e.g., amino) of the linker. In one embodiment, the cleavable linker comprises ("AOL" linker moiety), wherein Z is -O-allyl.

[0179] In certain embodiments, the length of the linker between the fluorescent dye (fluorophore) and the guanine base can be varied, for example, by introducing a polyethylene glycol spacer group, thereby increasing the fluorescence intensity compared to the same fluorophore attached to the guanine base by other bonds known in the art. Exemplary linkers and their properties are shown in PCT Publication WO2007020457 (incorporated herein by reference). The design of the linker, and in particular its increased length, can allow for improved brightness of the fluorophore attached to the guanine base of a guanosine nucleotide when incorporated into a polynucleotide such as DNA. Therefore, when the dye is used in any analytical method requiring detection of a fluorescent dye label attached to a nucleotide containing guanine, it is advantageous that the linker comprises the formula -((CH2)2O) n -a spacer group wherein n is an integer between 2 and 50 as described in WO 2007 / 020457.

[0180] Nucleoside and nucleotide can be marked at the site on sugar or nucleobase.As known in the art, "nucleotide" is composed of nitrogenous base, sugar and one or more phosphate groups.In RNA, the sugar is ribose, and in DNA it is deoxyribose, that is, the sugar lacking the hydroxyl group present in ribose.Nitrogenous base is a derivative of purine or pyrimidine.Purine is adenine (A) and guanine (G), and pyrimidine is cytosine (C) and thymine (T), or in the case of RNA it is uracil (U).The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine.Nucleotide is also the phosphate ester of nucleoside, wherein esterification occurs on the hydroxyl group of C-3 or C-5 attached to sugar.Nucleotide is usually monophosphate, diphosphate or triphosphate.

[0181] A "nucleoside" is structurally similar to a nucleotide, but lacks a phosphate moiety. An example of a nucleoside analog is one in which the label is linked to the base and there is no phosphate group attached to the sugar molecule.

[0182] Although bases are commonly referred to as purines or pyrimidines, it will be appreciated by the skilled artisan that derivatives and analogs that do not change the ability of a nucleotide or nucleoside to undergo Watson-Crick base pairing are available. "Derivatives" or "analogs" mean compounds or molecules whose core structures are identical or very similar to the core structures of the parent compound, but which have chemical or physical modifications that allow the derived nucleotide or nucleoside to be connected to another molecule, such as different or additional side groups. For example, the base can be a deazapurine. In a particular embodiment, the derivative should be able to undergo Watson-Crick pairing. "Derivatives" and "analogs" also include, for example, synthetic nucleotides or nucleoside derivatives with modified base moieties and / or modified sugar moieties. Such derivatives and analogs are discussed in, for example, Scheit, Nucleotide analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90: 543-584, 1990. Nucleotide analogs can also include modified phosphodiester bonds, including phosphorothioate bonds, phosphorodithioate bonds, alkylphosphonate bonds, aniline phosphate bonds, phosphoramidate bonds, and the like.

[0183] Dye can be attached to any position on the nucleotide base, for example, by a linker. In a particular embodiment, the analogue obtained can still be subjected to Watson-Crick base pairing. Specific core base labeling sites include the C5 position of pyrimidine bases or the C7 position of 7-deazapurine bases. As mentioned above, a linker group can be used for covalently attaching the dye to a nucleoside or nucleotide.

[0184] In a particular embodiment, the nucleotide or oligonucleotide of the label can be enzymatically incorporated and enzymatically extended. Therefore, the linker portion can have enough length to connect the nucleotide to the compound so that the compound does not significantly interfere with the overall binding and recognition of the nucleic acid replicator to the nucleotide. Therefore, the linker can also include a spacer unit. The spacer distance is, for example, the distance of the nucleotide base from the cleavage site or label.

[0185] Nucleosides or nucleotides labeled with dyes described herein may have the following formula:

[0186]

[0187] Wherein the dye is a diboron dye compound (label) moiety described herein (after covalent bonding between the functional group of the dye and the functional group of the linker "L"); B is a nucleobase, such as uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, 7-deazaguanine, etc.; L is an optional linker that may or may not be present; R' can be H, or -OR' is a monophosphate, diphosphate, triphosphate, thiophosphate, phosphate analog, -O- attached to a reactive phosphorus-containing group, or -O- protected by a blocking group; R" is H or OH; and R"' is H, a 3' blocking group described herein, or -OR"' forms a phosphoramidite. Wherein -OR"' is a phosphoramidite and R' is an acid-cleavable hydroxyl protecting group that allows subsequent monomer coupling under automated synthesis conditions. In some additional embodiments, B includes Or their optionally substituted derivatives and analogs. In some other embodiments, the labeled nucleobase comprises the structure

[0188] In a particular embodiment, the capping group is separate and independent of the dye compound, i.e., not attached to the latter. Alternatively, the dye may comprise all or part of a 3'-OH capping group. Thus, R"' may be a 3'OH capping group that may or may not constitute a dye compound.

[0189] In yet another alternative embodiment, there is no capping group on the 3' carbon of the pentose, and the dye (or dye and linker configuration) attached to the base can, for example, be of a size or structure sufficient to act as a barrier to the incorporation of additional nucleotides. Thus, the barrier can be due to steric hindrance or can be due to a combination of size, charge, and structure, regardless of whether the dye is attached to the 3' position of the sugar.

[0190] In yet another alternative embodiment, the capping group is present on the 2' or 4' carbon of the pentose sugar and may be of a size or structure sufficient to act as a barrier to the incorporation of additional nucleotides.

[0191] The use of capping groups allows for control of polymerization, such as by stopping extension when a labeled nucleotide is incorporated. If the capping effect is reversible, e.g., by way of non-limiting example, extension can be stopped at certain points by changing the chemical conditions or by removing the chemical barrier and then allowed to continue.

[0192] In a specific embodiment, connecting base (between dyestuff and nucleotide) and end-capping group both all exist and are independent parts.In a specific embodiment, connecting base and end-capping group are all cleavable under identical or substantially similar conditions.Therefore, deprotection and deblocking process may be more effective, because only need single processing just can remove dye compound and end-capping group these two.But, in some embodiments, connecting base and end-capping group do not need to can crack under similar conditions, but can crack separately under different conditions.

[0193] The present disclosure also encompasses polynucleotides incorporating dye compounds. Such polynucleotides can be DNA or RNA composed of deoxyribonucleotides or ribonucleotides joined with phosphodiester bonds, respectively. Polynucleotides can include naturally occurring nucleotides combined with at least one modified nucleotide (e.g., labeled with a dye compound) as shown herein, non-naturally occurring (or modified) nucleotides or any combination thereof except the nucleotides of the labeling described herein. Polynucleotides according to the present disclosure can also include non-natural backbone bonds and / or non-nucleotide chemical modifications. It is also envisioned that a chimeric structure consisting of a mixture of ribonucleotides and deoxyribonucleotides comprising at least one labeled nucleotide.

[0194] Non-limiting exemplary labeled nucleotides as described herein include:

[0195]

[0196] wherein L represents a linker and R represents a ribose or deoxyribose moiety as described above, or a ribose or deoxyribose moiety having the 5' position substituted with a monophosphate, diphosphate or triphosphate.

[0197] In some embodiments, non-limiting exemplary fluorescent dye conjugates are shown below:

[0198]

[0199]

[0200]

[0201]

[0202] wherein PG represents a 3'OH capping group as described herein; p is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and k is 0, 1, 2, 3, 4, or 5. In one embodiment, -O-PG is AOM. In another embodiment, -O-PG is -O-azidomethyl. In one embodiment, k is 5. In some additional embodiments, p is 1, 2, or 3; and k is 5. Refers to the point of attachment of the dye to the cleavable linker as a result of a reaction between an amino group of the linker moiety and a carboxyl group of the dye. In any of the embodiments of the labeled nucleotides described herein, the nucleotide is a nucleotide triphosphate.

[0203] Other aspects of the present disclosure relate to oligonucleotides or polynucleotides comprising or incorporating the labeled nucleotides described herein. In some embodiments, the oligonucleotide or polynucleotide hybridizes with at least a portion of the target polynucleotide. In some embodiments, the target polynucleotide is fixed on a solid support. In some other embodiments, the solid support comprises an array or a plurality of different fixed target polynucleotides. In other embodiments, the solid support comprises a patterned flow cell. In other embodiments, the patterned flow cell comprises a plurality of nanopores. In other embodiments, the solid support comprises at least 5,000,000 spatially distinguishable sites / cm 2 , which comprises multiple copies of the target polynucleotide.

[0204] Other aspects of the present disclosure relate to protein tags or antibodies comprising one or more biboron dyes described herein. Specifically, the protein tags or antibodies can comprise multiple copies of the same dye to increase fluorescence intensity. The protein tags or antibodies can be used as affinity reagents that specifically bind to specific types of unlabeled 3' blocked nucleotides.

[0205] Reagent test kit

[0206] Also provided herein is a kit comprising a first type of nucleotide (i.e., a first label) labeled with a diboron compound of the present disclosure. In some embodiments, the kit also includes a second type of labeled nucleotide, which is labeled with a second compound (i.e., a second label), and the second compound is different from the diboron dye in the first type of labeled nucleotide. In some embodiments, a single excitation source can be used to excite the first type of labeled nucleotide and the second type of labeled nucleotide, and the single excitation source can be a first light source with a first excitation wavelength. For example, the excitation bands of the first label and the second label can overlap at least partially, so that the excitation in the spectral overlap region causes the two labels to emit fluorescence. In some other embodiments, the second type of labeled nucleotide can be excited using a second excitation source, and the second excitation source can be a second light source with a second excitation wavelength different from the first excitation wavelength. In some other embodiments, the kit may include a third type of nucleotide, wherein the third nucleotide is labeled with a third compound (i.e., a third label) different from the first label and the second label. Alternatively, the third type of nucleotide is labeled with both the first label and the second label. In some such embodiments, the third type of labeled nucleotide can be excited using a first light source with a first excitation wavelength. That is, each of the first type of nucleotide, the second type of nucleotide and the third type of nucleotide can be excited using the same light source with a single wavelength. In some other embodiments, the nucleotide of the third type of labeling can be excited using the first light source with the first excitation wavelength or the second light source with the second excitation wavelength. In other embodiments, the nucleotide of the third type can be excited using the third light source with the third excitation wavelength. In some other embodiments, the kit can also include the nucleotide of the fourth type. In some such embodiments, the fourth nucleotide is unlabeled (dark). In other embodiments, compared with the first nucleotide, the second nucleotide and the third nucleotide, the fourth nucleotide is labeled with different compounds, and each label has different absorbance maxima that can be distinguished from other labels. In still other embodiments, the fourth nucleotide is unlabeled. In some embodiments, the first light source has an excitation wavelength of about 400nm to about 480nm, about 420nm to about 470nm or 450nm to about 460nm (for example, 450nm). The second excitation light source has a wavelength of about 500nm to about 550nm, about 510nm to about 540nm or about 520nm to about 530nm (for example, 520nm). The second light source has an excitation wavelength of about 400 nm to about 480 nm, about 420 nm to about 470 nm, or 450 nm to about 460 nm (eg, about 452 nm).In some embodiments, the emission of the first type of labeled nucleotides, the second type of labeled nucleotides, and the third type of labeled nucleotides is detectable in two detection channels with different wavelengths (e.g., at a wavelength range of about 472 nm to about 520 nm in the blue region, and at a wavelength range of about 540 nm to about 640 nm in the green region). In other embodiments, each of the first type of nucleotides, the second type of nucleotides, and the third type of nucleotides has an emission spectrum that can be collected in a single emission collection filter or channel.

[0207] In some embodiments, the kit may contain four types of labeled nucleotides (A, C, G and T or U), wherein the first nucleotide of the four nucleotides is labeled with a compound as disclosed herein. In this kit, each of the four nucleotides can be labeled with a compound that is the same or different from the label on the other three nucleotides. Alternatively, the first nucleotide of the four nucleotides is a labeled nucleotide described herein, the second nucleotide of the four nucleotides carries the second label, the third nucleotide carries the third label, and the fourth nucleotide is unlabeled (dark). As another example, the first nucleotide of the four nucleotides is a labeled nucleotide described herein, the second nucleotide of the four nucleotides carries the second label, the third nucleotide carries a mixture of two labels, and the fourth nucleotide is unlabeled (dark). Therefore, one or more of the labeled compounds can have different absorbance maxima and / or emission maxima, so that the compound can be distinguished from other compounds. For example, each compound can have different absorbance maxima and / or emission maxima, so that each of these compounds can be distinguished from the other three compounds (or if the fourth nucleotide is unlabeled, two compounds) in the spectrum. It will be appreciated that parts of the absorption and / or emission spectra other than the maximum may differ and these differences may be used to differentiate the compounds. The kit may be such that two or more of the compounds have different absorbance maxima.

[0208] The compounds, nucleotides or kits shown herein can be used to detect, measure or identify biological systems (including, for example, processes or components thereof). Exemplary techniques that can employ the compounds, nucleotides or kits include sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, cell assays (e.g., cell binding or cell function analysis) or protein assays (e.g., protein binding assays or protein activity assays). The use can be on an automated instrument (such as an automated sequencing instrument) for performing a particular technique. A sequencing instrument can include two light sources operating at different wavelengths.

[0209] In a specific embodiment, the nucleotides of the labeling described herein can be combined with unlabeled or natural nucleotides or any combination thereof. The combination of nucleotides can be provided as an independent individual component (e.g., one nucleotide type per container or tube) or as a nucleotide mixture (e.g., two or more nucleotides mixed in the same container or tube).

[0210] In the case where the test kit comprises a variety, particularly two or three or more particularly four nucleotides, different nucleotides can be marked with different dye compounds, or one can be dark, without dye compounds. In the case where different nucleotides are marked with different dye compounds, a feature of the test kit is that the dye compound is a spectrally distinguishable fluorescent dye. As used herein, the term "spectrally distinguishable fluorescent dye" refers to a fluorescent dye that emits fluorescent energy at a wavelength that can be distinguished by a fluorescence detection device (e.g., a commercial capillary-based DNA sequencing platform) when there are two or more such dyes in a sample. When two nucleotides marked with fluorescent dye compounds are provided in a test kit form, some embodiments are characterized in that the spectrally distinguishable fluorescent dye can be excited with the same wavelength, for example, by the same light source. When four nucleotides marked with fluorescent dye compounds are provided in a test kit form, some embodiments are characterized in that two fluorescent dyes in the spectrally distinguishable fluorescent dye can be excited with a wavelength, and two other spectrally distinguishable dyes can be excited with another wavelength. The specific excitation wavelength of the dye is between 450 nm and 460 nm, between 490 nm and 500 nm, or 520 nm or above (eg, 532 nm).

[0211] In some embodiments, the kit includes a first type of nucleotides labeled with a double boron dye of the present disclosure. In addition, the kit may contain a second type of mark, which has a long Stokes shift of about 70nm or more, higher than 80nm, about 90nm or higher than 100nm (wherein "Stokes shift" is the distance between the peak absorption wavelength and the peak emission wavelength). In addition, the kit may contain a third type of labeled nucleotides, which has a short Stokes shift of about or less than 50nm, 40nm or 30nm. In some such embodiments, the first type of labeled nucleotides with a double boron dye may have a Stokes shift range falling between the second type of nucleotides and the third type of nucleotides, for example, between about 30nm to about 70nm, between about 40nm to about 60nm, or between about 45nm to about 55nm. In other embodiments, each of the first type of nucleotides, the second type of nucleotides and the third type of nucleotides can be excited by a single light source (for example, a blue light with a wavelength between about 450nm to about 460nm). In some embodiments, the emission spectra of the first type of nucleotides, the second type of nucleotides, and the third type of nucleotides can be collected in two different detection channels or regions (e.g., in the blue region with a wavelength range of about 472 to about 520 nm, and in the green region with a wavelength range of about 540 nm to about 640 nm).

[0212] Although the kit is described herein as having different nucleotides labeled with different dye compounds, it should be understood that the kit can include 2, 3, 4 or more different nucleotides with the same dye compound.

[0213] In addition to the labeled nucleotides, the kit can also include at least one additional component together. The other components can be one or more components in the components identified in the method shown herein or in the Examples section below. Some non-limiting examples of components that can be incorporated into the kit of the present disclosure are shown below. In some embodiments, the kit also includes a DNA polymerase (such as a mutant DNA polymerase) and one or more buffer compositions. Non-limiting examples that can be used for DNA polymerases of the present disclosure include those disclosed in WO 2005 / 024010, the United States discloses 2020 / 0131484A1 and 2020 / 0181587A1, and each of these documents is incorporated herein by reference in its entirety. A buffer composition can include an antioxidant, such as ascorbic acid or sodium ascorbate, which can be used to protect dye compounds from light damage during detection. Other buffer compositions can include reagents that can be used to cleave 3' capping groups and / or cleavable linkers. For example, a water-soluble phosphine or water-soluble transition metal catalyst formed by a transition metal and at least a portion of a water-soluble ligand (such as a palladium complex). The various components of the test kit can be provided in a concentrated form to dilute before use. In such embodiments, a suitable dilution buffer may also be included. Similarly, one or more components of the components identified in the method shown herein may be included in the test kit of the present disclosure. In any embodiment of the nucleotides described herein or the nucleotides of the labeling, the nucleotides include a 3' blocking group.

[0214] Sequencing methods

[0215] Nucleotides containing dye compounds according to the present disclosure can be used in any analytical method, such as methods including detecting fluorescent labels attached to such nucleotides, whether used by themselves, incorporated into or associated with larger molecular structures or conjugates. In this context, the term "incorporated into a polynucleotide" can mean that the 5' phosphate is joined to the 3' hydroxyl group of the second nucleotide with a phosphodiester bond, and the second nucleotide itself can form a part of a longer polynucleotide chain. The 3' end of the nucleotide shown herein may or may not be joined to the 5' phosphate of another nucleotide with a phosphodiester bond. Therefore, in a non-limiting embodiment, the present disclosure provides a method for detecting labeled nucleotides incorporated into a polynucleotide, the method comprising: (a) incorporating at least one labeled nucleotide of the present disclosure into a polynucleotide, and (b) determining the identity of the nucleotide incorporated into the polynucleotide by detecting a fluorescent signal from a dye compound attached to the nucleotide. A specific embodiment of the sequencing method utilizes an excitation, dual-channel detection system (also referred to as 1Ex-2Ch). Detailed disclosure is provided in WO 2018 / 165099 and U.S. Application No. 17 / 338590, each of which is incorporated by reference in its entirety. Specifically, 1Ex-2Ch may contain a blue excitation light source with a wavelength between about 450 nm and about 460 nm, and two separate collection channels in both the blue region and the green region (e.g., in the blue region with a wavelength range of about 472 nm to about 520 nm and in the green region with a wavelength range of about 540 nm to about 640 nm).

[0216] The method may include: a synthesis step (a), in which one or more labeled nucleotides according to the present disclosure are incorporated into a polynucleotide; and a detection step (b), in which the nucleotides are detected by detecting or quantitatively measuring the fluorescence of the one or more labeled nucleotides incorporated into the polynucleotide.

[0217] Some embodiments of the present application relate to a method for determining the sequence of a plurality of different target polynucleotides, the method comprising:

[0218] (a) contacting a solid support with a solution containing sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;

[0219] (b) contacting the solid support with an aqueous solution comprising a DNA polymerase and one or more of four different types of nucleotides (e.g., dATP, dGTP, dCTP, and dTTP or dUTP) under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotide into the sequencing primer to produce an extended copy polynucleotide, wherein at least one type of nucleotide is a biboron dye-labeled nucleotide as described herein, and wherein each of the four types of nucleotides comprises a 3'-capping group covalently attached to the deoxyribose sugar of the nucleotide;

[0220] (c) imaging the solid support and performing one or more fluorescence measurements of the extended copy polynucleotide; and

[0221] (d) removing the 3' blocking group of the incorporated nucleotide. In some embodiments, step (d) also removes the label of the incorporated nucleotide (if the incorporated nucleotide is labeled). In some such embodiments, the label and 3' blocking group of the incorporated nucleotide are removed in a single chemical reaction. In some other embodiments, the method may also include (e) washing the solid support with an aqueous washing solution (e.g., washing away the removed label portion and 3' blocking group from the extended copy polynucleotide). In some embodiments, steps (b) to (e) are repeated for at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250 or 300 cycles to determine the target polynucleotide sequence. In some embodiments, four types of nucleotides include dATP, dCTP, dGTP and dTTP or dUTP or their non-natural nucleotide analogs. In some embodiments, sequence determination is performed after completing repeated cycles of sequencing steps described herein. In some embodiments, the bi-boron dye described herein can be used as any of the first label, the second label, or the third label described in the method.

[0224] In some other embodiments, the method is carried out on an automated sequencing instrument, and wherein the automated sequencing instrument includes a single light source operated with a blue laser of about 450nm to about 460nm. The incorporation of the first type of nucleotide is determined by detecting in a blue or green channel / region (e.g., at a wavelength range of about 472nm to about 520nm in a blue region, or at a wavelength range of about 540nm to about 640nm in a green region). The incorporation of the second type of nucleotide is determined by detecting in another of the blue or green detection channels / regions. The incorporation of the third type of nucleotide is determined by detecting both the blue and green channels / regions. The incorporation of the fourth type of nucleotide is determined by not detecting in the blue or green channels / regions.

[0225] In other embodiments, the automatic sequencer may include two light sources operating at different wavelengths (e.g., at 450nm-460nm and 520nm-530nm). The incorporation of the first type of nucleotide complex is determined by the signal state in the first imaging event and the dark state in the second imaging event. The incorporation of the second type of nucleotide conjugate is determined by the signal state in the dark state in the first imaging event and the second imaging event. The incorporation of the third type of nucleotide conjugate is determined by the signal state in both the first imaging event and the second imaging event. The incorporation of the fourth type of nucleotide conjugate is determined by the dark state in both the first imaging event and the second imaging event.

[0226] In some embodiments, at least one nucleotide is incorporated into a polynucleotide (such as a single-stranded primer polynucleotide described herein) by the action of a polymerase during a synthesis step. However, other methods of joining nucleotides to polynucleotides can be used, such as chemical oligonucleotide synthesis or connecting labeled oligonucleotides to unlabeled oligonucleotides. Therefore, when the term "incorporation" is used with respect to nucleotides and polynucleotides, polynucleotide synthesis by chemical methods as well as enzymatic methods can be encompassed.

[0227] In a particular embodiment, a synthesis step is performed and the synthesis step may optionally include incubating the template polynucleotide chain or the target polynucleotide chain with a reaction mixture comprising fluorescently labeled nucleotides of the present disclosure. A polymerase may also be provided under conditions that allow the formation of a phosphodiester bond between a free 3' hydroxyl group on the polynucleotide chain annealed to the template or target polynucleotide chain and a 5' phosphate group on the labeled nucleotide. Thus, the synthesis step may include directing the formation of a polynucleotide chain by complementary base pairing of nucleotides with the template / target chain.

[0228] In all embodiments of the method, the detection step can be performed when the polynucleotide chain into which the nucleotides of the mark are incorporated is annealed to the template / target chain, or after a denaturation step in which the two chains are separated. Additional steps, such as a chemical reaction step or an enzymatic reaction step, or a purification step may be included between the synthesis step and the detection step. Specifically, the polynucleotide chain into which the nucleotides of the mark are incorporated can be separated or purified, and then further processed or used for subsequent analysis. By way of example, the polynucleotide chain into which the nucleotides of the mark as described herein are incorporated in the synthesis step can be used as a probe or primer for labeling subsequently. In other embodiments, the product of the synthesis step shown herein can be subjected to further reaction steps, and if desired, the products of these subsequent steps are purified or separated.

[0229] Suitable conditions for the synthesis step will be well known to those familiar with standard molecular biology techniques. In one embodiment, the synthesis step can be similar to a standard primer extension reaction, which uses nucleotide precursors (including the nucleotides of labels as described herein) to form a polynucleotide chain (primer polynucleotide chain) complementary to the template / target chain in the presence of a suitable polymerase. In other embodiments, the synthesis step itself can form a part of the amplification reaction of the double-stranded amplification product of the mark, and the double-stranded amplification product of the mark is composed of the annealing complementary chain derived from the replication of the primer polynucleotide chain and the template polynucleotide chain. Other exemplary synthesis steps include nick translation, strand displacement polymerization, random DNA labeling, etc. The particularly useful polymerase for the synthesis step is a polymerase that can catalyze the incorporation of the nucleotides of the labels shown in this article. A variety of naturally occurring or mutated / modified polymerases can be used. By way of example, a thermostable polymerase can be used for the synthesis reaction performed using thermal cycling conditions, and a thermostable polymerase may not be desired by an isothermal primer extension reaction. Suitable thermostable polymerases capable of incorporating labeled nucleotides according to the present disclosure include those described in WO 2005 / 024010 or WO06120433, each of which is incorporated herein by reference. In synthesis reactions conducted at lower temperatures such as 37° C., the polymerase need not be a thermostable polymerase, and thus the choice of polymerase will depend on many factors, such as reaction temperature, pH, strand displacement activity, etc.

[0230] In specific non-limiting embodiments, the present disclosure encompasses methods of nucleic acid sequencing, resequencing, whole genome sequencing, single nucleotide polymorphism scoring, and any other application involving the detection of modified nucleotides or nucleosides labeled with the dyes set forth herein when incorporated into polynucleotides.

[0231] Specific embodiments of the present disclosure provide the purposes of the nucleotides comprising the labeling of the dye moiety according to the present disclosure in the sequencing reaction while synthesizing the polynucleotides.Synthesis sequencing generally involves using polymerase or ligase to sequentially add one or more nucleotides or oligonucleotides to the growing polynucleotide chain in 5' to 3' direction, so as to form an extended polynucleotide chain complementary to the template / target nucleic acid to be sequenced. The identity of the base present in the one or more added nucleotides can be determined in detection or "imaging" steps. The identity of the added base can be measured after each nucleotide incorporation step. Conventional Watson-Crick base pairing rules can then be used to infer the sequence of the template. It may be useful to use the nucleotides labeled with the dyes shown herein to measure the identity of a single base, for example, in the scoring of a single nucleotide polymorphism, and such single base extension reactions are within the scope of the present disclosure.

[0232] In one embodiment of the present disclosure, the sequence of the template / target polynucleotide is determined by detecting the incorporation of one or more nucleotides into a nascent chain complementary to the template polynucleotide to be sequenced by detecting a fluorescent label attached to the incorporated nucleotides. The sequencing of the template polynucleotide can be initiated with a suitable primer (or prepared as a hairpin construct, which will include a primer as part of the hairpin), and the nascent chain is extended in a one-by-one manner by adding nucleotides to the 3' end of the primer in a polymerase-catalyzed reaction.

[0233] In a particular embodiment, each of the different nucleotide triphosphates (A, T, G and C) can be labeled with a unique fluorophore, and a blocking group is also included at the 3' position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides can be unlabeled (dark). Polymerase incorporates nucleotides into a nascent chain complementary to the template polynucleotide / target polynucleotide, and the blocking group prevents further incorporation of nucleotides. Any unincorporated nucleotides can be washed away, and the fluorescent signal from each nucleotide incorporated can be optically "read" by a suitable device (such as a charge-coupled device using a light source to excite and a suitable emission filter). Then 3' blocking groups and fluorescent dye compounds can be removed (deprotected) (simultaneously or sequentially) to expose the nascent chain for further incorporation of nucleotides. Typically, the identity of the incorporated nucleotides will be determined after each incorporation step, but this is not strictly necessary. Similarly, U.S. Patent No. 5,302,509 (incorporated herein by reference) discloses a method for sequencing polynucleotides fixed on a solid support.

[0234] As illustrated above, the method utilizes the presence of DNA polymerase to incorporate fluorescently labeled 3'-capped nucleotides A, G, C and T into a growing chain complementary to a fixed polynucleotide. Polymerase incorporates bases complementary to the target polynucleotide, but is prevented from further addition by 3'-capping groups. The labeling of the incorporated nucleotides can then be determined, and the capping groups are removed by chemical cleavage to allow further polymerization to occur. The nucleic acid template to be sequenced in the synthesis sequencing reaction can be any polynucleotide that is desired to be sequenced. The nucleic acid template for sequencing reaction will generally include a double-stranded region with a free 3' hydroxyl group, which serves as a primer or starting point for adding additional nucleotides in the sequencing reaction. The region of the template to be sequenced will dangle the free 3' hydroxyl group on the complementary chain. The dangling region of the template to be sequenced can be single-stranded, but can also be double-stranded, provided that there is a "nick" on the chain complementary to the template chain to be sequenced, to provide a free 3' OH group for initiating sequencing reactions. In such embodiments, sequencing can be performed by strand displacement. In certain embodiments, primers with free 3' hydroxyl groups can be added as separate components (e.g., short oligonucleotides) hybridized with the single-stranded region of template to be sequenced. Alternatively, primers to be sequenced and template strands can each form a part of a partial self-complementary nucleic acid chain that can form an intramolecular duplex (such as a hairpin loop structure). Hairpin polynucleotides and methods that can be attached to solid supports are disclosed in PCT Publication No. WO0157248 and WO2005 / 047301, and each of these disclosures is incorporated herein by reference. Nucleotide can be continuously added to the growth primer, resulting in a polynucleotide chain synthesized in the 5' to 3' direction. The properties of the bases added can be determined, particularly but not necessarily after each addition of nucleotides, thereby providing the sequence information of nucleic acid templates. Therefore, by forming a phosphodiester bond with the 5' phosphate group of nucleotides and nucleotides are joined to the free 3' hydroxyl groups of nucleic acid chains, thereby the nucleotides are incorporated into nucleic acid chains (or polynucleotides).

[0235] The nucleic acid template to be sequenced can be DNA or RNA, or even a hybrid molecule composed of deoxynucleotides and ribonucleotides. The nucleic acid template can contain naturally occurring and / or non-naturally occurring nucleotides and natural or non-natural backbone bonds, provided that these do not prevent replication of the template in the sequencing reaction.

[0236] In certain embodiments, the nucleic acid template to be sequenced can be attached to a solid support via any suitable connection method known in the art (e.g., via covalent attachment). In certain embodiments, the template polynucleotide can be directly attached to a solid support (e.g., a silica-based support). However, in other embodiments of the present disclosure, the surface of the solid support can be modified in some way to allow direct covalent attachment of the template polynucleotide, or the template polynucleotide can be fixed by a hydrogel or polyelectrolyte multilayer that can itself be non-covalently attached to the solid support.

[0237] In some embodiments, the polynucleotides are directly attached to a carrier (e.g., a silica-based carrier, such as those disclosed in WO00 / 06770 (incorporated herein by reference)), wherein the polynucleotides are fixed to a glass carrier by reaction between an epoxy side group on the glass and an internal amino group on the polynucleotides. In addition, polynucleotides can be attached to a solid carrier by reaction of a sulfonyl nucleophile with a solid carrier, for example, as described in WO2005 / 047301 (incorporated herein by reference). Other additional examples of template polynucleotides supported by solids are template polynucleotides attached to a hydrogel loaded on silica or other solid carriers, for example, as described in WO00 / 31148, WO01 / 01143, WO02 / 12566, WO03 / 014392, U.S. Patent No. 6,465,178 and WO00 / 53812, each of which is incorporated herein by reference.

[0238] The specific surface on which the template polynucleotide can be fixed is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the references cited above and WO2005 / 065814, which is incorporated herein by reference. Specific hydrogels that can be used include those described in WO2005 / 065814 and U.S. Publication 2014 / 0079923. In one embodiment, the hydrogel is PAZAM (poly (N- (5-azidoacetamidopentyl) acrylamide-co-acrylamide)).

[0239] The DNA template molecules can be attached to beads or microparticles, for example, as described in U.S. Pat. No. 6,172,218 (incorporated herein by reference). Connection to beads or microparticles can be used for sequencing applications. Bead libraries can be prepared, wherein each bead contains a different DNA sequence. Exemplary libraries and methods for their generation are described in Nature, 437, 376-380 (2005); Science, 309, 5741, 1728-1732 (2005), each of which is incorporated herein by reference. It is within the scope of the present disclosure to sequence an array of such beads using the nucleotides shown herein.

[0240] The template to be sequenced can form part of an "array" on a solid support, in which case the array can take any convenient form. Therefore, the method of the present disclosure is applicable to all types of high-density arrays, including single-molecule arrays, clustered arrays, and bead arrays. Nucleotides labeled with dye compounds of the present disclosure can be used to sequence templates on substantially any type of array (including but not limited to those formed by fixing nucleic acid molecules on a solid support).

[0241] However, nucleotides labeled with dye compounds of the present disclosure are particularly advantageous in the context of sequencing clustered arrays. In clustered arrays, different regions on the array (commonly referred to as sites or features) include multiple polynucleotide template molecules. Generally speaking, the multiple polynucleotide molecules can not be distinguished individually by optical means, but are detected as a whole. Depending on the formation mode of the array, each site on the array can include multiple copies of a single polynucleotide molecule (for example, the site is homogeneous for specific single-stranded nucleic acid species or double-stranded nucleic acid species) or even a small amount of multiple copies of different polynucleotide molecules (for example, multiple copies of two different nucleic acid species). The clustered array of nucleic acid molecules can be produced using technology known in the art. By way of example, WO 98 / 44151 and WO00 / 18957 (each of these patents is incorporated herein by reference) describe a method for amplifying nucleic acids, wherein template and amplification product are both kept fixed on a solid support, so as to form an array consisting of a cluster or " colony" of fixed nucleic acid molecules. Nucleic acid molecules present on clustered arrays prepared according to these methods are suitable templates for sequencing using nucleotides labeled with the dye compounds of the present disclosure.

[0242] Nucleotides labeled with dye compounds of the present disclosure can also be used to sequence the templates on single molecule arrays. As used herein, the term "single molecule array" or "SMA" refers to a polynucleotide molecule population distributed (or arranged) on a solid support, wherein the spacing between any single polynucleotide and all other polynucleotides of the molecule population makes it possible to distinguish the single polynucleotide molecules individually. Therefore, in some embodiments, the target nucleic acid molecules fixed to the surface of the solid support can be distinguished by optical means. This means that one or more different signals (each signal represents a polynucleotide) will appear in the distinguishable region of the specific imaging device used.

[0243] Single molecule detection can be achieved, wherein the spacing between adjacent polynucleotide molecules on the array is at least 100 nm, more specifically at least 250 nm, more specifically at least 300 nm, and even 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 the single molecule fluorescent spot also exhibits single-step photobleaching.

[0244] The terms "individually resolved" and "individually resolved" are used herein to specify that, when visualized, it is possible to distinguish a molecule on an array from its adjacent molecules. The spacing between the 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 published applications WO 00 / 06770 and WO 01 / 57248, each of which is incorporated herein by reference. Although one use of the labeled nucleotides of the present disclosure is for use in synthetic sequencing reactions, the effectiveness 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 detection of fluorescent labels attached to nucleotides incorporated into the polynucleotides.

[0245] Specifically, nucleotides labeled with dye compounds of the present disclosure can be used in automated fluorescent sequencing protocols, especially 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 of labeled dideoxynucleotides.

[0246] Thus, the present disclosure also encompasses 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-type sequencing methods and the like.

[0247] It will be appreciated that nucleotides labeled with dye compounds of the present disclosure incorporating 3' blocking groups may also be used in Sanger methods and related protocols, since the same effect as that achieved by using dideoxy nucleotides may be achieved by using nucleotides with 3'OH blocking groups: both prevent incorporation of subsequent nucleotides. In cases where nucleotides according to the present disclosure and with 3' blocking groups are to be used in Sanger-type sequencing methods, it will be appreciated that the dye compound or detectable label attached to the nucleotide need not be attached via a cleavable linker, since in each case of incorporating a labeled nucleotide of the present disclosure; no subsequent incorporation of a nucleotide is required, and therefore no removal of the label from the nucleotide is required.

[0248] Alternatively, unlabeled nucleotides and affinity reagents containing fluorescent dyes described herein can also be used to carry out sequencing methods described herein. For example, in the incorporation mixture of step (a), one type, two types, three types or each type of four different types of nucleotides (e.g., dATP, dCTP, dGTP and dTTP or dUTP) can be unlabeled. Each type of four types of nucleotides (e.g., dNTP) has a 3' blocking group to ensure that only a single base can be added to the 3' end of the primer polynucleotide by a polymerase. After incorporating unlabeled nucleotides in step (b), the remaining unincorporated nucleotides are washed away. Then an affinity reagent is introduced, which specifically recognizes and binds to the incorporated dNTP to provide an extension product containing the label of the incorporated dNTP. The use of unlabeled nucleotides and affinity reagents in synthetic sequencing has been disclosed in WO 2018 / 129214 and WO 2020 / 097607. The sequencing method of the modification of the present disclosure using unlabeled nucleotides may include the following steps:

[0249] (a') contacting a solid support with a solution containing sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;

[0250] (b') contacting the solid support with an aqueous solution comprising a DNA polymerase and one or more of four different types of unlabeled nucleotides (e.g., dATP, dCTP, dGTP, and dTTP or dUTP) under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotide into the sequencing primer to produce an extended copy polynucleotide, and wherein each of the four types of nucleotides comprises a 3' blocking group;

[0251] (c') contacting the extended copy polynucleotide with a set of affinity reagents under conditions where one of the affinity reagents specifically binds to the incorporated unlabeled nucleotide to provide a labeled extended copy polynucleotide;

[0252] (d') imaging the solid support and performing one or more fluorescence measurements of the extended copy polynucleotide; and

[0253] (e') Removal of the 3' blocking group of the incorporated nucleotide.

[0254] In some embodiments of the modified sequencing methods described herein, the method further comprises removing the affinity reagent from the incorporated nucleotides. In still other embodiments, the 3' blocking group and the affinity reagent are removed in the same reaction. In some embodiments, the method further comprises step (f') washing the solid support with an aqueous washing solution. In other embodiments, steps (b') to (f') are repeated for at least 50, 100, 150, 200, 250 or 300 cycles to determine the target polynucleotide sequence. In some embodiments, the affinity reagent group may comprise a first affinity reagent that specifically binds to the first type of nucleotides, a second affinity reagent that specifically binds to the second type of nucleotides, and a third affinity reagent that specifically binds to the third type of nucleotides. In other embodiments, each of the first affinity reagent, the second affinity reagent, and the third affinity reagent comprises a spectrally distinguishable detectable label. In some embodiments, the affinity reagent may comprise a protein tag, an antibody (including but not limited to a binding fragment of an antibody, a single-chain antibody, a bispecific antibody, etc.), an aptamer, a knotting element, an affimer, or any other known reagent that binds to the incorporated nucleotides with suitable specificity and affinity. In one embodiment, at least one affinity reagent is an antibody or a protein tag. In another embodiment, at least one of the first type of affinity reagent, the second type of affinity reagent, and the third type of affinity reagent is an antibody or a protein tag comprising one or more detectable labels (e.g., multiple copies of the same detectable label), wherein the detectable label is or includes a biboron dye moiety as described herein.

[0255] Example

[0256] Additional embodiments are disclosed in more detail in the following examples, which are not intended to limit the scope of the claims in any way.

[0257] Example 1. Synthesis of dyes containing fused biboronic heterocycles

[0258] General Procedure A

[0259]

[0260] 3,8-DichloroBOPHY in acetonitrile was stirred with a nucleophile and K2CO3 under reflux until converted to the product. The reaction mixture was cooled, poured into DCM, and washed with water and brine. The crude product was dried over MgSO4, filtered, and the solvent was removed under reduced pressure.

[0261] Compound I-1 Synthesis

[0262]

[0263] Based on General Procedure A, a mixture of 3,8-dichloroBOPHY (1.0 eq.), tert-butyl 4-hydroxybenzoate (2.0 eq.) and K2CO3 (4.0 eq.) was refluxed in acetonitrile for 3 h, then the crude product was dissolved in DCM and TFA1% was added after treatment. The mixture was stirred at rt for 16 h. The solvent was removed under vacuum. The crude mixture was then diluted with 0.1 M TEAB and MeCN and then purified by reverse phase preparative HPLC to obtain compound I-1 (34%) as a bright yellow solid. 1 HNMR (400MHz, DMSO-d6) δ13.02(br s,1H),8.83(s,1H),8.68(s,1H),8.03(d,J=8.80Hz,2H),7.64(d,J=4.60Hz 1H),7.64(d,J=4.12Hz 1H), 7.45(d,J=8.8Hz 2H), 6.76(d,J=4.12Hz 1H), 6.11(d,J=4.48Hz 1H). 19 F NMR(377MHz, DMSO-d6)δ-140.84(s,2F),-142.33(s,2F). 11 B NMR (128MHz, DMSO-d6) δ0.38 (s, 2B). Mass spectrum: [MH] - =451.

[0264] Compound I-2 Synthesis

[0265]

[0266] Based on General Procedure A, a mixture of 3,8-dichloroBOPHY (1.0 equiv.), tert-butyl 4-hydroxybenzoate (10.0 equiv.) and K2CO3 (4.0 equiv.) was refluxed in acetonitrile for 16 h. After treatment, the crude product was purified by normal phase flash chromatography (DCM: MeOH), followed by dissolving the t-Bu diester in DCM and adding TFA 1%. The mixture was stirred at rt for 16 h. The solvent was removed under vacuum. The crude mixture was then diluted with 0.1 M TEAB and MeCN and then purified by reverse phase preparative HPLC to obtain compound I-2 (84%) in the form of a bright orange solid. 1 H NMR (400MHz, DMSO-d6) δ 13.07 (br s, 2H), 8.65 (s, 2H), 8.07 (d, J = 8.80, 4H), 7.59 (d, J = 4.40, 2H), 7.41 (d, J = 8.80, 4H), 6.13 (d, J = 4.40, 2H). 19F NMR(377MHz,DMSO-d6)δ-141.99(s,4F). 11 B NMR (128MHz, DMSO-d6) δ0.30 (s, 2B). Mass spectrum: [MH] - =553,[M-2H] 2- =276.

[0267] General Procedure B

[0268]

[0269] With the BCl3 (4.5 equivalents) of dropwise addition, process the relevant BOPHY in DCM.Reaction mixture is stirred for 30min.Then add nucleophile (8.0 equivalents) and TEA (12.0 equivalents).Reaction mixture is stirred under rt.Crude product is filtered through diatomite and washed with DCM.Desolventizing under vacuum, and gained residue is dissolved in 0.1M TEAB / MeCN mixture, and by reverse phase preparative HPLC purification.Calculate productive rate by the OD of dilution.

[0270] Compound I-3 Synthesis

[0271]

[0272] The reaction mixture was stirred at rt for 3 h to afford compound 1-3 (83%) as a bright orange solid based on General Procedure B. The yield was calculated by the OD of the dilution. 1 H NMR (400MHz, DMSO-d6) δ 8.44 (s, 2H), 8.03 (d, J = 8.80, 4H), 7.45 (d, J = 4.40, 2H), 7.22 (d, J = 8.80, 4H), 5.98 (d, J = 4.40, 2H), 1.88 (s, 12H). 11 BNMR (128MHz, DMSO-d6) δ0.70 (s, 2B). Mass spectrum: [MH] - =713,[M-2H] 2- =356.

[0273] Compound I-5 Synthesis

[0274]

[0275] Based on General Procedure B, the reaction mixture was stirred at rt for 16 h to give compound I-5 (29%) as a bright orange solid. The yield was calculated by the OD of the dilution. Mass Spectrum: [MH]- =601,[M-2H] 2- =300.

[0276] Synthesis of Compound I-6

[0277]

[0278] Based on General Procedure B, the reaction mixture was stirred at rt for 16 h to give compound I-6 (5%) as a bright orange solid. The yield was calculated by the OD of the dilution. Mass Spectrum: [MH] - =657,[M-2H] 2- =328.

[0279] Compound I-11 Synthesis

[0280]

[0281] Based on General Procedure B, the reaction mixture was stirred at rt for 3 h to give compound I-11 (4%) as a bright orange solid. The yield was calculated by the OD of the dilution. Mass Spectrum: [MH] - =761,[M-2H] 2- =380.

[0282] Compound I-12 Synthesis

[0283]

[0284] Based on General Procedure B, the reaction mixture was stirred at rt for 3 h to give compound I-12 (6%) as a bright orange solid. The yield was calculated by the OD of the dilution. Mass Spectrum: [MH] - =962,[M-2H] 2- =480.

[0285] General Procedure C

[0286]

[0287] After adding the activated dye 4-aminobutyric acid (1.20 eq.) 30 min, the relevant BOPHY in dry DMF was treated with TNTU (1.20 eq.) and DIPEA (10.0 eq.). The reaction mixture was stirred at rt for 16 h. The crude product was purified by reverse phase preparative HPLC.

[0288] Compound I-4 Synthesis

[0289]

[0290] Based on the general procedure D, the reaction mixture starting from I-3 was stirred at rt for 16 h to give compound I-4 (29%). The yield was calculated by the OD of the dilution. Mass spectrum: [MH] - =883,[M-2H] 2- =441.

[0291] Synthesis of Compound I-7

[0292]

[0293] Based on the general procedure C, the reaction mixture starting from compound I-2 was stirred at rt for 16 h to give compound I-7 (25%). The yield was calculated by the OD of the dilution. Mass spectrum: [MH] - =723,[M-2H] 2- =361.

[0294] General Procedure D - Asymmetric Dyes

[0295] Step A :

[0296]

[0297] Under nitrogen, a solution of 3,8-dichloroBOPHY (1 equivalent) in acetonitrile (10 mL / mg) was charged into a three-necked round-bottom flask equipped with a reflux condenser. Tert-butyl p-hydroxybenzoate (1.6 equivalents) was added to the stirred solution together with potassium carbonate (5 equivalents). The reaction was heated to 65 ° C and kept stirring. A mixture of DCM: petroleum ether was used as an eluent mixture and the reaction was monitored by LC-MS and TLC. After the starting material was completely consumed, phenol (1.6 equivalents) was added to the stirred solution, and the stirred solution was stirred at 65 ° C under an inert atmosphere until the monosubstituted intermediate was completely converted into the desired asymmetric product. The reaction was then cooled, concentrated to dryness under reduced pressure, and the crude product was dissolved in DCM and washed with water and brine. The organic layer was separated, and the aqueous phase was extracted with DCM again. The combined organic phase was concentrated under reduced pressure, and the crude material was purified by silica gel chromatography or processed to the next step without purification.

[0298]

[0299] Following step A of general procedure D, 3,8-dichloroBOPHY (0.050 g, 0.143 mmol) was dissolved in ACN (50 mL) and tert-butyl p-hydroxybenzoate (0.045 g, 0.229 mmol) and potassium carbonate (0.098 g, 0.715 mmol) were added. After complete conversion of the starting material, 3,4,5-trifluorophenol (0.034 g, 0.229 mmol) was added. Compound A was purified by silica gel chromatography using DCM:PE 9:1 as eluent mixture and isolated as a bright orange powder (0.032 g, 36% yield). 1 H NMR (400MHz, CDCl3) δ8.08(s,1H),8.06(s,1H),7.98(s,1H),7.29(s,1H),7.23(d,J=4.5Hz, 2H), 6.46 (dd, J = 8.7, 5.5Hz, 4H), 5.90 (d, J = 4.4Hz, 1H), 5.85 (d, J = 4.5Hz, 1H), 1.61 (s, 9H).

[0300]

[0301] Following step A of general procedure D, 3,8-dichloroBOPHY (0.010 g, 0.029 mmol) was dissolved in ACN (10 mL), and tert-butyl p-hydroxybenzoate (0.009 g, 0.046 mmol) and potassium carbonate (0.020 g, 0.145 mmol) were added. After complete conversion of the starting material, phenol (0.004 g, 0.046 mmol) was added. The crude product, Compound B, was carried forward to the next step without purification. 1 H NMR(400MHz, CDCl3)δ8.07(m,1H),8.05(m,1H),8.02(dd,1H),7.95(dd,1H),7.46-7.41,(m,2H),7.32-7.29(m,2H),7.26-7.21 (m,1H),7.19(t,J=4.2Hz,2H),6.97-6.89(m,1H),6.87-6.79(m,2H),5.84(d,J=4.4Hz,1H),5.78(d,J=4.4Hz,1H)1.61(s,9H).

[0302]

[0303] Following step A of general procedure D, 3,8-dichloroBOPHY (0.010 g, 0.029 mmol) was dissolved in ACN (10 mL) and tert-butyl p-hydroxybenzoate (0.009 g, 0.046 mmol) and potassium carbonate (0.020 g, 0.145 mmol) were added. After complete conversion of the starting material, 3,5-dimethoxyphenol (0.004 g, 0.046 mmol) was added. The crude product, Compound C, was carried forward to the next step without purification. [M+H2O] + =644.

[0304] Step B: Ester hydrolysis under acidic conditions

[0305]

[0306] Under nitrogen, a solution of starting material (1 equivalent) in DCM (10mL / eq) was loaded into a one-necked round-bottom flask, and trifluoroacetic acid (TFA) (2%) was added to the solution, and the solution was stirred for 8 hours at room temperature under an inert atmosphere. The reaction was monitored by LC-MS. After SM was completely consumed, the reaction was concentrated to dryness, and the crude material was purified by the gradient of ACN in 0.1M TEAB (triethylammonium bicarbonate) by preparative HPLC. The purity and properties of the final material were determined by UPLC and LC-MS. The yield of the reaction was determined by spectrophotometry.

[0307] Synthesis of Compound I-9

[0308]

[0309] Following step B of general procedure D, compound A (0.030 g, 0.048 mmol) was dissolved in DCM (10 mL) and TFA (2 mL) was added. Compound I-9 was purified by preparative HPLC using a 40%-60% gradient of ACN in 0.1 M TEAB (yield: 51.3%). [MH] - =563.

[0310] Compound I-13 Synthesis

[0311]

[0312] Following step B of general procedure D, compound B (0.077 g, 0.136 mmol) was dissolved in DCM (10 mL) and TFA (3 mL) was added. Compound I-13 was purified by preparative HPLC using a 40% to 60% gradient of ACN in 0.1 M TEAB (yield: 97%). [MH] - =509.

[0313] Compound I-15 Synthesis

[0314]

[0315] Following step B of general procedure D, compound C (crude) was dissolved in DCM (5 mL) and TFA (1 mL) was added. The product was purified by preparative HPLC using a 40% to 60% gradient of ACN in 0.1M TEAB to give compound I-15. [MH] - =569.

[0316] Compound I-10 Synthesis

[0317]

[0318] 5-Formyl-2,4-dimethyl-3-pyrrolecarboxylic acid (507 mg, 3.03 mmol) in EtOH (15 mL) was treated with hydrazine hydrate (0.11 mL, 1.7 mmol) and AcOH (1 drop) and heated at 50° C. for 5 h. The mixture was cooled to room temperature and the resulting precipitate was filtered under vacuum and washed with EtOH to give compound D (470 mg, 94%) as a yellow solid. 1 HNMR (400MHz, DMSO) δ8.43(s,1H),2.44(s,3H),2.32(s,3H).

[0319] Compound D (470 mg, 1.42 mmol) in PhMe (15 mL) was treated with BF3OEt (11.3 mL, 42.6 mmol) and Et3N (4.0 mL, 29 mmol) and heated at 110°C for 3 h. The reaction mixture was cooled to room temperature and the resulting solid was isolated by filtration and dried under vacuum. The crude material was dissolved in DCM / MeOH and purified by column chromatography. The isolated material was again placed under reaction conditions and stirred at 110°C for 2 h. The precipitate was filtered under vacuum, dissolved in 0.1 MTEAB and purified by reverse phase chromatography to give compound E (4%). Mass spectrum: [MH] - =425.

[0320]

[0321] Compound E (20 μmol) in DMA (2mL) was treated with DIPEA (0.4mL) and PyBOP (13mg, 24 μmol). The reaction mixture was stirred at room temperature for 20min, and C3 spacer (3.9mg, 20 μmol) was added. The reaction mixture was stirred for 2h, and then concentrated in vacuo. The residue was dissolved in DCM (5mL) and TFA (2mL) was added. The reaction mixture was concentrated in vacuo and purified by reverse phase preparative HPLC to obtain compound I-10 (14% yield, calculated by dilution OD). Mass spectrum: [MH] - =510.

[0322] Synthesis of Compound I-14

[0323]

[0324] Under nitrogen, a 50-mL round-bottom flask was charged with a solution of 3,8-dichloroBOPHY (0.200 g, 0.57 mmol) in ACN (20 mL), and tert-butyl 3-hydroxybenzoate (0.560 g, 2.83 mmol) was added to the stirred solution together with potassium carbonate (0.400 g, 2.9 mmol). The reaction was heated to reflux and stirred overnight under a nitrogen atmosphere. The reaction was monitored by TLC (DCM: PE 9: 1), and after the starting material was completed, the solution was cooled. The reaction was concentrated to dryness under reduced pressure and dissolved in ethyl acetate, and washed with water and brine. The organic layer was separated, and the aqueous phase was re-extracted with DCM. The combined organic phase was concentrated under reduced pressure to give a crude compound F, which was purified and separated into an orange powder by silica gel chromatography in a yield of 50%. 1 H NMR (400MHz, CDCl3) δ8.00(s,2H),7.93(t,J=1.4Hz,1H),7.91(t,J=1.4Hz,1H),7.87(t,J=2.0Hz,2H) ,7.49(t,J=7.9Hz,2H),7.46-7.39(m,2H),7.19(d,J=4.4Hz,2H),5.78(d,J=4.5Hz,2H),1.60(s,18H).

[0325] Under nitrogen, a solution of compound F (0.163 g, 0.245 mmol) in DCM (10 mL) was loaded into a 50-mL round-bottom flask, and TFA (2 mL) was added. The reaction was placed overnight under a nitrogen atmosphere at RT under stirring. The reaction was monitored by LC-MS and after the starting material was completely converted. The reaction was concentrated under reduced pressure to obtain compound I-14. The product obtained is pure and can be used without further purification (0.082 g, 61% yield).1 H NMR (400MHz, DMSO) δ13.29 (s, b, 2H), 8.64 (s, 2H) 7.90 (d, J = 1.7Hz, 2H), 7.73-7.69 (m, 2H), 7.65(t,J=7.8Hz,2H),7.62-7.58(m,2H),7.56(d,J=4.4Hz,2H),6.03(d,J=4.4Hz,2H).[MH] - =554.

[0326] Compound I-8 Synthesis

[0327]

[0328] Under nitrogen, a solution of compound I-14 (0.030 g, 0.054 mmol) in DMA (5 mL) was loaded into a 25-mL round-bottom flask. At this stage, TNTU (0.024 g, 0.065 mmol) was added together with DIPEA (0.100 mL, 0.054 mmol). The reaction was stirred at RT under an inert atmosphere, and its release was checked by UPLC and TLC. After the starting material was completely consumed, γ-aminobutyric acid (0.007 g, 0.069 mmol) was added to the reaction mixture, and the reaction mixture was stirred overnight at RT under nitrogen. LC-MS showed the presence of the desired material (monosubstituted) and disubstituted adducts. The reaction was quenched by adding 0.1 M TEAB (15 mL), and the crude material was purified by preparative HPLC using a 15%-35% gradient of ACN in 0.1 M TEAB. Resolution of the two species failed and this material was processed in the next step as a mixture of mono- and di-substituted products.

[0329] Synthesis of Compound I-25

[0330]

[0331] Under nitrogen, compound F (0.118 g, 1.76 × 10 -4 mol) was dissolved in 20 mL of anhydrous DCM, and Me2AlCl (0.9 M in hexane, 4.4 eq., 7.04×10 -4mol, 782 μmol) was slowly added to the stirred solution. The addition of the reagent caused the color of the solution to change from green-yellow to yellow-orange. The reaction was monitored by TLC (DCM AcOET 9: 1) to detect SM. After 5 minutes, the starting material disappeared from the reaction and the reaction was quenched by adding water (15mL). The addition of water resulted in the formation of an orange precipitate, which was collected by filtration and processed to the next step. The material was dissolved in HCl (aqueous solution) and stirred overnight at rt. The crude material (yield: 11%) was purified using an AXIA column on a preparative HPLC using a 30%-50% gradient of ACN in 0.1M TEAB, determined by OD in two steps. 1 H NMR (400 MHz, acetone) δ 8.11, s, 2H, 8.09-8.05m, 4H, 7.20d, 2H, 7.19-7.18m, 4H, 5.87-5.86d, 2H, 0.14, s, 12H. [MH] - =538.

[0332] The fluorescence spectra of some exemplary dyes disclosed herein are summarized in Table 1 below.

[0333] Table 1. Absorption and emission spectra of compounds of formula (I)

[0334]

[0335]

[0336] Example 2. Synthesis of fully functionalized nucleotides (ffN) conjugated with biboron dyes

[0337] General procedure E-ffN coupling

[0338]

[0339] Under nitrogen, a solution of FA (10 μmol) in N, N-dimethylacetamide (DMA, 3 mL) was loaded into a 25-mL round-bottom flask, and N, N-diisopropylamine (DIPEA) (10 equivalents) was added together with 2-(5-norbornene-2,3-dicarboxyimino)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU, 1.2 equivalents). The reactant was stirred at room temperature under nitrogen and monitored by UPLC and TLC. After conversion to the desired intermediate, LN3-WFT (1 equivalent) was added to the stirred solution together with triethylamine (7.2 equivalents). At room temperature, the reactant was stirred for 8 hours under an inert atmosphere. After the intermediate was completely consumed and the desired material was formed, the reactant was quenched by adding 20 mL of 0.1 M TEAB. The reactant was loaded onto a dextran gel column and treated with a gradient of 1 M TEAB in 0.1 M TEAB. The fractions containing the product were then processed onto a 60 g C18 column with a gradient of 0.1 M TEAB in ACN. Finally, the product was purified by preparative HPLC using a gradient of ACN in 0.1 M TEAB. The nature and purity of the product were confirmed by UPLC and LC-MS. The yield was determined by spectrophotometry and is summarized as follows:

[0340] ffT-LN3-I-1 was prepared in 5% yield. [MH] - =1359,[M-2H] 2- =679,[M-3H] 3- =452.0.

[0341] ffT-LN3-I-2 was prepared in 19% yield. [MH] - =1461,[M-2H] 2- =730,[M-3H] 3- =486.

[0342] ffT-LN3-I-4 was prepared in 20% yield. [MH] - =1791,[M-2H] 2- =895,[M-3H] 3- =596.

[0343] fT-LN3-I-9 was prepared at 32.7%. [M] - =1470.

[0344] fT-LN3-I-10 was prepared in 4% yield. [M-2H] 2- =708.

[0345] fT-LN3-I-13 was prepared in 16.7% yield. [M] - =1416.

[0346] fT-LN3-I-15 was prepared in 24.4% yield. [M]-=1576.

[0347]

[0348] Using 20 μmol of compound I-8 as the starting material, ffT-LN3-I-8 was obtained according to General Procedure E. The conjugated ffN was purified by preparative HPLC using a 32%-37% gradient of ACN in 0.1 M TEAB (yield: 12%). [M] - =1546.

[0349] The fluorescence spectra of exemplary ffNs disclosed herein are summarized in Table 2 below.

[0350] Table 2. Absorption and emission spectra of ffN conjugated with dyes of formula (I)

[0351]

[0352]

[0353] *Universal scanning mixture (USM, 1M Tris pH 7.5, 0.05%, TWEEN, 20 mM sodium ascorbate,

[0354] 10 mM ethyl gallate).

[0355] Example 3. Illumina MiSeq TM Sequencing experiments on the platform

[0356] In Illumina MiSeq TM The fft labeled with double boron dyes as described herein was tested on the instrument, which was set to take the first image under blue excitation light (about 450nm) and take the second image under blue excitation light (about 450nm). Images were taken simultaneously by the collection channels of blue (472nm-520nm) and green (540nm-640nm).

[0357] Comparative sequencing run A: For Figure 1AThe spiked mixture used in this experiment included the following five ffNs: 2 μM ffA labeled with blue cyanine dye A (ffA-sPA-dye A), 2 μM ffC labeled with blue coumarin dye B (ffC-sPA-dye B), 0.7 μM ffT labeled with blue coumarin dye C with strong fluorescence (ffT-LN3-dye C), 1.3 μM ffT labeled with known green cyanine dye D (ffT-LN3-dye D) and 2 μM unlabeled ffG (dark G) in 50 mM glycine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO4 and DNA polymerase. For Figure 1B , the spiked mixture contained the same ffN set, except that 1.5 μM ffT labeled with biboron dye I-2 (ffT-LN3-I-2) replaced ffT labeled with coumarin dye C, and ffT-LN3-dye D was 0.5 μM. It was observed that the ffN set with dye I-2 generated a good scatter plot that was comparable to the scatter plot generated with the ffN set containing coumarin dye C. For both figures, the scatter plots were generated at cycle 26.

[0358] Comparative sequencing run B: Comparative sequencing run A, for Figure 2A The spiked mixture used in this experiment included the following four ffNs: 2 μM ffA labeled with blue chromophore quinoline dye A (ffA-sPA-dye A), 2 μM ffC labeled with blue coumarin dye B (ffC-sPA-dye B), 2 μM ffT labeled with blue coumarin dye C with strong fluorescence (ffT-LN3-dye C), and 2 μM unlabeled ffG (dark G), in 50 mM glycine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO4, and DNA polymerase. For Figure 2B , the spiked mixture contained the same ffN set except that 2 μM ffT labeled with the biboron dye I-3 (ffT-LN3-I-3) was used instead of ffT labeled with coumarin dye C. It was observed that the ffN set with dye I-3 produced scatter plots of excellent quality with very high cloud separation, which was comparable to the scatter plots produced with the ffN set containing coumarin dye C. For both figures, the scatter plots were generated at cycle 26 at 22°C imaging temperature.

[0359] Comparative sequencing run C: For Figure 3AThe spiked mixture used in this experiment included the following five ffNs: 2 μM ffA labeled with blue cyanine quinoline dye E (ffA-sPA-dye E), 2 μM ffC labeled with blue COT-linked coumarin dye B (ffC-sPA-dye B-COT), 1 μM ffT labeled with blue coumarin dye C with strong fluorescence (ffT-LN3-dye C), 1 μM ffT labeled with known green cyanine dye D (ffT-LN3-dye D) and 2 μM unlabeled ffG (dark G) in 50 mM glycine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO4 and DNA polymerase. For Figure 3B , the spiked mixture contained the same ffN set except that 1 μM ffT labeled with the bisboron dye I-8 (ffT-LN3-I-8) was used instead of ffT labeled with coumarin dye C. Again, it was observed that the ffN set with dye I-8 generated scatter plots of good quality with high cloud separation that were comparable to those generated with the ffN set containing coumarin dye C. For both figures, the scatter plots were generated at cycle 26 at 22°C imaging temperature.

[0360] Figure 4A , Figure 4B and Figure 4C The signal decay percentage, error rate and the phasing percentage of comparative sequencing run A when circulating 151 cycles using the ffN group with ffT-LN3-coumarin dye C or ffT-LN3-I-2 are shown, wherein the blue laser of the different doses of 1x, 3x, 5x, 7.5x and 10x is assigned to the different areas of the same flow cell. For signal decay percentage, it is also observed with 0x light dose in the absence of light and light damage. For the ffN group with ffT-LN3-coumarin dye C, the phasing value is 0.77 to 0.148, and for the ffN group with ffT-LN3-I-2, the phasing value is 0.141 to 0.237. The average pre-phasing value independent of light is 0.26 and 0.07 respectively.

[0361] Figure 5A , Figure 5B and Figure 5CThe signal decay percentage, error rate and phasing percentage of comparative sequencing run C' (wherein ffA-sPA-dye A-COT is used to replace ffA-sPA-dye E) at cycle 151 using ffN groups with ffT-LN3-coumarin dye C or ffT-LN3-I-8, respectively, are shown, where different doses of blue laser light of 1x, 3x, 5x, 7.5x and 10x are assigned to different areas of the same flow cell. For the signal decay percentage, observations were also made with 0x light dose in the absence of light and light damage. The phasing values ​​are comparable, for the use of ffT-LN 3- The ffN set for coumarin dye C had phasing values ​​ranging from 0.094 to 0.151, and for the ffN set with ffT-LN3-I-8, the phasing values ​​ranged from 0.122 to 0.18. The light-independent average prephasing values ​​were 0.2 and 0.13, respectively.

[0362] Comparative sequencing run D: The first spike-in mixture used in this experiment included the following five ffNs: 2 μM ffA labeled with COT-linked chromene quinoline dye A (ffA-sPA-dye A-COT), 2 μM ffC labeled with COT-linked coumarin dye B (ffC-sPA-dye B-COT), 0.75 μM ffT labeled with a blue coumarin dye C with strong fluorescence (ffT-LN3-dye C), 1.25 μM ffT labeled with a known green cyanine dye D (ffT-LN3-dye D) and 2 μM unlabeled ffG (dark G), in 50 mM glycine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO4 and DNA polymerase. The second incorporation mixture contained the same ffN set except that 1.75 μM ffT labeled with bisboron dye I-9 (ffT-LN3-I-9) replaced ffT labeled with coumarin dye C, and ffT-LN3-dye D was 0.25 μM. Fig. 6A , Figure 6B and Figure 6C The signal decay percentage, error rate, and phasing percentage of comparative sequencing run D at cycle 151 are shown, where different doses of blue laser light, 1x, 3x, 5x, 7.5x, and 10x, were assigned to different areas of the same flow cell. The signal decay values ​​were comparable. 3- The ffN set for coumarin dye C had phasing values ​​ranging from 0.085 to 0.152, and for the ffN set with ffT-LN3-I-9, the phasing values ​​ranged from 0.141 to 0.181. The light-independent average prephasing values ​​were 0.2 and 0.176, respectively.

[0363] Chromene quinoline dye A and dye E are disclosed in U.S. Application No. 18 / 190531 and, when conjugated with FA, have the following structural moieties respectively: The structures of ffA-sPA-chromene quinoline dye A and ffA-sPA-chromene quinoline dye E are:

[0364]

[0365] The structure of ffA-sPA-coumarin dye A-COT is:

[0366]

[0367] Coumarin dye B is disclosed in U.S. Publication No. 2018 / 0094140 and, when conjugated with WC, has a structural moiety The structure of ffC-sPA-coumarin dye B is:

[0368]

[0369] The structure of ffC-sPA-coumarin dye B-COT is:

[0370]

[0371] Coumarin dye C is disclosed in U.S. Publication No. 2020 / 0277670A1 and, when conjugated with ffT, has a structural moiety The structure of ff-LN3-coumarin dye C is:

[0372]

[0373] Cyanine dye D is disclosed in U.S. Publication No. 2018 / 0282791A1 and, when conjugated to ffT, has a structural moiety Each of these documents is incorporated by reference in its entirety. The structure of WT-LN3-cyanine dye D is:

[0374]

[0375] The structures of WT-LN3-I-2, WT-LN3-I-3, WT-LN3-I-8 and WT-LN3-I-9 are:

[0376]

[0377]

[0378] Example 4. Chemical stability of methylated BOPHY dyes

[0379] Evaluation of methyl groups using HPLC Chemical stability of substituted BOPHY dyes. Figure 7 The stability of methylated BOPHY dye in various culture media is demonstrated. The samples were aliquoted at regular time intervals (indicated on the X-axis) and stored in the dark at room temperature between analyses. The Y-axis refers to the integrated area of ​​the signal in the channel shown in the legend of the figure. It can be seen that the methylated BOPHY dye shows excellent stability over several days in a variety of media.

Claims

1. A compound of formula (I): its salts or meso forms, Where R 1 , R 2 , R 3 , R 6 , R 7 and R 8 Each of the above is independently H, unsubstituted or substituted C1-C6 alkyl, C1-C6 alkoxy, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, (C1-C6 alkoxy)(C1-C6 alkyl), unsubstituted or substituted amino, halo, cyano, carboxyl, hydroxyl, nitro, sulfonyl, sulfinyl, sulfonyl, sulfonate, S-sulfonamido, N-sulfonamido, -C(=O)NR 9 R 10 、-XR 11 , unsubstituted or substituted C3-C 10 Carbocyclic, unsubstituted or substituted C6-C 10 aryl, unsubstituted or substituted 5- to 10-membered heteroaryl, or unsubstituted or substituted 3- to 10-membered heterocyclyl; R 4 and R 5 Each of the following is independently H, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C3-C 10 Carbocyclic, unsubstituted or substituted C6-C 10 aryl, unsubstituted or substituted 5- to 10-membered heteroaryl, or unsubstituted or substituted 3- to 10-membered heterocyclyl; R 9 and R 10 Each of is independently H, or unsubstituted or substituted C1-C6 alkyl; X is -O-, -S-, -CH=CH-, or -C≡C-; R 11 Each of which is independently unsubstituted or substituted C3-C 10 Carbocyclic, unsubstituted or substituted C6-C 10 aryl, unsubstituted or substituted 5- to 10-membered heteroaryl, or unsubstituted or substituted 3- to 10-membered heterocyclyl; R a , R b , R c and R d Each of the above is independently a halogen group, a cyano group, an unsubstituted C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, a C1-C6 haloalkoxy group, an unsubstituted or substituted C6-C 10 Aryl, unsubstituted or substituted C6-C 10 Aryloxy or -OC(=O)R 12 ; R 12 is an unsubstituted or substituted C1-C6 alkyl, an unsubstituted or substituted C2-C6 alkenyl, an unsubstituted or substituted C6-C 10 aryl or unsubstituted or substituted 5- to 10-membered heteroaryl; Alternatively, when R a and R b All are -OC(=O)R 12 When two R 12 Together with the atoms to which they are attached, they form an unsubstituted or substituted 6- to 10-membered heterocyclyl; Alternatively, when R c and R d All are -OC(=O)R 12 When two R 12 Together with the atoms to which they are attached, form an unsubstituted or substituted 6- to 10-membered heterocyclyl; and The prerequisite is R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 At least one of the comprises a carboxyl group.

2. The compound according to claim 1, wherein R 1 , R 2 and R 3 Each of is H or unsubstituted C1-C6 alkyl.

3. The compound according to claim 2, wherein R 1 and R 3 Each of R is methyl and R 2 It is ethyl.

4. The compound according to claim 1, wherein R 1 , R 2 and R 3 Both of them are H or unsubstituted C1-C6 alkyl, and R 1 , R 2 and R 3 One of them is a halogen group, a carboxyl group, a -C(=O)NR 9 R 10 , C1-C6 alkyl substituted by carboxyl, carboxylate, sulfonate or sulfonate, unsubstituted or substituted phenyl, unsubstituted or substituted 5- to 6-membered heteroaryl, or unsubstituted or substituted 5- to 6-membered heterocyclyl.

5. The compound according to claim 4, wherein R 1 and R 2 Yes H, R 3 is phenyl optionally substituted by carboxyl, or R 1 and R 3 Yes H, R 2 is phenyl optionally substituted by carboxyl.

6. The compound according to claim 1, wherein R 1 , R 2 and R 3 Both of them are H or unsubstituted C1-C6 alkyl, and R 1 , R 2 and R 3 One of them is -XR 11 , and wherein X is -O-, -S-, or -C≡C-.

7. The compound according to claim 6, wherein R 1 and R 2 are independently H or unsubstituted C1-C6 alkyl, and R 3 Yes - XR 11 , and wherein X is -O-, -S-, or -C≡C-.

8. The compound according to claim 6, wherein R 1 and R 3 are independently H or unsubstituted C1-C6 alkyl, and R 2 Yes - XR 11 , and wherein X is -O-, -S-, or -C≡C-.

9. A compound according to any one of claims 6 to 8, wherein R 11 is phenyl, which is unsubstituted or substituted by one or more substituents selected from the group consisting of halo, unsubstituted C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, carboxyl, carboxylate, sulfonate, sulfonate or -C(O)NR 13 R 14 , and each R 13 and R 14 and C1-C6 alkyl substituted with carboxyl, carboxylate, sulfonate or sulfonate.

10. The compound according to claim 9, wherein R 11 is phenyl, which is unsubstituted or substituted by halo, methoxy, carboxyl or -C(O)NHR 14 Substitution, where R 14 It is a C1-C6 alkyl group substituted by a carboxyl group, a carboxylate group, a sulfonate group or a sulfonate group.

11. A compound according to any one of claims 6 to 8, wherein R 11 is a 5-membered or 6-membered heterocyclic or heteroaryl group, which is unsubstituted or substituted by one or more substituents selected from the group consisting of halo, unsubstituted C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, carboxyl, carboxylate, sulfonate, sulfonate or -C(O)NR 13 R 14 , and each R 13 and R 14 and C1-C6 alkyl substituted with carboxyl, carboxylate, sulfonate or sulfonate.

12. A compound according to any one of claims 1 to 11, wherein R 6 , R 7 and R 8 Each of is independently H or unsubstituted C1-C6 alkyl.

13. The compound according to claim 12, wherein R 6 and R 8 Each of R is methyl and R 7 It is ethyl.

14. A compound according to any one of claims 1 to 11, wherein R 6 , R 7 and R 8 Both of them are H or unsubstituted C1-C6 alkyl, and R 6 , R 7 and R 8 One of them is a halogen group, a carboxyl group, a -C(=O)NR 9 R 10 , C1-C6 alkyl substituted by carboxyl, carboxylate, sulfonate or sulfonate, unsubstituted or substituted phenyl, unsubstituted or substituted 5- to 6-membered heteroaryl, or unsubstituted or substituted 5- to 6-membered heterocyclyl.

15. The compound according to claim 14, wherein R 6 and R 7 is H, and R 8 is phenyl optionally substituted by carboxyl, or R 6 and R 8 is H, and R 7 is phenyl optionally substituted by carboxyl.

16. A compound according to any one of claims 1 to 11, wherein R 6 , R 7 and R 8 Both of them are H or unsubstituted C1-C6 alkyl, and R 6 , R 7 and R 8 One of them is -XR 11 , and wherein X is -O-, -S-, or -C≡C-.

17. The compound according to claim 16, wherein R 6 and R 7 are independently H or unsubstituted C1-C6 alkyl, and R 8 Yes - XR 11 , and wherein X is -O-, -S-, or -C≡C-.

18. The compound according to claim 16, wherein R 6 and R 8 are independently H or unsubstituted C1-C6 alkyl, and R 7 Yes - XR 11 , and wherein X is -O-, -S-, or -C≡C-.

19. A compound according to any one of claims 16 to 18, wherein R 11 is phenyl, which is unsubstituted or substituted by one or more substituents selected from the group consisting of halo, unsubstituted C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, carboxyl, carboxylate, sulfonate, sulfonate or -C(O)NR 13 R 14 .

20. The compound according to claim 19, wherein R 11 is phenyl, which is unsubstituted or substituted by halo, methoxy, carboxyl or -C(O)NHR 14 Substitution, where R 14 It is a C1-C6 alkyl group substituted by a carboxyl group, a carboxylate group, a sulfonate group or a sulfonate group.

21. A compound according to any one of claims 16 to 18, wherein R 11 is a 5-membered or 6-membered heterocyclic or heteroaryl group, which is unsubstituted or substituted by one or more substituents selected from the group consisting of halo, unsubstituted C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, carboxyl, carboxylate, sulfonate, sulfonate or -C(O)NR 13 R 14 .

22. A compound according to any one of claims 1 to 21, wherein R 4 and R 5 Each of is H.

23. A compound according to any one of claims 1 to 22, wherein R a and R b Each of the groups is independently fluoro, cyano, methyl, trifluoromethyl, methoxy or -OC(=O)R 12 , where R 12 It is an unsubstituted C1-C6 alkyl group, an unsubstituted C2-C6 alkenyl group or an unsubstituted phenyl group.

24. The compound according to claim 23, wherein R a and R b All are fluorine, methyl, trifluoromethyl, methoxy, ethoxy, -OAc(-OC(=O)CH3), -OC(=O)CH=CH2 or -OC(=O)Ph.

25. A compound according to any one of claims 1 to 22, wherein R a and R b All are -OC(=O)R 12 , and the two R 12 Together with the atoms to which they are attached, they form structures A 6-membered heterocyclic group.

26. A compound according to any one of claims 1 to 25, wherein R c and R d Each of the groups is independently fluoro, cyano, methyl, trifluoromethyl, methoxy or -OC(=O)R 12 , where R 12 It is an unsubstituted C1-C6 alkyl group, an unsubstituted C2-C6 alkenyl group or an unsubstituted phenyl group.

27. The compound according to claim 26, wherein R c and R d All are fluorine, methyl, trifluoromethyl, methoxy, ethoxy, -OAc, -OC(=O)CH=CH2 or -OC(=O)Ph.

28. A compound according to any one of claims 1 to 25, wherein R c and R d All are -OC(=O)R 12 , and the two R 12 Together with the atoms to which they are attached, they form structures A 6-membered heterocyclic group.

29. The compound according to claim 1, selected from the group consisting of: and their salts and meso forms.

30. A nucleotide labeled with a compound according to any one of claims 1 to 29.

31. The labeled nucleotide of claim 30, wherein the compound is attached to the nucleotide via the carboxyl group of the compound of formula (I).

32. The labeled nucleotide of claim 30 or 31, wherein the compound is attached to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base via a linker moiety.

33. The labeled nucleotide according to any one of claims 30 to 32, further comprising a 3' blocking group covalently attached to the ribose or deoxyribose of the nucleotide.

34. An oligonucleotide or polynucleotide comprising a labeled nucleotide according to any one of claims 30 to 33 incorporated therein.

35. The oligonucleotide or polynucleotide of claim 34, wherein the oligonucleotide or polynucleotide is at least partially complementary to and hybridizes to a target polynucleotide immobilized on the surface of a solid support.

36. The oligonucleotide or polynucleotide of claim 35, wherein the solid support comprises an array of a plurality of target polynucleotides immobilized thereon.

37. A kit comprising a first type of labeled nucleotides according to any one of claims 30 to 33.

38. The kit of claim 37, further comprising a second type of labeled nucleotides, wherein the second type of labeled nucleotides is labeled with a different compound than the first type of labeled nucleotides.

39. A kit according to claim 38, further comprising a third type of nucleotides and a fourth type of nucleotides, wherein each of the second type of nucleotides, the third type of nucleotides and the fourth type of nucleotides is labeled with a different compound, wherein each label has a different absorbance maximum value that can be distinguished from the other labels.

40. A kit according to claim 37, wherein the kit comprises four types of nucleotides, wherein a first type of nucleotides are labeled nucleotides according to any one of claims 30 to 33, a second type of nucleotides carries a second label, a third type of nucleotides carries a third label, and a fourth type of nucleotides is unlabeled (dark).

41. A kit according to claim 37, wherein the kit comprises four types of nucleotides, wherein a first type of nucleotides are labeled nucleotides according to any one of claims 30 to 33, a second type of nucleotides carries a second label, a third type of nucleotides comprises a mixture of third type nucleotides carrying two labels, and a fourth type of nucleotides is unlabeled (dark).

42. The kit according to any one of claims 37 to 41, further comprising a DNA polymerase and one or more buffer compositions.

43. A method for determining the sequence of a plurality of target polynucleotides, the method comprising: (a) contacting a solid support with a solution containing sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (b) contacting the solid support with an aqueous solution comprising a DNA polymerase and one or more of four different types of nucleotides under conditions suitable for DNA polymerase-mediated primer extension, wherein at least one type of nucleotide is a labeled nucleotide according to any one of claims 30 to 33, and wherein each of the one or more of the four different types of nucleotides comprises a 3' blocking group; (c) incorporating one type of nucleotide into the sequencing primer to produce an extended copy polynucleotide; and (d) performing one or more fluorescence measurements on the extended copy polynucleotide.

44. The method of claim 43, further comprising: (e) removing the 3' blocking group from the nucleotide incorporated into the extended copy polynucleotide.

45. The method of claim 44, wherein the label and the 3' capping group from the nucleotide incorporated into the extended copy polynucleotide are removed in a single chemical reaction.

46. ​​The method according to claim 44 or 45, further comprising: (f) washing the solid support after removing the 3' blocking group from the incorporated nucleotide.

47. The method of claim 46, further comprising repeating steps (b) to (f) until the sequence of at least a portion of the target polynucleotide is determined.

48. The method of claim 47, wherein steps (b) to (f) are repeated for at least 50, 100, 150, 200, 250 or 300 cycles.

49. The method of any one of claims 43 to 48, wherein the method is performed on an automated sequencing instrument, and wherein the automated sequencing instrument comprises two light sources operating at different wavelengths.

50. The method of claim 49, wherein a light source operates at about 450 nm to about 460 nm.

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