Alkylpyridinium cation coumarin dyes and their use in sequencing applications

Abstract

The present application relates to coumarin dyes substituted with alkylpyridinium cations of formula (I) and their use as fluorescent markers. For example, these dyes can be used to label nucleotides for nucleic acid sequencing applications. Wherein R1 is or and wherein R1 is substituted with one or more C1-C6 alkyl groups.

Description

Technical Field

[0001] The present disclosure relates to alkylpyridinium cation substituted coumarin derivatives and their use as fluorescent markers. Specifically, these compounds can be used as nucleotide markers for nucleic acid sequencing applications. Background Art

[0002] The nonradioactive detection of nucleic acids carrying fluorescent labels is an important technique in molecular biology. Many procedures employed in recombinant DNA technology have previously relied on the use of nucleotides or polynucleotides carrying radioactive labels, such as 32 P. Radioactive compounds allow for 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, lack of sensitivity, and more importantly safety considerations. Eliminating the need for radiolabeling reduces safety risks as well as environmental impacts and costs associated with, for example, reagent handling. By way of non-limiting example, methods suitable for non-radioactive fluorescence detection include 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 to enable independent detection of multiple spatially overlapping analytes. In such multiplex approaches, 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, multiplexed fluorescence detection allows 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 there are many important factors that limit the selection of appropriate fluorescent labels. First, it may be difficult to find dye compounds with significantly resolvable 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 simultaneous excitation, because the absorption bands of these dyes are usually widely separated, so even two dyes are difficult to reach a suitable fluorescence excitation efficiency. Many excitation methods use high-power light sources, such as lasers, and therefore dyes must have enough photostability to withstand such excitations. The final 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, buffers, polymerases and ligases.

[0005] As sequencing technology advances, there is a need for additional fluorescent dye compounds, their nucleic acid conjugates, 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.

[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 properties 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" of 532nm). Therefore, it is necessary to use blue dye labels for fluorescence detection in sequencing applications.

[0009] The coumarin dye family has attracted the attention of chemists due to their remarkable spectral properties. However, only several light-stable fluorescent dyes with large Stokes shift (LSS) are commercially available. Most of these dyes also contain coumarin fragments as scaffolds. Therefore, designing dyes with customized adsorption wavelengths and fluorescence Stokes shifts and good stability remains a key challenge in dye development. Summary of the invention

[0010] Alkylpyridinium cation substituted coumarin dyes with long Stokes shift and improved fluorescence intensity and chemical stability suitable for nucleotide labeling are described herein. These coumarin dyes have strong fluorescence under both blue and green light excitation (e.g., these coumarin dyes can have an absorption wavelength of about 450nm to about 530nm, about 460nm to about 520nm, about 475nm to about 510nm, or about 490nm to about 500nm).

[0011] Some aspects of the present disclosure relate to compounds of formula (I) or salts or meso forms thereof:

[0012]

[0013] Where R 1 yes And where R 1 Substituted with one or more C1-C6 alkyl groups;

[0014] Each R 2 , R 5 and R 7 is independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted amino, amino(C1-C6 alkyl), halo, cyano, hydroxy, hydroxy(C1-C6 alkyl), nitro, sulfonyl, sulfo, sulfinyl, sulfonate, S-sulfonamido or N-sulfonamido;

[0015] R 3 and R 4 Each of is independently H, C1-C6 alkyl or substituted C1-C6 alkyl;

[0016] Alternatively, R 2 and R 3 Together with the atoms to which they are attached, they form a ring or ring system selected from the group consisting of optionally substituted 5- to 10-membered heteroaryl or optionally substituted 5- to 10-membered heterocyclyl;

[0017] Alternatively, R 4 and R 5 Together with the atoms to which they are attached, they form a ring or ring system selected from the group consisting of optionally substituted 5- to 10-membered heteroaryl or optionally substituted 5- to 10-membered heterocyclyl;

[0018] R 6 is H, C1-C6 alkyl, substituted C1-C6 alkyl or optionally substituted C6-C10 aryl; and

[0019] R a , R b and R c Each of is independently a C1-C6 alkyl group or a substituted C1-C6 alkyl group.

[0020] In some embodiments, the compound of formula (I) is also represented by formula (Ia) or a salt or meso form thereof:

[0021]

[0022] Each R 8 , R 9 , R 10 and R 11 are independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted amino, amino(C1-C6 alkyl), halo, cyano, hydroxy, hydroxy(C1-C6 alkyl), nitro, sulfonyl, sulfo, sulfinyl, sulfonate, S-sulfonamido or N-sulfonamido; and are represented by solid and dashed lines. The bond represented by is selected from the group consisting of single bonds and double bonds, provided that when When it is a double bond, R 11 Does not exist.

[0023] In some embodiments of the compound of Formula (I) or Formula (Ia), R 1 (For example, R a , R b or R c ) comprises a carboxyl group (-C(O)OH). In other embodiments, R 3 or R 4 Contains a carboxyl group.

[0024] 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 performed via a carboxyl group, which can react with an amino or hydroxyl group on a moiety (e.g., a nucleotide) or a linker bound thereto to form an amide or ester using methods known in the art.

[0025] Some other aspects of the present disclosure relate to dye compounds that include a linker group to achieve, for example, covalent attachment to a substrate moiety. Attachment can be made at any position of the dye, including at any R group. In some embodiments, the R 1 (For example, R a , R b or R c ) or R 3 or R 4 In some other embodiments, R of formula (Ia) can be used to connect. 1 (For example, R a , R b or R c ) or R 3 to connect.

[0026] Some further aspects of the present disclosure provide labeled nucleoside or nucleotide compounds defined by the following formula:

[0027] NL-Dye

[0028] wherein N is a nucleoside or a nucleotide;

[0029] L is an optional linker moiety; and

[0030] The dye is part of a fluorescent compound of formula (I) or formula (Ia) according to the present disclosure, wherein a functional group (e.g., a carboxyl group) of the compound of formula (I) or formula (Ia) reacts with a linker moiety or an amino or hydroxyl group of a nucleoside / nucleotide to form a covalent bond.

[0031] Some additional aspects of the disclosure relate to nucleotides or oligonucleotides labeled with compounds of Formula (I) or Formula (Ia).

[0032] Some other aspects of the present disclosure relate to a kit comprising dye compounds (free or in labeled form) that can be used for various immunological assays, oligonucleotide or nucleic acid labeling, or for DNA sequencing by synthesis. In yet another aspect, the present disclosure provides a kit comprising a dye "set" that is particularly suitable for sequencing by synthesis cycles on automated instrument platforms. In some aspects, a kit comprising one or more nucleotides, wherein at least one nucleotide is a labeled nucleotide as described herein.

[0033] Another aspect of the present disclosure is a method of determining the sequence of a target polynucleotide, the method comprising:

[0034] (a) contacting the primer polynucleotide / target polynucleotide complex with one or more labeled nucleotides (e.g., A, G, C, and T or dATP, dGTP, dCTP, and dTTP), wherein at least one of the labeled nucleotides is a coumarin dye-labeled nucleotide substituted with an alkylpyridinium cation of Formula (I) or Formula (Ia) as described herein, and wherein the primer polynucleotide is complementary to at least a portion of the target polynucleotide;

[0035] (b) incorporating a labeled nucleotide into the primer polynucleotide / target nucleotide complex to produce an extended primer polynucleotide / target nucleotide complex; and

[0036] (c) performing one or more fluorescence measurements on the extended primer polynucleotide / target nucleotide complex to determine the identity of the incorporated nucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1A and Figure 1B Shown are the fluorescence emission spectra of coumarin dye I-1 compared with reference dye A in buffer solution at excitation wavelengths of 450 nm and 520 nm, respectively.

[0038] Figure 2A and Figure 2B Shown are the fluorescence emission spectra of coumarin dye I-5 compared with reference dye C in buffer solution at excitation wavelengths of 450 nm and 520 nm, respectively.

[0039] Figure 3A and Figure 3B Shown are the fluorescence emission spectra of coumarin dye I-8 compared with reference dye B in buffer solution at excitation wavelengths of 450 nm and 520 nm, respectively.

[0040] Figure 4A and Figure 4B Shown are the fluorescence emission spectra of fully functionalized A nucleotide (ffA) labeled with coumarin dye 1-1 compared with reference dye A labeled with ffA in buffer solution at excitation wavelengths of 450 nm and 520 nm, respectively.

[0041] Figure 5A and Figure 5B Shown are the fluorescence emission spectra of ffA labeled with coumarin dye I-5 compared with ffA labeled with reference dye C in buffer solution at excitation wavelengths of 450 nm and 520 nm, respectively.

[0042] Fig. 6A and Figure 6BShown are the fluorescence emission spectra of ffA labeled with coumarin dye 1-8 compared with ffA labeled with reference dye B in buffer solution at excitation wavelengths of 450 nm and 520 nm, respectively.

[0043] Fig. 7A and Figure 7B Shown are the fluorescence emission spectra of ffA labeled with coumarin dye I-3 compared with ffA labeled with reference dye D in buffer solution at excitation wavelengths of 450 nm and 520 nm, respectively.

[0044] Fig. 8A and Figure 8B The scatter plots obtained for the spiking mixture containing ffA labeled with coumarin dye 1-1 and the scatter plots obtained from the spiking mixture containing ffA labeled with reference dye A are shown, respectively.

[0045] Figure 8C and Fig.8D The scatter plots obtained for the spiking mixture containing ffA labeled with coumarin dye 1-3 and the scatter plots obtained from the spiking mixture containing ffA labeled with reference dye D are shown, respectively. DETAILED DESCRIPTION

[0046] Embodiments of the present disclosure relate to alkylpyridinium cation substituted coumarin dyes with enhanced fluorescence intensity and long Stokes shift. These coumarin dyes also have a wide excitation wavelength and can be excited by both a blue light source and a green light source. In some embodiments, the alkylpyridinium cation coumarin dyes described herein can be used in Illumina's iSeq with two-channel CMOS detection (green light excitation and blue light excitation). TM On the platform.

[0047] definition

[0048] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0049] 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 teachings of the present invention. Therefore, it is intended that the various embodiments described herein cover other modifications and variations within the scope of the appended claims and their equivalents.

[0050] 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 term "include" as well as other forms such as "include / includes / included" is not limiting. The use of the term "have" as well as other forms such as "have / has / had" is not limiting. As used in this specification, the terms "comprise(s)" 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.

[0051] As used herein, common organic abbreviations are defined as follows:

[0052] ℃ Temperature in degrees Celsius

[0053] dATP deoxyadenosine triphosphate

[0054] dCTP deoxycytidine triphosphate

[0055] dGTP deoxyguanosine triphosphate

[0056] dTTP deoxythymidine triphosphate

[0057] ddNTP dideoxynucleoside triphosphate

[0058] ffA fully functionalized A nucleotide

[0059] ffC fully functionalized C nucleotide

[0060] ffG fully functionalized G nucleotide

[0061] ffN Fully functionalized nucleotides

[0062] ffT fully functionalized T nucleotide

[0063] h hour

[0064] RT Room temperature

[0065] SBS Sequencing by Synthesis

[0066] As used herein, the term "array" refers to a group of different probe molecules that are connected to one or more substrates so that different probe molecules can be distinguished from each other based on relative position. The array can include different probe molecules at different addressable positions on the substrate. Alternatively or additionally, the array can include separate substrates with different probe molecules, where different probe molecules can be identified based on the position of the substrate on the surface to which the substrate is connected or based on the position of the substrate in the liquid. Exemplary arrays in which the separated substrate is located on the surface include, but are not limited to, arrays that include beads in holes, such as, for example, described in U.S. Patent No. 6,355,431B1, US 2002 / 0102578, and PCT Publication No. WO 00 / 63437. For example, an exemplary form that can be used in the present invention, for example, using a microfluidic device, such as a fluorescence activated cell sorter (FACS) to distinguish beads in a liquid array is described in U.S. Patent No. 6,524,793. 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; EP742287; and those described in EP 799897.

[0067] 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 means other than covalent attachment.

[0068] 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.

[0069] As used herein, wherein "a" and "b" are integers, a To Cb " 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 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2C H2-, 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 no reference is made to an alkyl, alkenyl, alkynyl, cycloalkyl or aryl group, the cycloalkyl radical is preferably substituted with a cycloalkyl radical having 3 to 4 carbon atoms, i.e. cyclopropyl and cyclobutyl. If "a" and "b" are not 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.

[0070] 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). An alkyl group can 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 can 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 having 1 to 9 carbon atoms. The alkyl group can also be a low-sized alkyl group having 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 in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like.

[0071] 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 "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, etc.

[0072] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. An alkenyl group can 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 can also be a medium-sized alkenyl group having from 2 to 9 carbon atoms. An alkenyl group can 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, and the like.

[0073] As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. Alkynyl groups can have from 2 to 20 carbon atoms, but this definition also encompasses the term "alkynyl" where it appears without a specified numerical range. Alkynyl groups can also be medium-sized alkynyl groups having from 2 to 9 carbon atoms. Alkynyl groups can also be lower alkynyl groups having from 2 to 6 carbon atoms. By way of example only, "C 2- "C-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.

[0074] 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.

[0075] 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.

[0076] "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 (ie, C 1-6 alkylene group).

[0077] 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 as "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.

[0078] "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., a C1-6 alkylene group).

[0079] As used herein, "carbocyclyl" means a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system backbone. When a carbocyclyl is a ring system, two or more rings may be joined together in a fused, bridged or spiroed manner. A carbocyclyl may have any degree of saturation, provided that at least one ring in the ring system is not aromatic. Thus, carbocyclyl includes cycloalkyl, cycloalkenyl and cycloalkynyl. A carbocyclyl group may have 3 to 20 carbon atoms, but this definition also encompasses the term "carbocyclyl" in which no numerical range is specified. A carbocyclyl group may also be a medium-sized carbocyclyl group having 3 to 10 carbon atoms. A carbocyclyl group may also be a carbocyclyl group having 3 to 6 carbon atoms. A carbocyclyl group may be designated as "C 3-6 "carbocyclyl", "C3-C6carbocyclyl" 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.

[0080] As used herein, "cycloalkyl" means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0081] 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) that constitute 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.

[0082] 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.

[0083] 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.

[0084] 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 A "-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).

[0085] A "sulfonyl" group refers to 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 is as defined herein.

[0086] A "sulfinyl" group refers to a "-S(=O)OH" group.

[0087] A "sulfo" group refers to a "-S(=O)2OH" or "-SO3H" group.

[0088] A "sulfonate" group refers to a "-SO3 - "Group.

[0089] A "sulfate" group refers to a "-SO4 - "Group.

[0090] An "S-sulfonamido" group refers to a 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 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclic group"-SO2NR A R B "Group, as defined herein.

[0091] An "N-sulfonylamino" group refers to a 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 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclic group"-N(R A )SO2R B "Group, as defined herein.

[0092] A "C-amido" 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 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl--C(=O)NR A R B "Group, as defined herein.

[0093] An "N-amido" group refers to a 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 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclic group"-N(R A )C(=O)R B "Group, as defined herein.

[0094] An "amino" group refers to a 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 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclic group"-NR A R B "group, as defined herein. Non-limiting examples include free amino groups (ie, -NH2).

[0095] An "aminoalkyl" group refers to an amino group attached through an alkylene group.

[0096] An "alkoxyalkyl" group refers to an alkoxy group attached via an alkylene group, such as "C2-C8 alkoxyalkyl" and the like.

[0097] When a group is described as "optionally substituted," the group may be unsubstituted or substituted. Likewise, when a group is described as "substituted," the substituents may be selected from one or more of the indicated substituents. As used herein, a substituted group is derived from an unsubstituted parent group in which there has been 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 by one or more substituents independently selected from the group consisting of 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-membered heterocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 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 (thiol), halo(C1-C6)alkoxy alkyl, -Ci-C6 alkyl, and oxo (=O). Wherever a group is described as "optionally substituted", the group may be substituted with a substituent as defined above.In some embodiments, when an alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl or heterocyclyl group is substituted, each of these groups is 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 , oxygen,

[0098] -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.

[0099] 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 positively or negatively charged substituent groups, e.g. SO3 - , it may also contain negatively or positively charged counterions so that the compound is neutral overall. In other aspects, the compound may exist in the form of a salt, wherein the counterion is provided by a conjugate acid or base.

[0100] It should be understood that certain group naming conventions may include either a monoradical or a diradical, depending on the context. 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".

[0101] 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:

[0102]

[0103] And R 1 and R2 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:

[0104]

[0105] wherein A is an aromatic ring or a carbocyclic group containing a double bond as depicted.

[0106] 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 Substituents of include those 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 and L is defined as an optionally present linker moiety; when L is absent (or missing), such group or substituent is equal to

[0107] The compounds described herein can be represented as several meso forms. Where a single structure is drawn, any relevant meso forms are contemplated. The coumarin compounds described herein are represented by a single structure, but can likewise be shown as any relevant meso forms. Exemplary meso structures of Formula (I) and Formula (Ia) are shown below, respectively:

[0108]

[0109] In each case where a single meso form of a compound described herein is shown, the alternative meso forms are also contemplated.

[0110] As used herein, "nucleotides" include a nitrogenous heterocyclic base, a sugar, and one or more phosphate groups. They are the 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 group present in ribose. The nitrogenous heterocyclic base can be a purine, a deazapurine, or a pyrimidine base. 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.

[0111] As used herein, a "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.

[0112] The term "purine base" is used herein with the ordinary meaning understood by those skilled in the art, and includes tautomers thereof. Similarly, the term "pyrimidine base" is used herein with the ordinary meaning understood by those skilled in the art, and includes tautomers thereof. A 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. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine).

[0113] As used herein, when an oligonucleotide or polynucleotide is described as "comprising" a nucleoside or nucleotide as described herein, this means that the nucleoside or nucleotide as described herein forms a covalent bond with the oligonucleotide or polynucleotide. Similarly, when a nucleoside or nucleotide is described as a part of an oligonucleotide or polynucleotide, such as "incorporated into" an oligonucleotide or polynucleotide, this means that the nucleoside or nucleotide as described herein forms a covalent bond with the oligonucleotide or polynucleotide. In some such embodiments, the covalent bond is formed between the 3' hydroxyl group of the oligonucleotide or polynucleotide and the 5' phosphate group of the nucleotide described herein as a phosphodiester bond between the 3' carbon atom of the oligonucleotide or polynucleotide and the 5' carbon atom of the nucleotide.

[0114] 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.

[0115] As used herein, "derivatives" or "analogs" means 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, and phosphoramidate bonds. As used herein, "derivatives", "analogs" and "modified" can be used interchangeably and are covered by the terms "nucleotides" and "nucleosides" defined herein.

[0116] 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.

[0117] As used herein, the term "phasing" refers to a phenomenon in SBS that is caused by incomplete removal of 3' terminators and fluorophores and / or failure to complete incorporation of a portion of the DNA chain within the cluster by a polymerase under a given sequencing cycle. Prephasing is caused by incorporating 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 trace amounts of unprotected or unblocked 3'-OH nucleotides during sequencing by synthesis (SBS). Unprotected 3'-OH nucleotides can be produced during the manufacturing process or may be produced during storage and reagent handling processes. Therefore, the discovery of nucleotide analogs that reduce the incidence of prephasing is surprising, and provides a huge advantage in SBS applications relative to existing nucleotide analogs. For example, the provided nucleotide analogs can produce faster SBS cycle times, lower phasing and prephasing values, and longer sequencing read lengths.

[0118] Fluorescent dye of formula (I)

[0119] Some aspects of the present disclosure relate to coumarin dyes of formula (I) and their salts and meso forms:

[0120]

[0121] Where R 1 yes And where R 1 Substituted with one or more C1-C6 alkyl groups;

[0122] Each R 2 , R 5 and R 7 is independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted amino, amino(C1-C6 alkyl), halo, cyano, hydroxy, hydroxy(C1-C6 alkyl), nitro, sulfonyl, sulfo, sulfinyl, sulfonate, S-sulfonamido or N-sulfonamido;

[0123] R 3 and R 4 Each of is independently H, C1-C6 alkyl or substituted C1-C6 alkyl;

[0124] Alternatively, R 2 and R 3 Together with the atoms to which they are attached, they form a ring or ring system selected from the group consisting of optionally substituted 5- to 10-membered heteroaryl or optionally substituted 5- to 10-membered heterocyclyl;

[0125] Alternatively, R 4 and R 5 Together with the atoms to which they are attached, they form a ring or ring system selected from the group consisting of optionally substituted 5- to 10-membered heteroaryl or optionally substituted 5- to 10-membered heterocyclyl;

[0126] R 6 is H, C1-C6 alkyl, substituted C1-C6 alkyl or optionally substituted C6-C 10 aryl; and

[0127] R a , R b and R c Each of is independently a C1-C6 alkyl group or a substituted C1-C6 alkyl group.

[0128] In some embodiments of the compound of Formula (I), R 3 and R 4 At least one of is H. In some other embodiments, R 3 and R 4 are all H. In other embodiments, R 3is H, and R 4 is C1-C6 alkyl or substituted C1-C6 alkyl. In other embodiments, R 3 and R 4 Each of the above is independently a C1-C6 alkyl or a substituted C1-C6 alkyl. Substituted C1-C6 alkyl includes, but is not limited to, methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl, n-hexyl, etc., which are substituted by one or more substituents such as carboxyl, carboxylate (-C(O)O - ), sulfonic acid (-SO3H), sulfonic acid (-SO3 - ), sulfate (-O-SO3 - ), optionally substituted amino (such as Boc protected amino group), -C(O)OR 12 or -C(O)NR 13 R 14 , where R 12 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, and wherein R 13 and R 14 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. 3 and R 4 In another embodiment, each of R 3 is H, and R 4 It is a propyl group substituted by a carboxyl group.

[0129] Some embodiments of compounds of formula (I) are also represented by formula (Ia), wherein R 4 and R 5 Together with the atoms to which they are attached, they form an optionally substituted 6-membered heterocyclyl having the structure:

[0130]

[0131] Its salts or meso forms:

[0132] Each R 8 , R 9 , R 10 and R 11is independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted amino, amino(C1-C6 alkyl), halo, cyano, hydroxy, hydroxy(C1-C6 alkyl), nitro, sulfonyl, sulfo, sulfinyl, sulfonate, S-sulfonamido or N-sulfonamido;

[0133] By solid and dotted lines The bond represented by is selected from the group consisting of single bonds and double bonds, provided that when When it is a double bond, R 11 Does not exist.

[0134] In some embodiments of the compound of Formula (I) or (Ia), R 1 is substituted with a C1-C6 alkyl group (e.g., methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl, or n-hexyl). 1 is independently substituted with two C1-C6 alkyl groups. 1 is independently substituted with three C1-C6 alkyl groups. In some other embodiments, R 1 yes In other embodiments, R 1 yes In some such embodiments, each R a and R b R is independently C1-C6 alkyl (e.g., methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl or n-hexyl, etc.). In some other embodiments, each R a and R b is independently substituted C1-C6 alkyl (e.g., methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl or n-hexyl), which is substituted with one or more substituents such as carboxyl, carboxylate (-C(O)O), sulfonate (-SO3H), sulfonate (-SO3 - ), sulfate (-O-SO3 - ), optionally substituted amino (such as Boc protected amino group), -C(O)OR 12 or -C(O)NR 13 R 14 , where R 12 is an optionally substituted C1-C6 alkyl, an optionally substituted C6-C 10aryl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted C3-C7 cycloalkyl, and wherein R 13 and R 14 Each of the above 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. a and R b Each of is independently a n-propyl, n-butyl or n-pentyl substituted by a carboxyl, carboxylate, sulfonate or sulfonate. In some embodiments, the substitution is at the end of a linear C2 alkyl, C3 alkyl, C5 alkyl, C6 alkyl or C6 alkyl.

[0135] In some embodiments of the compound of Formula (Ia), the solid and dashed lines The bond represented by is a double bond. In some such embodiments, R 10 is H or C1-C6 alkyl. In one example, R 10 In some other embodiments, the solid and dashed lines The bond represented by is a single bond. In some such embodiments, R 10 is H, and R 11 is C1-C6 alkyl. In other embodiments, R 10 and R 11 Each of is H. In some embodiments of the compound of Formula (Ia), R 8 and R 9 Each of is H. In other embodiments, R 8 and R 9 In another embodiment, at least one of R 8 and R 9 Each of R is a C1-C6 alkyl group. 8 and R 9 In some embodiments, each of R 3 is H. In other embodiments, R 3 is C1-C6 alkyl (e.g., methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl or n-hexyl, etc.). In another embodiment, R 3 is a substituted C1-C6 alkyl group (e.g., methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl or n-hexyl), which is substituted with one or more substituents such as carboxyl, carboxylate (-C(O)O - ), sulfonic acid (-SO3H), sulfonic acid (-SO3 -), sulfate (-O-SO3 - ), optionally substituted amino, -C(O) or 12 or -C(O)NR 13 R 14 , where R 12 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, and wherein R 13 and R 14 Each of the above 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. 3 is ethyl, n-propyl, n-butyl or n-pentyl, each optionally substituted with carboxyl, carboxylate, sulfonate or sulfonate. As another example, R 3 Is -C(O)NR 13 R 14 substituted ethyl, n-propyl, n-butyl or n-pentyl, and wherein each R 13 and R 14 are independently carboxyl, carboxylate, -C(O)OR 12 , sulfonyl or sulfonate substituted C1-C6 alkyl.

[0136] In some embodiments of the compound of Formula (I) or (Ia), R 2 is H. In other embodiments, R 2 and R 3 and the atoms to which they are attached are joined together to form an optionally substituted 5-, 6-, or 7-membered heterocyclyl. 2 and R 3 Together with the atoms to which they are attached, they form a 6-membered heterocyclic group substituted with one or more C1-C6 alkyl groups.

[0137] In some embodiments of the compound of Formula (I) or (Ia), R 6 is H or optionally substituted phenyl.

[0138] In some embodiments of the compound of Formula (I) or (Ia), R 7 It's H.

[0139] Additional embodiments of compounds of formula (I) or (Ia) include the following:

[0140]

[0141] and salts and meso forms thereof. Non-limiting examples are corresponding C1-C6 alkyl carboxylates (such as methyl, ethyl, isopropyl and tert-butyl esters formed from the carboxyl groups of the compounds); corresponding imine analogs (wherein instead, the coumarin core -C(=O) portion is -C(=NH)), and salts and meso forms thereof.

[0142] Cyclooctatetraene (COT) photoprotective moiety

[0143] In some embodiments, the fluorescent compounds described herein (Formula (I) or (Ia)) can be further modified to introduce photoprotective moieties covalently bonded thereto, for example, cyclooctatetraene moieties including the structure

[0144] in

[0145] R 1A and R 2A Each of the following is independently H, hydroxy, halogen, azido, thiol, nitro, cyano, optionally substituted amino, carboxyl, -C(O)OR 5A 、-C(O)NR 6A R 7A , optionally substituted C 1-6 Alkyl, optionally substituted C 1-6 Alkoxy, optionally substituted C 1-6 haloalkyl, optionally substituted C 1-6 haloalkoxy, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted C2-6 6-10 Aryl, optionally substituted C 7-14 Arylalkyl, optionally substituted C 3-7 carbocyclyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 3- to 10-membered heterocyclyl;

[0146] X 1 and Y 1 are each independently a bond, -O-, -S-, -NR 3A -, -C(=O)-, -C(=O)-O-,

[0147] -C(=O)-NR 4A -、-S(O)2-、-NR 3A -C(=O)-NR 4A 、-NR 3A -C(=S)-NR 4A -, optionally substituted C 1-6 alkylene or optionally substituted heteroalkylene, wherein at least one carbon atom is replaced by O, S or N;

[0148] Z is absent and is optionally substituted C 2-6 Alkenylene or optionally substituted C 2-6 Alkyne;

[0149] R 3A and R 4A Each of which is independently H, optionally substituted C 1-6 Alkyl or optionally substituted C 6-10 Aryl;

[0150] R 5A is optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 7-14 Arylalkyl, optionally substituted C 3-7 carbocyclyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 3- to 10-membered heterocyclyl;

[0151] R 6A and R 7A Each of which is independently H, optionally substituted C 1-6 Alkyl, optionally substituted C 6-10 Aryl, optionally substituted C 7-14 Arylalkyl, optionally substituted C 3-7 carbocyclyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 3- to 10-membered heterocyclyl;

[0152] exist Medium R 1A and R 2A The carbon atom to which it is attached is optionally replaced by O, S or N, provided that when the carbon atom is replaced by O or S, then R 1A and R 2A When the carbon atom is replaced by N, R 2A is absent; and m is an integer between 0 and 10. In some embodiments, neither X nor Y is a bond.

[0153] In some embodiments, the cyclooctatetraene moiety comprises the structure In some such embodiments, R 1A and R 2A In some other embodiments, at least one of R 1A and R 2A In some other embodiments, R 1A H and R 2A is an optionally substituted amino, carboxyl or -C(O)NR 6A R 7AIn some embodiments, m is 1, 2, 3, 4, 5 or 6, and R 1A and R 2A Each of the following is independently hydrogen, optionally substituted amino, carboxyl, -C(O)NR 6A R 7A or a combination thereof. In some further embodiments, when m is 2, 3, 4, 5 or 6, one R 1A is amino, carboxyl or -C(O)NR 6A R 7A , and the remaining R 1A and R 2A is hydrogen. In some embodiments, Medium R 1A and R 2A At least one carbon atom of the connection is replaced by O, S or N. In some such embodiments, One of the carbon atoms in is replaced by an oxygen atom, and R connected to the replaced carbon atom 1A and R 2A In some other embodiments, when When one of the carbon atoms in is replaced by a nitrogen atom, the R 2A is not present and is attached to the substituted carbon atom R 1A is hydrogen or C 1-6 Alkyl. In R 1A and R 2A In any embodiment of the present invention, when R 1A or R 2A Yes-C(O)NR 6A R 7A When R 6A and R 7A Can be H, C independently 1-6 Alkyl or substituted C 1-6 Alkyl (e.g., -CO2H, -NH2, -SO3H or -SO3 - Substituted C 1-6 alkyl).

[0154] In some additional embodiments, the fluorescent dyes described herein comprise a cyclooctatetraene moiety having the following structure:

[0155]

[0156] The COT moieties described herein can be generated by the reaction between a functional group (eg, a carboxyl group) of a fluorescent 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).

[0157] Labeled nucleotides or oligonucleotides

[0158] According to one aspect of the present disclosure, dye compounds as described herein are applicable to be connected to substrate moiety, particularly comprise linker group so that it is possible to be connected to substrate moiety.Substrate moiety 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 examples, can include nucleosides, nucleotides, polynucleotides, carbohydrates, ligands, particles, solid surfaces, organic polymers and inorganic polymers, chromosomes, nuclei, living cells, and their combinations or assemblies.Dye can be put together by various means (including hydrophobic attraction, ion attraction and covalent attachment) by optional linker.In some respects, dye is put together to substrate by covalent attachment.More specifically, covalent attachment is realized by means of linker group.In some cases, the nucleotide of such labeling is also referred to as " modified nucleotide ".

[0159] Some aspects of the present disclosure relate to nucleotides or oligonucleotides labeled with dyes of formula (I) or formula (Ia) as described herein or salts of their meso form, or derivatives thereof containing the light protection moiety COT as described herein. The labeled nucleotides or oligonucleotides can be connected to dye compounds disclosed herein to form amides or alkyl-amide bonds via carboxyl (-CO2H) or alkyl-carboxyl groups. In some other embodiments, the carboxyl group can be in the activated form of a carboxyl group, such as an amide or ester, which can be used to connect to an amino or hydroxyl group of a nucleotide or oligonucleotide. The term "activated ester" as used herein refers to a carboxyl group derivative that can react with, for example, a compound containing an amino group under mild conditions. Non-limiting examples of activated esters include, but are not limited to, p-nitrophenyl, pentafluorophenyl, and succinimide esters.

[0160] For example, the dye compound of formula (I) can be 1 (For example, R a , R b or R c ) or R 3 / R 4 In some such embodiments, R of formula (I) 1 Contains -CO2H or (CH2) 1-6 -CO2H, and this connection forms R 1 The amide portion between the carboxyl functional group of the nucleotide and the amino functional group of the nucleotide or nucleotide linker. As an example, the labeled nucleotide or oligonucleotide may include a dye portion having the following structure:

[0161]

[0162] In other embodiments, R of formula (I) 3 or R4 Including -CO2H or (CH2) 1-6 -CO2H, and the linkage forms an amide using the -CO2H group. For example, a labeled nucleotide or oligonucleotide may comprise the following dye moieties:

[0163]

[0164] Similarly, the dye compound of formula (Ia) can be obtained via R 1 (For example, R a , R b or R c ) or R 3 , by using R 1 or R 3 The carboxyl functional group of the labeled nucleotide or oligonucleotide forms an amide moiety with the amino functional group of the nucleotide or nucleotide linker to attach to the nucleotide or oligonucleotide. For example, the labeled nucleotide or oligonucleotide may include the following dye moieties:

[0165]

[0166] In other embodiments, R of Formula (I) or Formula (Ia) b or R c Including -CO2H or -(CH2) 1-6 -CO2H, and the linkage forms an amide using the -CO2H group.

[0167] In some embodiments, dye compound can be covalently connected to oligonucleotide or nucleotide via nucleotide base.In some such embodiments, the nucleotide or oligonucleotide of labeling can have the dye of C5 position or C7 position of 7-deazapurine base optionally connected to pyrimidine base by connecting base part.For example, core base can be 7-deazaadenine, and dye is optionally connected to 7-deazaadenine by connecting base at C7 position.Core base can be 7-deazaguanine, and dye is optionally connected to 7-deazaguanine by connecting base at C7 position.Core base can be cytosine, and dye is optionally connected to cytosine by connecting base at C5 position.As another example, core base can be thymine or uracil, and dye is optionally connected to thymine or uracil by connecting base at C5 position.

[0168] 3′-OH end group

[0169] The nucleotide or oligonucleotide of labeling can also have the blocking group of the ribose or deoxyribose of nucleotide covalently connected.This blocking group can be connected to any position on ribose or deoxyribose.In a particular embodiment, this blocking group is located at the 3'-OH position of the ribose or deoxyribose of nucleotide.Various 3'-OH blocking groups are disclosed in WO2004 / 018497 and WO2014 / 139596, and these applications are hereby incorporated by reference.For example, the blocking group can be the azidomethyl (-CH2N3) or substituted azidomethyl (for example, -CH(CHF2)N3 or CH(CH2F)N3) or allyl group connected to the 3' oxygen atom of ribose or deoxyribose moiety.In some embodiments, 3' blocking group is azidomethyl, forms 3'-OCH2N3 with the 3' carbon of ribose or deoxyribose.

[0170] In some other embodiments, the 3' blocking group and the 3' oxygen atom form a structure covalently linked to the 3' carbon of ribose or deoxyribose. An acetal group, wherein:

[0171] R 1a and R 1b each independently H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, cyano, halogen, optionally substituted phenyl, or optionally substituted aralkyl;

[0172] R 2a and R 2b Each is independently H, C1-C6 alkyl, C1-C6 haloalkyl, cyano or halogen;

[0173] Alternatively, R 1a and R 2a together with the atoms to which they are attached form an optionally substituted five- to eight-membered heterocyclyl group;

[0174] R F is H, optionally substituted C2-C6 alkenyl, optionally substituted C3-C7 cycloalkenyl, optionally substituted C2-C6 alkynyl or optionally substituted (C1-C6 alkylene)Si(R 3a )3; and

[0175] Each R 3a are independently H, C1-C6 alkyl or optionally substituted C6-C 10 Aryl.

[0176] Additional 3′-OH blocking groups are disclosed in U.S. Publication No. 2020 / 0216891A1, which is incorporated by reference in its entirety. Non-limiting examples of acetal blocking groups are Each is covalently linked to the 3' carbon of ribose or deoxyribose.

[0177] Deprotection of 3′-OH blocking group

[0178] In some embodiments, the azidomethyl 3' hydroxy protecting group can be removed or deprotected by using a water-soluble phosphine reagent. 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 acetal capping groups containing vinyl or alkenyl moieties, 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, e.g., 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).

[0179] Palladium cleavage reagent

[0180] In some embodiments, the 3'hydroxyl end-capping group described herein 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"-phosphinyltri(benzenesulfonate)palladium(0) nonahydrate). In some cases, the Pd(0) complex can be generated in situ by reducing a 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(CH3CN)2Cl2. d(TFA)2. In one such embodiment, the Pd(0) complex is generated in situ from Na2PdCl4. 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 tri(hydroxypropyl)phosphine (THP), tri(hydroxymethyl)phosphine (THMP), 1,3,5-triaza-7-phosphaadamantane (PTA), bis(p-sulfonylphenyl)phenylphosphine dihydrate potassium salt, tri(carboxyethyl)phosphine (TCEP), and triphenylphosphine-3,3',3'-trisulfonic acid trisodium salt.

[0181] 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 carbonates, phosphates or borates, or a combination thereof. In one embodiment, the inorganic salt is a sodium salt.

[0182] Connector

[0183] 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 connected 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.

[0184] 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 solutions, the latter form 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.

[0185] 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:

[0186]

[0187] (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, e.g., a dye compound described herein.

[0188] 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:

[0189]

[0190] (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.

[0191] Other examples of linking groups include moieties of the formula:

[0192]

[0193]

[0194] 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 bonded to the linker by reacting a functional group (e.g., carboxyl) of the dye compound with a functional group (e.g., amino) of the linker. In one embodiment, the cleavable linker comprises ("AOL" linker moiety), wherein Z is -O-allyl.

[0195] 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 connected 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, especially its increased length, can allow for improved brightness of the fluorophore connected 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 connected 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.

[0196] Nucleosides and nucleotides can be labeled at sites on sugars or nucleobases. As known in the art, "nucleotides" are composed of nitrogenous bases, sugars, and one or more phosphate groups. In RNA, the sugar is ribose, and in DNA it is deoxyribose, i.e., a sugar lacking the hydroxyl group present in ribose. Nitrogenous bases are derivatives of purines or pyrimidines. Purines are adenine (A) and guanine (G), and pyrimidines are cytosine (C) and thymine (T), or uracil (U) in the case of RNA. The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine. Nucleotides are also phosphate esters of nucleosides, wherein esterification occurs on the hydroxyl group of the C-3 or C-5 connected to the sugar. Nucleotides are typically monophosphates, diphosphates, or triphosphates.

[0197] 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 attached to the base and there is no phosphate group attached to the sugar molecule.

[0198] Although bases are often referred to as purines or pyrimidines, the skilled artisan will appreciate that derivatives and analogs are available that do not alter the ability of a nucleotide or nucleoside to undergo Watson-Crick base pairing. "Derivative" or "analog" means a compound or molecule whose core structure is identical or very similar to that of the parent compound, but which has a chemical or physical modification, such as a different or additional side group, that allows the derived nucleotide or nucleoside to be attached to another molecule. For example, the base may 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 nucleotide or nucleoside derivatives having a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are discussed, for example, in 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.

[0199] 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.

[0200] 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.

[0201] Nucleosides or nucleotides labeled with dyes described herein may have the following formula:

[0202]

[0203] Wherein the dye is a 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'OH blocking group described herein, or -OR"' forms a phosphoramidite. Wherein -OR"' is a phosphoramidite, and R' is an acid-cleavable hydroxyl protecting group, which 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

[0204] In a particular embodiment, the blocking group is separate and independent of the dye compound, i.e., not connected to the latter. Alternatively, the dye may comprise all or part of a 3′-OH blocking group. Thus, R″ may be a 3′-OH blocking group that may or may not constitute a dye compound.

[0205] In yet another alternative embodiment, there is no blocking group present 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.

[0206] In yet another alternative embodiment, the blocking 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.

[0207] The use of blocking groups allows for control of polymerization, such as by stopping extension when a labeled nucleotide is incorporated. If the blocking 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.

[0208] In a specific embodiment, linking base (between dyestuff and nucleotide) and blocking group all exist and are independent parts.In a specific embodiment, linking base and blocking 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 blocking group these two.But, in some embodiments, linking base and blocking group do not need to can crack under similar conditions, but can crack separately under different conditions.

[0209] 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.

[0210] Non-limiting exemplary labeled nucleotides as described herein include:

[0211]

[0212] 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.

[0213] In some embodiments, non-limiting exemplary fluorescent dye conjugates are shown below:

[0214]

[0215]

[0216]

[0217] 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.

[0218] Other aspects of the present disclosure relate to oligonucleotides comprising the nucleotides of the labeling described herein. In some embodiments, the oligonucleotide 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 of multiple fixed target polynucleotides. In other embodiments, the solid support comprises a patterned flow cell. In other embodiments, the patterned flow cell is manufactured on a CMOS chip. In other embodiments, the patterned flow cell comprises a plurality of nanopores. In still other embodiments, a plurality of nanopores are directly aligned on each CMOS photodiode (pixel).

[0219] Reagent test kit

[0220] Provided herein is a kit including a first nucleotide labeled with an alkylpyridinium cation coumarin compound (i.e., a first label) of the present disclosure. In some embodiments, the kit also includes a second labeled nucleotide, which is labeled with a second compound (i.e., a second label), and the second compound is different from the alkylpyridinium cation coumarin compound in the nucleotide of the first label. In some embodiments, a single excitation source can be used to excite the nucleotide of the first label and the nucleotide of the second label, 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 in part, so that the excitation in the spectral overlap region causes the two labels to emit fluorescence. In some other embodiments, the kit can include a third 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. In some such embodiments, the nucleotide of the first label and the nucleotide of the third label 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. For example, the excitation bands of the first label and the third label can overlap at least in part, so that the excitation in the spectral overlap region causes the two labels to emit fluorescence. In some other embodiments, the kit can also include a fourth nucleotide. 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 mark has a different absorbance maximum that can be distinguished from other marks. In still other embodiments, the fourth nucleotide is unlabeled. In some embodiments, the first 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 400nm to about 480nm, about 420nm to about 470nm or 450nm to about 460nm (for example, 450nm). In alternative 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 (e.g., 520nm). The second light source has an excitation wavelength of about 400nm to about 480nm, about 420nm to about 470nm or 450nm to about 460nm (e.g., 450nm). In other embodiments, each of the first label, the second label, and the third label has an emission spectrum that can be collected in a single emission collection filter or channel.

[0221] In some embodiments, the kit may include four kinds of labeled nucleotides (A, C, G and T or U), wherein the first nucleotide in the four kinds of nucleotides is labeled with a compound as disclosed herein. In this kit, each of the four kinds of nucleotides can be labeled with a compound identical or different from the labeling on the other three kinds of nucleotides. Alternatively, the first nucleotide in the four kinds of nucleotides is a labeled nucleotide described herein, the second nucleotide in the four kinds of nucleotides carries the second labeling, the third nucleotide carries the third labeling, and the fourth nucleotide is unlabeled (dark). As another example, the first nucleotide in the four kinds of nucleotides is a labeled nucleotide described herein, the second nucleotide in the four kinds of nucleotides carries the second labeling, the third nucleotide carries a mixture of two labels, and the fourth nucleotide is unlabeled (dark). Therefore, one or more compounds in the labeled compound 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 the compounds can be distinguished from other three compounds (or if the fourth nucleotide is unlabeled, two compounds). It should be understood that the parts except the maximum in the absorption spectrum and / or emission spectrum can be different, and these differences can be utilized to distinguish compounds. The kit can be such that two or more compounds in these compounds have different absorbance maxima. Alkyl pyridinium cation coumarin dyes disclosed herein absorb light in the region lower than 500nm usually. For example, these coumarin dyes can have an absorption wavelength of about 450nm to about 530nm, about 460nm to about 520nm, about 475nm to about 510nm or about 490nm to about 500nm.

[0222] 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 these 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.

[0223] In a specific embodiment, the nucleotides of the labels described herein can be supplied in combination with unlabeled or natural nucleotides or any combination thereof. The combination of nucleotides can be provided as independent individual components (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).

[0224] In the case where the test kit includes multiple, specifically two or three or more specifically four nucleotides, different nucleotides can be labeled with different dye compounds, or a nucleotide can be dark, without dye compounds. In the case where different nucleotides are labeled 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 labeled 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 labeled 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 450nm-460nm, 490nm-500nm, or 520nm or higher (eg, 532nm).

[0225] In some embodiments, the kit includes the first nucleotide labeled with a compound of the present disclosure and the second nucleotide labeled with a second dye, wherein the dye has at least 10nm, specifically 20nm to 50nm or 30nm to 40nm absorbance maximum difference. More specifically, the first label can have a Stokes shift higher than 40nm, higher than 50nm or higher than 60nm. The second label can have a Stokes shift (wherein "Stokes shift" is the distance between the peak absorption wavelength and the peak emission wavelength) of about 80nm, higher than 90nm or higher than 100nm. In addition, the first label can have an absorption maximum of about 460nm to about 520nm, about 475nm to about 510nm or about 490nm to about 500nm. The second label can have an absorption maximum of about 400nm to about 470nm or about 450nm to about 460nm. In other embodiments, the kit can also be the nucleotide of the third label, wherein the third label has an absorption maximum higher than 520nm. The 3rd mark can have the Stokes shift higher than 20nm, higher than 30nm or higher than 40nm, or the Stokes shift between 20nm-40nm.Kit can also include the 4th unlabeled Nucleotide.In other embodiments, each mark in the first mark, the second mark and the 3rd mark has the emission maximum greater than 540nm, greater than 550nm, greater than 560nm, greater than 570nm, greater than 580nm, greater than 590nm or greater than 600nm.In some embodiments, can detect or collect the emission spectrum of the first mark, the second mark and the 3rd mark in single emission collection channel or optical filter (for example, from about 580nm to the collection area of ​​about 700nm).

[0226] In alternative embodiments, the kit of the present disclosure may include nucleotides in which the same base is labeled with two different compounds. The first nucleotide may be labeled with a compound of the present disclosure. The second nucleotide may be labeled with a spectrally different compound, such as a "green" dye that absorbs less than 600nm. The third nucleotide may be labeled with a mixture of a compound of the present disclosure and a spectrally different compound, and the fourth nucleotide may be "dark" and unlabeled. Therefore, in brief, nucleotides 1 to 4 may be labeled with "blue", "green", "blue / green" and dark. In order to further simplify the instrument, four nucleotides may be labeled with two dyes excited by a single light source, and therefore the labels of nucleotides 1 to 4 may be "blue 1", "blue 2", "blue 1 / blue 2" and dark.

[0227] 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.

[0228] In addition to the labeled nucleotides, the kit can also include at least one additional component together. The additional component 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. A buffer composition can include an antioxidant, such as ascorbic acid or sodium ascorbate, which can be used to protect the dye compound from light damage during detection. Other buffer compositions can include reagents that can be used to crack 3' blocking groups and / or cleavable linking groups. For example, a water-soluble phosphine or a water-soluble transition metal catalyst (such as a palladium complex) formed by a transition metal and at least a portion of a water-soluble ligand. The various components of the kit can be provided in a concentrated form to dilute before use. In such embodiments, a suitable dilution buffer can also be included. Similarly, one or more components in the components identified in the method shown herein can be included in the kit of the present disclosure. In any embodiment of the nucleotide or labeled nucleotide described herein, the nucleotide or labeled nucleotide includes a 3' hydroxyl blocking group.

[0229] Sequencing methods

[0230] 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 larger molecular structures or conjugates or associated with larger molecular structures or conjugates. In this context, the term "incorporated into a polynucleotide" can mean that the 5' phosphate is connected to the 3' hydroxyl group of the second nucleotide with a phosphodiester bond, and the second nucleotide itself can form part of a longer polynucleotide chain. The 3' end of the nucleotide shown herein may or may not be connected 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.

[0231] 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 one or more labeled nucleotides are detected by detecting or quantitatively measuring the fluorescence of the one or more labeled nucleotides incorporated into the polynucleotide.

[0232] Some embodiments of the present application relate to a method for determining the sequence of a target polynucleotide, the method comprising: (a) contacting a primer polynucleotide / target polynucleotide complex with one or more labeled nucleotides (such as nucleoside triphosphates A, G, C and T), wherein at least one of the labeled nucleotides is a labeled nucleotide described herein, and wherein the primer polynucleotide is complementary to at least a portion of the target polynucleotide; (b) incorporating the labeled nucleotide into the primer polynucleotide to produce an extended primer polynucleotide; and (c) performing one or more fluorescence measurements to determine the identity of the incorporated nucleotide. In some such embodiments, the primer polynucleotide / target polynucleotide complex is formed by contacting a target polynucleotide with a primer polynucleotide complementary to at least a portion of the target polynucleotide. In some embodiments, the method further comprises (d) removing the label portion and 3' blocking group from the nucleotides incorporated into the primer polynucleotide. In some additional embodiments, the method may also comprise (e) washing away the removed label portion and 3' blocking group from the primer polynucleotide chain. In some embodiments, repeat step (a) to (d) or step (a) to (e), until determining the sequence of at least a portion of the target polynucleotide chain. In some cases, step (a) to (d) or step (a) to (e) repeats at least 30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,250 or 300 cycles. In some embodiments, remove the labeling part and 3' blocking group from the nucleotides incorporated into the primer polynucleotide chain in a single chemical reaction. In some other embodiments, the method is carried out on an automated sequencing instrument, and wherein the automated sequencing instrument includes two light sources operating with different wavelengths. In some embodiments, sequence determination is carried out after completing the repeated cycles of sequencing steps as described herein.

[0233] Some embodiments of the present disclosure relate to a method for determining the sequence of a target polynucleotide (e.g., a single-stranded target polynucleotide), the method comprising: (a) contacting a primer polynucleotide with an incorporation mixture comprising one or more nucleotide conjugates of four different types of nucleotide conjugates, wherein the first type of nucleotide conjugate comprises a first label, the second type of nucleotide conjugate comprises a second label, and the third type of nucleotide conjugate comprises a third label, wherein each of the first label, the second label, and the third label is spectrally different from each other, and wherein the primer polynucleotide is complementary to at least a portion of the single-stranded target polynucleotide; (b) incorporating a nucleotide conjugate from the mixture into the primer polynucleotide to produce an extended primer polynucleotide; (c) performing a first imaging event using a first excitation light source and detecting a first emission signal from the extended polynucleotide; and (d) performing a second imaging event using a second excitation light source and detecting a second emission signal from the extended polynucleotide; wherein the first excitation light source and the second excitation light source have different wavelengths; and wherein the first emission signal and the second emission signal are detected or collected in a single emission detection channel. In some embodiments, the chromene-quinoline dye described herein can be used as any of the first label, the second label, or the third label described in the method. In some embodiments, the method does not include chemical modification of any nucleotide conjugate in the mixture after the first imaging event and before the second imaging event. In some other embodiments, the incorporation mixture also includes a fourth type of nucleotides, wherein the fourth type of nucleotides are unlabeled or labeled with a fluorescent moiety that does not emit a signal from the first imaging event or the second imaging event. In this sequencing method, the identity of each incorporated nucleotide conjugate is determined based on the detection mode of the first imaging event and the second imaging event. For example, the incorporation of the first type of nucleotide conjugate 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 dark state in the first imaging event and the signal state in the second imaging event. The incorporation of the third type of nucleotide conjugate is determined by the signal state in 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 the first imaging event and the second imaging event. In other embodiments, steps (a) to (d) are performed in a repeated cycle (e.g., at least 30, 50, 100, 150, 200, 250, 300, 400, or 500 times), and the method further comprises sequentially determining the sequence of at least a portion of the single-stranded target polynucleotide based on the identity of each sequentially incorporated nucleotide conjugate. In some embodiments, the wavelength of the first excitation light source is shorter than the wavelength of the second excitation light source. In some such embodiments, the first excitation light source has a wavelength of about 400 nm to about 480 nm, about 420 nm to about 470 nm, or about 450 nm to about 460 nm (i.e., "blue light").In one embodiment, the first excitation light source has a wavelength of about 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 (i.e., "green light"). In one embodiment, the second excitation light source has a wavelength of about 520nm. In other embodiments, the wavelength of the first excitation light source is longer than the wavelength of the second excitation light source. In some such embodiments, the first excitation light source has a wavelength of about 500nm to about 550nm, about 510nm to about 540nm, or about 520nm to about 530nm (i.e., "green light"). In one embodiment, the second excitation light source has a wavelength of about 520nm. The second excitation light source has a wavelength of about 400nm to about 480nm, about 420nm to about 470nm, or about 450nm to about 460nm (i.e., "blue light"). In one embodiment, the second excitation light source has a wavelength of about 450nm.

[0234] In one embodiment, at least one nucleotide is incorporated into a polynucleotide (such as a single-stranded primer polynucleotide as described herein) by the action of a polymerase during a synthesis step. However, other methods of linking nucleotides to polynucleotides can be used, for example, chemical oligonucleotide synthesis or linking 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.

[0235] In certain embodiments, a synthesis step is performed and the synthesis step may optionally include incubating the template or 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 for 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.

[0236] In all embodiments of these methods, the detection step can be carried out when the polynucleotide chain of the nucleotides into which the mark is incorporated is annealed to the template / target chain, or after the denaturation step in which the two chains are separated. Additional steps, such as chemical reaction steps or enzymatic reaction steps, or purification steps can be included between the synthesis step and the detection step. Specifically, the polynucleotide chain into which the nucleotides into which the mark is incorporated can be separated or purified, and then further processed or used for subsequent analysis. By way of example, the polynucleotide 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 product of these subsequent steps is purified or separated.

[0237] 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 mutant / 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.

[0238] In specific non-limiting embodiments, the present disclosure encompasses methods for 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 a polynucleotide.

[0239] 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 polynucleotides. Sequencing while synthesizing generally involves using a polymerase or a ligase to sequentially add one or more nucleotides or oligonucleotides to the growing polynucleotide chain in the 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 bases present in the one or more added nucleotides can be determined in the detection or "imaging" step. The identity of the added bases can be determined 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 determine the purposes of 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.

[0240] In embodiments 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 nucleotide. 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 reaction catalyzed by a polymerase.

[0241] In a specific embodiment, each nucleoside triphosphate in different nucleoside triphosphates (A, T, G and C) can be labeled with a unique fluorophore, and also includes a blocking group at the 3' position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides can be unlabeled (dark). Polymerase incorporates nucleotides into the nascent chain complementary to the template / target polynucleotide, and the blocking group prevents the further incorporation of nucleotides. Any unincorporated nucleotides can be washed away, and the fluorescent signal from the nucleotides of each incorporation 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 (deprotection) 3' blocking groups and fluorescent dye compounds can be removed (simultaneously or sequentially) to expose the nascent chain for further incorporation of nucleotides. Usually, the identity of the nucleotides incorporated will be determined after each incorporation step, but this is not strictly necessary. Similarly, U.S. Patent No. 5,302,509 (the patent is incorporated herein by reference) discloses a method for sequencing the polynucleotides fixed on a solid support.

[0242] As illustrated above, the method utilizes the incorporation of fluorescently labeled 3'-enclosed nucleotides A, G, C and T into a growing chain complementary to a fixed polynucleotide in the presence of a DNA polymerase. The polymerase incorporates a base complementary to the target polynucleotide, but is prevented from further addition by a 3'-enclosed group. The label of the nucleotides incorporated can then be determined, and the enclosed group is 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 overhanging 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 the sequencing reaction. 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 the template to be sequenced. Alternatively, primers to be sequenced and template strands can each form a part of a partial self-complementary nucleic acid strand capable of forming an intramolecular duplex (such as a hairpin loop structure). Hairpin polynucleotides and methods in which they can be connected to a solid support are disclosed in PCT Publication No. WO0157248 and No. WO2005 / 047301, each of which is incorporated herein by reference. Nucleotides can be continuously added to the growing primer, resulting in the synthesis of polynucleotide chains in the 5' to 3' direction. The properties of the added bases 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 the nucleotide and the nucleotide is joined to the free 3' hydroxyl group of the nucleic acid chain, the nucleotide is incorporated into the nucleic acid chain (or polynucleotide).

[0243] 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.

[0244] In certain embodiments, the nucleic acid template to be sequenced can be connected 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, which itself can be non-covalently attached to the solid support.

[0245] In polynucleotide directly connected to carrier (for example, based on silica carrier, as disclosed in WO00 / 06770 (incorporated herein by reference) those carriers) array, wherein by the reaction between the epoxy side group on glass and the internal amino group on polynucleotide, polynucleotide is fixed on the glass carrier.In addition, polynucleotide can be connected to solid carrier by the reaction of sulfenyl nucleophilic substance and solid carrier, for example, as described in WO2005 / 047301 (incorporated herein by reference).Still other example of the template polynucleotide of solid load is that template polynucleotide is connected to the hydrogel based on silica or other solid carrier, for example, as described in WO00 / 31148, WO01 / 01143, WO02 / 12566, WO03 / 014392, U.S. Patent No. 6,465,178 and WO00 / 53812, each document in these documents is incorporated herein by reference.

[0246] The specific surface on which the template polynucleotide can be fixed is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the references described above and WO2005 / 065814, which are incorporated herein by reference. Specific hydrogels that can be used include those described in WO2005 / 065814 and U.S. Publication No. 2014 / 0079923. In one embodiment, the hydrogel is PAZAM (poly (N- (5-azidoacetamidopentyl) acrylamide-co-acrylamide)).

[0247] The DNA template molecule can be attached to a bead or microparticle, for example, as described in U.S. Pat. No. 6,172,218 (incorporated herein by reference). Attachment 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 presented herein.

[0248] The template to be sequenced may form part of an "array" on a solid support, in which case the array may take any convenient form. Thus, the methods of the present disclosure are 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 may be used to sequence templates on essentially any type of array, including but not limited to those formed by immobilizing nucleic acid molecules on a solid support.

[0249] 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 (commonly referred to as sites or features) on the array 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 a 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 documents is incorporated herein) describe a method for amplifying nucleic acids, wherein template and amplification product are both kept fixed on a solid carrier, 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.

[0250] Nucleotides labeled with dye compounds disclosed herein can also be used to sequence templates on single molecule arrays. The term "single molecule array" or "SMA" as used herein refers to a population of polynucleotide molecules distributed (or arranged) on a solid support, wherein the spacing between any individual polynucleotide and all other polynucleotides of the population of molecules makes it possible to individually distinguish individual polynucleotide molecules. 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 representing a polynucleotide) will appear in the distinguishable region of the specific imaging device used.

[0251] 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.

[0252] The terms "individually resolved" and "individually resolved" are used herein to specify that, when visualized, it is possible to distinguish one molecule on an array from its neighboring molecules. The spacing between individual molecules on the array will be determined in part by the specific techniques used to resolve the individual molecules. The general characteristics of single molecule arrays will be understood by reference to published applications WO 00 / 06770 and WO 01 / 57248, each of which is incorporated herein by reference. Although one use of the labeled nucleotides disclosed herein is for sequencing-by-synthesis reactions, the utility of such nucleotides is not limited to such methods. In fact, the labeled nucleotides described herein can be advantageously used in any sequencing method that requires detection of fluorescent labels attached to nucleotides incorporated into a polynucleotide.

[0253] 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 generally 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.

[0254] Thus, the present disclosure also encompasses nucleotides labeled with dye compounds, which 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.

[0255] 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 dideoxynucleotides can 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.

[0256] Alternatively, unlabeled nucleotides and affinity reagents containing fluorescent dyes described herein can also be used to carry out the sequencing methods described herein. For example, one, two, three or each of four different types of nucleotides (e.g., dATP, dCTP, dGTP and dTTP or dUTP) in the incorporation mixture of step (a) can be unlabeled. Each of the four types of nucleotides (e.g., dNTP) has a 3' hydroxyl 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. An affinity reagent is then introduced that specifically recognizes and binds to the incorporated dNTP to provide an extension product containing a label of the incorporated dNTP. The use of unlabeled nucleotides and affinity reagents in sequencing by synthesis is disclosed in WO 2018 / 129214 and WO 2020 / 097607. The modified sequencing method of the present disclosure using unlabeled nucleotides can include the following steps:

[0257] (a') contacting a primer polynucleotide / target polynucleotide complex with one or more unlabeled nucleotides (e.g., dATP, dCTP, dGTP, and dTTP or dUTP), wherein the primer polynucleotide is complementary to at least a portion of the target polynucleotide;

[0258] (b') incorporating the nucleotide into the primer polynucleotide to produce an extended primer polynucleotide (ie, an extended primer polynucleotide / target polynucleotide complex);

[0259] (c') contacting the extended primer polynucleotide with a set of affinity reagents under conditions where one affinity reagent specifically binds to the incorporated unlabeled nucleotide to provide a labeled extended primer polynucleotide (i.e., a labeled extended primer polynucleotide / target polynucleotide complex);

[0260] (d') performing one or more fluorescence measurements on the labeled extended primer polynucleotide to determine the identity of the incorporated nucleotide.

[0261] In some embodiments of the modified sequencing methods described herein, each unlabeled nucleotide in the unlabeled nucleotides incorporated into the mixture contains a 3' hydroxyl blocking group. In other embodiments, the 3' hydroxyl blocking group of the incorporated nucleotide is removed before the next incorporation cycle. In still other embodiments, the method also includes removing the affinity reagent from the incorporated nucleotide. In still other embodiments, the 3' hydroxyl blocking group and the affinity reagent are removed in the same reaction. In some embodiments, the group of affinity reagents may include a first affinity reagent that specifically binds to a first type of nucleotide, a second affinity reagent that specifically binds to a second type of nucleotide, and a third affinity reagent that specifically binds to a third type of nucleotide. In some other embodiments, each of the first affinity reagent, the second affinity reagent, and the third affinity reagent includes a spectrally distinguishable detectable label. In some embodiments, the affinity reagent may include 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 knottin, an affimer, or any other known reagent that binds to the incorporated nucleotide with suitable specificity and affinity. In one embodiment, one or more affinity reagents in the group are antibodies or protein tags. 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 protein tag comprising one or more detectable labels (e.g., multiple copies of the same detectable label), and the detectable label comprises or is an alkylpyridinium cation coumarin dye moiety as described herein.

[0262] Example

[0263] 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.

[0264] Example 1. Synthesis of Alkylpyridinium Cationic Coumarin Dyes

[0265] Synthesis of Alkylpyridinium Cation Intermediates

[0266]

[0267] 2,4-lutidine (5.8mL, 50mmol) was dissolved in anhydrous THF (20mL) in a dry flask under nitrogen. A solution of 1M lithium diisopropylamide in THF / hexane (50mL, 50mmol) was slowly added at 0°C. The solution turned dark red and was stirred at room temperature for 4 hours. Then, anhydrous diethyl carbonate (14.7mL, 2.5mmol) dissolved in 10mL anhydrous THF was slowly added, and the reactant was stirred at room temperature overnight. The mixture was then quenched with saturated NH4Cl aqueous solution (10mL), diluted with 200mL ethyl acetate and washed with 200mL water. The organic phase was dried over MgSO4, filtered and evaporated under reduced pressure. The residue was purified on silica gel by flash chromatography. The intermediate was then dissolved in 10mL concentrated HCl and heated at 100°C for 1 hour. Volatiles were removed under reduced pressure, and the residue was then dissolved in ethanol (50mL), and a few drops of concentrated sulfuric acid were added. The solution was stirred at room temperature until complete. The reaction was quenched with 5 mL of saturated NaHCO3 aqueous solution and volatiles were removed under reduced pressure. The residue was distributed between saturated NaHCO3 (50 mL) and ethyl acetate (100 mL). The organic phase was dried over MgSO4, filtered and evaporated under reduced pressure. Compound 1 was obtained as a colorless viscous oil in a yield of 34%.

[0268] 6-Bromohexanoic acid (228 mg, 1.17 mmol) and compound 1 (210 mg, 1.17 mmol) were mixed and then heated at 100°C overnight. The mixture was then partitioned between water and dichloromethane. The aqueous phase was evaporated to give compound 2 with a yield of 70% (312 mg, 0.838 mmol). LC-MS (ESI): (positive ion) m / z 294 (M+H + ).

[0269]

[0270] Compound 1 (199 mg, 1.1 mmol) and 1,3-propane sultone (101 μL, 1.15 mmol) were dissolved in 1 mL of butyronitrile and heated at 100 °C for 2 hours. The volatiles were removed under reduced pressure, and the residue was dissolved in 50 mL of water and washed with 2×20 mL of DCM. The aqueous phase was evaporated to dryness. Compound 3 was obtained as an off-white solid in 88% yield (297 mg, 0.98 mmol). LC-MS (ESI): (positive ion) m / z 302 (M+H + ).

[0271]

[0272] 2,4,6-trimethylpyridine (2.5mL, 20mmol) is dissolved in anhydrous THF (20mL) in a dry flask under nitrogen. A solution of 1M lithium diisopropylamide in THF / hexane (22mL, 22mmol) is slowly added. The solution becomes dark red and turbid, and is stirred at room temperature for 2 hours. Then it is slowly added to anhydrous diethyl carbonate (5mL, 40mmol) dissolved in 20mL anhydrous THF at -70°C, and the reactant is slowly warmed to room temperature overnight. The mixture is then quenched with saturated NH4Cl aqueous solution (10mL) and diluted with 200mL ethyl acetate. The organic phase is separated, dried over MgSO4, filtered and evaporated under reduced pressure. The crude product is purified on silica gel by flash chromatography. The intermediate is then dissolved in 20mL concentrated HCl and left to stand overnight at room temperature. Volatiles are removed under reduced pressure, and the residue is then dissolved in ethanol (50mL), and 0.5mL concentrated sulfuric acid is added. The solution was refluxed for 4 hours, then quenched with 5 mL of saturated NaHCO3 aqueous solution, and volatiles were removed under reduced pressure. The residue was distributed between saturated NaHCO3 (50 mL) and ethyl acetate (100 mL). The organic phase was dried over MgSO4, filtered and evaporated under reduced pressure. Compound 4 was obtained as a colorless viscous oil with a yield of 12% (474 ​​mg, 2.44 mmol).

[0273] Acetonitrile (300 μL) and ethyl trifluoromethanesulfonate (67 μL, 0.518 mmol) were added to compound 4 (100 mg, 0.518 mmol). The solution was stirred at room temperature for 3 days, and then the volatiles were evaporated under reduced pressure to give compound 5 as a colored solid, which was used in the next step without further purification. LC-MS (ESI): (positive ion) m / z 222 (M+H + ).

[0274]

[0275] CuCN (2g, 23.2mmol) and anhydrous THF (75mL) were added to a flask under nitrogen and then cooled to -78°C while stirring. Isopropylmagnesium bromide (3M solution in 2-methyltetrahydrofuran, 15.5mL, 46.5mmol) was added dropwise while stirring vigorously. The suspension was left to stand at -78°C for 20 minutes, and then 2-bromo-4-methylpyridine (1g, 5.81mmol) was added. The reaction was stirred at -78°C for 3 hours, and then warmed to room temperature overnight. The suspension was then cooled in an ice bath and slowly quenched with concentrated aqueous ammonium hydroxide solution. The resulting suspension was extracted with 2×100mL dichloromethane. The organic phase was dried over MgSO4, filtered and evaporated under reduced pressure. The crude product was purified on silica gel by flash column chromatography. Compound 6 was obtained in a yield of 30% (240mg, 1.77mmol). LC-MS (ESI): (positive ion) m / z 135 (M+H + ).

[0276] Compound 6 (240mg, 1.77mmol) is dissolved in anhydrous THF (5mL) in a dry flask under nitrogen, and is cooled to -78°C. A solution of 1M lithium diisopropylamide in THF / hexane (3.6mL, 3.54mmol) is added dropwise. The solution becomes light red, and is stirred at -78°C for 1 hour. Then anhydrous diethyl carbonate (0.417mL, 3.54mmol) dissolved in 5mL anhydrous THF is slowly added, and the reactant is warmed to room temperature for 3.5 hours. The mixture is then quenched with saturated NH4Cl aqueous solution (5mL), diluted with 100mL ethyl acetate. The organic phase is dried over MgSO4, filtered and evaporated under reduced pressure. The residue is purified on silica gel by flash chromatography. The intermediate is then dissolved in 1mL concentrated HCl and heated at 100°C for 1 hour. Volatiles are removed under reduced pressure, and the residue is then dissolved in ethanol (5mL), and a few drops of concentrated sulfuric acid are added. The solution is stirred at room temperature until completion. The reaction was quenched with 5 mL of saturated NaHCO3 aqueous solution and volatiles were removed under reduced pressure. The residue was distributed between saturated NaHCO3 (50 mL) and ethyl acetate (100 mL). The organic phase was dried over MgSO4, filtered and evaporated under reduced pressure. Compound 7 was obtained as a light yellow viscous oil in a yield of 29%.

[0277] 6-Bromohexanoic acid (103 mg, 0.53 mmol), compound 7 (100 mg, 0.48 mmol) and tetrabutylammonium iodide (several mg, catalytic amount) were dissolved in 1 mL of acetonitrile and heated in a sealed tube at 100° C. for 2 days. The mixture was then partitioned between water and ethyl acetate. The aqueous phase was evaporated to give compound 8 in approximately 60% yield (86 mg, 0.318 mmol). LC-MS (ESI): (positive ion) m / z 322 (M+H + ).

[0278]

[0279] Compound 9 was prepared using the same procedure described for 5. The crude product was used in the next step without purification. LC-MS (ESI): (positive ion) m / z 236 (M+H + ).

[0280] Synthesis of Coumarin Dyes

[0281]

[0282] The starting material and a catalytic amount of pyridine acetate were dissolved in ethanol and refluxed until the reaction was complete. The crude product was evaporated to dryness and purified by flash column chromatography on reverse phase C18. Compound I-1 was obtained as a brown solid in a yield of 43% (56 mg, 0.129 mmol). 1 H NMR (400MHz, MeOD): δ (ppm) 8.71 (d, J=6.9Hz, 1H, Ar-H pyr), 8.57 (s, 1H, Ar-H), 8.48 (d, J=2.2Hz, 1H, Ar-H pyr), 8.42 (dd, J=6.8, 2.3Hz, 1H, Ar-H pyr), 7.30 (s, 1H, Ar-H), 6.57 (s, 1H, Ar-H), 4.51 (d, J=7.8Hz, 2H, CH2-N + ), 3.58-3.48 (m, 4H, CH2-N), 2.88 (s, 3H, CH3 pyr), 2.83 (t, J=6.2Hz, 2H, CH2-Ar), 2.22 (t, J=7.1Hz, 2H, CH2-COO), 2.04-1.95 (m, 4H, CH2-CH2-N, CH2-CH2-N +), 1.71 (p, J = 7.2 Hz, 2H, CH2-CH2-COO), 1.58-1.44 (m, 2H, CH2-CH2-CH2-COO), 1.27 (d, J = 7.1 Hz, 3H, CH3Et). LC-MS (ESI): (positive ion) m / z 435 (M+H + ); (negative ion) m / z 433 (M-).

[0283]

[0284] The starting material and the pyridine acetate of catalytic amount are dissolved in ethanol and refluxed until the reaction is complete. The crude product is evaporated to dryness, and the residue is then dissolved in 1mL water containing 1mL trifluoroacetic acid, and heated to 90 DEG C for 1 hour. The reaction is evaporated under reduced pressure, and the crude product is then purified by flash column chromatography on reversed phase C18. Compound I-2 is obtained as a brown solid with a yield of 75% (74mg, 0.123mmol). 1 H NMR (400MHz, MeOD): δ (ppm) 8.73 (d, J=6.1Hz, 1H, Ar-H pyr), 8.56 (s, 1H, Ar-H), 8.49 (br s, 1H, Ar-H pyr), 8.43 (br s, 1H, Ar-H pyr), 7.28 (s, 1H, Ar-H), 6.67 (s, 1H, Ar-H), 4.73 (t, J=8.0Hz, 2H, CH2-N + ), 3.58-3.45 (m, 4H, CH2-N), 2.98 (t, J=6.6Hz, 2H, CH2-SO3 - ), 2.91 (s, 3H, CH3 pyr), 2.82 (t, J=6.2Hz, 2H, CH2-Ar), 2.39 (m, 2H,, CH2-CH2-N + ), 2.32(t, J=7.1Hz, 2H, CH2-COO), 2.02-1.91(m, 4H, CH2-CH2-N, CH2-CH2-N + ). LC-MS (ESI): (positive ion) m / z 501 (M+H + ); (negative ion) m / z 499 (M - ).

[0285]

[0286] Compound 1-3 was prepared following the same procedure described for 1-2. Compound 1-3 was obtained as a brown solid in 59% yield (32 mg, 0.064 mmol). 1H NMR (400MHz, MeOD): δ (ppm) 8.50 (d, J=6.9Hz, 1H, Ar-Hpyr), 8.41 (s, 1H, Ar-H), 8.27 (d, J=2.3Hz, 1H, Ar-H pyr), 8.20 (dd, J=6.8, 2.3Hz, 1H, Ar-H pyr), 7.25 (d, J=1.5Hz, 1H, Ar-H), 6.45 (s, 1H, H-Ar), 4.50 (d, J=8.2Hz, 2H, CH2-N + ), 3.38 (m, 1H, HA CH2-N), 3.17 (m, 1H, HB CH2-N), 2.74 (m, 3H, CH-Ar, CH2-SO3 - ), 2.68(s, 3H, CH3 pyr), 2.21-2.09(m, 4H, CH2-CH2-N + , CH2-COO), 1.71 (m, 4H, CH2-CH2-CH2-&CH2-CH), 1.24 (s, 3H, CH3), 1.20 (d, J=6.5 Hz, 3H, CH3), 1.12 (s, 3H, CH3). LC-MS (ESI): (positive ion) m / z 543 (M+H + );(negative ion)m / z 541(M - ).

[0287]

[0288] Compound 1-4 was prepared following the same procedure described for 1-2. Compound 1-4 was obtained as a brown solid in 84% yield (77 mg, 0.135 mmol). 1 H NMR (400MHz, MeOD): δ (ppm) 8.57 (s, 1H, Ar-H), 8.35 (s, 2H, Ar-H pyr), 7.43 (d, J=1.5Hz, 1H, Ar-H), 6.62 (s, 1H, H-Ar), 4.58 (q, J=7.3Hz, 2H, CH2-N +), 3.62 (m, 1H, HA CH2-N), 3.34 (m, 1H, HB CH2-N), 2.91 (s, 7H, CH3 pyr, CH3-CH-Ar), 2.29 (t, J=7.1Hz, 2H, CH2-COO), 1.92 (m, 4H, CH2-CH2-N, CH2-CH), 1.56 (t, J=7.3Hz, 3H, CH3Et), 1.46 (s, 3H, CH3), 1.41 (d, J=6.5Hz, 3H, CH3), 1.34 (s, 3H, CH3). LC-MS(ESI): (positive ion) m / z 463(M+H + ); (negative ion) m / z 461 (M-).

[0289]

[0290] Compound 1-5 was prepared following the same procedure described for 1-2. Compound 1-4 was obtained as a brown solid in 66% yield (79 mg, 0.139 mmol). 1 H NMR (400MHz, MeOD): δ (ppm) 8.48 (s, 1H, Ar-H), 8.33 (s, 2H, Ar-H pyr), 7.24 (s, 1H, Ar-H pyr), 6.60 (s, 1H, Ar-H), 4.57 (q, J=7.3Hz, 2H, CH2-N + ), 3.55-3.43(m, 4H, CH2-N), 2.90(s, 6H, CH3 pyr), 2.80 (t, J=6.3Hz, 2H, CH2-Ar), 2.27 (t, J=7.2Hz, 2H, CH2-COO), 2.02-1.89 (m, 4H, CH2-CH2-N, CH2-CH2-COO), 1.55 (t, J=7.3Hz, 3H, CH3Et). LC-MS(ESI): (positive ion) m / z 421 (M+H + ).

[0291]

[0292] Compound 1-6 was prepared following the same procedure described for 1-1. Compound 1-6 was obtained as a brown solid in 33% yield (47 mg, 0.101 mmol). 1H NMR (400MHz, MeOD): δ (ppm) 8.69 (d, J=6.9Hz, 1H, Ar-Hpyr), 8.62 (m, 2H, Ar-H), 8.35 (dd, J=6.9, 2.3Hz, 1H, Ar-H pyr), 7.33 (d, J=1.2Hz, 1H, Ar-H), 6.58 (s, 1H, Ar-H), 4.62-4.54 (m, 2H, CH2-N + ), 3.60-3.48 (m, 5H, CH2-N, CH iPr), 2.84 (t, J=6.2Hz, 2H, CH2-Ar), 2.22 (t, J=7.1Hz, 2H, CH2-COO), 2.05-1.92 (m, 4H, CH2-CH2-N, CH2-CH2-N + ), 1.71 (p, J = 7.2 Hz, 2H, CH2-CH2-COO), 1.51 (m, 8H, CH3 iPr, CH2-CH2-CH2-COO), 1.27 (t, J = 7.1 Hz, 3H, CH3 Et). LC-MS (ESI): (positive ion) m / z 463 (M+H + ).

[0293]

[0294] Compound 1-7 was prepared following the same procedure described for 1-2. Compound 1-7 was obtained as a brown solid in 33% yield (48 mg, 0.1 mmol). 1 H NMR (400MHz, MeOD): δ (ppm) 8.68 (d, J=6.9Hz, 1H, Ar-H), 8.66 (s, 1H, Ar-H), 8.62 (d, J=2.3Hz, 1H, Ar-H), 8. 38 (dd, J=6.9, 2.3Hz, 1H, Ar-H), 7.47 (d, J=1.5Hz, 1H, Ar-H), 6.64 (s, 1H, Ar-H), 4.65 (q, J=7.3Hz, 2H, CH2-N +), 3.62 (m, 1H, HA CH2-N), 3.44-3.35 (m, 1H, HB CH2-N), 2.91 (m, 1H, CH iPr), 2.32 (t, J = 7.2 Hz, 2H, CH2-COO), 2.00-1.87 (m, 4H, CH2-CH2-N, CH2-CN), 1.62 (t, J = 7.3 Hz, 3H, CH3 Et), 1.52 (d, J = 6.8 Hz, 6H, CH3 iPr), 1.47 (s, 3H, C-CH3), 1.42 (d, J = 6.5 Hz, 3H, CH-CH3), 1.36 (s, 3H, C-CH3). LC-MS (ESI): (positive ion) m / z 477 (M+H + ).

[0295]

[0296] Compound 1-8 was prepared following the same procedure described for 1-1. Compound 1-8 was obtained as a brown solid in 61% yield (69 mg, 0.16 mmol). 1 H NMR (400MHz, MeOD): δ (ppm) 8.74 (d, J=6.9Hz, 1H, Ar-Hpyr), 8.65 (s, 1H, Ar-H), 8.50 (d, J=2.3Hz, 1H, Ar-H pyr), 8.43 (dd, J=6.9, 2.3Hz, 1H, Ar-H pyr), 7.61 (d, J=9.0Hz, 1H, Ar-H), 6.88 (dd, J=9.1, 2.5Hz, 1H, Ar-H), 6.63 (d, J=2.4Hz, 1H, Ar-H), 4.52 (d, J=7.5Hz, 2H, CH2-N + ), 3.67-3.53 (m, 4H, CH2-N), 2.89 (s, 3H, CH3pyr), 2.22 (t, J=7.1Hz, 2H, CH2-COO), 1.98 (p, J=7.7Hz, 2H, CH2-CH2-N + ), 1.71 (p, J = 7.3 Hz, 2H, CH2-CH2-COO), 1.59-1.46 (m, 2H, CH2-CH2-CH2-), 1.28 (t, J = 7.1 Hz, 6H, CH3 Et). LC-MS (ESI): (positive ion) m / z 423 (M+H + );(negative ion)m / z421(M - ).

[0297] Example 2. General Synthesis of Nucleotides Labeled with Alkylpyridinium Coumarin Dyes

[0298] Alkyl pyridinium cation coumarin dye (0.015mmol) is co-evaporated with 2×2mL anhydrous N, N′-dimethylformamide (DMF), then dissolved in 1mL anhydrous N, N′-dimethylacetamide (DMA). Add N, N-diisopropylethylamine (17μL, 0.1mmol), followed by N, N, N′, N′-tetramethyl-O-(N-succinimidyl) urea tetrafluoroborate (TSTU, 4.8mg, 0.016mmol). Stir the reaction at room temperature for 30 minutes under nitrogen. Meanwhile, the aqueous solution of nucleoside triphosphate (0.01mmol) is evaporated to dryness under reduced pressure, and resuspended in 100μL of 0.1M triethylammonium bicarbonate (TEAB) aqueous solution. Activated dye solution is added to triphosphate, and the reaction is stirred at room temperature for up to 18 hours and monitored by RP-HPLC. The crude product was purified by ion exchange chromatography on DEAE-Sephadex A25 (25 g) using a linear gradient elution of triethylammonium bicarbonate aqueous solution (TEAB, from 0.1 M to 1 M). The fractions containing triphosphate were pooled and the solvent was evaporated to dryness under reduced pressure. The crude material was further purified by preparative HPLC using a YMC-Pack-Pro C18 column.

[0299]

[0300] ffA-sPA-LN3-(I-1): Yield: 8.5 μmol, (53%). LC-MS (ES): (negative ion) m / z 1360 (MH + ), 680(M-2H + ). UV-Visλ max :507nm. Fluorescence λ max :566nm.

[0301]

[0302] ffA-LN3-(I-2): Yield: 1.9 μmol, (38%). LC-MS (ES): (negative ion) m / z 1428 (MH + ). UV-Visλ max :502nm. Fluorescence λ max :563nm.

[0303]

[0304] ffA-LN3-(I-3): Yield: 30.7 μmol, (61%). LC-MS (ES): (negative ion) m / z 1470 (MH + ). UV-Visλ max:501nm. Fluorescence λ max :565nm.

[0305]

[0306] ffA-LN3-(I-4): Yield: 1.4 μmol, (29%). LC-MS (ES): (negative ion) m / z 1390 (MH + ). UV-Visλ max :493nm. Fluorescence λ max :551nm.

[0307]

[0308] ffA-LN3-(I-5): Yield: 2.7 μmol, (54%). LC-MS (ES): (negative ion) m / z 1348 (MH + ). UV-Visλ max :491nm. Fluorescence λ max :555nm.

[0309]

[0310] ffA-sPA-LN3-(I-8): Yield: 7.8 μmol, (78%). LC-MS (ES): (negative ion) m / z 1348 (MH + ). UV-Visλ max :495nm. Fluorescence λ max :556nm.

[0311] Example 3. Spectral properties of alkylpyridinium cation coumarin dyes

[0312] In this example, the spectral properties of several alkylpyridinium coumarin dyes described herein are compared to corresponding reference dyes that are not methylated. Figure 1A and Figure 1B In the present invention, the fluorescence emission of the methylpyridinium cation coumarin dye I-1 in a solution of universal scanning mixture (USM, 1M Tris pH7.5, 0.05% TWEEN, 20mM sodium ascorbate, 10mM ethyl gallate) was compared with the fluorescence emission of reference dye A at excitation wavelengths of 450nm ("blue light") and 520nm ("green light"), respectively. Spectra were acquired on an Agilent Cary 100UV-Vis spectrophotometer and on a CaryEclipse fluorescence spectrophotometer using quartz cuvettes. It was observed that dye I-1 showed an increase of about 2 times in fluorescence emission when excited by green light, and an increase of about 3 times in fluorescence emission when excited by blue light, compared to reference dye A.

[0313] Similarly, Figure 2A and Figure 2B The fluorescence emission of methylpyridinium coumarin dye I-5 in USM solution is shown in comparison with the fluorescence emission of reference dye C at excitation wavelengths of 450 nm and 520 nm, respectively. Figure 3A and Figure 3B The fluorescence emission of the methylpyridinium cation coumarin dye I-8 in USM solution is shown at 450nm and 520nm excitation wavelengths, respectively, compared with the fluorescence emission of the reference dye B. Compared with the reference dye C, the coumarin dye I-5 shows that the fluorescence emission increases by about 2.5 times when excited by green light, and increases by about 8 times when excited by blue light. Compared with the reference dye B, the coumarin dye I-8 shows that the fluorescence emission is similar when excited by green light, and increases by about 2.5 times when excited by blue light (450nm).

[0314]

[0315] Example 4. Spectral properties of alkylpyridinium coumarin-conjugated ffA nucleotides

[0316] In this example, the spectral properties of several fully functionalized A nucleotides (ffA) conjugated to the picolinium coumarin dyes described herein are compared to corresponding reference dyes without methylation. Figure 4A and Figure 4B In the , the fluorescence emission of ffA (in the form of a 2 μM solution in USM) conjugated with the picolinium coumarin dye I-1 was compared to that of the reference dye A at excitation wavelengths of 450 nm ("blue light") and 520 nm ("green light"), respectively. Spectra were acquired on an Agilent Cary 100 UV-Vis spectrophotometer and on a Cary Eclipse fluorescence spectrophotometer using quartz cuvettes. It was observed that ffA-sPA-LN3-(I-1) showed similar fluorescence emission upon green light excitation and an approximately 3-fold increase in fluorescence emission upon blue light excitation compared to ffA-sPA-LN3-(reference dye A).

[0317] Similarly, Figure 5A and Figure 5B The fluorescence emission of ffA conjugated with picolinyl coumarin dye 1-5 (as a 2 μM solution in USM) is shown compared to that of reference dye C at excitation wavelengths of 450 nm and 520 nm, respectively. Fig. 6A and Figure 6BThe fluorescence emission of ffA conjugated with picolinyl coumarin dye 1-8 (as a 2 μM solution in USM) is shown compared to that of reference dye B at excitation wavelengths of 450 nm and 520 nm, respectively. Fig. 7A and Figure 7B The fluorescence emission of ffA (in the form of a 2 μM solution in USM) conjugated with methylpyridinium coumarin dye I-3 is shown at 450 nm and 520 nm excitation wavelengths, respectively, compared to the fluorescence emission of reference dye D. Compared to ffA-sPA-LN3- (reference dye C), ffA-LN3- (I-5) shows that the fluorescence emission is similar when excited by green light (520 nm), and the fluorescence emission is increased by about 2.5 times when excited by blue light (450 nm). Compared to ffA-sPA-LN3- (reference dye B), ffA-sPA-LN3- (I-8) shows that the fluorescence emission is similar when excited by green light (520 nm), and the fluorescence emission is increased by about 1.6 times when excited by blue light (450 nm). Compared to ffA-sPA-LN3-(reference dye D), ffA-sPA-LN3-(I-3) showed similar fluorescence emission upon excitation with green light (520 nm) and an approximately 1.2-fold increase in fluorescence emission upon excitation with blue light (450 nm).

[0318]

[0319] Example 5. Illumina iSeq TM 100 sequencing experiments on instruments

[0320] In this example, the Illumina iSeq TMThe WA-linker-dye compounds described herein were tested on a 100 instrument that had been set up to take a first image with green excitation light (about 520 nm) and a second image with blue excitation light (about 450 nm). The sequencing recipe was modified to perform a standard SBS cycle (incorporation, followed by imaging, followed by cleavage). The spiked mixture used in each of these experiments contained the following four ffNs: 1) ffA conjugated to an alkylpyridinium cation dye described herein or ffA conjugated to a reference dye described herein; 2) ffC (e.g., ffC-linker-coumarin dye E) that can be excited with 450 nm blue light in the case of using ffA conjugated to an alkylpyridinium cation dye described herein, and ffC-linker-coumarin dye F in the case of using ffA conjugated to a reference dye described herein; 3) ffT (e.g., ffT-LN3-NR550s0) that can be excited with green light; and 4) unlabeled ffG (dark ffG) in 50 mM ethanolamine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO4, and DNA polymerase. Coumarin dyes E and F are disclosed in U.S. Publication No. 2018 / 0094140 and have structural moieties when conjugated to ffC, respectively. Fig. 8A and Figure 8B Shown are scatter plots obtained for the spiked mixtures containing ffA-sPA-LN3-(I-1) and ffA-sPA-LN3-(reference dye A), respectively. Figure 8C and Fig.8D Scatter plots obtained for spiked mixtures containing ffA-sPA-LN3-(I-3) and ffA-sPA-LN3-(reference dye D), respectively, are shown. In both cases, it was observed that the spiked mixture containing ffA conjugated with an alkylpyridinium coumarin dye provided increased intensity and better A-cloud separation compared to the corresponding reference dye without methyl substitution at the pyridinium moiety.

[0321] Table 1 shows the results of the standard reagents and recipes used in the Illumina iSeq TM Compared to the metrics obtained from 2 × 151 cycles run on the 100 instrument, the ffA-sPA-LN3-(I-1), ffA-LN3-(I-3), and ffA-sPA-(reference dye D) on the iSeq TMPhasing, prephasing, and PhiX error rate metrics for 2×151 cycles run on 100. The spike-in mixture used in each of these experiments contained the following four ffNs: 1) ffA conjugated to an alkylpyridinium dye described herein or ffA conjugated to a reference dye described herein; 2) ffC (e.g., ffC-linker-coumarin dye E) that can be excited with 450 nm blue light in the case of using ffA conjugated to an alkylpyridinium dye described herein, and ffC-linker-coumarin dye F in the case of using ffA conjugated to a reference dye described herein; 3) ffT (e.g., ffT-LN3-NR550s0) that can be excited with green light; and 4) unlabeled ffG (dark ffG) in 50 mM ethanolamine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO4, and DNA polymerase. Compared with ffA-sPA-(reference dye D) and standard iSeq TM An improvement in the PhiX error rate metric was observed with compounds ffA-sPA-LN3-(I-1) and ffA-LN3-(I-3) compared to 100 reagents.

[0322] Table 1: iSeq TM 100 sequencing metrics comparison (2×151 cycles)

[0323]

[0324] In addition, compound ffA-sPA-LN3-(I-1) was selected to be used in the Illumina iSeq TM 2×300 cycles were run on a 100 instrument. The instrument was set to take the first image with green excitation light and the second image with blue excitation light, and the recipe was modified to perform standard SBS cycles (incorporation, subsequent imaging, and subsequent cleavage) for 2×300 cycles. The incorporation mixture used in these experiments contained the following four ffNs: 1) ffA-sPA-LN3-(I-1); 2) 1 ffC (e.g., ftC-linker-coumarin dye E) that can be excited with 450nm blue light; 3) ffT (e.g., ffT-linker-NR550s0) that can be excited with green light; and 4) dark ffG in 50mM ethanolamine buffer, pH 9.6, 50mM NaCl, 1mM EDTA, 0.2% CHAPS, 4mM MgSO4, and DNA polymerase. Phasing, prephasing, PhiX error rate, and %Q30 metrics are shown in Table 2 below.

[0325] Table 2: iSeq TM 100 sequencing metrics (2×300 cycles)

[0326]

Claims

1. A compound of formula (I), a salt or meso form thereof: Where R 1 yes And where R 1 substituted with one or more C1 alkyl groups; R 2 , R 5 , R 6 and R 7 Each of is independently H; R 3 and R 4 Each of which is independently H, C1-C6 alkyl or substituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is a C1-C6 alkyl substituted by one or more substituents selected from the group consisting of carboxyl (-C(O)OH), carboxylate (-C(O)Oˉ), sulfonate (-SO3H), sulfonate (-SO3ˉ); Or, R 4 and R 5 Together with the atoms to which they are attached, they form a ring or ring system selected from the group consisting of a 6-membered heterocyclyl, wherein the 6-membered heterocyclyl is an unsubstituted 6-membered heterocyclyl or a 6-membered heterocyclyl substituted with a C1-C6 alkyl group; and R a , R b and R c Each of the R is independently C1-C6 alkyl or substituted C1-C6 alkyl; wherein R a and R b Each of the above is independently a C1-C6 alkyl group or a C1-C6 alkyl group substituted with one or more substituents selected from the group consisting of: carboxyl (-C(O)OH), carboxylate (-C(O)Oˉ), sulfonate (-SO3H), sulfonate (-SO3ˉ); wherein R c It is a C1-C6 alkyl group or a C1-C6 alkyl group substituted by one or more substituents selected from the group consisting of a carboxyl group (-C(O)OH), a sulfo group (-SO3H), and a sulfonate group (-SO3ˉ).

2. The compound according to claim 1, wherein R 3 is H, and R 4 It is a C1-C6 alkyl group or a substituted C1-C6 alkyl group.

3. The compound according to claim 1, wherein R 3 and R 4 Each of is independently a C1-C6 alkyl.

4. The compound according to claim 1, wherein the compound of formula (I) is also represented by formula (Ia): Its salt or meso form means wherein: Each R 8 , R 9 , R 10 and R 11 are independently H, C1-C6 alkyl; and By solid and dotted lines The bond represented by is selected from the group consisting of single bonds and double bonds, provided that when When it is a double bond, R 11 Does not exist.

5. A compound according to any one of claims 1 to 4, wherein R 1 yes 6. A compound according to any one of claims 1 to 4, wherein R 1 yes 7. The compound according to claim 5 or 6, wherein each R a and R b are independently C1-C6 alkyl.

8. The compound according to claim 5 or 6, wherein each R a and R b Independently, it is a C1-C6 alkyl group substituted by a carboxyl group (-C(O)OH), a carboxylate group (-C(O)Oˉ), a sulfonate group (-SO3H) or a sulfonate group (-SO3ˉ).

9. A compound according to any one of claims 4 to 8, wherein the solid and dotted lines The bond indicated is a double bond.

10. The compound according to claim 9, wherein R 10 It is H or C1-C6 alkyl.

11. The compound according to claim 10, wherein R 10 It's methyl.

12. A compound according to any one of claims 4 to 8, wherein the solid and dotted lines The bond represented is a single bond.

13. The compound according to claim 12, wherein R 10 is H, and R 11 It is a C1-C6 alkyl group.

14. The compound according to claim 12, wherein R 10 and R 11 Each of is H.

15. A compound according to any one of claims 4 to 14, wherein R 8 and R 9 Each of is H.

16. A compound according to any one of claims 4 to 14, wherein R 8 and R 9 At least one of them is a C1-C6 alkyl group.

17. The compound according to claim 16, wherein R 8 and R 9 Each of is a C1-C6 alkyl group.

18. The compound according to claim 17, wherein R 8 and R 9 Each of is methyl.

19. A compound according to any one of claims 4 to 18, wherein R 3 It is a C1-C6 alkyl group.

20. A compound according to any one of claims 4 to 18, wherein R 3 It is a substituted C1-C6 alkyl group; the substituted C1-C6 alkyl group is a C1-C6 alkyl group substituted by one or more substituents selected from the group consisting of: carboxyl (-C(O)OH), carboxylate (-C(O)Oˉ), sulfonate (-SO3H), and sulfonate (-SO3ˉ).

21. The compound according to claim 20, wherein R 3 It is a C1-C6 alkyl group substituted by one or more substituents selected from the group consisting of carboxyl (-C(O)OH), carboxylate (-C(O)Oˉ), and sulfonyl (-SO3H).

22. The compound according to claim 21, wherein R 3 It is a C1-C6 alkyl group substituted by a carboxyl group.

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

24. A nucleotide or oligonucleotide labelled with a compound according to any one of claims 1 to 23.

25. A nucleotide or oligonucleotide according to claim 24, wherein the compound is represented by R of formula (Ia) 8a , R 8b or R 8c The carboxyl group is attached to the nucleotide or oligonucleotide.

26. A nucleotide or oligonucleotide according to claim 24, wherein the compound is represented by R of formula (I) 3 or R 4 The carboxyl group is attached to the nucleotide or oligonucleotide.

27. A nucleotide or oligonucleotide according to any one of claims 24 to 26, wherein the compound is linked to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base via a linker moiety.

28. The nucleotide or oligonucleotide according to any one of claims 24 to 27, wherein the labeled nucleotide or oligonucleotide further comprises a 3'OH blocking group covalently linked to the ribose or deoxyribose of the nucleotide.

29. The nucleotide or oligonucleotide of claim 24, wherein the nucleotide or oligonucleotide is an oligonucleotide that hybridizes to at least a portion of a target polynucleotide.

30. The oligonucleotide of claim 29, wherein the target polynucleotide is immobilized on a solid support.

31. The oligonucleotide of claim 30, wherein the solid support comprises an array of a plurality of immobilized target polynucleotides.

32. A kit comprising a first nucleotide labelled with a first compound according to any one of claims 1 to 23.

33. The kit of claim 32, further comprising a second nucleotide labeled with a second compound, wherein the second compound is different from the first compound of the first labeled nucleotide.

34. The kit of claim 33, wherein the first labeled nucleotide and the second labeled nucleotide are capable of being excited using a first light source wavelength.

35. The kit according to claim 33 or 34, further comprising a third nucleotide, wherein the third nucleotide is labeled with a third compound different from the first compound and the second compound, and wherein the first labeled nucleotide and the third labeled nucleotide can be excited using a second light source wavelength.

36. The kit of claim 35, further comprising a fourth nucleotide, and wherein the fourth nucleotide is unlabeled.

37. The kit according to any one of claims 32 to 36, wherein each of the first labeled nucleotide, the second labeled nucleotide, and the third labeled nucleotide has an emission spectrum that can be detected in a single detection channel.

38. The kit according to any one of claims 32 to 37, further comprising a DNA polymerase and one or more buffer compositions.

39. A method for determining the sequence of a target polynucleotide, the method comprising: (a) contacting a primer polynucleotide / target polynucleotide complex with one or more labeled nucleotides, wherein at least one of the labeled nucleotides is a nucleotide according to any one of claims 24 to 28, and wherein the primer polynucleotide is complementary to at least a portion of the target polynucleotide; (b) incorporating a labeled nucleotide into the primer polynucleotide to generate an extended primer polynucleotide / target polynucleotide complex; and (c) performing one or more fluorescence measurements on the extended primer polynucleotide / target polynucleotide complex to determine the identity of the incorporated nucleotide.

40. The method of claim 39, wherein the primer polynucleotide / target polynucleotide complex is formed by contacting the target polynucleotide and a primer polynucleotide that is complementary to at least a portion of the target polynucleotide.

41. The method of claim 39 or 40, wherein the method further comprises (d) removing a label and a 3' blocking group from the nucleotide incorporated into the primer polynucleotide.

42. The method of claim 40, wherein the method further comprises (e) washing the removed label and the 3' blocking group from the extended primer polynucleotide.

43. The method of claim 42, wherein the method further comprises repeating steps (a) to (e) until the sequence of at least a portion of the template polynucleotide chain is determined.

44. The method of claim 43, wherein steps (a) to (e) are repeated at least 50 times.

45. The method of any one of claims 39 to 44, wherein the label and the 3' blocking group from the nucleotide incorporated into the primer polynucleotide are removed in a single chemical reaction.

46. ​​The method of any one of claims 39 to 45, wherein the method is performed on an automated sequencing instrument, and wherein the automated sequencing instrument comprises two light sources operating at different wavelengths.

Images