Alkylpyridinium coumarin dyes and their use in sequencing applications

Abstract

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

Description

[Technical Field]

[0001] The present disclosure relates to alkylpyridinium-substituted coumarin derivatives and their use as fluorescent labels. Specifically, the compounds can be used as nucleotide labels for nucleic acid sequencing applications. [Background technology]

[0002] Non-radioactive detection of nucleic acids bearing fluorescent labels is an important technique in molecular biology. Many procedures used in recombinant DNA technology have previously been developed, e.g. 32 These methods rely on the use of nucleotides or polynucleotides radioactively labeled with P. Radioactive compounds allow for sensitive detection of nucleic acids and other molecules of interest. However, the use of radioisotopes has significant limitations, including their cost, limited shelf life, poor sensitivity, and, more importantly, safety considerations. Eliminating the need for radioactive labeling reduces both safety risks and the environmental impact and costs associated with, for example, reagent disposal. Methods suitable for non-radioactive fluorescence detection include, but are not limited to, automated DNA sequencing, hybridization methods, real-time detection of polymerase chain reaction products, and immunoassays.

[0003] For many applications, it is desirable to use multiple spectrally distinguishable fluorescent labels to achieve independent detection of multiple spatially overlapping analytes. Such multiplexing methods can reduce the number of reaction vessels, simplifying experimental protocols and facilitating the creation of application-specific reagent kits. For example, in multicolor automated DNA sequencing systems, multiplexed fluorescent detection allows for the analysis of multiple nucleotide bases in a single electrophoretic lane, thereby increasing throughput compared to single-color methods and reducing the uncertainty associated with lane-to-lane electrophoretic mobility variation.

[0004] However, multiplexed fluorescence detection can be problematic, and several important factors constrain the selection of appropriate fluorescent labels. First, it can be difficult to find a dye compound with substantially resolved absorption and emission spectra for a given application. In addition, when several fluorescent dyes are used together, generating fluorescent signals in distinguishable spectral regions through simultaneous excitation can be complicated because the dye absorption bands are usually widely separated, making it difficult to achieve comparable fluorescence excitation efficiency even for two dyes. Because many excitation methods use high-power light sources such as lasers, the dyes must be sufficiently photostable to withstand such excitation. A final consideration, particularly important for molecular biology methods, is how compatible the fluorescent dye needs to be with reagent chemicals, such as DNA synthesis solvents and reagents, buffers, polymerase enzymes, and ligase enzymes.

[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 the above constraints and are particularly suitable for high-throughput molecular methods such as solid-phase sequencing.

[0006] Fluorescent dye molecules with improved fluorescence properties, such as favorable fluorescence intensity, shape, and wavelength maximum of the fluorescence band, can improve the speed and accuracy of nucleic acid sequencing. A strong fluorescent signal is particularly important when measurements are performed in aqueous biological buffers and at high temperatures, because the fluorescence intensity of most organic dyes is significantly lower under such conditions. Furthermore, the nature of the base to which the dye is attached also affects the fluorescence maximum, fluorescence intensity, and other spectral properties of the dye. The sequence-specific interaction between nucleic acid bases and fluorescent dyes can be tailored by specific design of the fluorescent dye. Optimizing the fluorescent dye structure can improve the efficiency of nucleotide incorporation, reduce the level of sequencing errors, and reduce the amount of reagents used in nucleic acid sequencing, thereby reducing the cost of nucleic acid sequencing.

[0007] Several optical and technological developments have already significantly improved image quality, but ultimately have been limited by insufficient optical resolution. Optical microscopes generally have a limited optical resolution, capable of distinguishing objects separated by approximately half the wavelength of the light used. In practical terms, only objects very far apart (at least 200–350 nm) can be distinguished by optical microscopy. One way to improve image resolution and increase the number of distinguishable objects per unit surface area is to use shorter wavelength excitation light. For example, using the same optical system, shortening the wavelength by approximately 100 nm (Δλ) results in better resolution (approximately Δ50 nm / (approximately 15%)), recording images with less distortion, and increasing the density of objects over the recognizable area by approximately 35%.

[0008] Certain nucleic acid sequencing methods use laser light to excite and detect dye-labeled nucleotides. These instruments use longer wavelength light, such as a red laser, with an appropriate dye excitable at 660 nm. To detect denser nucleic acid sequencing clusters while maintaining useful resolution, a shorter wavelength blue light source (450-460 nm) can be used. In this case, optical resolution is limited not by the emission wavelength of the long-wavelength red fluorescent dye, but rather by the emission of the dye excitable by the next longest wavelength light source, e.g., a 532 nm "green laser." Therefore, there is a need for blue dye labels for use in fluorescence detection in sequencing applications.

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

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

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

[0012] [ka] In the formula, R 1 teeth,

[0013] [ka] and R 1 is substituted with one or more C1-C6 alkyl groups; Each R 2 , R 5 and R 7 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, sulfino, sulfonate, S-sulfonamido, or N-sulfonamido; R 3 and R 4 each is independently H, C1-C6 alkyl, or substituted C1-C6 alkyl; Alternatively, R 2 and R 3together with the atoms to which they are attached 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; Alternatively, R 4 and R 5 together with the atoms to which they are attached 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; R 6 is H, C1-C6 alkyl, substituted C1-C6 alkyl, or optionally substituted C6-C 10 is aryl, R a , R b and R c Each of is independently C1-C6 alkyl or substituted C1-C6 alkyl.

[0014] In some embodiments, the compound of Formula (I) is also represented by Formula (Ia), or a salt or mesomeric form thereof:

[0015] [ka] In the formula, 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, sulfino, sulfonate, S-sulfonamido, or N-sulfonamido, and include solid and dashed lines

[0016] [ka] is selected from the group consisting of a single bond and a double bond, with the proviso that

[0017] [ka] If is a double bond, R 11 is provided that it does not exist.

[0018] In some embodiments of compounds of Formula (I) or (Ia), R 1 (For example, R a , R b or R c ) contains a carboxyl group (—C(O)OH). In other embodiments, R 3 or R 4 contains a carboxyl group.

[0019] In some aspects, the compounds of the present disclosure are labeled or conjugated with a substrate moiety, such as, for example, a nucleoside, a nucleotide, a polynucleotide, a polypeptide, a carbohydrate, a ligand, a particle, a cell, a semi-solid surface (e.g., a gel), or a solid surface. Labeling or conjugation can be carried out via a carboxyl group, which can react with an amino or hydroxyl group on an attached moiety (such as a nucleotide) or linker to form an amide or ester, using methods known in the art.

[0020] Some other aspects of the present disclosure relate to dye compounds that include a linker group to enable covalent attachment to, for example, a substrate moiety. The attachment can be at any position on the dye, including any of the R groups. In some embodiments, the attachment is at the R 1 (For example, R a , R b or R c ) or R 3 or R 4 In some further embodiments, the linkage can be achieved via R 1 (For example, R a , R b or R c ) or R3 This can be implemented via:

[0021] Some further aspects of the present disclosure provide labeled nucleoside or nucleotide compounds defined by the following formula: NL-dye wherein N is a nucleoside or nucleotide; L is an optional linker moiety; The dye is a moiety of a fluorescent compound of formula (I) or (Ia) according to the present disclosure, wherein a functional group (e.g., a carboxyl group) of the compound of formula (I) or (Ia) reacts with an amino or hydroxyl group of a linker moiety or a nucleoside / nucleotide to form a covalent bond.

[0022] Some additional aspects of the present disclosure pertain to nucleotides or oligonucleotides labeled with compounds of Formula (I) or (Ia).

[0023] Some additional aspects of the present disclosure relate to kits containing dye compounds (free or labeled) that can be used in various immunoassays, oligonucleotide or nucleic acid labeling, or for DNA sequencing by synthesis. In yet another aspect, the present disclosure provides kits containing dye "sets" particularly suited for sequencing-by-synthesis cycles on automated instrument platforms. In some aspects, the kits contain one or more nucleotides, at least one of which is a labeled nucleotide as described herein.

[0024] A further aspect of the present disclosure is a method for determining the sequence of a target polynucleotide, comprising: (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 nucleotide described herein labeled with an alkylpyridinium-substituted coumarin dye of Formula (I) or (Ia), 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 / target nucleotide complex to form an extended primer polynucleotide / target nucleotide complex; (c) performing one or more fluorescence measurements of the extended primer polynucleotide / target nucleotide complex to determine the identity of the incorporated nucleotide. [Brief explanation of the drawings]

[0025] [Figure 1A] Fluorescence emission spectra of coumarin dye I-1 compared to reference dye A in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 1B] Fluorescence emission spectra of coumarin dye I-1 compared to reference dye A in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 2A] Fluorescence emission spectra of coumarin dye I-5 compared to reference dye C in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 2B] Fluorescence emission spectra of coumarin dye I-5 compared to reference dye C in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 3A] Fluorescence emission spectra of coumarin dye I-8 compared to reference dye B in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 3B]Fluorescence emission spectra of coumarin dye I-8 compared to reference dye B in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 4A] Fluorescence emission spectra of ffA labeled with coumarin dye I-1 compared to a fully functionalized A nucleotide (ffA) labeled with reference dye A in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 4B] Fluorescence emission spectra of ffA labeled with coumarin dye I-1 compared to a fully functionalized A nucleotide (ffA) labeled with reference dye A in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 5A] Fluorescence emission spectra of ffA labeled with coumarin dye I-5 compared to ffA labeled with reference dye C in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 5B] Fluorescence emission spectra of ffA labeled with coumarin dye I-5 compared to ffA labeled with reference dye C in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 6A] Fluorescence emission spectra of ffA labeled with coumarin dye I-8 compared to ffA labeled with reference dye B in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 6B] Fluorescence emission spectra of ffA labeled with coumarin dye I-8 compared to ffA labeled with reference dye B in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 7A] Fluorescence emission spectra of ffA labeled with coumarin dye I-3 compared to ffA labeled with reference dye D in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 7B]Fluorescence emission spectra of ffA labeled with coumarin dye I-3 compared to ffA labeled with reference dye D in buffer at excitation wavelengths of 450 nm and 520 nm, respectively. [Figure 8A] Shown are scatter plots obtained for uptake mixtures containing ffA labeled with coumarin dye I-1 and reference dye A, respectively. [Figure 8B] Shown are scatter plots obtained for uptake mixtures containing ffA labeled with coumarin dye I-1 and reference dye A, respectively. [Figure 8C] Shown are scatter plots obtained for uptake mixtures containing ffA labeled with coumarin dye I-3 and reference dye D, respectively. [Figure 8D] Shown are scatter plots obtained for uptake mixtures containing ffA labeled with coumarin dye I-3 and reference dye D, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0026] Embodiments of the present disclosure relate to alkylpyridinium-substituted coumarin dyes with enhanced fluorescence intensity and long Stokes shifts. These coumarin dyes also have broad excitation wavelengths and can be excited by both blue and green light sources. In some embodiments, the alkylpyridinium coumarin dyes described herein can be used with Illumina's iSeq™ platform, which features two-channel CMOS detection (green light excitation and blue light excitation).

[0027] definition The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0028] 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 unambiguously limited to one referent. It will be apparent to those skilled in the art that various modifications and variations can be made to the various embodiments described herein without departing from the spirit or scope of the present teachings. Accordingly, it is intended that the various embodiments described herein encompass other modifications and variations that come within the scope of the appended claims and their equivalents.

[0029] 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 "including" and other forms, such as "include," "includes," and "included," is not limiting. Furthermore, the use of the term "having" and other forms, such as "have," "has," and "had," is not limiting. As used herein, in transitional phrases or the body of claims, the terms "comprise" and "comprising" should be interpreted as having an open-ended meaning. That is, the above terms should be interpreted as synonymous with the phrases "having at least" or "including at least." For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may 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 recited features or components, but may include additional features or components.

[0030] As used herein, common organic abbreviations are defined as follows: ℃ Celsius temperature dATP deoxyadenosine triphosphate dCTP deoxycytidine triphosphate dGTP deoxyguanosine triphosphate dTTP deoxythymidine triphosphate ddNTP Dideoxynucleotide Triphosphate ffA fully functionalized A nucleotide ffC fully functionalized C nucleotide ffG fully functionalized G nucleotide ffN fully functionalized nucleotides ffT fully functionalized T nucleotide h time RT room temperature SBS sequencing by synthesis

[0031] As used herein, the term "array" refers to a collection of different probe molecules bound to one or more substrates such that the different probe molecules can be distinguished from one another according to their relative positions. An array can include different probe molecules each located at a different addressable location on the substrate. Alternatively, or additionally, an array can include separate substrates, each bearing a different probe molecule, and the different probe molecules can be identified according to the position of the substrate on the surface to which the substrate is bound or according to the position of the substrate in a liquid. Exemplary arrays in which separate substrates are located on a surface include, but are not limited to, those containing beads in wells, as described, for example, in U.S. Pat. No. 6,355,431 (B1), U.S. Patent Application Publication No. 2002 / 0102578, and PCT Publication No. WO 00 / 63437. An exemplary format that can be used in the present invention to distinguish beads in a liquid array using a microfluidic device, such as a fluorescent activated cell sorter (FACS), is described, for example, in U.S. Pat. No. 6,524,793. Further examples of arrays that can be used in the present invention include U.S. Patent 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, and 6,288,230. 97,006, 6,291,193, 6,346,413, 6,416,949, 6,482,591, 6,514,751 and 6,610,482, and those described in WO 93 / 17126, WO 95 / 11995, WO 95 / 35505, EP 742287, and EP 799897.

[0032] As used herein, the terms "covalently attached" or "covalently bonded" refer to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently bonded polymer coating refers to a polymer coating that forms a chemical bond with the functionalized surface of a substrate, as compared to attaching to the surface by other means, such as adhesion or electrostatic interactions. It will be understood that a polymer covalently bonded to a surface can be attached by other means in addition to covalent bonds.

[0033] As used herein, the term "halogen" or "halo" means any one of Group 7 of the Periodic Table of the Elements, for example, fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred.

[0034] As used herein, "C" is a generalized expression in which "a" and "b" are integers. a ~C b" refers to the number of carbon atoms in an alkyl, alkenyl, or alkynyl group, or the number of ring atoms in a cycloalkyl or aryl group. That is, the alkyl, alkenyl, alkynyl, cycloalkyl ring, and aryl ring can contain "a" to "b" carbon atoms, inclusive. For example, a "C1-C4 alkyl" group refers to all alkyl groups having 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-, and a C3-C4 cycloalkyl group refers 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. When "a" and "b" are not specified with respect to an alkyl, alkenyl, alkynyl, cycloalkyl, or aryl group, the broadest range described by those definitions is to 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 the two numbers. For example, C1-C6 alkyl includes C1, C2, C3, C4, C5, and C6 alkyl, C2-C6 alkyl, C1-C3 alkyl, etc. Similarly, C2-C6 alkenyl includes C2, C3, C4, C5, and C6 alkenyl, C2-C5 alkenyl, C3-C4 alkenyl, etc., and C2-C6 alkynyl includes C2, C3, C4, C5, and C6 alkynyl, C2-C5 alkynyl, C3-C4 alkynyl, etc. C3-C8 cycloalkyl includes hydrocarbon rings containing 3, 4, 5, 6, 7, and 8 carbon atoms, respectively, or a range defined by any two numbers, such as C3-C7 cycloalkyl or C5-C6 cycloalkyl.

[0035] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group may have 1 to 20 carbon atoms (wherever provided herein, a numerical range such as "1 to 20" refers to each integer within the given range. For example, "1 to 20 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms, although this definition also covers the occurrence of the term "alkyl" without a specified numerical range). The alkyl group may also be a medium-sized alkyl having 1 to 9 carbon atoms. The alkyl group may also be a lower alkyl having 1 to 6 carbon atoms. By way of example only, "C 1~6 "Alkyl" or "C1-C6 alkyl" indicates that there are 1 to 6 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.

[0036] As used herein, "alkoxy" refers to a group in which R is alkyl as defined above, such as "C 1~9 "C1-C9 alkoxy" refers to groups of the formula -OR, such as "alkoxy" or "C1-C9 alkoxy," including, but not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy.

[0037] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. Alkenyl groups can have from 2 to 20 carbon atoms, although this definition also covers the occurrence of the term "alkenyl" where no numerical range is specified. Alkenyl groups can also be medium-sized alkenyls having from 2 to 9 carbon atoms. Alkenyl groups can also be lower alkenyls having from 2 to 6 carbon atoms. By way of example only, "C 2~ C6 alkenyl" or "C 2~6 "Alkenyl" indicates that there are 2 to 6 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 not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

[0038] 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, although this definition also covers occurrences of the term "alkynyl" where no numerical range is specified. Alkynyl groups can also be medium-sized alkynyls having from 2 to 9 carbon atoms. Alkynyl groups can also be lower alkynyls having from 2 to 6 carbon atoms. By way of example only, "C 2~6 alkynyl" or "C 2~"C6 alkenyl" indicates that there are 2 to 6 carbon atoms in the alkynyl chain, i.e., 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 not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0039] The term "aromatic" refers to a ring or ring system having a conjugated π-electron system and including both carbocyclic aromatic (e.g., phenyl) and heterocyclic aromatic (e.g., pyridine) groups. The term includes monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of atoms) groups, so long as the entire ring system is aromatic.

[0040] 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, all rings in the system are aromatic. Aryl groups can have 6 to 18 carbon atoms, although this definition also covers occurrences of the term "aryl" where no numerical range is specified. In some embodiments, aryl groups have 6 to 10 carbon atoms. An aryl group is defined as "C 6~ C 10 aryl," "C6 or C 10 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.

[0041] "Aralkyl" or "arylalkyl" refers to "C 7~14 An aryl group bonded as a substituent via an alkylene group, such as "aralkyl," includes, but is not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group may be joined to a lower alkylene group (i.e., C 1~6 alkylene group).

[0042] As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent atoms) containing one or more heteroatoms, i.e., elements other than carbon, in the ring backbone, including, but not limited to, nitrogen, oxygen, and sulfur. When heteroaryl is a ring system, all rings in the system are aromatic. Heteroaryl groups can have 5 to 18 ring members (i.e., the number of atoms comprising the ring backbone, including carbon atoms and heteroatoms), although this definition also covers occurrences of the term "heteroaryl" where no numerical range is specified. In some embodiments, heteroaryl groups have 5 to 10 ring members or 5 to 7 ring members. Heteroaryl groups can be designated as "5- to 7-membered heteroaryl," "5- to 10-membered heteroaryl," or similar designations. Examples of heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.

[0043] A "heteroaralkyl" or "heteroarylalkyl" is a heteroaryl group bonded as a substituent via an alkylene group. Examples include, but are not limited to, 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. In some cases, an alkylene group may be joined to a lower alkylene group (i.e., C 1~6 alkylene group).

[0044] As used herein, "carbocyclyl" refers to a non-aromatic ring or ring system containing only carbon atoms in the ring system backbone. When a carbocyclyl is a ring system, two or more rings can be joined together in a fused, bridged, or spiro-connected manner. A carbocyclyl can 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 can have 3 to 20 carbon atoms, although this definition also covers the occurrence of the term "carbocyclyl" where no numerical range is specified. A carbocyclyl group can also be a medium-sized carbocyclyl having 3 to 10 carbon atoms. A carbocyclyl group can also be a carbocyclyl having 3 to 6 carbon atoms. A carbocyclyl group can also be a "C 3~6 Carbocyclyl, C 3~ C6 carbocyclyl," or similar designations. Examples of carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

[0045] As used herein, "cycloalkyl" means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0046] As used herein, "heterocyclyl" refers to a non-aromatic ring or ring system containing at least one heteroatom in the ring backbone. Heterocyclyls may be joined together in a fused, bridged, or spiro-linked fashion. Heterocyclyls may have any degree of saturation, provided that at least one ring in the ring system is not aromatic. The heteroatom may be present in either the non-aromatic or aromatic ring in the ring system. Heterocyclyl groups may have 3 to 20 ring members (i.e., the number of atoms comprising the ring backbone, including carbon atoms and heteroatoms), although this definition also covers the occurrence of the term "heterocyclyl" where no numerical range is specified. Heterocyclyl groups may also be medium-sized heterocyclyls having 3 to 10 ring members. Heterocyclyl groups may also be heterocyclyls having 3 to 6 ring members. Heterocyclyl groups may also be designated as "3- to 6-membered heterocyclyl" or similar notations. In preferred 6-membered monocyclic heterocyclyls, the heteroatoms are selected from 1 to 3 of O, N, or S. In preferred 5-membered monocyclic heterocyclyls, the heteroatoms are selected from 1 or 2 heteroatoms selected from O, N, or S.Examples of heterocyclyl rings include azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiapanyl, piperidinyl, piperazinyl, dioxapiperazinyl, pyrrolidinyl, pyrrolidionyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathinyl, 1,4-oxathinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl, hexahydro-1,3, These include, but are not limited to, 5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.

[0047] As used herein, "alkoxyalkyl" or "(alkoxy)alkyl" refers to a C2-C8 alkoxyalkyl, or (C1-C6 alkoxy)C1-C6 alkyl, such as -(CH2) 1~3 Refers to an alkoxy group connected via an alkylene group such as -OCH3.

[0048] An "O-carboxy" group refers to a "-OC(=O)R" group, where R is hydrogen, C, 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~7 Carbocyclyl, C 6~10 It is selected from aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl.

[0049] A "C-carboxy" group refers to a "-C(=O)OR" group, where R is hydrogen and C 1~6 Alkyl and C 2~6 Alkenyl and C 2~6 Alkynyl and C 3~7 Carbocyclyl and C 6~10 It is selected from the group consisting of aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl. Non-limiting examples include carboxyl (i.e., -C(=O)OH).

[0050] A "sulfonyl" group refers to a "-SO2R" group, where R is hydrogen, C, 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~7 Carbocyclyl, C 6~10 It is selected from aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl.

[0051] A "sulfino" group refers to a "-S(=O)OH" group.

[0052] A "sulfo" group refers to a "-S(=O)2OH" or "-SO3H" group.

[0053] The "sulfonate" group is "-SO3 - " refers to the group.

[0054] The "sulfate" group is "-SO4 - " refers to the group.

[0055] The "S-sulfonamide" group is "-SONR A R B " group, where R A and R B are each independently hydrogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~7 Carbocyclyl, C 6~10It is selected from aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl.

[0056] An "N-sulfonamido" group is defined as "-N(R A )SO2R B " group, where R A and R b are each independently hydrogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~7 Carbocyclyl, C 6~10 It is selected from aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl.

[0057] A "C-amido" group is defined as "-C(=O)NR A R B " group, where R A and R B are each independently hydrogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~7 Carbocyclyl, C 6~10 It is selected from aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl.

[0058] An "N-amido" group is defined as "-N(R A )C(=O)R B " group, where R A and R B are each independently hydrogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~7 Carbocyclyl, C 6~10 It is selected from aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl.

[0059] The "amino" group is "-NR A R B " group, where R Aand R B are each independently hydrogen, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~7 Carbocyclyl, C 6~10 It is selected from aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl. Non-limiting examples include free amino (i.e., -NH2).

[0060] An "aminoalkyl" group refers to an amino group linked via an alkylene group.

[0061] An "alkoxyalkyl" group refers to an alkoxy group connected via an alkylene group, such as, for example, "C2-C8 alkoxyalkyl."

[0062] When a group is described as "optionally substituted," it can be either unsubstituted or substituted. Similarly, when a group is described as "substituted," the substituents can be selected from one or more of the indicated substituents. As used herein, a substituent is derived from an unsubstituted parent group in which one or more hydrogen atoms have been replaced for another atom or group. Unless otherwise stated, when a group is considered to be "substituted," it means that the group is selected from C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C3-C7 carbocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), C3-C7-carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), C3-C7-carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkoxy), C3-C7-carbocyc ... haloalkyl, and C1-C6 haloalkoxy), 3-10 membered heterocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3-10 membered heterocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl (optionally aryl(C1-C6)alkyl (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); 5-10 membered heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy); substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), halo, -CN, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy(C1-C6)alkyl (i.e., ether), aryloxy, sulfhydryl (mercapto), halo(C1-C6)alkyl (e.g., -CF3),"Optionally substituted" means substituted with one or more substituents independently selected from halo(C1-C6)alkoxy (e.g., -OCF3), C1-C6 alkylthio, arylthio, amino, amino(C1-C6)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl, cyanate, isocyanate, thiocyanate, isothiocyanate, sulfinyl, sulfonyl, -SO3H, sulfonate, sulfate, sulfino, -OSO2C1-C4 alkyl, and oxo (=O). Whenever a group is described as "optionally substituted," that group can be substituted with the above substituents. In some embodiments, if the alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl group is substituted, each independently is selected from halo, —CN, —SO 3 , - , -OSO3 - , -SO3H, -SR A , -OR A , -NR B R C , oxo, -CONR B R C , -SO2NR B R C , -COOH, and -COOR B wherein R is substituted with one or more substituents selected from the group consisting of A , R B and R C are each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.

[0063] As will be appreciated by those skilled in the art, the compounds described herein may be in ionized form, e.g., -CO2 - , -SO3 - or -O-SO3 - The compound may contain positively or negatively charged substituents, e.g., SO3 -In other embodiments, the compound may contain a negatively or positively charged counterion such that the compound is overall neutral. In other embodiments, the compound may exist in a salt form, with the counterion being provided by a conjugate acid or base.

[0064] It is understood that certain radical nomenclature can include either a monoradical or a diradical, depending on the context. For example, if a substituent requires two points of attachment to the rest of the molecule, the substituent is understood to be a diradical. For example, a substituent specified as alkyl, which requires two points of attachment, includes diradicals such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc. Other radical nomenclature clearly indicates that the radical is a diradical, such as "alkylene" or "alkenylene."

[0065] When two "adjacent" R groups are said to form a ring together with the atoms to which they are attached, it is meant that the collective unit of atoms, the intervening bond, and the two R groups is the recited ring. For example, if the following substructure is present:

[0066] [ka] R 1 and R 2 is defined as selected from the group consisting of hydrogen and alkyl, or R 1 and R 2 together with the atom to which they are attached form an aryl or carbocyclyl, which is represented by R 1 and R 2 can be selected from hydrogen or alkyl, or alternatively, the substructure means having the structure:

[0067] [ka] wherein A is an aryl ring or a carbocyclyl containing the indicated double bond.

[0068] Where a substituent is depicted as a diradical (i.e., having two points of attachment to the rest of the molecule), it is understood that the substituent can be attached in any directional configuration unless otherwise indicated. Thus, for example, -AE- or

[0069] [ka] Substituents depicted as: represent substituents oriented such that A is attached at the left-most point of attachment of the molecule, as well as when A is attached at the right-most point of attachment of the molecule. In addition, groups or substituents may be

[0070] [ka] where L is defined as an optionally present linker moiety, and when L is not present (or absent), such a group or substituent is

[0071] [ka] is equivalent to

[0072] The compounds described herein can be represented in several mesomeric forms. When a single structure is depicted, any of the related mesomeric forms is intended. The coumarin compounds described herein are represented by a single structure, but can also be represented in any of the related mesomeric forms. Exemplary mesomeric structures are shown below for formula (I) and formula (Ia), respectively.

[0073] [ka]

[0074] In each instance where a single mesomeric form of a compound described herein is shown, the alternative mesomeric form is contemplated as well.

[0075] As used herein, a "nucleotide" comprises a nitrogen-containing 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 nitrogen-containing heterocyclic base can be a purine, deazapurine, or pyrimidine base. Purine bases include adenine (A) and guanine (G), as well as modified derivatives or analogs thereof, such as 7-deazaadenine or 7-deazaguanine. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), as well as modified derivatives or analogs thereof. The C-1 atom of deoxyribose is linked to the N-1 atom of a pyrimidine or the N-9 atom of a purine.

[0076] As used herein, a "nucleoside" is structurally similar to a nucleotide but lacks a phosphate moiety. An example of a nucleoside analog is one in which a label is linked to the base and there is no phosphate group attached to the sugar molecule. The term "nucleoside" is used herein in its ordinary sense as understood by those skilled in the art. Examples include, but are not limited to, ribonucleosides containing a ribose moiety and deoxyribonucleosides containing a deoxyribose moiety. Modified pentose moieties are those in which an oxygen atom is replaced with a carbon and / or a carbon is replaced with a sulfur or oxygen atom. A "nucleoside" is a monomer that may have a substituted base and / or sugar moiety. Furthermore, nucleosides can be incorporated into larger DNA and / or RNA polymers and oligomers.

[0077] The term "purine base" is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers. 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).

[0078] As used herein, when an oligonucleotide or polynucleotide is described as "comprising" a nucleoside or nucleotide described herein, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. Similarly, when a nucleoside or nucleotide is described as being part of an oligonucleotide or polynucleotide, such as being "incorporated" into an oligonucleotide or polynucleotide, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. In some such embodiments, the covalent bond is formed between the 3' hydroxy group of the oligonucleotide or polynucleotide and the 5' phosphate group of a nucleotide described herein, or as a phosphodiester bond between the 3' carbon atom of the oligonucleotide or polynucleotide and the 5' carbon atom of the nucleotide.

[0079] As used herein, the term "cleavable linker" does not imply that the entire linker must be removed. The cleavage site may be located on the linker at a position that ensures that a portion of the linker remains attached to the detectable dye and / or nucleoside or nucleotide moiety after cleavage.

[0080] As used herein, "derivative" or "analog" refers to a synthetic nucleotide or nucleoside derivative 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 contain modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl-phosphonate, phosphoranilidate, and phosphoramidate linkages. As used herein, "derivative," "analog," and "modified" can be used interchangeably and are encompassed by the terms "nucleotide" and "nucleoside," as defined herein.

[0081] As used herein, the term "phosphate" is used in its ordinary sense as understood by one of ordinary skill in the art and includes its protonated form (e.g.,

[0082] [ka] As used herein, the terms "monophosphate," "diphosphate," and "triphosphate" are used in their ordinary sense as understood by those of skill in the art and include protonated forms.

[0083] As used herein, the term "phasing" refers to a phenomenon in SBS caused by incomplete removal of 3' terminators and fluorophores and / or incomplete incorporation of a portion of the DNA strand within a cluster by the polymerase in a given sequencing cycle. Prephasing is caused by the incorporation of a nucleotide without an effective 3' terminator, and the incorporation event advances one cycle due to incomplete termination. Phasing and prephasing cause the measured signal intensity for a particular cycle to consist of signal from the current cycle and noise from the previous and next cycles. As the number of cycles increases, the proportion of sequences per cluster affected by phasing and prephasing increases, preventing the identification of the correct base. Prephasing can be caused by the presence of traces of unprotected or unblocked 3'-OH nucleotides during sequencing by synthesis (SBS). Unprotected 3'-OH nucleotides can be generated during the manufacturing process or, in some cases, during storage and reagent handling processes. Therefore, the discovery of nucleotide analogs that reduce the incidence of prephasing is surprising and offers significant advantages over existing nucleotide analogs in SBS applications. For example, the provided nucleotide analogs may result in faster SBS cycle times, lower phasing and prephasing values, and longer sequencing read lengths.

[0084] Fluorescent dye of formula (I) Some aspects of the present disclosure relate to coumarin dyes of formula (I), and salts and mesomeric forms thereof:

[0085] [ka] In the formula, R 1 teeth,

[0086] [ka] and R 1 is substituted with one or more C1-C6 alkyl groups; Each R 2 , R 5 and R 7 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, sulfino, sulfonate, S-sulfonamido, or N-sulfonamido; R 3 and R 4 each is independently H, C1-C6 alkyl, or substituted C1-C6 alkyl; Alternatively, R 2 and R 3 together with the atoms to which they are attached 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; Alternatively, R 4 and R 5 together with the atoms to which they are attached 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; R 6 is H, C1-C6 alkyl, substituted C1-C6 alkyl, or optionally substituted C6-C 10 is aryl, R a , R b and R c Each of is independently C1-C6 alkyl or substituted C1-C6 alkyl.

[0087] In some embodiments of the compounds of Formula (I), R 3 and R 4 At least one of R 3 and R4 and R are H. In other embodiments, R 3 is H and R 4 is C1-C6 alkyl or substituted C1-C6 alkyl. In other embodiments, R 3 and R 4 Each of the is independently a C1-C6 alkyl or a substituted C1-C6 alkyl. Substituted C1-C6 alkyl includes carboxyl, carboxylate (-C(O) - ), sulfo (-SO3H), sulfonate (-SO3 - ), sulfate (-O-SO3 - ), optionally substituted amino (such as a Boc-protected amino group), —C(O)OR 12 or -C(O)NR 13 R 14 and the like, substituted with one or more substituents such as, but not limited to, methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl, n-hexyl, and the like, wherein R 12 optionally substituted C1-C6 alkyl, optionally substituted C6-C 10 aryl, optionally substituted 5-10 membered heteroaryl, or optionally substituted C3-C7 cycloalkyl; R 13 and R 14 each independently selected from H, optionally substituted C1-C6 alkyl, optionally substituted C6-C 10 aryl, optionally substituted 5-10 membered heteroaryl, or optionally substituted C3-C7 cycloalkyl. 3 and R 4 Each of R is ethyl. 3 is H and R 4 is n-propyl substituted with carboxyl.

[0088] Some embodiments of the compounds of formula (I) are also represented by formula (Ia), wherein R 4 and R 5 together with the atom to which they are attached form an optionally substituted 6-membered heterocyclyl of the structure

[0089] [ka] forming its salts or mesomeric forms, Each R 8 , R 9 , R 10 and R 11 are independently 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, sulfino, sulfonate, S-sulfonamido, or N-sulfonamido; Solid and dashed lines

[0090] [ka] is selected from the group consisting of a single bond and a double bond, with the proviso that

[0091] [ka] If is a double bond, R 11 is provided that it does not exist.

[0092] In some embodiments of compounds of Formula (I) or (Ia), R 1 is substituted with one C1-C6 alkyl (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. In other embodiments, R 1 is independently substituted with three C1-C6 alkyl. In some further embodiments, R 1 teeth,

[0093] [ka] In other embodiments, R 1 teeth,

[0094] [ka] In some such embodiments, each R a and R b 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 further embodiments, each R a and R b are independently substituted C1-C6 alkyl (e.g., carboxyl, carboxylate (-C(O)O - ), sulfo (-SO3H), sulfonate (-SO3 - ), sulfate (-O-SO3 - ), optionally substituted amino (such as a Boc-protected amino group), —C(O)OR 12 or -C(O)NR 13 R 14 methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl, or n-hexyl, substituted with one or more substituents such as 12 is an optionally substituted C1-C6 alkyl, an optionally substituted C6-C 10 aryl, optionally substituted 5-10 membered heteroaryl, or optionally substituted C3-C7 cycloalkyl; R 13 and R 14 each independently represents H, optionally substituted C1-C6 alkyl, optionally substituted C6-C 10 aryl, optionally substituted 5-10 membered heteroaryl, or optionally substituted C3-C7 cycloalkyl. For example, R a and R bEach of is independently n-propyl, n-butyl, or n-pentyl substituted with carboxyl, carboxylate, sulfo, or sulfonate. In some embodiments, the substitution is 2、 C 3、 C 5、 C 6、 Or at the end of a C6 alkyl.

[0095] In some embodiments of the compound of Formula (Ia), the solid and dashed lines

[0096] [ka] 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 is methyl. In some other embodiments, the solid and dashed lines

[0097] [ka] The bond represented by is a single bond. In some such embodiments, R 10 is H and R 11 is C1-C6 alkyl. In another embodiment, R 10 and R 11 is H. In some embodiments of compounds of Formula (Ia), R 8 and R 9 Each of is H. In other embodiments, R 8 and R 9 At least one of R is C1-C6 alkyl. 8 and R 9 Each of R is a C1-C6 alkyl. 8 and R 9 Each of R is methyl. 3 is H. In other embodiments, R 3is C1-C6 alkyl (e.g., methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl, or n-hexyl, etc.). 3 is a substituted C1-C6 alkyl (e.g., carboxyl, carboxylate (-C(O) - ), sulfo (-SO3H), sulfonate (-SO3 - ), sulfate (-O-SO3 - ), optionally substituted amino, -C(O)OR 12 or -C(O)NR 13 R 14 methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl, or n-hexyl substituted with one or more substituents such as 12 is an optionally substituted C1-C6 alkyl, an optionally substituted C6-C 10 aryl, optionally substituted 5-10 membered heteroaryl, or optionally substituted C3-C7 cycloalkyl; R 13 and R 14 each independently represents H, optionally substituted C1-C6 alkyl, optionally substituted C6-C 10 aryl, optionally substituted 5-10 membered heteroaryl, or optionally substituted C3-C7 cycloalkyl. For example, R 3 is ethyl, n-propyl, n-butyl, or n-pentyl, each of which is substituted with carboxyl, carboxylate, sulfo, or sulfonate. 3 teeth,

[0098] -C(O)NR 13 R 14 ethyl, n-propyl, n-butyl, or n-pentyl substituted with 13 and R 14 are independently carboxyl, carboxylate, -C(O)OR 12 , C1-C6 alkyl substituted with sulfo or sulfonate.

[0099] In some embodiments of compounds of Formula (I) or (Ia), R 2 is H. In other embodiments, R 2 and R 3 are joined together with the atoms to which they are attached to form an optionally substituted 5-, 6-, or 7-membered heterocyclyl. In some such embodiments, R 2 and R 3 are joined together with the atoms to which they are attached to form a 6-membered heterocyclyl substituted with one or more C1-C6 alkyls.

[0100] In some embodiments of compounds of Formula (I) or (Ia), R 6 is H or optionally substituted phenyl.

[0101] In some embodiments of compounds of Formula (I) or (Ia), R 7 is H.

[0102] Additional embodiments of compounds of Formula (I) or (Ia) include:

[0103] [ka] and their salts and mesomeric forms. Non-limiting examples of the corresponding C1-C6 alkyl carboxylic acid esters (such as methyl esters, ethyl esters, isopropyl esters, and t-butyl esters formed from the carboxylic acid group of the compound), the corresponding imine analogs (where the coumarin core -C(=O) moiety is instead -C(=NH)), and their salts and mesomeric forms.

[0104] Cyclooctatetraene (COT) photoprotective moiety In some embodiments, the fluorescent compounds described herein (Formula (I) or (Ia)) can be further modified to introduce a photoprotective moiety covalently attached thereto, for example, a cyclooctatetraene moiety having the structure

[0105] [ka] wherein R 1A and R 2A each independently represents H, hydroxyl, halogen, azide, 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 C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, optionally substituted C 6~10 Aryl, optionally substituted C 7~14 Aralkyl, optionally substituted C 3~7 carbocyclyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 3- to 10-membered heterocyclyl; X 1 and Y 1 each independently represents a bond, -O-, -S-, or -NR 3A -, -C(=O)-, -C(=O)-O-, -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 in which at least one carbon atom is replaced with O, S, or N; Z is absent or optionally substituted C 2~6 Alkenylene or optionally substituted C 2~6 is alkynylene, R 3A and R 4A each independently represents H, optionally substituted C 1~6 Alkyl or optionally substituted C 6~10 is aryl, R 5A is an arbitrarily substituted C 1~6 Alkyl, optionally substituted C 6~10 Aryl, optionally substituted C 7~14 Aralkyl, optionally substituted C 3~7 carbocyclyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 3- to 10-membered heterocyclyl; R 6A and R 7A each independently represents H, optionally substituted C 1~6 Alkyl, optionally substituted C 6~10 Aryl, optionally substituted C 7~14 Aralkyl, optionally substituted C 3~7 carbocyclyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 3- to 10-membered heterocyclyl;

[0106] [ka] R in 1A and R 2A The carbon atom to which R is attached is optionally replaced with O, S, or N, provided that when the carbon atom is replaced with O or S, R 1A and R 2A provided that both are absent, and if the carbon atom is replaced by N, then R 2A is absent, and m is an integer from 0 to 10. In some embodiments, X and Y are not both bonds.

[0107] In some further embodiments, the cyclooctatetraene moiety has the structure

[0108] [ka] In some such embodiments, R 1A and R 2A In some further embodiments, at least one of R 1A and R2A and R are hydrogen. 1A is H and R 2A is an optionally substituted amino, carboxyl, or —C(O)NR 6A R 7A In some embodiments, m is 1, 2, 3, 4, 5, or 6, and R 1A and R 2A each independently represents hydrogen, an optionally substituted amino, a carboxyl, or —C(O)NR 6A R 7A 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,

[0109] [ka] R in 1A and R 2A is attached to is replaced with O, S, or N. In some such embodiments,

[0110] [ka] One carbon atom in 1A and R 2A In some other embodiments, both of

[0111] [ka] When one carbon atom in is replaced by a nitrogen atom, R bonded to the replaced carbon atom 2A is absent, and R bonded to the replaced carbon atom1A is hydrogen or C 1~6 It is alkyl. R 1A and R 2A In any embodiment of the present invention, R 1A or R 2A is -C(O)NR 6A R 7A and R 6A and R 7A are independently H, C 1~6 Alkyl or substituted C 1~6 Alkyl (e.g., -CO2H, -NH2, -SO3H, or -SO3 - C replaced with 1~6 alkyl).

[0112] In some further embodiments, the fluorescent dyes described herein comprise a cyclooctatetraene moiety of the following structure:

[0113] [ka] The COT moieties described herein are obtained by reaction between a functional group (e.g., a carboxyl group) of a fluorescent dye described herein and an amino group of a COT derivative to form an amide bond (the carbonyl group of the amide bond is not shown).

[0114] Labeled nucleotides or oligonucleotides According to one aspect of the present disclosure, the dye compounds described herein are suitable for attachment to a substrate moiety, and in particular, include a linker group that enables attachment to the substrate moiety. The substrate moiety can be virtually any molecule or substance to which the dyes of the present disclosure can be conjugated, including, but not limited to, nucleosides, nucleotides, polynucleotides, carbohydrates, ligands, particles, solid surfaces, organic and inorganic polymers, chromosomes, nuclei, living cells, and combinations or aggregates thereof. The dyes can be conjugated via any linker by various means, including hydrophobic attraction, ionic attraction, and covalent bonding. In some embodiments, the dyes are conjugated to the substrate by a covalent bond. More specifically, the covalent bond is via a linker group. In some cases, such labeled nucleotides are also referred to as "modified nucleotides."

[0115] Some aspects of the present disclosure relate to nucleotides or oligonucleotides labeled with the dyes of formula (I) or (Ia) described herein, or their salts in mesomeric form, or derivatives thereof containing the photoprotective moiety COT described herein. The labeled nucleotide or oligonucleotide may be attached to the dye compounds disclosed herein via a carboxyl group (—COH) or an alkyl-carboxyl group to form an amide or alkyl-amide. In some further embodiments, the carboxyl group may be in an activated form, e.g., an amide or ester form, which can be used for attachment to amino or hydroxyl groups of nucleotides or oligonucleotides. As used herein, the term “activated ester” refers to a carboxyl group derivative that can react with, for example, an amino-containing compound under mild conditions. Non-limiting examples of activated esters include, but are not limited to, p-nitrophenyl, pentafluorophenyl, and succinimide esters.

[0116] For example, the dye compound of formula (I) can be represented by the formula (I) R 1 (For example, R a , R b or R c) or R 3 / R 4 In some such embodiments, R of formula (I) may be attached to the nucleotide or oligonucleotide via one of 1 is -CO2H or -(CH2) 1~6 -CO2H and the bond is R 1 and the amino functional group of the nucleotide or nucleotide linker. As an example, the labeled nucleotide or oligonucleotide may include a dye moiety of the following structure:

[0117] [ka] In other embodiments, R of formula (I) 3 or R 4 is -CO2H or -(CH2) 1~6 For example, a labeled nucleotide or oligonucleotide may contain the following dye moiety:

[0118] [ka]

[0119] Similarly, the dye compound of formula (Ia) can be prepared by reacting R 1 (For example, R a , R b or R c ) or R 3 Via R 1 or R 3 The labeled nucleotide or oligonucleotide may be attached to the nucleotide or oligonucleotide by forming an amide moiety between the carboxyl functional group of the nucleotide or nucleotide linker and the amino functional group of the nucleotide or nucleotide linker. For example, the labeled nucleotide or oligonucleotide may be attached to the nucleotide or oligonucleotide by forming an amide moiety between the carboxyl functional group of the nucleotide or nucleotide linker and the amino functional group of the nucleotide or nucleotide linker.

[0120] [ka] In other embodiments, R of formula (I) or (Ia) may include b or R c is -CO2H or -(CH2) 1~6 It contains -CO2H, and the bond forms an amide using the -CO2H group.

[0121] In some embodiments, the dye compound may be covalently bound to the oligonucleotide or nucleotide via the nucleotide base. In some such embodiments, the labeled nucleotide or oligonucleotide may have a dye attached to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base, optionally via a linker moiety. For example, the nucleobase may be 7-deazaadenine, and the dye is attached to the 7-deazaadenine at the C7 position, optionally via a linker. The nucleobase may be 7-deazaguanine, and the dye is attached to the 7-deazaguanine at the C7 position, optionally via a linker. The nucleobase may be cytosine, and the dye is attached to the cytosine at the C5 position, optionally via a linker. As another example, the nucleobase may be thymine or uracil, and the dye is attached to the thymine or uracil at the C5 position, optionally via a linker.

[0122] 3'-OH blocking group The labeled nucleotide or oligonucleotide may also have a blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide. The blocking group may be attached to any position on the ribose or deoxyribose sugar. In certain embodiments, the blocking group is at the 3'OH position of the ribose or deoxyribose sugar of the nucleotide. Various 3'OH blocking groups are disclosed in WO 2004 / 018497 and WO 2014 / 139596, which are incorporated herein by reference. For example, the blocking group can be azidomethyl (-CHN) or substituted azidomethyl (e.g., -CH(CHF)N or CH(CHF)N), or allyl attached to the 3' oxygen atom of the ribose or deoxyribose sugar. In some embodiments, the 3' blocking group is azidomethyl, forming 3'-OCHN with the 3' carbon of the ribose or deoxyribose.

[0123] In some other embodiments, the 3' blocking group and the 3' oxygen atom are covalently attached to the 3' carbon of the ribose or deoxyribose.

[0124] [ka] to form an acetal group of the formula In the formula, each R 1a and R 1b are independently H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, cyano, halogen, optionally substituted phenyl, or optionally substituted aralkyl; Each R 2a and R 2b are independently H, C1-C6 alkyl, C1-C6 haloalkyl, cyano, or halogen; Alternatively, R 1a and R 2a together with the atom to which they are attached form an optionally substituted 5- to 8-membered heterocyclyl group, R Fis 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, Each R 3a are independently H, C1-C6 alkyl, or optionally substituted C6-C 10 It is aryl.

[0125] Additional 3'-OH blocking groups are disclosed in U.S. Patent Application Publication No. 2020 / 0216891(A1), which is incorporated by reference in its entirety. Non-limiting examples of acetal blocking groups include:

[0126] [ka] (AOM),

[0127] [ka] and are covalently attached to the 3' carbon of ribose or deoxyribose, respectively.

[0128] Deprotection of the 3'-OH blocking group In some embodiments, the azidomethyl 3' hydroxyl 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(hydroxylpropyl)phosphine (THP or THPP). The 3'-acetal blocking groups described herein can be removed or cleaved under a variety of chemical conditions. Acetal blocking groups containing vinyl or alkenyl moieties

[0129] [ka] In the case of (III), non-limiting cleavage conditions include Pd(II) complexes such as Pd(OAc)2 or allylPd(II) chloride dimer in the presence of a phosphine ligand, such as tris(hydroxymethyl)phosphine (THMP) or tris(hydroxypropyl)phosphine (THP or THPP). In the case of those blocking groups containing alkynyl groups (e.g., ethynyl), they can also be removed with Pd(II) complexes (e.g., Pd(OAc)2 or allylPd(II) chloride dimer) in the presence of a phosphine ligand (e.g., THP or THMP).

[0130] Palladium Cleavage Reagent In some embodiments, the 3' hydroxyl blocking groups described herein can be cleaved by a palladium catalyst. In some such embodiments, the Pd catalyst is water soluble. In some such embodiments, the Pd catalyst is a Pd(0) complex (e.g., tris(3,3',3''-phosphinidinetris(benzenesulfonato)palladium(0) nonasodium salt nonahydrate). In some cases, Pd(0) can be generated in situ from the reduction of a Pd(II) complex with a reagent such as an alkene, alcohol, amine, phosphine, or metal hydride. Suitable palladium sources include NaPdCl, Pd(CHCN)Cl, and the like. 2、Examples of suitable palladium sources include (PdCl(C3H5))2, [Pd(C3H5)(THP)]Cl, [Pd(C3H5)(THP)2]Cl, Pd(OAc)2, Pd(Ph3)4, Pd(dba)2, Pd(Acac)2, PdCl2(COD), and Pd(TFA)2. In one such embodiment, the Pd(0) complex is generated in situ from Na2PdCl4. 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 a Pd(II) complex with a phosphine. Suitable phosphines include water-soluble phosphines such as tris(hydroxypropyl)phosphine (THP), tris(hydroxymethyl)phosphine (THMP), 1,3,5-triaza-7-phosphaadamantane (PTA), bis(p-sulfonatophenyl)phenylphosphine dihydrate potassium salt, tris(carboxyethyl)phosphine (TCEP), and triphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt.

[0131] In some embodiments, Pd(0) is prepared in situ by mixing a Pd(II) complex [(PdCl(C3H5))2] with THP. The molar ratio of 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 further embodiments, one or more reducing agents, such as ascorbic acid or a salt thereof (e.g., sodium ascorbate), may be added. In some embodiments, the cleavage mixture may contain additional buffering reagents, such as primary amines, secondary amines, tertiary amines, carbonates, phosphates, or borates, or combinations thereof. In some further embodiments, the buffering reagent comprises 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 buffering reagent is DEEA. In another embodiment, the buffering reagent contains one or more inorganic salts, such as carbonate, phosphate, or borate, or a combination thereof. In one embodiment, the inorganic salt is a sodium salt.

[0132] Linker The dye compounds disclosed herein may include a reactive linker group at one of the substitution positions for covalently attaching the compound to a substrate or another molecule. A reactive linking group is a moiety that can form a bond (e.g., covalent or non-covalent), particularly a covalent bond. In certain embodiments, the linker may be a cleavable linker. The use of the term "cleavable linker" does not imply that the entire linker must be removed. The cleavage site may be located on the linker at a position that ensures that a portion of the linker remains attached to the dye and / or substrate moiety after cleavage. The cleavable linker may be, by way of non-limiting example, an electrophilically cleavable linker, a nucleophilically cleavable linker, a photocleavable linker, a linker cleavable under reducing conditions (e.g., a disulfide- or azide-containing linker), a linker cleavable under oxidative conditions, a linker cleavable by use of a safety catch linker, or a linker that is cleavable by a removal mechanism. By using a cleavable linker to attach the dye compound to the substrate moiety, the label can be removed after detection, if desired, avoiding any interfering signals in downstream processes.

[0133] Useful linker groups can be found in PCT Publication No. WO 2004 / 018493 (incorporated herein by reference), and examples include linkers that can be cleaved using a water-soluble phosphine or a water-soluble transition metal catalyst formed from a transition metal and an at least partially water-soluble ligand. In aqueous solution, the latter forms an at least partially water-soluble transition metal complex. Such cleavable linkers can be used to connect the base of a nucleotide to a label, such as the dyes described herein.

[0134] Particular linkers include those disclosed in PCT Publication No. WO 2004 / 018493 (hereby incorporated by reference), such as those containing a moiety of the formula:

[0135] [ka]

[0136] wherein X is selected from the group including O, S, NH, and NQ, Q is a C1-10 substituted or unsubstituted alkyl group, Y is selected from the group including O, S, NH, and N(allyl), and T is hydrogen or C1-C 10 A linker is a substituted or unsubstituted alkyl group, and the * indicates where the moiety is attached to the remainder of the nucleotide or nucleoside. In some embodiments, the linker connects the base of the nucleotide to a label, such as, for example, a dye compound described herein.

[0137] Additional examples of linkers include those disclosed in U.S. Patent Application Publication No. 2016 / 0040225 (herein incorporated by reference), such as those containing a moiety of the formula:

[0138] [ka] (where * indicates where the moiety is attached to the remainder of the nucleotide or nucleoside.) The linker moieties shown herein may comprise a full or partial linker structure between the nucleotide / nucleotide and the label. The linker moieties shown herein may comprise a full or partial linker structure between the nucleotide / nucleotide and the label.

[0139] Additional examples of linkers include those of the formula

[0140] [ka] wherein B is a nucleobase, Z is -N3 (azide), -O-C1-C6 alkyl, -O-C2-C6 alkenyl, or -O-C2-C6 alkynyl, and F1 comprises a dye moiety that may contain an additional linker structure. Those skilled in the art will appreciate that the dye compounds described herein are covalently attached to a 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 is

[0141] [ka] (an "AOL" linker moiety), where Z is -O-allyl.

[0142] In certain embodiments, the length of the linker between the fluorescent dye (fluorophore) and the guanine base can be modified, for example, by introducing a polyethylene glycol spacer group, thereby increasing the fluorescence intensity compared to the same fluorophore attached to the guanine base via other linkages known in the art. Exemplary linkers and their properties are described in PCT Publication WO 2007 / 020457 (incorporated herein by reference). Such linker designs, particularly their increased length, can allow for improved brightness of fluorophores attached to the guanine base of guanosine nucleotides when incorporated into polynucleotides such as DNA. Thus, when the dye is intended for use in any analytical method requiring the detection of a fluorescent dye label attached to a guanine-containing nucleotide, the linker may be a linker of the formula -((CH2)2O) n It is advantageous if the compound contains a spacer group of the formula - (wherein n is an integer from 2 to 50).

[0143] Nucleosides and nucleotides may be labeled at sites on the sugar or nucleobase. As known in the art, a "nucleotide" consists of a nitrogenous base, a sugar, and one or more phosphate groups. In RNA, the sugar is ribose, and in DNA, the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present in ribose. The nitrogenous base is a derivative of a purine or pyrimidine. Purines are adenine (A) and guanine (G), and pyrimidines are cytosine (C) and thymine (T), or, in the context of RNA, uracil (U). The C-1 atom of deoxyribose is linked to the N-1 atom of a pyrimidine or the N-9 atom of a purine. Nucleotides are also phosphate esters of nucleosides, where esterification occurs at the hydroxyl group attached to C-3 or C-5 of the sugar. Nucleosides are usually monophosphate, diphosphate, or triphosphate.

[0144] A "nucleoside" is structurally similar to a nucleotide, but lacks the phosphate moiety. An example of a nucleotide analog is one in which the label is linked to the base and there is no phosphate group attached to the sugar molecule.

[0145] While bases are typically referred to as purines or pyrimidines, those skilled in the art 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. A "derivative" or "analog" refers to a compound or molecule whose core structure is the same as or closely resembles that of the parent compound, but which has chemical or physical modifications, such as different or additional side groups, that allow the derivative nucleotide or nucleoside to be linked to other molecules. For example, the base may be a deazapurine. In certain embodiments, the derivative should be able to undergo Watson-Crick pairing. "Derivative" and "analog" also include synthetic nucleotide or nucleoside derivatives, for example, with modified base moieties and / or modified sugar moieties. Such derivatives and analogs are discussed, for example, in Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman, Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also contain modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl-phosphonate, phosphoranilidate, phosphoramidate linkages, and the like.

[0146] The dye may be attached to any position on the nucleotide base, for example, via a linker. In certain embodiments, Watson-Crick base pairing can still be performed on the resulting analog. Specific nucleobase labeling sites include the C5 position of pyrimidine bases or the C7 position of 7-deazapurine bases. As noted above, a linker group can be used to covalently attach the dye to the nucleoside or nucleotide.

[0147] In certain embodiments, the labeled nucleoside or nucleotide may be enzymatically incorporable and enzymatically extendable. Thus, the linker portion may be long enough to connect the nucleotide to the compound so that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by the nucleic acid replication enzyme. Thus, the linker may also include a spacer unit. The spacer may, for example, distance the nucleotide base from the cleavage site or label.

[0148] A nucleoside or nucleotide labeled with the dyes described herein can have the formula:

[0149] [ka]

[0150] wherein dye is a dye compound (label) moiety as described herein (after the covalent bond between the functional group of the dye and the functional group of the linker "L"), B is a nucleobase, e.g., 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 ester analog, -O- attached to a reactive phosphorus-containing group, or -O- protected by a blocking group, R" is H or OH, R'" is H, a 3'OH blocking group as described herein, or -OR'" forms a phosphoramidite. When -OR'" is a phosphoramidite, R' is an acid-cleavable hydroxyl protecting group that allows for subsequent monomer coupling under automated synthesis conditions. In some further embodiments, B is

[0151] [ka] or

[0152] [ka] or optionally substituted derivatives and analogs thereof. In some further embodiments, the labeled nucleobase has the structure

[0153] [ka] Includes.

[0154] In certain embodiments, the blocking group is separate and independent from the dye compound, i.e., not attached to the dye compound. Alternatively, the dye may include all or part of the 3'-OH blocking group. Thus, R''' can be a 3'-OH blocking group that may or may not include a dye compound.

[0155] In yet another alternative embodiment, there is no blocking group on the 3' carbon of the pentose sugar, and for example, the dye (or dye and linker structure) attached to the base may be of sufficient size or structure to act as a block to the incorporation of additional nucleotides. Thus, whether or not the dye is attached to the 3' position of the sugar, blocking may be due to steric hindrance or a combination of size, charge, and structure.

[0156] In yet another alternative embodiment, the blocking group may be present on the 2' or 4' carbon of the pentose sugar and may be of sufficient size or structure to act as a block for the incorporation of additional nucleotides.

[0157] The use of blocking groups allows for controlled polymerization, e.g., by terminating elongation when a labeled nucleotide is incorporated. If the blocking effect is reversible, e.g., by changing chemical conditions or by removing the chemical block (including, but not limited to, examples), elongation can be halted at a specific point and then allowed to continue.

[0158] In certain embodiments, both the linker (between the dye and the nucleotide) and the blocking group are present and are separate from each other. In certain embodiments, the linker and the blocking group are both cleavable under the same or substantially similar conditions. Thus, the deprotection and deblocking process may be more efficient because only a single treatment is required to remove both the dye compound and the blocking group. However, in some embodiments, the linker and the blocking group do not need to be cleavable under the same conditions, but instead can be individually cleaved under different conditions.

[0159] The present disclosure also encompasses polynucleotides incorporating dye compounds. Such polynucleotides may be DNA or RNA composed of deoxyribonucleotides or ribonucleotides, respectively, joined by phosphodiester linkages. Polynucleotides can include naturally occurring nucleotides, non-naturally occurring (or modified) nucleotides other than the labeled nucleotides described herein, or any combination thereof, in combination with at least one modified nucleotide (e.g., labeled with a dye compound) as described herein. Polynucleotides according to the present disclosure can also include non-naturally occurring backbone linkages and / or non-nucleotide chemical modifications. Chimeric structures composed of a mixture of ribonucleotides and deoxyribonucleotides containing at least one labeled nucleotide are also contemplated.

[0160] Non-limiting exemplary labeled nucleotides described herein include:

[0161] [ka] wherein L represents a linker and R represents a ribose or deoxyribose moiety as described above, or a ribose or deoxyribose moiety substituted at the 5' position with a monophosphate, diphosphate, or triphosphate.

[0162] In some embodiments, non-limiting exemplary fluorescent dye conjugates are shown below:

[0163] [ka]

[0164] [ka]

[0165] [ka] wherein PG represents a 3'OH blocking 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 further embodiments, p is 1, 2, or 3 and k is 5.

[0166] [ka] refers to the point of attachment of the dye with 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 embodiment of the labeled nucleotide described herein, the nucleotide is a nucleotide triphosphate.

[0167] Additional aspects of the present disclosure relate to oligonucleotides comprising the labeled nucleotides described herein. In some embodiments, the oligonucleotides are hybridized to at least a portion of a target polynucleotide. In some embodiments, the target polynucleotide is immobilized on a solid support. In some further embodiments, the solid support comprises an array of a plurality of immobilized target polynucleotides. In further embodiments, the solid support comprises a patterned flow cell. In further embodiments, the patterned flow cell is fabricated on a CMOS chip. In further embodiments, the patterned flow cell comprises a plurality of nanowells. In yet another embodiment, the plurality of nanowells is aligned directly over each CMOS photodiode (pixel).

[0168] kit Provided herein are kits comprising a first nucleotide labeled with an alkylpyridinium coumarin compound of the present disclosure (i.e., a first label). In some embodiments, the kit also comprises a second labeled nucleotide labeled with a second compound (i.e., a second label) that is different from the alkylpyridinium coumarin compound in the first labeled nucleotide. In some embodiments, the first and second labeled nucleotides are excitable using a single excitation source, which may be a first light source having a first excitation wavelength. For example, the excitation bands of the first and second labels may at least partially overlap such that excitation in an overlapping region of the spectrum causes both labels to fluoresce. In some further embodiments, the kit may comprise a third nucleotide, which is labeled with a third compound (i.e., a third label) that is different from the first and second labels. In some such embodiments, the first and third labeled nucleotides are excitable using a second excitation source, which may be a second light source having a second excitation wavelength that is different from the first excitation wavelength. For example, the excitation bands of the first and third labels may at least partially overlap such that excitation in the overlapping region of the spectrum causes both labels to fluoresce. In some further embodiments, the kit may further include a fourth nucleotide. In some such embodiments, the fourth nucleotide is unlabeled (dark). In other embodiments, the fourth nucleotide is labeled with a compound different from the first, second, and third nucleotides, and each label has a distinct absorbance maximum that is distinguishable from the other labels. In still other embodiments, the fourth nucleotide is unlabeled. In some embodiments, the first excitation light source has a wavelength of about 500 nm to about 550 nm, about 510 to about 540 nm, or about 520 to about 530 nm (e.g., 520 nm). The second light source has an excitation wavelength of about 400 nm to about 480 nm, about 420 nm to about 470 nm, or 450 nm to about 460 nm (e.g., 450 nm). In alternative embodiments, the first light source has an excitation wavelength of about 400 nm to about 480 nm, about 420 nm to about 470 nm, or 450 nm to about 460 nm (eg, 450 nm).The second excitation light source has a wavelength of about 500 nm to about 550 nm, about 510 to about 540 nm, or about 520 to about 530 nm (e.g., 520 nm). The second light source has an excitation wavelength of about 400 nm to about 480 nm, about 420 nm to about 470 nm, or 450 nm to about 460 nm (e.g., 450 nm). In further embodiments, the first label, second label, and third label each have an emission spectrum that can be collected on a single emission collection filter or channel.

[0169] In some embodiments, the kit can include four labeled nucleotides (A, C, G, and T or U), with the first of the four nucleotides labeled with a compound disclosed herein. In such kits, each of the four nucleotides can be labeled with the same or a different compound as the labels on the other three nucleotides. Alternatively, the first of the four nucleotides is a labeled nucleotide described herein, the second of the four nucleotides has a second label, the third nucleotide has a third label, and the fourth nucleotide is unlabeled (dark). As another example, the first of the four nucleotides is a labeled nucleotide described herein, the second of the four nucleotides has a second label, the third nucleotide carries a mixture of two labels, and the fourth nucleotide is unlabeled (dark). Thus, one or more of the labeled compounds can have different absorbance and / or emission maxima so that the compound is distinguishable from the other compounds. For example, each compound may have a different absorbance and / or emission maximum such that each compound is distinguishable from the other three compounds (or two compounds if the fourth nucleotide is unlabeled). It will be understood that portions of the absorbance and / or emission spectra other than the maximum may differ, and these differences can be utilized to distinguish the compounds. The kit may also be such that two or more compounds have different absorbance maxima. The alkylpyridinium coumarin dyes disclosed herein typically absorb light in the sub-500 nm region. For example, these coumarin dyes may have an absorption wavelength of about 450 nm to about 530 nm, about 460 nm to about 520 nm, about 475 nm to about 510 nm, or about 490 nm to about 500 nm.

[0170] The compounds, nucleotides, or kits described herein may be used to detect, measure, or identify biological systems (e.g., including processes or components thereof). Exemplary techniques in which the compounds, nucleotides, or kits can be used include sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, cellular assays (e.g., cell binding or cell function assays), or protein assays (e.g., protein binding assays or protein activity assays). The use may also be in an automated instrument for performing a particular technique, such as an automated sequencing instrument. The sequencing instrument may include two light sources operating at different wavelengths.

[0171] In certain embodiments, the labeled nucleotides described herein may be provided in combination with unlabeled or natural nucleotides, or any combination thereof. The combination of nucleotides may be provided as separate 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).

[0172] When the kit includes multiple nucleotides, particularly two, three, or more specifically four nucleotides, the different nucleotides may be labeled with different dye compounds or may be dark and contain no dye compounds. When different nucleotides are labeled with different dye compounds, it is a feature of the kit that the dye compounds are spectrally distinguishable fluorescent dyes. As used herein, the term "spectrally distinguishable fluorescent dyes" refers to fluorescent dyes that emit fluorescent energy at wavelengths that can be distinguished by a fluorescence detection device (e.g., a commercially available capillary-based DNA sequencing platform) when two or more dyes are present in a sample. When two nucleotides labeled with fluorescent dye compounds are provided in kit form, it is a feature of some embodiments that the spectrally distinguishable fluorescent dyes can be excited at the same wavelength, e.g., by the same light source. When four nucleotides labeled with fluorescent dye compounds are provided in kit form, it is a feature of some embodiments that two of the spectrally distinguishable fluorescent dyes can be excited at one wavelength, and the other two spectrally distinguishable dyes can both be excited at another wavelength. The specific excitation wavelength of the dye is 450 to 460 nm, 490 to 500 nm, or 520 nm or longer (for example, 532 nm).

[0173] In some embodiments, the kit includes a first nucleotide labeled with a compound of the present disclosure and a second nucleotide labeled with a second dye, wherein the dyes have a difference in absorbance maxima of at least 10 nm, particularly 20 to 50 nm or 30 to 40 nm. More particularly, the first label may have a Stokes shift of greater than 40 nm, greater than 50 nm, or greater than 60 nm. The second label may have a Stokes shift of greater than about 80 nm, greater than 90 nm, or greater than 100 nm ("Stokes shift" is the distance between the peak absorption wavelength and the peak emission wavelength). Furthermore, the first label may have an absorption maximum of about 460 nm to about 520 nm, about 475 nm to about 510 nm, or about 490 nm to about 500 nm. The second label may have an absorption maximum of about 400 nm to about 470 nm, or about 450 nm to about 460 nm. In further embodiments, the kit may further include a third labeled nucleotide, wherein the third label has an absorption maximum greater than 520 nm. The third label may have a Stokes shift greater than 20 nm, greater than 30 nm, or greater than 40 nm, or a Stokes shift between 20 and 40 nm. The kit may further include an unlabeled fourth nucleotide. In further embodiments, the first label, second label, and third label each have an emission maximum greater than 540 nm, greater than 550 nm, greater than 560 nm, greater than 570 nm, greater than 580 nm, greater than 590 nm, or greater than 600 nm. In some embodiments, the emission spectra of the first label, second label, and third label may be detected or collected in a single emission collection channel or filter (e.g., a collection region of about 580 to about 700 nm).

[0174] In an alternative embodiment, the disclosed kit may contain 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 distinct compound, such as a "green" dye that absorbs at less than 600 nm. The third nucleotide may be labeled with a mixture of a compound of the present disclosure and a spectrally distinct compound, and the fourth nucleotide may be "dark" and may contain no label. Thus, for convenience, nucleotides 1-4 may be labeled "blue," "green," "blue / green," and dark. To further simplify the instrumentation, the four nucleotides may be labeled with two dyes excited by a single light source, and thus the labels of nucleotides 1-4 may be "blue 1," "blue 2," "blue 1 / blue 2," and dark.

[0175] Although the kits are exemplified herein in configurations having different nucleotides labeled with different dye compounds, it will be understood that the kits may include two, three, four or more different nucleotides having the same dye compound.

[0176] In addition to labeled nucleotides, the kit may also include at least one additional component. The additional component may be one or more of the components identified in the methods described herein or in the Examples section below. Some non-limiting examples of components that can be combined into the kits of the present disclosure are described below. In some embodiments, the kit further includes a DNA polymerase (such as a mutant DNA polymerase) and one or more buffer compositions. One buffer composition may include an antioxidant, such as ascorbic acid or sodium ascorbate, which may be used to protect the dye compound from photodamage during detection. An additional buffer composition may include a reagent that can be used to cleave the 3' blocking group and / or the cleavable linker. For example, a water-soluble phosphine or water-soluble transition metal catalyst formed from a transition metal, such as a palladium complex, and an at least partially water-soluble ligand. Various components of the kit may be provided in a concentrated form that is diluted before use. In such embodiments, a suitable dilution buffer may also be included. Again, one or more of the components identified in the methods described herein may be included in the kits of the present disclosure. In any embodiment of the nucleotide or labeled nucleotide described herein, the nucleotide or labeled nucleotide comprises a 3' hydroxyl blocking group.

[0177] Sequencing methods Nucleotides containing dye compounds according to the present disclosure can be used, by themselves or incorporated into or associated with a larger molecular structure or conjugate, in any analytical method, such as a method involving the detection of a fluorescent label attached to such a nucleotide. In this context, the term "incorporated into a polynucleotide" can mean that the 5' phosphate is attached via a phosphodiester linkage to the 3' hydroxyl group of a second nucleotide, which itself may form part of a longer polynucleotide chain. The 3' end of a nucleotide described herein may or may not be attached via a phosphodiester linkage to the 5' phosphate of an additional nucleotide. Thus, in one non-limiting embodiment, the present disclosure provides a method for detecting a labeled nucleotide incorporated into a polynucleotide, the method comprising: (a) incorporating at least one labeled nucleotide of the present disclosure into the 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.

[0178] The method may include (a) a synthesis step in which one or more labeled nucleotides according to the present disclosure are incorporated into a polynucleotide, and (b) a detection step in which the one or more labeled nucleotides incorporated into the polynucleotide are detected by detecting or quantitatively measuring their fluorescence.

[0179] Some embodiments of the present application relate to a method for determining the sequence of a target polynucleotide, comprising: (a) contacting a primer polynucleotide / target polynucleotide complex with one or more labeled nucleotides (such as nucleoside triphosphates A, G, C, and T), where at least one of the labeled nucleotides is a labeled nucleotide described herein, and the primer polynucleotide is complementary to at least a portion of the target polynucleotide; (b) incorporating the labeled nucleotide into the primer polynucleotide to generate 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 the 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 a label moiety and a 3' blocking group from the nucleotide incorporated into the primer polynucleotide. In some further embodiments, the method may also comprise (e) washing the removed label moiety and 3' blocking group from the primer polynucleotide strand. In some embodiments, steps (a)-(d) or steps (a)-(e) are repeated until the sequence of at least a portion of the target polynucleotide strand is determined. In some cases, steps (a)-(d) or steps (a)-(e) are repeated 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, the label moiety and the 3' blocking group from the nucleotide incorporated into the primer polynucleotide strand are removed in a single chemical reaction. In some further embodiments, the method is performed on an automated sequencing instrument, wherein the automated sequencing instrument includes two light sources operating at different wavelengths. In some embodiments, sequencing is performed after completing the repeated cycles of the sequencing steps described herein.

[0180] 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), comprising: (a) contacting a primer polynucleotide with an incorporation mixture comprising one or more of four different types of nucleotide conjugates, wherein a first type of nucleotide conjugate comprises a first label, a second type of nucleotide conjugate comprises a second label, and a third type of nucleotide conjugate comprises a third label, wherein each of the first label, the second label, and the third label are spectrally distinct from one another, and wherein the primer polynucleotide is in contact with at least one of the single-stranded target polynucleotides. (b) contacting a nucleotide conjugate from the mixture with a primer polynucleotide complementary to a portion of the nucleotide conjugate; (c) incorporating one nucleotide conjugate from the mixture into a primer polynucleotide to generate 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 the first emission signal and the second emission signal are detected or collected in a single emission detection channel. In some embodiments, the chromenoquinoline dyes described herein can be used as any one of the first, second, or third labels described in the method. In some embodiments, the method does not include chemical modification of any nucleotide conjugates in the mixture after the first imaging event and before the second imaging event. In some further embodiments, the incorporation mixture further comprises a fourth type of nucleotide, wherein the fourth type of nucleotide is unlabeled and labeled with a fluorescent moiety that does not emit a signal from either the first or second imaging event. In this sequencing method, the identity of each incorporated nucleotide conjugate is determined based on the detection patterns of the first imaging event and the second imaging event. For example, the incorporation of the first type of nucleotide conjugate is determined by a signal state in the first imaging event and a dark state in the second imaging event.Incorporation of a second type of nucleotide conjugate is determined by a dark state in the first imaging event and a signal state in the second imaging event. Incorporation of a third type of nucleotide conjugate is determined by a signal state in both the first imaging event and the second imaging event. Incorporation of a fourth type of nucleotide conjugate is determined by a dark state in both the first imaging event and the second imaging event. In further embodiments, steps (a)-(d) are performed in repeated cycles (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 successively incorporated nucleotide conjugate. In some embodiments, the first excitation light source has a shorter wavelength than 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 450 nm. The second excitation light source has a wavelength of about 500 nm to about 550 nm, about 510 nm to about 540 nm, or about 520 nm to about 530 nm (i.e., "green light"). In one embodiment, the second excitation light source has a wavelength of about 520 nm. In other embodiments, the first excitation light source has a longer wavelength than the second excitation light source. In some such embodiments, the first excitation light source has a wavelength of about 500 nm to about 550 nm, about 510 nm to about 540 nm, or about 520 nm to about 530 nm (i.e., "green light"). In one embodiment, the second excitation light source has a wavelength of about 520 nm. The second 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 second excitation light source has a wavelength of about 450 nm.

[0181] In one embodiment, at least one nucleotide is incorporated into a polynucleotide (such as a single-stranded primer polynucleotide described herein) during synthesis by the action of a polymerase enzyme. However, other methods of attaching nucleotides to a polynucleotide may be used, such as, for example, chemical oligonucleotide synthesis or ligation of a labeled oligonucleotide to an unlabeled oligonucleotide. Thus, the term "incorporate," when used with respect to nucleotides and polynucleotides, can encompass chemical and enzymatic polynucleotide synthesis.

[0182] In certain embodiments, a synthesis step is performed, and may optionally include incubating a template or target polynucleotide strand with a reaction mixture containing the fluorescently labeled nucleotides of the present disclosure. A polymerase may also be provided under conditions that allow the formation of a phosphodiester linkage between a free 3' hydroxyl group on a polynucleotide strand annealed to the template or target polynucleotide strand and a 5' phosphate group on the labeled nucleotide. Thus, the synthesis step may include the formation of a polynucleotide strand directed by complementary base pairing of the nucleotides to the template / target strand.

[0183] In all embodiments of the present method, the detection step can be performed while the polynucleotide strand incorporating the labeled nucleotide is annealing to the template / target strand, or after the denaturation step in which the two strands are separated. Additional steps, such as chemical or enzymatic reaction steps or purification steps, may be included between the synthesis step and the detection step. In particular, the polynucleotide strand incorporating the labeled nucleotide can be isolated or purified and then further processed or used in subsequent analyses. For example, a polynucleotide incorporating a labeled nucleotide described herein in a synthesis step can subsequently be used as a labeled probe or primer. In other embodiments, the product of the synthesis step described herein can be subjected to additional reaction steps, the products of these subsequent steps being purified or isolated, if desired.

[0184] Suitable conditions for the synthesis step are well known to those familiar with standard molecular biology techniques. In one embodiment, the synthesis step may resemble a standard primer extension reaction, in which nucleotide precursors containing labeled nucleotides as described herein are used to form an extended target strand (primer polynucleotide) complementary to the template / target strand in the presence of a suitable polymerase enzyme. In other embodiments, the synthesis step may itself form part of an amplification reaction, generating a labeled, double-stranded amplification product consisting of a primer and an annealed complementary strand derived from a copy of the template polynucleotide strand. Other exemplary synthesis steps include nick translation, strand displacement polymerization, random-primed DNA labeling, and the like. Polymerase enzymes particularly useful for the synthesis step are those capable of catalyzing the incorporation of labeled nucleotides as described herein. A variety of naturally occurring or mutated / modified polymerases may be used. By way of example, thermostable polymerases can be used in synthesis reactions performed using thermal cycling conditions, but thermostable polymerases may not be desirable for isothermal primer extension reactions. Suitable thermostable polymerases capable of incorporating labeled nucleotides according to the present disclosure include those described in WO 2005 / 024010 or WO 06 / 120433, each of which is incorporated herein by reference. For synthesis reactions performed at low temperatures, such as 37°C, the polymerase enzyme does not necessarily need to be a thermostable polymerase; therefore, the choice of polymerase will depend on several factors, such as reaction temperature, pH, and strand displacement activity.

[0185] In certain non-limiting embodiments, the present disclosure includes 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 described herein when incorporated into a polynucleotide.

[0186] Certain embodiments of the present disclosure provide for the use of labeled nucleotides containing dye moieties according to the present disclosure in polynucleotide sequencing-by-synthesis reactions. Sequencing by synthesis generally involves sequentially adding one or more nucleotides or oligonucleotides to a growing polynucleotide chain in a 5' to 3' direction using a polymerase or ligase to form an extended polynucleotide chain complementary to the template / target nucleic acid to be sequenced. The identity of the base present in one or more of the added nucleotides can be determined in a detection or "imaging" step. The identity of the added base can be determined after each nucleotide incorporation step. The sequence of the template can then be inferred using conventional Watson-Crick base-pairing rules. The use of dye-labeled nucleotides described herein to determine the identity of a single base can be useful, for example, in single-nucleotide polymorphism scoring, and such single-base extension reactions are within the scope of the present disclosure.

[0187] In one embodiment of the present disclosure, the sequence of a template / target polynucleotide is determined by detecting the incorporation of one or more nucleotides into a nascent strand complementary to the template polynucleotide to be sequenced via detection of a fluorescent label attached to the incorporated nucleotide. The sequencing of the template polynucleotide can be primed with a suitable primer (or a primer prepared as a hairpin structure containing the primer as part of the hairpin), and the nascent strand is extended stepwise by adding nucleotides to the 3' end of the primer in a polymer-catalyzed reaction.

[0188] In certain embodiments, each of the different nucleotide triphosphates (A, T, G, and C) may be labeled with a unique fluorophore and contain a blocking group at the 3' position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides may be unlabeled (dark). The polymerase enzyme incorporates the nucleotide into a nascent strand complementary to the template / target polynucleotide, and the blocking group prevents further incorporation of the nucleotide. Any unincorporated nucleotides can be washed away, and the fluorescent signal from each incorporated nucleotide can be optically "read" by suitable means, such as a charge-coupled device using a light source excitation and suitable emission filters. The 3' blocking group and fluorescent dye compound can then be removed (deprotected) (simultaneously or sequentially) to expose the nascent strand for incorporation of additional nucleotides. Typically, the identity of the incorporated nucleotide is determined after each incorporation step, although this is not strictly required. Similarly, US Pat. No. 5,302,509 (hereby incorporated by reference) discloses a method for sequencing polynucleotides immobilized on a solid support.

[0189] The method, as exemplified above, incorporates fluorescently labeled 3'-blocked nucleotides A, G, C, and T into a growing strand complementary to an immobilized polynucleotide in the presence of a DNA polymerase. The polymerase incorporates the base complementary to the target polynucleotide, but further addition is prevented by the 3'-blocking group. The label of the incorporated nucleotide can then be determined, after which the blocking group can be removed by chemical cleavage to allow further polymerization. The nucleic acid template to be sequenced in a sequencing-by-synthesis reaction can be any polynucleotide for which sequencing is desired. Nucleic acid templates for sequencing reactions typically contain a double-stranded region with a free 3' hydroxyl group that serves as a primer or starting point for the addition of additional nucleotides in the sequencing reaction. The region of the template to be sequenced has this free 3' hydroxyl group overhanging the complementary strand. The overhang region of the template to be sequenced may be single-stranded, but can also be double-stranded, so long as a "nick" exists on the strand complementary to the template strand to be sequenced, providing a free 3'OH group for initiating the sequencing reaction. In such embodiments, sequencing may proceed by strand displacement. In certain embodiments, a primer with a free 3' hydroxyl group may be added as a separate component (e.g., a short oligonucleotide) that hybridizes to a single-stranded region of the template to be sequenced. Alternatively, the primer and template strand to be sequenced may each form part of a partially self-complementary nucleic acid strand that can form an intramolecular duplex, such as a hairpin loop structure. Hairpin polynucleotides and methods by which they can be attached to solid supports are disclosed in WO 01 / 57248 and WO 2005 / 047301, each of which is incorporated herein by reference. Nucleotides can be added sequentially to the growing primer to synthesize a polynucleotide chain in the 5' to 3' direction. The nature of the added base may, but need not, be determined particularly after each nucleotide addition, and such determination provides sequence information about the nucleic acid template.Thus, a nucleotide is incorporated into a nucleic acid strand (or polynucleotide) by linking the nucleotide to the free 3' hydroxyl group of the nucleic acid strand through formation of a phosphodiester bond with the 5' phosphate group of the nucleotide.

[0190] The nucleic acid template to be sequenced may be DNA or RNA, or even a hybrid molecule composed of deoxynucleotides and ribonucleotides. The nucleic acid template may contain naturally occurring and / or non-naturally occurring nucleotides and naturally occurring or non-naturally occurring backbone linkages, as long as they do not prevent copying of the template in the sequencing reaction.

[0191] In certain embodiments, the nucleic acid template to be sequenced can be attached to a solid support via any suitable linking method known in the art, for example, via covalent bonding. 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 either direct covalent attachment of the template polynucleotide or immobilization of the template polynucleotide via a hydrogel or polyelectrolyte multilayer, which itself can be non-covalently attached to the solid support.

[0192] Arrays in which polynucleotides are directly attached to a support (e.g., a silica-based support such as those disclosed in WO 00 / 06770, incorporated herein by reference), where the polynucleotides are immobilized on the glass support by reaction between pendant epoxide groups on the glass and internal amino groups on the polynucleotide. Additionally, polynucleotides can be immobilized on the glass support by reaction with sulfur-based nucleophiles, as described, for example, in WO 2005 / 047301, incorporated herein by reference. The template polynucleotide may be bound to the solid support by reaction of the agent with the solid support. A still further example of a solid-supported template polynucleotide is when the template polynucleotide is bound to a hydrogel supported on a silica-based or other solid support, such as those described in WO 00 / 31148, WO 01 / 01143, WO 02 / 12566, WO 03 / 014392, U.S. Patent No. 6,465,178, and WO 00 / 53812, each of which is incorporated herein by reference.

[0193] Specific surfaces onto which template polynucleotides can be immobilized include polyacrylamide hydrogels. Polyacrylamide hydrogels are described in the above references and in International Publication No. 2005 / 065814, which are incorporated herein by reference. Specific hydrogels that can be used include those described in International Publication No. 2005 / 065814 and U.S. Patent Application Publication No. 2014 / 0079923. In one embodiment, the hydrogel is PAZAM (poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide)).

[0194] DNA template molecules can be bound to beads or microparticles, for example, as described in U.S. Patent No. 6,172,218 (incorporated herein by reference). Binding to beads or microparticles can be useful for sequencing applications. Bead libraries can be prepared, with each bead containing a different DNA sequence. Exemplary libraries and methods for their creation are described in Nature, 437, 376-380 (2005), Science, 309, 5741, 1728-1732 (2005), each of which is incorporated herein by reference. Sequencing of such bead arrays using the nucleotides described herein is within the scope of this disclosure.

[0195] The templates to be sequenced may form part of an "array" on a solid support, where the array can 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 the dye compounds of the present disclosure can 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.

[0196] However, the nucleotides labeled with the dye compounds of the present disclosure are particularly advantageous in the context of clustered array sequencing. In clustered arrays, distinct regions (often referred to as sites or features) on the array contain multiple polynucleotide template molecules. Generally, multiple polynucleotide molecules cannot be individually distinguished by optical means, but are instead detected as an ensemble. Depending on how the array is formed, each site on the array may contain multiple copies of one individual polynucleotide molecule (e.g., the site is homogeneous for a specific single-stranded or double-stranded nucleic acid species), or even multiple copies of a small number of different polynucleotide molecules (e.g., multiple copies of two different nucleic acid species). Clustered arrays of nucleic acid molecules can be generated using techniques commonly known in the art. By way of example, WO 98 / 44151 and WO 00 / 18957, each incorporated herein, describe methods for amplifying nucleic acids in which both the template and the amplification product remain immobilized on a solid support to form arrays consisting of clusters or "colonies" of immobilized nucleic acid molecules. The 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.

[0197] Nucleotides labeled with the dye compounds of the present disclosure are also useful for sequencing templates on single molecule arrays. As used herein, the term "single molecule array" or "single molecule array (SMA)" refers to a population of polynucleotide molecules distributed (or arrayed) on a solid support, where the separation distance of any individual polynucleotide from every other polynucleotide in the population is such that each individual polynucleotide molecule can be individually distinguished. Thus, in some embodiments, target nucleic acid molecules immobilized on the surface of a solid support can be resolved by optical means. This means that one or more distinct signals, each representing a polynucleotide, are generated within a discernible area of ​​the particular imaging device used.

[0198] Single molecule detection can be achieved with spacing between adjacent polynucleotide molecules on the array of at least 100 nm, more specifically at least 250 nm, even more specifically at least 300 nm, and even more specifically at least 350 nm. Thus, each molecule can be individually resolved and distinguished and detected as a single molecule fluorescent spot, and the fluorescence from this single molecule fluorescent spot also exhibits single-step photobleaching.

[0199] The terms "individually resolved" and "individual resolution" are used herein to specify that, when visualized, it is possible to distinguish one molecule on an array from its neighboring molecules. The separation between individual molecules on an array is determined, in part, by the particular technique used to resolve the individual molecules. General characteristics of single molecule arrays can be understood by reference to International Publication Nos. WO 00 / 06770 and WO 01 / 57248, each of which is incorporated herein by reference. One use of the labeled nucleotides of the present disclosure is in sequencing-by-synthesis reactions, although the utility of such nucleotides is not limited to such methods. Indeed, the labeled nucleotides described herein can be advantageously used in any sequencing method requiring detection of a fluorescent label attached to a nucleotide incorporated into a polynucleotide.

[0200] In particular, nucleotides labeled with the dye compounds of the present disclosure can be used in automated fluorescent sequencing protocols, particularly fluorescent dye terminator cycle sequencing, which is based on the chain termination sequencing method of Sanger and coworkers. Such methods generally incorporate fluorescently labeled dideoxynucleotides in primer extension sequencing reactions using enzymes and cycle sequencing. So-called Sanger sequencing and related protocols (Sanger-type) utilize randomized chain terminations with labeled dideoxynucleotides.

[0201] Thus, the present disclosure also encompasses nucleotides labeled with dye compounds that are dideoxynucleotides lacking hydroxyl groups at both the 3' and 2' positions, such modified deoxynucleotides being suitable for use in Sanger-type sequencing and the like.

[0202] It will be appreciated that the same effect achieved by using a dideoxy base can be achieved by using a nucleotide having a 3'-OH blocking group (both of which can prevent the incorporation of a subsequent nucleotide), and therefore nucleotides labeled with dye compounds of the present disclosure incorporating a 3'-blocking group can be useful in Sanger sequencing and related protocols. When a nucleotide according to the present disclosure having a 3'-blocking group is used in a Sanger-type sequencing method, it will be understood that the dye compound or detectable label attached to the nucleotide need not be connected via a cleavable linker, because in each instance in which a labeled nucleotide of the present disclosure is incorporated, the nucleotide need not be subsequently incorporated, and therefore the label need not be removed from the nucleotide.

[0203] Alternatively, the sequencing methods described herein can also be performed using unlabeled nucleotides and affinity reagents containing the fluorescent dyes described herein. For example, one, two, three, or each of the four different types of nucleotides in the incorporation mixture in step (a) (e.g., dATP, dCTP, dGTP, and dTTP or dUTP) can be unlabeled. Each of the four types of nucleotides (e.g., dNTPs) has a 3' hydroxyl blocking group to ensure that only one base can be added to the 3' end of the primer polynucleotide by the polymerase. After incorporation of the 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 dNTPs, providing labeled extension products containing the incorporated dNTPs. The use of unlabeled nucleotides and affinity reagents in sequencing by synthesis is disclosed in International Publication Nos. WO 2018 / 129214 and WO 2020 / 097607. The modified sequencing method of the present disclosure using unlabeled nucleotides may include the following steps.

[0204] (a') contacting the 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; (b') incorporating nucleotides into the primer polynucleotide to generate an extended primer polynucleotide (i.e., an extended primer polynucleotide / target polynucleotide complex); (c') contacting the extended primer polynucleotide with a set of affinity reagents, wherein 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); (d') performing one or more fluorescence measurements of the labeled extended primer polynucleotide to determine the identity of the incorporated nucleotide.

[0205] In some embodiments of the modified sequencing methods described herein, each unlabeled nucleotide in the incorporation mixture contains a 3' hydroxyl blocking group. In further embodiments, the 3' hydroxyl blocking group of the incorporated nucleotide is removed before the next incorporation cycle. In still further embodiments, the method further comprises removing the affinity reagent from the incorporated nucleotide. In still further embodiments, the 3' hydroxyl blocking group and the affinity reagent are removed in the same reaction. In some embodiments, the set of affinity reagents can 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 further embodiments, each of the first, second, and third affinity reagents comprises a detectable label that is spectrally distinguishable. In some embodiments, the affinity reagent can include a protein tag, an antibody (including, but not limited to, an antibody binding fragment, a single-chain antibody, a bispecific antibody, etc.), an aptamer, a knottin, an affimer, or any other known agent that binds to the incorporated nucleotide with appropriate specificity and affinity. In one embodiment, one or more affinity reagents in the set are antibodies or protein tags. In another embodiment, at least one of the first, second, and third types of affinity reagents is an antibody or protein tag that includes one or more detectable labels (e.g., multiple copies of the same detectable label), and the detectable label is or includes an alkylpyridinium coumarin dye moiety described herein.

[0206] Example Additional embodiments are disclosed in more detail in the following examples, which are not intended to limit the scope of the claims.

[0207] Example 1. Synthesis of alkylpyridinium coumarin dyes Synthesis of alkylpyridinium intermediates

[0208] [ka]

[0209] 2,4-Lutidine (5.8 mL, 50 mmol) was dissolved in anhydrous THF (20 mL) in a dry flask under nitrogen. A 1 M solution of lithium diisopropylamide (50 mL, 50 mmol) in THF / hexane was slowly added at 0 °C. The solution turned dark red and was stirred at room temperature for 4 hours. Anhydrous diethyl carbonate (14.7 mL, 2.5 mmol) dissolved in 10 mL of anhydrous THF was then slowly added, and the reaction was stirred at room temperature overnight. The mixture was then quenched with saturated aqueous NH4Cl (10 mL), diluted with 200 mL of ethyl acetate, and washed with 200 mL of water. The organic phase was dried over MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel. The intermediate was then dissolved in 10 mL of concentrated HCl and heated at 100 °C for 1 hour. The volatiles were removed under reduced pressure, and the residue was then dissolved in ethanol (50 mL) and a few drops of concentrated sulfuric acid were added. The solution was stirred at room temperature until completion. The reaction was quenched with 5 mL of saturated aqueous NaHCO3, and the volatiles were removed under reduced pressure. The residue was partitioned 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 in 34% yield as a colorless viscous oil.

[0210] 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 in 70% yield (312 mg, 0.838 mmol). LC-MS (ESI): (positive ion) m / z 294 (M+H + ).

[0211] [ka]

[0212] 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 h. 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 + ).

[0213] [ka]

[0214] 2,4,6-Collidine (2.5 mL, 20 mmol) was dissolved in anhydrous THF (20 mL) in a dry flask under nitrogen. 1 M lithium diisopropylamide solution (22 mL, 22 mmol) in THF / hexane was slowly added. The solution turned dark red and cloudy, and it was stirred at room temperature for 2 hours. It was then slowly added to anhydrous diethyl carbonate (5 mL, 40 mmol) dissolved in 20 mL of anhydrous THF at -70 °C, and the reaction was allowed to warm slowly to room temperature overnight. The mixture was then quenched with saturated aqueous NH4Cl (10 mL) and diluted with 200 mL of ethyl acetate. The organic phase was separated, dried over MgSO4, filtered, and evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel. The intermediate was then dissolved in 20 mL of concentrated HCl and allowed to stand at room temperature overnight. The volatiles were removed under reduced pressure, and the residue was then dissolved in ethanol (50 mL) and 0.5 mL of concentrated sulfuric acid was added. The solution was refluxed for 4 hours, then quenched with 5 mL of saturated aqueous NaHCO3, and the volatiles were removed under reduced pressure. The residue was partitioned 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 in 12% yield (474 ​​mg, 2.44 mmol) as a colorless viscous oil.

[0215] To compound 4 (100 mg, 0.518 mmol) was added acetonitrile (300 μL) and ethyl trifluoromethanesulfonate (67 μL, 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 white solid, which was used in the next step without further purification. LC-MS (ESI): (positive ion) m / z 222 (M+H + ).

[0216] [ka]

[0217] CuCN (2 g, 23.2 mmol) and anhydrous THF (75 mL) were added to a flask under nitrogen and then cooled to −78° C. with stirring. Isopropylmagnesium bromide (3 M solution in 2-methyltetrahydrofuran, 15.5 mL, 46.5 mmol) was added dropwise with vigorous stirring. The suspension was allowed to stand at −78° C. for 20 minutes, and then 2-bromo-4-methylpyridine (1 g, 5.81 mmol) was added. The reaction was stirred at −78° C. for 3 hours and then allowed to warm 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 2×100 mL of dichloromethane. The organic phase was dried over MgSO4, filtered, and evaporated under reduced pressure. The crude product was purified by flash column chromatography on silica gel. Compound 6 was obtained in 30% yield (240 mg, 1.77 mmol). LC-MS(ESI):(Positive ion) m / z135(M+H + ).

[0218] Compound 6 (240 mg, 1.77 mmol) was dissolved in anhydrous THF (5 mL) in a dry flask under nitrogen and cooled to -78 °C. A 1 M solution of lithium diisopropylamide (3.6 mL, 3.54 mmol) in THF / hexane was added dropwise. The solution turned pale red and was stirred at -78 °C for 1 h. Anhydrous diethyl carbonate (0.417 mL, 3.54 mmol) dissolved in 5 mL of anhydrous THF was then slowly added, and the reaction was allowed to warm to room temperature for 3.5 h. The mixture was then quenched with saturated aqueous NH4Cl (5 mL) and diluted with 100 mL of ethyl acetate. The organic phase was dried over MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel. The intermediate was then dissolved in 1 mL of concentrated HCl and heated at 100 °C for 1 h. The volatiles were removed under reduced pressure, and the residue was then dissolved in ethanol (5 mL) and a few drops of concentrated sulfuric acid were added. The solution was stirred at room temperature until completion. The reaction was quenched with 5 mL of saturated aqueous NaHCO3, and the volatiles were removed under reduced pressure. The residue was partitioned 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 in 29% yield as a yellowish viscous oil.

[0219] 6-Bromohexanoic acid (103 mg, 0.53 mmol), compound 7 (100 mg, 0.48 mmol), and tetrabutylammonium iodide (a few 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 + ).

[0220] [ka]

[0221] 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 + ).

[0222] Synthesis of coumarin dyes [ka]

[0223] The starting material and a catalytic amount of piperidinium 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 a reversed-phase C18 column. Compound I-1 was obtained in 43% yield (56 mg, 0.129 mmol) as a brown solid. 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, CH3pyr), 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=7.2Hz, 2H, CH2-CH2-COO), 1.58-1.44(m, 2H, CH2-CH2-CH2-COO), 1.27(d, J=7.1Hz, 3H, CH3Et). LC-MS(ESI):(Positive ion) m / z435(M+H + (negative ion) m / z 433 (M - ).

[0224] [ka]

[0225] The starting material and a catalytic amount of piperidinium acetate were dissolved in ethanol and refluxed until the reaction was complete. The crude product was evaporated to dryness, and the residue was then dissolved in 1 mL of trifluoroacetic acid in 1 mL of water and heated to 90°C for 1 hour. The reaction mixture was evaporated under reduced pressure, and the crude product was purified by flash column chromatography on a reversed-phase C18 column. Compound I-2 was obtained as a brown solid in 75% yield (74 mg, 0.123 mmol). 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(brs, 1H, Ar-H pyr), 8.43(brs, 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, CH3pyr), 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 - ).

[0226] [ka]

[0227] Compound I-3 was prepared according to the same procedure described for I-2. Compound I-3 was obtained in 59% yield (32 mg, 0.064 mmol) as a brown solid. 1H NMR (400MHz, MeOD): δ(ppm)8.50(d, J=6.9Hz, 1H, Ar-H pyr), 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, H-ACH2-N), 3.17(m, 1H, H-BCH2-N), 2.74(m, 3H, CH-Ar, CH2-SO3 - ), 2.68(s, 3H, CH3pyr), 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=6.5Hz, 3H, CH3), 1.12(s, 3H, CH3). LC-MS(ESI):(Positive ion)m / z543(M+H + ); (negative ion) m / z 541 (M - ).

[0228] [ka]

[0229] Compound I-4 was prepared according to the same procedure described for I-2. Compound I-4 was obtained in 84% yield (77 mg, 0.135 mmol) as a brown solid. 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, H-ACH2-N), 3.34 (m, 1H, H-BCH2-N), 2.91 (s, 7H, CH3pyr, 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 / z463(M+H + ); (negative ion) m / z 461 (M - ).

[0230] [ka]

[0231] Compound I-5 was prepared according to the same procedure described for I-2. Compound I-4 was obtained in 66% yield (79 mg, 0.139 mmol) as a brown solid. 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, CH3pyr), 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 / z421(M+H + ).

[0232] [ka]

[0233] Compound I-6 was prepared according to the same procedure described for I-1. Compound I-6 was obtained in 33% yield (47 mg, 0.101 mmol) as a brown solid. 1 H NMR (400MHz, MeOD): δ (ppm) 8.69 (d, J=6.9Hz, 1H, Ar-H pyr), 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=7.2Hz, 2H, CH2-CH2-COO), 1.51(m, 8H, CH3iPr, CH2-CH2-CH2-COO), 1.27(t, J=7.1Hz, 3H, CH3Et). LC-MS(ESI):(Positive ion) m / z463(M+H + ).

[0234] [ka]

[0235] Compound I-7 was prepared according to the same procedure described for I-2. Compound I-7 was obtained in 33% yield (48 mg, 0.1 mmol) as a brown solid. 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, H-ACH2-N), 3.44-3.35(m, 1H, H-BCH2-N), 2.91(m, 1H, CH iPr), 2.32(t, J=7.2Hz, 2H, CH2-COO), 2.00-1.87(m, 4H, CH2-CH2-N, CH2-CN), 1.62(t, J=7.3Hz, 3H, CH3Et ), 1.52(d, J=6.8Hz, 6H, CH3iPr), 1.47(s, 3H, C-CH3), 1.42(d, J=6.5Hz, 3H, CH-CH3), 1.36(s, 3H, C-CH3). LC-MS(ESI):(Positive ion) m / z477(M+H + ).

[0236] [ka]

[0237] Compound I-8 was prepared according to the same procedure described for I-1. Compound I-8 was obtained in 61% yield (69 mg, 0.16 mmol) as a brown solid. 1 H NMR (400MHz, MeOD): δ(ppm)8.74(d, J=6.9Hz, 1H, Ar-H pyr), 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=7.3Hz, 2H, CH2-CH2-COO), 1.59-1.46 (m, 2H, CH2-CH2-CH2-), 1.28 (t, J=7.1Hz, 6H, CH3Et). LC-MS(ESI):(Positive ion) m / z423(M+H + ), (negative ion) m / z 421 (M- ).

[0238] Example 2. General synthesis of alkylpyridinium coumarin dye-labeled nucleotides Alkylpyridinium coumarin dye (0.015 mmol) was coevaporated with 2 × 2 mL of anhydrous N,N'-dimethylformamide (DMF) and then dissolved in 1 mL of anhydrous N,N'-dimethylacetamide (DMA). N,N-Diisopropylethylamine (17 μL, 0.1 mmol) was added, followed by N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU, 4.8 mg, 0.016 mmol). The reaction was stirred under nitrogen at room temperature for 30 min. During this time, an aqueous solution of nucleotide triphosphates (0.01 mmol) was evaporated to dryness under reduced pressure and resuspended in 100 μL of a 0.1 M triethylammonium bicarbonate (TEAB) solution in water. The activated dye solution was added to the triphosphate, and the reaction was stirred at room temperature for up to 18 h and monitored by RP-HPLC. The crude product was purified by ion-exchange chromatography on DEAE-Sephadex A25 (25 g) eluting with a linear gradient of aqueous triethylammonium bicarbonate (TEAB, 0.1 M to 1 M). Fractions containing the triphosphate were pooled, and the solvent was evaporated to dryness under reduced pressure. The crude product was further purified by preparative-scale HPLC using a YMC-Pack-ProC18 column.

[0239] [ka]

[0240] ffA-sPA-LN3-(I-1): Yield: 8.5 μmol (53%). LC-MS (ES): (negative ion) m / z 1360 (MH + ), 680(M-2H + ). UV-visible λ max :507nm. Fluorescence λ max :566nm.

[0241]

change

[0242] ffA-LN3-(I-2): Yield: 1.9 μmol (38%). LC-MS (ES): (negative ion) m / z 1428 (MH) + UV-visible λ max :502nm. Yung Guang λ max 563nm.

[0243]

change

[0244] ffA-LN3-(I-3): Yield: 30.7 μmol (61%). LC-MS (ES): (negative ion) m / z 1470 (MH) + UV-visible λ max 501nm. Laser light (λ). max 565nm.

[0245]

change

[0246] ffA-LN3-(I-4): Yield: 1.4 μmol (29%). LC-MS (ES): (negative ion) m / z 1390 (MH) + UV-visible λ max :493nm. Yung Guang λ max 551nm.

[0247]

change

[0248] ffA-LN3-(I-5): Yield: 2.7 μmol (54%). LC-MS (ES): (negative ion) m / z 1348 (MH) + UV-visible λ max :491nm. Yung Guang λmax :555nm.

[0249] [ka]

[0250] ffA-sPA-LN3-(I-8): Yield: 7.8 μmol (78%). LC-MS (ES): (negative ion) m / z 1348 (MH + ). UV-visible λ max :495nm. Fluorescence λ max :556nm.

[0251] Example 3. Spectral properties of alkylpyridinium coumarin dyes In this example, the spectral characteristics of several alkylpyridinium coumarin dyes described herein were compared with the corresponding unmethylated reference dyes. In Figures 1A and 1B, the fluorescence emission of methylpyridinium coumarin dye I-1 in a solution of Universal Scan Mix (USM, 1 M Tris pH 7.5, 0.05% TWEEN, 20 mM sodium ascorbate, 10 mM ethyl gallate) was compared with that of 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-Visible Spectrophotometer and a Cary Eclipse Fluorescence Spectrophotometer using a quartz cuvette. It was observed that dye I-1 exhibited an approximately two-fold increase in fluorescence emission upon green light excitation and an approximately three-fold increase in fluorescence emission upon blue light excitation compared to reference dye A.

[0252] Similarly, Figures 2A and 2B show the fluorescence emission of methylpyridinium coumarin dye I-5 in USM solution compared to that of Reference Dye C at excitation wavelengths of 450 nm and 520 nm, respectively. Figures 3A and 3B show the fluorescence emission of methylpyridinium coumarin dye I-8 in USM solution compared to that of Reference Dye B at excitation wavelengths of 450 nm and 520 nm, respectively. Coumarin dye I-5 exhibited an approximately 2.5-fold increase in fluorescence emission upon green light excitation and an approximately 8-fold increase in fluorescence emission upon blue light excitation compared to Reference Dye C. Coumarin dye I-8 exhibited similar fluorescence emission upon green light excitation and an approximately 2.5-fold increase in fluorescence emission upon blue (450 nm) light excitation compared to Reference Dye B.

[0253] [ka]

[0254] Example 4. Spectral properties of alkylpyridinium coumarin-conjugated ffA nucleotides In this example, the spectral characteristics of several fully functionalized A nucleotides (ffAs) conjugated with the methylpyridinium coumarin dyes described herein were compared with the corresponding unmethylated reference dyes. In Figures 4A and 4B, the fluorescence emission of ffAs conjugated with the methylpyridinium coumarin dye I-1 as a 2 μM solution in USM solution was compared with that of 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-Visible Spectrophotometer and a Cary Eclipse Fluorescence Spectrophotometer using a quartz cuvette. It was observed that ffA-sPA-LN3-(I-1) exhibited similar fluorescence emission upon green light excitation and an approximately threefold increase in fluorescence emission upon blue light excitation compared to ffA-sPA-LN3-(Reference Dye A).

[0255] Similarly, Figures 5A and 5B show the fluorescence emission of ffA conjugated with methylpyridinium coumarin dye I-5 as a 2 μM solution in USM solution compared to that of Reference Dye C at excitation wavelengths of 450 nm and 520 nm, respectively. Figures 6A and 6B show the fluorescence emission of ffA conjugated with methylpyridinium coumarin dye I-8 as a 2 μM solution in USM solution compared to that of Reference Dye B at excitation wavelengths of 450 nm and 520 nm, respectively. Figures 7A and 7B show the fluorescence emission of ffA conjugated with methylpyridinium coumarin dye I-3 as a 2 μM solution in USM solution compared to that of Reference Dye B at excitation wavelengths of 450 nm and 520 nm, respectively. Compared with ffA-sPA-LN3-(Reference Dye C), ffA-LN3-(I-5) exhibited similar fluorescence emission upon excitation with green light (520 nm) and an approximately 2.5-fold increase in fluorescence emission upon excitation with blue light (450 nm). Compared with ffA-sPA-LN3-(Reference Dye B), ffA-sPA-LN3-(I-8) exhibited similar fluorescence emission upon excitation with green light (520 nm) and an approximately 1.6-fold increase in fluorescence emission upon excitation with blue light (450 nm). Compared with ffA-sPA-LN3-(Reference Dye D), ffA-sPA-LN3-(I-3) exhibited 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).

[0256] [ka]

[0257] Example 5. Sequencing experiments on an Illumina iSeq™ 100 instrument In this example, the ffA-linker dye compounds described herein were tested on an Illumina iSeq™ 100 instrument configured to take a first image with green excitation light (approximately 520 nm) and a second image with blue excitation light (approximately 450 nm). The sequencing strategy was modified to perform a standard SBS cycle (incorporation, followed by imaging, followed by cleavage). The incorporation mixtures used in each of these experiments contained the following four ffNs: 1) ffA conjugated with an alkylpyridinium dye described herein or a reference dye described herein, 2) ffC excitable with blue light at 450 nm when ffA conjugated with an alkylpyridinium dye described herein is used (e.g., ffC-linker-coumarin dye E), and when ffA conjugated with a reference dye described herein is used (ffC-linker-coumarin dye F), 3) ffT excitable with green light (e.g., ffT-LN3-NR550s0), 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 MgSO, and DNA polymerase. Coumarin dyes E and F are disclosed in U.S. Patent Application Publication No. 2018 / 0094140 and, when conjugated with ffC, each have the structural moiety:

[0258] [ka] Figures 8A and 8B show the scatter plots obtained for incorporation mixtures containing ffA-sPA-LN3-(I-1) and ffA-sPA-LN3-(Reference Dye A), respectively. Figures 8C and 8D show the scatter plots obtained for incorporation mixtures containing ffA-sPA-LN3-(I-3) and ffA-sPA-LN3-(Reference Dye D), respectively. In both examples, incorporation mixtures containing ffA conjugated with alkylpyridinium coumarin dyes were observed to provide increased intensity and better separation of the A cloud compared to the corresponding reference dyes without methyl substitutions on the pyridinium moiety.

[0259] Table 1 shows metrics of phasing, prephasing, and PhiX error rates for ffA-sPA-LN3-(I-1), ffA-LN3-(I-3), and ffA-sPA-(Reference Dye D) for 2 x 151 cycles on an Illumina iSeq™ 100 instrument compared to metrics obtained from 2 x 151 cycles run on an iSeq™ 100 instrument using standard reagents and standard methods. The integration mixtures used in each of these experiments contained the following four ffNs: 1) ffA conjugated to an alkylpyridinium dye described herein or conjugated to a reference dye described herein, 2) ffC that is excitable with blue light at 450 nm when ffA conjugated to an alkylpyridinium dye described herein is used (e.g., ffC-linker-coumarin dye E), and when ffA conjugated to a reference dye described herein is used, ffC-linker-coumarin dye F, 3) ffT that is excitable with green light (e.g., ffT-LN3-NR550s0), 4) unlabeled ffG (dark ffG) in 50 mM ethanolamine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO and DNA polymerase. Improved PhiX error rate metrics were observed with compounds ffA-sPA-LN3-(I-1) and ffA-LN3-(I-3) compared to ffA-sPA-(Reference Dye D) and standard iSeq™ 100 reagents.

[0260] [Table 1] Additionally, compound ffA-sPA-LN3-(I-1) was selected for 2 x 300 cycle runs on an Illumina iSeq™ 100 instrument. The instrument was set to capture a first image with green excitation light and a second image with blue excitation light, and the strategy was modified to perform 2 x 300 standard SBS cycles (incorporation, followed by imaging, followed by cleavage). The incorporation mixture used in these experiments contained the following four ffNs: 1) ffA-sPA-LN3-(I-1), 2) ffC excitable with 450 nm blue light (e.g., ffC-linker-coumarin dye E), 3) ffT excitable with green light (e.g., ffT-linker-NR550s0), and 4) dark ffG in 50 mM ethanolamine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO, and DNA polymerase. Quantification of phasing, prephasing, PhiX error rate, and Q30% is shown in Table 2 below.

[0261] [Table 2]

Claims

1. A compound of formula (I), a salt thereof, or a mesomeric form thereof, 【Chemistry 1】 In the formula, R １ teeth, 【Chemistry 2】 and R １ is one or more C １ ~C ６ is substituted with alkyl, Each R ２ , R ５ , R6 and R ７ is H, R ３ and R ４ each independently represents H, C １ ~C ６ Alkyl or substituted C １ ~C ６ is alkyl, Alternatively, R ４ and R ５ together with the atom to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 6- to 10-membered heterocyclyl; R ａ , R ｂ and R ｃ Each of the groups is independently １ ~C ６ Alkyl or substituted C １ ~C ６ The compound, its salt, or mesomeric form, which is alkyl.

2. R ３ is H, and R ４ But, C １ ~C ６ Alkyl or substituted C １ ~C ６ 2. The compound of claim 1, wherein R 3 and R 4 are independently C 1 -C 6 alkyl.

3. The compound of formula (I) has the formula (Ia) 【Chemistry 3】 Also represented by its salts or mesomeric forms, wherein Each R ８ , R ９ , R １０ and R １１ are independently H, C １ ~C ６ Alkyl, substituted C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, C ２ ~C ６ Alkenyl, C ２ ~C ６ Alkynyl, C １ ~C ６ Haloalkyl, C １ ~C ６ Haloalkoxy, (C １ ~C ６ Alkoxy)C １ ~C ６ Alkyl, optionally substituted amino, amino (C １ ~C ６ alkyl), halo, cyano, hydroxy, hydroxy(C １ ~C ６ alkyl), nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido, or N-sulfonamido; Solid and dashed lines 【Chemistry 4】 is selected from the group consisting of a single bond and a double bond, with the proviso that 【Chemistry 5】 When is a double bond, R １１ The compound of claim 1 , provided that: is not present.

4. R １ but, 【Chemistry 6】 and optionally each R a and R b is independently C 1 to C 6 alkyl substituted with carboxyl (-C(O)OH), carboxylate (-C(O)O - ), sulfo (-SO 3 H) or sulfonate (-SO 3 - ).

5. Solid and dashed lines 【Chemistry 7】 5. The compound according to claim 3 or 4, wherein the bond represented by is a single bond or a double bond.

6. R １０ is H or C １ ~C ６ The compound of claim 3, wherein the aryl group is an alkyl group.

7. R １０ is H, and R １１ But, C １ ~C ６ 7. The compound of claim 6, wherein each of R10 and R11 is H or alkyl.

8. R ８ and R ９ The compound of any one of claims 3 to 7, wherein each of R 8 and R 9 is H or at least one of R 8 and R 9 is C 1 -C 6 alkyl.

9. R ８ and R ９ The compound of claim 3 , wherein each of is methyl.

10. R ３ But, C １ ~C ６ alkyl or substituted C 1 -C 6 alkyl; or R 3 is C 1 -C 6 alkyl substituted with one or more substituents selected from the group consisting of carboxyl (-C(O)OH), carboxylate (-C(O)O - ), sulfo (-SO 3 H), sulfonate (-SO 3 - ), -C(O)OR 12 , and -C(O)NR 13 R 14 , R 12 is optionally substituted C 1 -C 6 alkyl, optionally substituted C 6 -C 10 aryl, optionally substituted 5-10 membered heteroaryl, or optionally substituted C 3 -C 7 cycloalkyl, and each of R 13 and R 14 is independently H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 6 -C 10 aryl, optionally substituted 5-10 membered heteroaryl, or optionally substituted C 3 -C 7 cycloalkyl; The compound according to any one of claims 3 to 9.

11. R ３ is carboxyl or -C(O)NR １３ R １４ C substituted with １ ~C ６ alkyl, and each R １３ and R １４ are independently carboxyl, carboxylate, -C(O)OR １２ , C substituted with sulfo or sulfonate １ ~C ６ The compound of claim 10 which is alkyl.

12. 【Fig. 8】 2. The compound of claim 1 selected from: and their salts and mesomeric forms.

13. Optionally, the compound is linked to the nucleotide or oligonucleotide via a carboxyl group of R 8a , R 8b or R 8c of formula (I) or via a carboxyl group of R 3 or R 4 of formula (I); Optionally, the compound is attached via a linker moiety to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base; Optionally, further comprising a 3'OH blocking group covalently attached to the ribose or deoxyribose sugar of said nucleotide. A nucleotide or oligonucleotide labeled with the compound according to any one of claims 1 to 12.

14. the nucleotide or oligonucleotide is an oligonucleotide hybridized to at least a portion of a target polynucleotide; the target polynucleotide is immobilized on a solid support; the solid support comprises an array of a plurality of immobilized target polynucleotides; The oligonucleotide of claim 13.

15. A kit comprising a first nucleotide labeled with a first compound according to any one of claims 1 to 12.

16. The kit of claim 15, a second nucleotide labeled with a second compound, said second compound being different from said first compound of said first labeled nucleotide; Optionally, the first labeled nucleotide and the second labeled nucleotide are excitable using a first light source wavelength; Optionally, the kit further comprises a third nucleotide, said third nucleotide being labeled with a third compound different from said first and said second compounds, said first and third labeled nucleotides being excitable using a second light source wavelength; Optionally, the kit further comprises a fourth nucleotide, said fourth nucleotide being unlabeled (dark colored); kit.

17. The kit according to claim 15 or 16, each of the first labeled nucleotide, the second labeled nucleotide, and the third labeled nucleotide has an emission spectrum that is detectable in a single detection channel; or The kit further comprises a DNA polymerase and one or more buffer compositions. kit.

18. 1. A method for determining a sequence of a target polynucleotide, comprising: (a) contacting a primer polynucleotide / target polynucleotide complex with one or more labeled nucleotides, the primer polynucleotide / target polynucleotide complex being formed by contacting the target polynucleotide with a primer polynucleotide complementary to at least a portion of the target polynucleotide, at least one of the labeled nucleotides being a nucleotide according to claim 13 or 14, the primer polynucleotide being complementary to at least a portion of the target polynucleotide; (b) incorporating a labeled nucleotide into said primer polynucleotide to generate an extended primer polynucleotide / target polynucleotide complex; (c) performing one or more fluorescence measurements of the extended primer polynucleotide / target polynucleotide complex to determine the identity of the incorporated nucleotide; (d) optionally removing the label and the 3' blocking group from the nucleotides incorporated into the primer polynucleotide; (e) optionally washing the removed label and the 3' blocking group from the extended primer polynucleotide; Including, Optionally, repeating steps (a) through (e) until the sequence of at least a portion of the template polynucleotide strand is determined; Optionally, repeating steps (a) to (e) at least 50 times; Optionally, the method is carried out in an automated sequencing instrument, the automated sequencing instrument comprising two light sources operating at different wavelengths; Optionally, the method is performed in an array format, wherein a plurality of target polynucleotides are immobilized on a solid support. method.

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