Long Stokes shift chromenoquinoline dyes and their use in sequencing applications.

Abstract

This application relates to long Stokes shift chromenoquinoline dyes and their use as fluorescent labels. For example, these dyes can be used to label nucleotides for nucleic acid sequencing applications. The chromenoquinoline dyes have the formula (I):

Description

[Technical Field]

[0001] This disclosure relates to chromenoquinoline dyes and their use as fluorescent labels. Specifically, the compounds may be used as nucleotide labels for nucleic acid sequencing applications. [Background technology]

[0002] Non-radioactive detection of fluorescently labeled nucleic acids is an important technique in molecular biology. Many of the procedures used in recombinant DNA technology were previously, for example, 32 The method relied on the use of nucleotides or polynucleotides radiolabeled with 3P. Radioactive compounds enable highly sensitive detection of nucleic acids and other molecules of interest. However, the use of radioisotopes has significant limitations, including their cost, limited shelf life, insufficient sensitivity, and, more importantly, safety considerations. Eliminating the need for radiolabeling reduces both safety risks and environmental impacts, as well as costs associated with, for example, reagent disposal. Suitable non-radioactive fluorescence detection methods, in non-limiting examples, include 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 spectroscopically distinguishable fluorescent labels to achieve independent detection of multiple spatially overlapping analytes. Such multiplexing methods can reduce the number of reaction vessels, simplify experimental protocols, and facilitate the creation of application-specific reagent kits. For example, in multicolor automated DNA sequencing systems, multiplex fluorescence detection allows for the analysis of multiple nucleotide bases in a single electrophoresis lane, thereby increasing throughput and reducing uncertainty associated with variations in inter-lane electrophoresis mobility compared to monocolor methods.

[0004] However, multiplex fluorescence detection can present challenges, and several key factors constrain the selection of appropriate fluorescent labels. Firstly, in a given application, it can be difficult to locate dye compounds with substantially degraded absorption and emission spectra. In addition, when several fluorescent dyes are used together, the absorption bands of the dyes are usually widely separated, making it complex to generate fluorescent signals in a spectral region distinguishable by simultaneous excitation, and thus difficult to achieve equivalent fluorescence excitation efficiency even for two dyes. Since many excitation methods use high-power light sources such as lasers, the dyes must have sufficient photostability to withstand such excitation. A particularly important final consideration for molecular biology methods is the degree to which the fluorescent dyes must be compatible 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 further fluorescent dye compounds, their nucleic acid conjugates, and sets of dyes that satisfy all of 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 suitable fluorescence intensity, shape, and maximum wavelength of the fluorescence band, can improve the speed and accuracy of nucleic acid sequencing. Strong fluorescence signals are particularly important when measurements are performed in aqueous biological buffers and at high temperatures, as the fluorescence intensity of most organic dyes is significantly lower under such conditions. Furthermore, the properties of the base to which the dye is bound also affect the maximum fluorescence value, fluorescence intensity, and other spectral properties of the dye. Sequence-specific interactions between nucleic acid bases and fluorescent dyes can be tuned through the specific design of the fluorescent dye. Optimizing the structure of fluorescent dyes can improve nucleotide incorporation efficiency, reduce the level of sequencing errors, decrease the amount of reagents used in nucleic acid sequencing, and consequently lower the cost of nucleic acid sequencing.

[0007] While several optical and technological advancements have significantly improved image quality, they have ultimately been limited by insufficient optical resolution. Generally, the optical resolution of an optical microscope is limited to the level at which objects separated by approximately half the wavelength of the light used can be distinguished. In practice, only objects that are very far apart from each other (at least 200-350 nm) can be distinguished by optical microscopy. One way to improve image resolution and increase the number of objects that can be resolved per unit surface area is to use excitation light with a shorter wavelength. For example, using the same optical system, shortening the wavelength by Δλ of approximately 100 nm results in better resolution (approximately Δ50 nm / (approximately 15%)), recording a less distorted image, and increasing the density of objects on 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, along with a suitable dye that can be excited 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, the optical resolution is limited not by the emission wavelength of the long-wavelength red fluorescent dye, but rather by the emission of the dye that can be excited by the next longer wavelength light source, for example, a 532 nm "green laser." Therefore, blue dye labeling is required for use in fluorescence detection for sequencing applications.

[0009] Chromenoquinoline dyes have been reported in the literature for use as fluorescent probes or mitochondrial labels. See Geng et al., Sensors & Actuators: B. Chemical 273 (2018) 1670 - 1675 and Liu et al., Chemical Communications 2018, 54(12): 1509 - 1512. Nevertheless, most of these chromenoquinoline dyes have red emission, and their stability under aqueous conditions for nucleic acid sequencing remains unconfirmed. Therefore, designing chromenoquinoline dyes with tailored adsorption wavelengths and a good fluorescence Stokes shift with good stability remains an important issue in dye development. SUMMARY OF THE INVENTION

[0010] This specification describes chromenoquinoline dyes having a long Stokes shift, as well as improved fluorescence intensity and chemical stability suitable for nucleotide labeling and sequencing applications. These chromenoquinoline dyes have strong fluorescence under excitation by both blue light and green light (e.g., these chromenoquinoline dyes can 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). Further, these chromenoquinoline dyes have higher stability in high pH buffers compared to commercially available dyes used in synthetic sequencing.

[0011] Some aspects of the present disclosure relate to the compound of formula (I), or a salt thereof, or a mesomeric form,

[0012]

Chemical formula

[0013] [ka] To have R 1 and R 2 Together with the atoms to which they are bonded, they form piperidinyl and R, 3 and R 4 Together with the atoms to which they are bonded, they form piperidinyl, R 5 , R 7 , R 8 , R 10 , R 11 , R 12a and R 12b Each of them is H, and R 6 However, it is methyl, and therefore, R 9 However, it is a substituted C1-C6 alkyl group containing a carboxyl molecule.

[0014] In some embodiments, the compound of formula (I) is also represented by formula (Ia) or its salt or mesomeric form.

[0015] [ka] In the formula, R 13a , R 13b , R 14a and R 14b Each of these is independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted amino, amino(C1-C6 alkyl), halo, cyano, hydroxy, hydroxy(C1-C6 alkyl), nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamide, or N-sulfonamide, and is represented by solid and dashed lines.

[0016] [ka] The bond represented by is selected from the group consisting of single bonds and double bonds, however,

[0017] [ka] If it is a double bond, R 14b This is conditional on it not existing.

[0018] In some embodiments of the compounds of formula (I) or (Ia), R 6 , R 7 , R 8 , R 9 , R 10 and R 11 One of them contains a carboxyl group (-C(O)OH). In other embodiments, R 2 or R 3 Contains a carboxyl group.

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

[0020] Some other aspects of this disclosure relate, for example, to dye compounds comprising a linker group for enabling covalent bonding to a substrate portion. Linking can be carried out at any position of the dye, comprising any of the R groups. In some embodiments, linking is made of the R of formula (I). 6 , R 7 , R 8 , R 9 , R 10 and R11 via one of the following, or R 2 or R 3 This can be carried out via R of equation (Ia). In some further embodiments, the concatenation is performed by R of equation (Ia). 6 , R 7 , R 8 , R 9 , R 10 and R 11 via one of the following, or R 2 It can be implemented through [method].

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

[0022] Some additional aspects of this disclosure relate to nucleotides or oligonucleotides labeled with compounds of formula (I) or (Ia).

[0023] Some additional aspects of this disclosure relate to kits comprising dye compounds (in free or labeled form) that can be used in various immunoassays, oligonucleotide or nucleic acid labeling, or for synthetic DNA sequencing. In yet another aspect, this disclosure provides kits comprising dye “sets” particularly suited for synthetic sequencing cycles on automated instrument platforms. In some aspects, the kits comprise one or more nucleotides, wherein at least one nucleotide is a labeled nucleotide as described herein.

[0024] Further aspects of this disclosure are methods for determining the sequence of a target polynucleotide, (a) Contacting a 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 a chromoquinoline dye of formula (I) or (Ia), 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 / target polynucleotide complex, (c) A method comprising performing one or more fluorescence measurements on an extended primer polynucleotide / target polynucleotide complex to determine the identity of the incorporated nucleotide. [Brief explanation of the drawing]

[0025] [Figure 1] The absorption spectra of ffA nucleotides conjugated with chromenoquinoline dyes I-1 to I-6 and I-8 as a 2 μM solution in Universal Scan Mix (USM) are shown. [Figure 2A] The fluorescence emission spectra of ffA nucleotides conjugated with chromenoquinoline dyes I-1, I-2, I-5, and I-6, obtained in USM using either 520 nm or 450 nm as the excitation wavelength, are shown. [Figure 2B] The fluorescence emission spectra of ffA nucleotides conjugated with chromenoquinoline dyes I-1, I-2, I-5, and I-6, obtained in USM using either 520 nm or 450 nm as the excitation wavelength, are shown. [Figure 2C] The fluorescence emission spectra of ffA nucleotides conjugated with chromenoquinoline dyes I-3, I-4, and I-8, obtained in USM using either 520 nm or 450 nm as the excitation wavelength, are shown. [Figure 2D] The fluorescence emission spectra of ffA nucleotides conjugated with chromenoquinoline dyes I-3, I-4, and I-8, obtained in USM using either 520 nm or 450 nm as the excitation wavelength, are shown. [Figure 3A] The residual fluorescence percentages of two ffA samples conjugated with chromenoquinoline dyes I-3 and I-4 in ethanolamine buffer at 45°C are shown, compared to ffC conjugated with coumarin reference dye A under the same conditions. [Figure 3B] The residual fluorescence percentages of two ffA samples conjugated with chromenoquinoline dyes I-3 and I-4 in glycine buffer at 45°C are shown, compared to ffC conjugated with coumarin reference dye A under the same conditions. [Figure 3C] The residual fluorescence percentages of two ffA samples conjugated with chromenoquinoline dyes I-3 and I-4 in TRIS buffer at 40°C are shown, compared to ffC conjugated with coumarin reference dye A under the same conditions. [Figure 3D] This shows the residual fluorescence percentage of two ffA samples conjugated with chromenoquinoline dyes I-3 and I-4 in MOPS buffer at 40°C, compared to ffC conjugated with coumarin reference dye A under the same conditions. [Figure 4A] The scattering plots obtained using an Illumina iSeq™ 100 instrument are shown, using an incorporation mixture of ffA nucleotides labeled with chrome quinoline dyes I-1, I-2, I-3, I-4, and I-5, respectively. [Figure 4B] The scattering plots obtained using an Illumina iSeq™ 100 instrument are shown, using an incorporation mixture of ffA nucleotides labeled with chrome quinoline dyes I-1, I-2, I-3, I-4, and I-5, respectively. [Figure 4C]The scattering plots obtained using an Illumina iSeq™ 100 instrument are shown, using an incorporation mixture of ffA nucleotides labeled with chrome quinoline dyes I-1, I-2, I-3, I-4, and I-5, respectively. [Figure 4D] The scattering plots obtained using an Illumina iSeq™ 100 instrument are shown, using an incorporation mixture of ffA nucleotides labeled with chrome quinoline dyes I-1, I-2, I-3, I-4, and I-5, respectively. [Figure 4E] The scattering plots obtained using an Illumina iSeq™ 100 instrument are shown, using an incorporation mixture of ffA nucleotides labeled with chrome quinoline dyes I-1, I-2, I-3, I-4, and I-5, respectively. [Figure 4F] The phasing and pre-phasing metricing over 1 × 300 cycles is demonstrated using an incorporation mixture of ffA nucleotides labeled with chrome quinoline dyes I-1, I-2, I-3, I-4, and I-5. [Modes for carrying out the invention]

[0026] Embodiments of this disclosure relate to chromenoquinoline dyes having enhanced fluorescence intensity, a long Stokes shift, and improved chemical stability, particularly in high pH aqueous environments. These chromenoquinoline dyes also have a broad excitation wavelength and can be excited by both blue and green light sources. In some embodiments, the chromenoquinoline dyes described herein can be used with Illumina's iSeq® platform, which has two-channel CMOS detection (green light excitation and blue light excitation).

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

[0028] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless explicitly and clearly limited to a single subject. It will be apparent to those skilled in the art that various modifications and changes can be made to the various embodiments described herein without departing from the spirit or scope of this teaching. Accordingly, the various embodiments described herein are intended to encompass other modifications and variations 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 those commonly understood by those skilled in the art. The use of the term “including,” and other forms such as “include,” “includes,” and “included,” is not limited. Furthermore, the use of the term “having,” and other forms such as “have,” “has,” and “had,” is not limited. When used herein, in transitional clauses or in the text 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 “at least have” or “at least include.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the listed steps, but may include additional steps. When used in the context of compounds, compositions, or devices, the term "comprising" means that the compound, composition, or device includes at least the listed features or components, but may also include additional features or components.

[0030] As used herein, common organic abbreviations are defined as follows:

[0031] [Table 1]

[0032] 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 may include different probe molecules, each located at different addressable positions on a substrate. Alternatively, or additionally, an array may include separate substrates, each having a different probe molecule, which may be identified according to the position of the substrate on the surface to which it 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 in U.S. Patent No. 6,355,431(B1), U.S. Patent Application Publication No. 2002 / 0102578, and PCT International Publication No. 00 / 63437. Exemplary forms that may 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) are described, for example, in U.S. Patent No. 6,524,793. Further examples of arrays that may 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 6,2 Examples include, but are not limited to, those described in Patent Nos. 97,006, 6,291,193, 6,346,413, 6,416,949, 6,482,591, 6,514,751 and 6,610,482, as well as International Publication Nos. 93 / 17126, 95 / 11995, 95 / 35505, European Patent No. 742287 and European Patent No. 799897.

[0033] As used herein, the term “covalently attached” or “covalently bonded” refers to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently bonded polymer coating refers to a polymer coating that forms a chemical bond with the functionalized surface of a substrate, compared to bonding to the surface by other means, such as adhesion or electrostatic interaction. It will be understood that polymers covalently bonded to a surface may be bonded by means other than covalent bonding.

[0034] As used herein, the term "halogen" or "halo" means one of the elements of Group 7 of the periodic table, for example, fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred.

[0035] As used herein, "a" and "b" are integers. a ~C b"C1-C4 alkyl" 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, alkyl, alkenyl, alkynyl, cycloalkyl rings, and aryl rings can contain carbon atoms at both ends, "a" to "b". For example, "C1-C4 alkyl" groups refer to all alkyl groups having 1 to 4 carbon atoms, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-, and "C3-C4 cycloalkyl" refers to all cycloalkyl groups having 3 to 4 carbon atoms, i.e., cyclopropyl and cyclobutyl. Similarly, "4-6 membered heterocyclyl" groups refer to all heterocyclyl groups having 4 to 6 whole-ring atoms, such as azetidine, oxetane, oxazoline, pyrrolidine, piperidine, piperazine, morpholine, etc. If “a” and “b” are not specified with respect to alkyl, alkenyl, alkynyl, cycloalkyl, or aryl groups, the broadest range described in those definitions should be assumed. As used herein, the term “C1-C6” includes the range defined by C1, C2, C3, C4, C5, and C6, and any 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. Each C3-C8 cycloalkyl group includes a hydrocarbon ring containing 3, 4, 5, 6, 7, and 8 carbon atoms, or a range defined by either two numbers, such as C3-C7 cycloalkyl or C5-C6 cycloalkyl.

[0036] As used herein, “alkyl” refers to a fully saturated (i.e., non-double or non-triple) straight or branched hydrocarbon chain. Alkyl groups may have 1 to 20 carbon atoms (wherein always, numerical ranges such as “1 to 20” refer to each integer within a given range. For example, “1 to 20 carbon atoms” means that an alkyl group may consist of up to 20 carbon atoms, such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., but this definition also means that it covers the occurrence of the term “alkyl” (without specifying a numerical range). Alkyl groups may also be medium alkyl groups having 1 to 9 carbon atoms. Alkyl groups may also be lower alkyl groups having 1 to 6 carbon atoms. For example, “C1-6 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, and hexyl.

[0037] As used herein, “alkoxy” means “C1-9 alkoxy” or “C 1- The term "-OR" refers to formulas such as "C9 alkoxy," and includes, but is not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy.

[0038] As used herein, “alkenyl” refers to a straight or branched hydrocarbon chain containing one or more double bonds. An alkenyl group may have 2 to 20 carbon atoms, but this definition also covers occurrences of the term “alkenyl” where no numerical range is specified. An alkenyl group may also be a medium alkenyl having 2 to 9 carbon atoms. An alkenyl group may also be a lower alkenyl having 2 to 6 carbon atoms. For example, “C 2- "C6 alkenyl" or "C 2-6 The term "alkenyl" indicates that the alkenyl chain contains 2 to 6 carbon atoms. Specifically, 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, and hexenyl.

[0039] As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. While an alkynyl group may have 2 to 20 carbon atoms, this definition also covers occurrences of the term "alkynyl" where no numerical range is specified. An alkynyl group may also be a medium-sized alkynyl having 2 to 9 carbon atoms. An alkynyl group may also be a lower alkynyl having 2 to 6 carbon atoms. For example, "C 2-6 "Alkinyl" or "C 2-"C6 alkynyl" indicates that there are 2 to 6 carbon atoms in the alkynyl chain, meaning the alkynyl chain is selected from the group consisting of ethynyl, propyne-1-yl, propyne-2-yl, butyne-1-yl, butyne-3-yl, butyne-4-yl, and 2-butynyl. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0040] The term "aromatic" refers to a ring or ring system that has a conjugated π-electron system and contains both carbocyclic aromatic groups (e.g., phenyl) and heterocyclic aromatic groups (e.g., pyridine). This term includes monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of atoms) groups, as long as the entire ring system is aromatic.

[0041] As used herein, “aryl” refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent carbon atoms) containing only carbon atoms in its ring skeleton. If an aryl is a ring system, all rings in the system are aromatic rings. While an aryl group may have 6 to 18 carbon atoms, this definition also covers occurrences of the term “aryl” where no numerical range is specified. In some embodiments, an aryl group has 6 to 10 carbon atoms. 6- C 10 "Aryl", "C6 or C 10 It may be specified as "aryl" or a similar notation. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azlenyl, and anthracenyl.

[0042] "Aralkyl" or "arylalkyl" is "C 7-14 These are aryl groups bonded as substituents via an alkylene group, such as "aralkyl," and include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, and naphthylalkyl groups. In some cases, the alkylene group is a lower alkylene group (i.e., C 1-6 (Alkylene group)

[0043] As used herein, “heteroaryl” refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent atoms) containing one or more heteroatoms, i.e., elements other than carbon, including but not limited to nitrogen, oxygen, and sulfur in its ring skeleton. If the heteroaryl is a ring system, all rings in the system are aromatic rings. A heteroaryl group may have 5 to 18 ring members (i.e., the number of atoms constituting the ring skeleton, including carbon atoms and heteroatoms), but this definition also covers occurrences of the term “heteroaryl” where no numerical range is specified. In some embodiments, a heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. A heteroaryl group may be designated as a “5-7 membered heteroaryl,” a “5-10 membered heteroaryl,” or similar notation. Examples of heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridadinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.

[0044] 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, isoxazolylylalkyl, and imidazolylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., C 1-6 (Alkylene group)

[0045] As used herein, "carbocyclic" means a non-aromatic ring or ring system containing only carbon atoms in the ring system skeleton. When the carbocyclic is a ring system, two or more rings can be integrally bonded in a fused, bridged, or spiro bonding manner. The carbocyclic may have any degree of saturation, provided that at least one ring in the ring system is not aromatic. Thus, examples of carbocyclics include cycloalkyl, cycloalkenyl, and cycloalkynyl. The carbocyclic group may have 3 to 20 carbon atoms, but this definition also covers the occurrence of the term "carbocyclic" for which no numerical range is specified. The carbocyclic group may also be a medium-sized carbocyclic having 3 to 10 carbon atoms. The carbocyclic group may also be a carbocyclic having 3 to 6 carbon atoms. The carbocyclic group may be specified by " 3-6 carbocyclic", " 3- C6 carbocyclic", or a similar notation. Examples of carbocyclic rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicyclo[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

[0046] As used herein, "cycloalkyl" means a fully saturated carbocyclic ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0047] As used herein, “heterocyclyl” means a non-aromatic ring or ring system containing at least one heteroatom in its ring skeleton. Heterocyclyls may be joined together integrally by condensation, bridging, or spirobonding. Heterocyclyls may have any degree of saturation, provided that at least one ring in the ring system is not aromatic. Heteroatoms may be present in either the non-aromatic or aromatic ring in the ring system. A heterocyclyl group may have 3 to 20 ring members (i.e., the number of atoms constituting the ring skeleton, including carbon atoms and heteroatoms), but this definition also covers occurrences of the term “heterocyclyl” without a specified numerical range. A heterocyclyl group may also be a medium-sized heterocyclyl having 3 to 10 ring members. A heterocyclyl group may also be a heterocyclyl having 3 to 6 ring members. A heterocyclyl group may be designated as a “3-6 membered heterocyclyl” or similar notation. In a preferred six-membered monocyclic heterocycline, the heteroatoms are selected from one to three of O, N, or S. In a preferred five-membered monocyclic heterocycline, the heteroatoms are selected from one or two heteroatoms selected from O, N, or S.Examples of heterocyclic rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxapiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathiinyl, 1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-dioxinyl, trioxanyl, hexahydro-1,3,5-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.

[0048] As used herein, "alkoxyalkyl" or "(alkoxy)alkyl" refers to C 2- C8 alkoxyalkyl, or (C1-C6 alkoxy)C1-C6 alkyl, for example, -(CH2) 1-3 -OCH3 and other alkoxy groups connected via an alkylene group.

[0049] The "O-carboxy" group refers to the "-OC(=O)R" group, where R is selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-7 carbocyclic, C 6-10 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclic as defined herein.

[0050] The "C-carboxyl" group refers to the "-C(=O)OR" group, where R is hydrogen, C as defined herein. 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkinyl, C 3-7 Carbocyclyl, C 6-10 The group is selected from aryls, 5-10 membered heteroaryls, and 3-10 membered heterocyclines. A non-restrictive example is carboxyl (i.e., -C(=O)OH).

[0051] The "sulfonyl" group refers to the "-SO2R" group, where R is hydrogen, C as defined herein. 1-6 Alkyl, C 2-6 Alkenil, C 2-6 Alkinyl, C 3-7 Carbocyclyl, C 6-10 The selection is made from aryls, 5-10 membered heteroaryls, and 3-10 membered heterocyclines.

[0052] The "sulfino" group refers to the "-S(=O)OH" group.

[0053] The "sulfino" group refers to the "-S(=O)2OH" or "-SO3H" group.

[0054] The "sulfonate" group refers to the "-SO3-" group.

[0055] The "sulfate" group refers to the "-SO4-" group.

[0056] The "S-sulfonamide" group is "-SO2NR A R B " refers to the base, and in the formula, R A and R B These are, as defined herein, independently of each other, hydrogen and C 1-6 Alkyl, C 2-6 Alkenil, C 2-6 Alkinyl, C 3-7 Carbocyclyl, C 6-10 The selection is made from aryls, 5-10 membered heteroaryls, and 3-10 membered heterocyclines.

[0057] The "N-sulfonamide" group is "-N(R A )SO2R B " refers to the base, and in the formula, R A and R b These are, as defined herein, independently of hydrogen and C 1-6 Alkyl, C 2-6 Alkenil, C 2-6 Alkinyl, C 3-7 Carbocyclyl, C 6-10 The selection is made from aryls, 5-10 membered heteroaryls, and 3-10 membered heterocyclines.

[0058] The "C-amide" group is -C(=O)NR A R B " refers to the base, and in the formula, R A and R B These are, as defined herein, independently of hydrogen and C 1-6 Alkyl, C 2-6 Alkenil, C 2-6 Alkinyl, C 3-7 Carbocyclyl, C 6-10 The selection is made from aryls, 5-10 membered heteroaryls, and 3-10 membered heterocyclines.

[0059] The "N-amide" group is -N(R A )C(=O)R B " refers to the base, and in the formula, R A and R B These are, as defined herein, independently of hydrogen and C 1-6 Alkyl, C 2-6 Alkenil, C 2-6 Alkinyl, C 3-7 Carbocyclyl, C 6-10 The selection is made from aryls, 5-10 membered heteroaryls, and 3-10 membered heterocyclines.

[0060] The "amino" group is "-NR A R B " refers to the base, and in the formula, R A and R B These are, as defined herein, independently of hydrogen and C1-6 Alkyl, C 2-6 Alkenil, C 2-6 Alkinyl, C 3-7 Carbocyclyl, C 6-10 The compounds are selected from aryls, 5- to 10-membered heteroaryls, and 3- to 10-membered heterocyclines. A non-limiting example is free aminos (i.e., -NH2).

[0061] The "aminoalkyl" group refers to an amino group linked via an alkylene group.

[0062] The "alkoxyalkyl" group is "C 2- This refers to an alkoxy group connected via an alkylene group, such as a "C8 alkoxyalkyl" group.

[0063] Where a group is described as "optionally substituted," it may be either unsubstituted or substituted. Similarly, where a group is described as "substituted," the substituent may be selected from one or more of the substituents shown. As used herein, substituents are derived from an unsubstituted parent group in which one or more hydrogen atoms are exchanged for another atom or group. Unless otherwise stated, where a group is considered "substituted," it means that the group is C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C3-C7 carbocyrill (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), C3-C7-carbocyrill-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 (substituted with haloalkyl and C1-C6 haloalkoxy), 3-10 member heterocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3-10 member heterocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl (optionally, 5-10 member heteroaryl (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 member heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy) (substituted with coxy), 5-10 member heteroaryl(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), halo, -CN, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy(C1-C6)alkyl (i.e., ether), aryloxy, sulfhydryl (mercapto), halo(C1-C6)alkyl (e.g., -CF3),Halo(C1-C6)alkoxy (e.g., -OCF3), C1-C6 alkylthio, arylthio, amino, amino(C1-C6)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, O-carboxy, acyl, cyanate, isocyanate, thiocyanate, isothiocyanate, sulfinyl, sulfonyl, -SO3H, sulfonate, sulfate, sulfino, -OSO2C, 1- This means that the group is substituted with one or more substituents independently selected from C4 alkyl and oxo (=O). Whenever a group is described as "optionally substituted," that group may be substituted with the substituents described above. In some embodiments, when an alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocykyl, or heterocyclyl group is substituted, each independently means halo, -CN, -SO3 - , -OSO3 - -SO3H, -SR A , -OR A , -NR B R C , oxo, -CONR B R C -SO2NR B R C -COOH, and -COOR B Substituted with one or more substituents selected from the group consisting of, where R A , R B , and R C Each of these is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl.

[0064] As those skilled in the art will understand, the compounds described herein are in ionized form, for example, -CO2 - , -SO3 - Alternatively, it may exist as -O-SO3-. The compound may have positively or negatively charged substituents, for example, -SO3 -If the compound contains a counterion, it may contain a negatively or positively charged counterion so that the compound as a whole is neutral. In other embodiments, the compound may exist in salt form, and the counterion is provided by a conjugate acid or base.

[0065] It should be understood that certain radical nomenclature can include either monoradicals or diradicals depending on the context. For example, a substituent is understood to be a diradical if it requires two bonding sites to the rest of the molecule. Examples of substituents identified as alkyls that require two bonding sites include diradicals such as -CH2-, -CH2CH2-, and -CH2CH(CH3)CH2-. Other radical nomenclature explicitly indicates that the radical is a diradical, such as an "alkylene" or "alkenylene".

[0066] When two "adjacent" R groups are said to form a ring "together with the atom to which they are bonded," it means a set of atoms, an intervening bond, and a ring in which the two R groups are enumerated. For example, if the following substructure exists,

[0067] [ka] R 1 and R 2 It is defined as being selected from the group consisting of hydrogen and alkyl, or R 1 and R 2 These, together with the atoms to which they are bonded, form an aryl or carbocykrine, and this is R 1 and R 2 However, this can be selected from hydrogen or alkyl, or alternatively, the substructure means that the following structure is present:

[0068] [ka] In the formula, A is a carbocyclyl containing an aryl ring or the indicated double bond.

[0069] When a substituent is shown as a diradical (i.e., having two bonding points to the rest of the molecule), it should be understood that unless otherwise specified, the substituent can be bonded in any directional configuration. For example, -AE- or

[0070] [ka] The substituents shown as indicate substituents that are oriented so that A is attached at the leftmost bond point of the molecule, as well as the case where A is attached at the rightmost bond point of the molecule. In addition, the group or substituent is

[0071] [ka] As shown, L is defined as an optionally present linker portion, and if L is absent (or not present), such a group or substituent is

[0072] [ka] It is equivalent to [this].

[0073] In each instance where a single mesomelic form of a compound described herein is shown, alternative mesomelic forms are also intended.

[0074] As used herein, “nucleotide” comprises a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. They are monomeric units of nucleic acid sequences. In RNA, the sugar is ribose, and in DNA, it is deoxyribose, i.e., a sugar lacking the hydroxyl group present in ribose. Nitrogen-containing heterocyclic bases can be purines, deazapurines, or pyrimidine bases. Purine bases include adenine (A) and guanine (G), and their modified derivatives or analogs such as 7-deazaadenine or 7-deazaguanine. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and their modified derivatives or analogs. The C-1 atom of deoxyribose is bonded to N-1 of pyrimidine or N-9 of purine.

[0075] As used herein, “nucleoside” refers to a molecule structurally similar to a nucleotide but lacking a phosphate moiety. Examples of nucleoside analogs include those in which the label is linked to a base and there is no phosphate group attached to a sugar molecule. The term “nucleoside” is used herein in the 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. A modified pentose moiety is a pentose moiety in which an oxygen atom is replaced by a carbon atom and / or a carbon atom is replaced by 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.

[0076] The term “purine base” is used herein in the ordinary sense as understood by those skilled in the art, and includes its tautomers. Similarly, the term “pyrimidine base” is used herein in the ordinary sense as understood by those skilled in the art, and includes its tautomers. An unrestricted list of optionally substituted purine bases includes purines, adenines, guanines, deazapurines, 7-deazaadenines, 7-deazaguanines, hypoxanthines, xanthines, alloxanthines, 7-alkylguanines (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanines. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine).

[0077] When used herein, if an oligonucleotide or polynucleotide is described as “containing” 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, if a nucleoside or nucleotide is described as part of an oligonucleotide or polynucleotide, such as “integrated” into the 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' hydroxyl group of the oligonucleotide or polynucleotide and the 5' phosphate group of the nucleotide described herein, as a phosphate diester linkage between the 3' carbon atom of the oligonucleotide or polynucleotide and the 5' carbon atom of the nucleotide.

[0078] As used herein, the term “cleavable linker” does not mean that the entire linker must be removed. The cleavage site may be located on the linker in a position that ensures that a portion of the linker remains bound to a detectable dye and / or nucleoside or nucleotide portion after cleavage.

[0079] As used herein, “derivative” or “analog” means 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. Nucleoside analogs may also include modified phosphate diester linkages, including phosphorothioates, phosphorodithioates, alkyl phosphonates, phosphoranilidates, and phosphoramidate linkages. As used herein, “derivative,” “analog,” and “modified” may be used interchangeably and are encompassed by the terms “nucleotide” and “nucleoside” as defined herein.

[0080] As used herein, the term "phosphate" is used in the ordinary sense as understood by those skilled in the art, and refers to its protonated form.

[0081] [ka] This includes. As used herein, the terms “monophosphate,” “diphosphate,” and “triphosphate” are used in the ordinary sense as understood by those skilled in the art, and include protonated forms.

[0082] As used herein, the term "phasing" refers to a phenomenon in SBS caused by incomplete removal of the 3' terminator and fluorophore, and / or poor completion of incorporation of a portion of the DNA strands within a cluster by polymerase in a given sequencing cycle. Prefasing is caused by incorporating nucleotides without an effective 3' terminator, and the incorporation event proceeds one cycle ahead due to poor termination. Phasing and prefasing cause the measured signal intensity for a particular cycle to consist of the signal from the current cycle, as well as noise from the previous and next cycles. As the number of cycles increases, the proportion of sequences per cluster affected by phasing and prefasing increases, hindering the identification of the correct bases. Prefasing can be caused by the presence of trace amounts of unprotected or unblocked 3'-OH nucleotides during sequencing by synthesis (SBS). Unprotected 3'-OH nucleotides can be 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 prefasing is surprising and provides a significant advantage over existing nucleotide analogs in SBS applications. For example, the nucleotide analogs provided can result in faster SBS cycle times, lower phasing and prefasing values, and longer sequencing read lengths.

[0083] Chromenoquinoline dyes of formula (I) Some aspects of the present disclosure relate to chromenoquinoline dyes of formula (I), as well as salts and mesomeric forms thereof,

[0084] [Chemical formula] wherein R 1 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10, R 11 , R 12a and R 12b each independently is 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-sulfonamide, or N-sulfonamide, R 2 and R 3 each independently is H, C1-C6 alkyl, or substituted C1-C6 alkyl, R 6 is C1-C6 alkyl or substituted C1-C6 alkyl, Alternatively, R 1 and R 2 together with the atoms to which they are attached form an optionally substituted 5- to 10-membered heteroaryl or an optionally substituted 5- to 10-membered heterocyclyl, and / or Alternatively, R 3 and R 4 together with the atoms to which they are attached form an optionally substituted 5- to 10-membered heteroaryl or an optionally substituted 5- to 10-membered heterocyclyl, and / or Alternatively, R 8 and R 9 together with the atoms to which they are attached form an optionally substituted C6-C 10 aryl, an optionally substituted 3- to 10-membered carbocyclyl, an optionally substituted 5- to 10-membered heteroaryl or an optionally substituted 3- to 10-membered heterocyclyl, Provided that when each of R 2 and R 3 is ethyl, R 1 , R 4 , R 5 , R 7 , R 8 , R 10 , R 11 , R 12aand R 12b Each of them is H, and R 6 However, it is methyl, and R 9 However, this is conditional on it being a substituted C1-C6 alkyl group containing a carboxyl molecule. However, the compound's structure

[0085] [ka] To have R 1 and R 2 Together with the atoms to which they are bonded, they form piperidinyl, and R 3 and R 4 When they form piperidine with the atoms to which they are bonded, R 5 , R 7 , R 8 , R 10 , R 11 , R 12a and R 12b Each of them is H, and R 6 However, it is methyl, and R 9 However, this is conditional on the compound being a substituted C1-C6 alkyl group containing a carboxyl group.

[0086] In any embodiment of the compound of formula (I), if defined as a group-substituted C1-C6 alkyl, it is a carboxyl, carboxylate, sulfo, sulfonate, -C(O)OR 15 or -C(O)NR 16 R 17 It may be a C1, C2, C3, C4, C5, or C6 alkyl substituted with, where each R 16 and R 17 However, independently, H, or carboxyl, carboxylate, -C(O)OR 15 These are C1-C6 alkyl groups substituted with sulfo or sulfonate.

[0087] In some embodiments of the compound of formula (I), R 2 and R 3 At least one of them is H. In some further embodiments, R 2 and R3 Both are H. In other embodiments, R 2 H is R 3 is a C1-C6 alkyl or a substituted C1-C6 alkyl. In other embodiments, R 2 and R 3 Each of these is independently a C1-C6 alkyl or a substituted C1-C6 alkyl. The substituted C1-C6 alkyls include carboxyl, carboxylate (-C(O) - ), sulfo(-SO3H), sulfonate(-SO3 - ), sulfate (-O-SO3-), optionally substituted amino (such as a Boc-protected amino group), -C(O)OR 15 or -C(O)NR 16 R 17 This includes, but is not limited to, methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl, n-hexyl, etc., substituted with one or more substituents such as R. 15 However, arbitrarily substituted C1-C6 alkyl, arbitrarily substituted C6-C 10 The aryl group is an aryl group, an optionally substituted 5-10 member heteroaryl group, or an optionally substituted C3-C7 cycloalkyl group. 16 and R 17 Each of these independently consists of H, an optionally substituted C1-C6 alkyl, and an optionally substituted C6-C 10 The aryl group is an optionally substituted 5-10 member heteroaryl group, or an optionally substituted C3-C7 cycloalkyl group. In one embodiment, R 2 and R 3 Each of them is ethyl. In other embodiments, R 2 H is R 3 It is a carboxyl-substituted n-propyl.

[0088] In any embodiment of the compound of formula (I), if defined as a group-substituted C1-C6 alkyl, it is a carboxyl, carboxylate, sulfo, sulfonate, -C(O)OR 15 or -C(O)NR 16 R 17It may be a C1, C2, C3, C4, C5, or C6 alkyl substituted with, where each R 16 and R 17 However, independently, H, or carboxyl, carboxylate, -C(O)OR 15 These are C1-C6 alkyl groups substituted with sulfo or sulfonate.

[0089] Some embodiments of the compound of formula (I) are also represented by formula (Ia), and R of formula (I) 3 and R 4 Together with the atoms to which they are bonded, they form a 6-membered heterocycline with any substitutions in the following structure.

[0090] [ka] Salt or its mesomeric form: In the formula, R 13a , R 13b , R 14a and R 14b Each of these is independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, (C1-C6 alkoxy)C1-C6 alkyl, optionally substituted amino, amino(C1-C6 alkyl), halo, cyano, hydroxy, hydroxy(C1-C6 alkyl), nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamide, or N-sulfonamide. Solid and dashed lines

[0091] [ka] The bond represented by is selected from the group consisting of single bonds and double bonds, however,

[0092] [ka] If it is a double bond, R 14b This is conditional on it not existing.

[0093] In some embodiments of the compound of formula (Ia), in practice and the dashed line

[0094]

Chemical formula

[0095]

Chemical formula

[0096] In some embodiments of the compound of formula (Ia), R 2 is H. In other embodiments, R 2R is a C1-C6 alkyl or a substituted C1-C6 alkyl (e.g., methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl, or n-hexyl). In some further embodiments, R 2 These are carboxyl (-C(O)OH), carboxylate (-C(O)O-), sulfo (-SO3H), sulfonate (-SO3-), and -C(O)OR 15 , and -C(O)NR 16 R 17 A C1-C6 alkyl group substituted with one or more substituents selected from the group consisting of, R 15 However, arbitrarily substituted C1-C6 alkyl, arbitrarily substituted C6-C 10 The aryl group is an aryl group, an optionally substituted 5-10 member heteroaryl group, or an optionally substituted C3-C7 cycloalkyl group. 16 and R 17 Each of these independently consists of H, an optionally substituted C1-C6 alkyl, and an optionally substituted C6-C 10 The aryl group is an optionally substituted 5- to 10-membered heteroaryl group, or an optionally substituted C3-C7 cycloalkyl group. In some further embodiments, R 2 However, carboxyl or -C(O)NR 16 R 17 It is a C1-C6 alkyl substituted with, and each R 16 and R 17 However, independently, carboxyl, carboxylate, -C(O)OR 15 , C1-C6 alkyl substituted with sulfo or sulfonate. In some embodiments, the substitution is linear C 2、 C 3、 C 5、 C 6、 Or it is located at the end of the C6 alkyl group. In one embodiment, R 3 It is a carboxyl-substituted n-propyl.

[0097] In some embodiments of the compounds of formula (I) or (Ia), R 1 In other embodiments, R 1 and R 2These bond together with the atoms to which they are bonded to form optionally substituted 5, 6, or 7-membered heterocyclines. In some such embodiments, R 1 and R 2 These atoms bond together with the atoms they are attached to to form a six-membered heterocycline substituted with one or more C1-C6 alkyl groups.

[0098] In some embodiments of the compounds of formula (I) or (Ia), R 6 is a C1-C6 alkyl group. In one embodiment, R 6 is methyl. In other embodiments, R 6 However, carboxyl or -C(O)NR 16 R 17 It is a C1-C6 alkyl substituted with, and each R 16 and R 17 However, independently, carboxyl, carboxylate, -C(O)OR 15 These are C1-C6 alkyl groups substituted with sulfo or sulfonate.

[0099] In some embodiments of the compounds of formula (I) or (Ia), R 5 In some further embodiments, R 11 In some further embodiments, R 12a and R 12b Each of these is H.

[0100] In some embodiments of the compounds of formula (I) or (Ia), R 7 , R 8 , R 9 and R 10 At least one of them is H. In one such embodiment, R 7 , R 8 , R 9 and R 10 Each of them is H. In another such embodiment, R 7 , R 8 , and R 10 Each of them is H. In another such embodiment, R 7 and R 10Each of these is H. In other embodiments, R 7 , R 8 , R 9 and R 10 At least one of them is independently a C1-C6 alkyl (e.g., methyl, ethyl, isopropyl, n-propyl, n-butyl, 2-butyl, n-pentyl, 2-pentyl, or n-hexyl) or a C1-C6 alkoxy (e.g., methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, 2-butoxy, n-pentoxy, 2-pentoxy, n-hexoxy). In such an embodiment, R 9 is methyl or methoxy. In another such embodiment, R 8 and R 9 Each of them is methyl or methoxy. In yet another embodiment, R 8 , R 9 and R 10 Each of them is methyl or methoxy. In other embodiments, R 7 , R 8 , R 9 and R 10 At least one of them is carboxyl, carboxylate, sulfo, sulfonate-C(O)OR 15 or -C(O)NR 16 R 17 It is a C1-C6 alkyl substituted with, and each R 16 and R 17 However, independently, H, or carboxyl, carboxylate, -C(O)OR 15 , a C1-C6 alkyl substituted with sulfo or sulfonate. In other embodiments, R 8 and R 9 This includes, along with the bonded atom, an optionally substituted 5-membered or 6-membered heterocycline, for example,

[0101] [ka] It forms structures such as those described above.

[0102] Additional embodiments of the compound of formula (I) or (Ia) include the following:

[0103] [ka] as well as their salts and mesomelic forms. Non-limiting examples include 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).

[0104] Cycloctatetraene (COT) light-protective portion In some embodiments, the fluorescent compounds described herein (formula (I) or (Ia)) may be further modified to introduce a covalently bonded photoprotective moiety thereto, for example, the cyclooctatetraene moiety, structure

[0105] [ka] Including, in the formula, R 1A and R 2A Each of these is independently H, hydroxyl, halogen, azide, thiol, nitro, cyano, optionally substituted amino, carboxyl, -C(O)OR 5A -C(O)NR 6A R 7A , arbitrarily 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 Alkinyl, optionally substituted C 6-10 Aryl, arbitrarily substituted C 7-14 Aralkyl, arbitrarily substituted C 3-7 Carbocyclyl, optionally substituted 5-10 member heteroaryl, or optionally substituted 3-10 member heterocyclyl, X 1 and Y 1 However, each is independent of the others, and they combine as -O-, -S-, and -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 -, arbitrarily substituted C 1-6 Alkylene, or any substituted heteroalkylene in which at least one carbon atom is replaced by O, S, or N, Z is either nonexistent or arbitrarily substituted with C. 2-6 Alkenylene, or optionally substituted C 2-6 It is alkynylene, R 3A and R 4A Each of these independently corresponds to H and C, which can be arbitrarily substituted. 1-6 Alkyl or optionally substituted C 6-10 It is Ariel, R 5A However, C is arbitrarily substituted. 1-6 Alkyl, optionally substituted C 6-10 Aryl, arbitrarily substituted C 7-14 Aralkyl, arbitrarily substituted C 3-7 Carbocyclyl, optionally substituted 5-10 member heteroaryl, or optionally substituted 3-10 member heterocyclyl, R 6A and R 7A Each of these independently corresponds to H and C, which can be arbitrarily substituted. 1-6 Alkyl, optionally substituted C 6-10 Aryl, arbitrarily substituted C 7-14 Aralkyl, arbitrarily substituted C 3-7 Carbocyclyl, optionally substituted 5-10 member heteroaryl, or optionally substituted 3-10 member heterocyclyl,

[0106] [ka] In R 1A and R 2AThe carbon atom to which is bonded can be arbitrarily replaced with O, S, or N, provided that if the carbon atom is replaced with O or S, R 1A and R 2A Provided that neither exists, if the carbon atom is replaced by N, then R 2A The condition is that does not exist, and m is an integer between 0 and 10. In some embodiments, both X and Y are not conjugate.

[0107] In some further embodiments, the cyclooctatetraene portion is structure

[0108] [ka] This includes R 1A and R 2A At least one of them is hydrogen. In some further embodiments, R 1A and R 2A Both are hydrogen. In some other embodiments, R 1A H is R 2 This 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 of these independently consists of hydrogen, optionally substituted amino, carboxyl, and -C(O)NR 6A R 7A , or a combination thereof. In some further embodiments, if m is 2, 3, 4, 5, or 6, then 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] In R1A and R 2A At least one carbon atom to which is bonded is replaced with O, S, or N. In some such embodiments,

[0110] [ka] In this, one carbon atom is replaced by an oxygen atom, and R is bonded to the replaced carbon atom. 1A and R 2A Neither exists. In some other embodiments,

[0111] [ka] When one carbon atom in is replaced by a nitrogen atom, the R bonded to the replaced carbon atom 2 It does not exist, and the R bonded to the replaced carbon atom is not present. 1 is hydrogen or C 1-6 It is alkyl. 1A and R 2A In any embodiment, R 1A or R 2A is -C(O)NR 6A R 7A And R 6A and R 7A H and C are independent of each other. 1-6 Alkyl or substituted C 1-6 Alkyl (e.g., C substituted with -CO2H, -NH2, -SO3H, or -SO3-) 1-6 It may be alkyl.

[0112] In some further embodiments, the fluorescent dyes described herein include a cyclooctatetraene moiety with the following structure.

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

[0114] Labeled nucleotides or oligonucleotides According to one aspect of this disclosure, the dye compounds described herein are suitable for conjugation to a substrate portion and, in particular, include a linker group that enables conjugation to the substrate portion. The substrate portion may be substantially any molecule or substance to which the dyes of this disclosure can be conjugated, and non-limiting examples include 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 by any linker by various means, including hydrophobic attraction, ion attraction, and covalent bonding. In some aspects, the dyes are conjugated to the substrate by covalent bonding. More specifically, the covalent bonding is by a linker group. In some cases, such labeled nucleotides are also referred to as “modified nucleotides.”

[0115] Several aspects of this disclosure relate to nucleotides or oligonucleotides labeled with dyes of formula (I) or (Ia) as described herein, or salts of their mesomeric forms, or derivatives thereof containing the photoprotective moiety COT as described herein. The labeled nucleotides or oligonucleotides may be bonded to the dye compounds disclosed herein via a carboxyl group (-CO2H) or an alkyl-carboxyl group to form amides or alkyl-amides. In some further embodiments, the carboxyl group may be an activated form of the carboxyl group, such as an amide or ester, which can be used to bond the nucleotide or oligonucleotide to an amino or hydroxyl group. As used herein, the term “activated ester” refers to a carboxyl group derivative that can react with, for example, a compound containing an amino group under mild conditions. Non-limiting examples of activated esters include, but are not limited to, p-nitrophenyl, pentafluorophenyl, and succinimide esters.

[0116] For example, the dye compound of formula (I) is R of formula (I). 6 , R 7 , R 8 , R 9 , R 10 and R 11 one of the following, or R 2 / R 3 It can be bound to a nucleotide or oligonucleotide via one of the following. In some such embodiments, R of formula (I) 9 is -CO2H or -(CH2) 1-6 -Contains CO2H, and the bond is R 9 An amide moiety is formed between the carboxyl functional group and the amino functional group of the nucleotide or nucleotide linker. As an example, the labeled nucleotide or oligonucleotide has the following structure as its dye moiety:

[0117] [ka] It may include R of formula (I). In other embodiments, R of formula (I) 2 or R 3is -CO2H or -(CH2) 1-6 -Contains CO2H, and the bond uses the CO2H group to form an amide. For example, labeled nucleotides or oligonucleotides have the following dye moieties:

[0118] [ka] It may include.

[0119] Similarly, the dye compound of formula (Ia) is R of formula (Ia). 6 , R 7 , R 8 , R 9 , R 10 and R 11 One of them (for example, R 9 ), or R 2 Through this, the compound can be bonded to a nucleotide or oligonucleotide by forming an amide moiety between the carboxyl functional group of the compound and the amino functional group of the nucleotide or nucleotide linker. For example, a labeled nucleotide or oligonucleotide may have the following dye moiety:

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

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

[0122] 3' Hydroxyl blocking group The labeled nucleotide or oligonucleotide may also have a blocking group covalently bonded to the ribose or deoxyribose sugar of the nucleotide. The blocking group may be bonded at any position on the ribose or deoxyribose sugar. In certain embodiments, the blocking group is located at the 3'OH position of the ribose or deoxyribose sugar of the nucleotide. Various 3'OH blocking groups are disclosed in International Publication 2004 / 018497 and International Publication 2014 / 139596, which are incorporated herein by reference. For example, the blocking group may be an azidomethyl (-CH2N3) or a substituted azidomethyl (e.g., -CH(CHF2)N3 or CH(CH2F)N3), or an allyl bonded to the 3' oxygen atom of the ribose or deoxyribose moiety. In some embodiments, the 3' blocking group is an azidomethyl, forming 3'-OCH2N3 using the 3' carbon of the ribose or deoxyribose.

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

[0124] [ka] It forms an acetal group, in the formula, Each R 1a and R 1b These are independently H, C1-C6 alkyl, and C 1- C6 haloalkyl, C 1- C6alkyl, C 1- C6 haloalkoxy, cyano, halogen, optionally substituted phenyl, or optionally substituted aralkyl, Each R 2a and R 2b H and C are independent of each other. 1- C6 alkyl, C 1- It is a C6 haloalkyl, cyano, or halogen. Alternatively, R 1a and R 2a Together with the atoms to which they are bonded, they form optionally substituted 5-8 membered heterocyclyl groups. R F H, and C as arbitrarily substituted. 2- C6 alkenyl, optionally substituted C 3- C7 cycloalkenyl, optionally substituted C 2- C6 alkynyl, or optionally substituted (C 1- C6 Alkylene)Si(R 3a )3, Each R 3a H and C are independent of each other. 1- C6 alkyl, or optionally substituted C 6- C 10 It is Ariel.

[0125] An additional 3'-OH blocking group is disclosed in U.S. Patent Application Publication No. 2020 / 0216891(A1), which is incorporated in whole by reference. Acetal blocking group

[0126] [ka] These are non-restrictive examples, each covalently bonded to the 3' carbon of ribose or deoxyribose.

[0127] Deprotection of the 3'-hydroxyl 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 various chemical conditions. Acetal blocking groups containing vinyl or alkenyl moieties

[0128] [ka] In this case, non-restrictive cleavage conditions include Pd(II) complexes such as Pd(OAc)2 or allyl Pd(II) chloride dimers in the presence of a phosphine ligand, e.g., tris(hydroxymethyl)phosphine (THMP) or tris(hydroxypropyl)phosphine (THP or THPP). In the case of these blocking groups containing an alkynyl group (e.g., ethynyl), they can also be removed by a Pd(II) complex (e.g., Pd(OAc)2 or allyl Pd(II) chloride dimer) in the presence of a phosphine ligand (e.g., THP or THMP).

[0129] Palladium cleavage reagent In some embodiments, the 3'-hydroxyl blocking group described herein can be cleaved by a palladium catalyst. In some such embodiments, the Pd catalyst is water-soluble. In some such embodiments, is a Pd(0) complex (e.g., tris(3,3',3''-phosphinidin tris(benzenesulfonate)palladium(0)) salt nonasodium nonahydrate). In some cases, Pd(0) can be produced 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 Na2PdCl4 and Pd(CH3CN)2Cl 2, Examples 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 produced in situ from Na2PdCl4. In another embodiment, the palladium source is the allylpalladium(II) chloride dimer [(PdCl(C3H5))2]. In some embodiments, the Pd(0) complex is produced in aqueous solution by mixing the Pd(II) complex with 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.

[0130] In some embodiments, Pd(0) is prepared by in-situ mixing a Pd(II) complex [(PdCl(C3H5))2] with THP. The molar ratio of the Pd(II) complex to THP may 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 agents, such as primary amines, secondary amines, tertiary amines, carbonates, phosphates, or borates, or combinations thereof. In some further embodiments, the buffering reagent includes ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, 2-dimethylethanolamine (DMEA), 2-diethylethanolamine (DEEA), N,N,N',N'-tetramethylethylenediamine (TEMED), or N,N,N',N'-tetraethylethylenediamine (TEEDA), or a combination thereof. In one embodiment, the buffering reagent is DEEA. In another embodiment, the buffering reagent contains one or more inorganic salts, such as carbonates, phosphates, or borates, or a combination thereof. In one embodiment, the inorganic salt is a sodium salt.

[0131] Linker The dye compounds disclosed herein may include a reactive linker group at one of the substitution sites for covalent bonding of the compound to a substrate or another molecule. A reactive linker is a portion capable of forming 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 in such a position that a portion of the linker remains bonded to the dye and / or substrate portion after cleavage. Cleavable linkers may, in non-limiting examples, be electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, linkers cleavable under reducing conditions (e.g., disulfide or azide-containing linkers), linkers cleavable under oxidative conditions, linkers cleavable by the use of a safety device linker, or linkers cleavable by a removal mechanism. By using a cleavable linker to bond the dye compound to the substrate, the label can be removed after detection if necessary, thus avoiding any interference signals in downstream processes.

[0132] Useful linker groups can be found in PCT International Publication 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 at least partially water-soluble ligands. In aqueous solution, the latter forms at least partially water-soluble transition metal complexes. Such cleavable linkers can be used to link the bases of nucleotides to labels such as dyes described herein.

[0133] Specific linkers include those disclosed in PCT International Publication No. 2004 / 018493 (incorporated herein by reference), including the part of the formula below.

[0134] [ka] In the formula, X is selected from groups including O, S, NH, and NQ, Q is a C1-10 substituted or unsubstituted alkyl group, Y is selected from groups including O, S, NH, and N (allyl), and T is hydrogen or C1-C 10 A substituted or unsubstituted alkyl group, * This indicates the location where the portion is attached to the rest of the nucleotide or nucleoside. In some embodiments, the linker attaches the bases of the nucleotide to a label, such as a dye compound as described herein.

[0135] Examples of additional linkers include those disclosed in U.S. Patent Application Publication 2016 / 0040225 (incorporated herein by reference), including those containing the following formula.

[0136] [ka] (In the formula, * (The symbol indicates the location where that portion is connected to the rest of the nucleotide or nucleoside). The linker portions shown herein may include a whole or partial linker structure between the nucleotide / nucleotide and the label. The linker portions shown herein may include a whole or partial linker structure between the nucleotide / nucleotide and the label.

[0137] An example of adding a linker is the following part of the expression:

[0138] [ka]

[0139] [ka] In the formula, B is a nucleic acid base, Z is -N3 (azide), -O-C1-C6 alkyl, -O-C2-C6 alkenyl, or -O-C2-C6 alkynyl, and Fl comprises a dye moiety which may contain an additional linker structure. Those skilled in the art will understand that the dye compounds described herein are covalently bonded to the linker by reacting a functional group of the dye compound (e.g., carboxyl) with a functional group of the linker (e.g., amino). In one embodiment, the cleavable linker is

[0140] [ka] The formula includes the ("AOL" linker part), where Z is an -O-allyle.

[0141] 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 bound to the guanine base via other linkages known in the art. Exemplary linkers and their properties are described in PCT International Publication 2007 / 020457 (incorporated herein by reference). Such design of linkers, in particular by increasing their length, can enable improved brightness of the fluorophore bound to the guanine base of guanosine nucleotides when incorporated into polynucleotides such as DNA. Therefore, if the dye is for use in any analytical method requiring detection of a fluorescent dye label bound to a guanine-containing nucleotide, the linker may be of the formula -((CH2)2O) as described in International Publication 2007 / 020457. n -It is advantageous if the formula includes a spacer base of (where n is an integer between 2 and 50).

[0142] Nucleosides and nucleotides may be labeled at a site on a sugar or nuclear base. As is known in the art, a “nucleotide” consists of a nitrogen 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 nitrogen 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 bonded to N-1 of the pyrimidine or N-9 of the purine. Nucleosides are also phosphate esters of nucleosides, with esterification occurring at the hydroxyl group bonded to C-3 or C-5 of the sugar. Nucleosides are usually monophosphate, diphosphate, or triphosphate.

[0143] A "nucleoside" is structurally similar to a nucleotide but lacks a phosphate group. Examples of nucleotide analogs are those in which a label is linked to a base and there is no phosphate group attached to a sugar molecule.

[0144] Bases are typically referred to as purines or pyrimidines, but those skilled in the art will understand that derivatives and analogues are available that alter the ability of the nucleotide or nucleoside to undergo Watson-Crick base pairing. “Derivative” or “analog” means a compound or molecule whose core structure is the same as or closely similar to the parent compound, but which has undergone chemical or physical modifications, such as different or additional side groups (allowing 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 capable of Watson-Crick pairing. “Derivatives” and “analogues” also include, for example, synthetic nucleotide or nucleoside derivatives having modified base moieties and / or modified sugar moieties. Such derivatives and analogues are discussed, for example, in Scheit, Nucleotide analogs (John Wiley & Son, 1980) and Uhlman, Chemical Reviews 90:543-584, 1990. Nucleotide analogs may also include modified phosphate diester linkages, such as phosphorothioates, phosphorodithioates, alkyl phosphonates, phosphoranilidates, and phosphoramidate linkages.

[0145] The dye may be bound to any position on the nucleotide base, for example, via a linker. In certain embodiments, Watson-Crick base pairing may still be performed on the resulting analogue. Specific nuclear base labeling sites include the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base. As described above, the dye can be covalently bonded to a nucleoside or nucleotide using a linker group.

[0146] In certain embodiments, the labeled nucleoside or nucleotide may be enzymatically embeddable and enzymatically elongable. Therefore, the linker portion may be long enough to connect the nucleotide to the compound, and as a result, the compound does not significantly interfere with the overall binding and recognition of the nucleotide by nucleic acid replication enzymes. Thus, the linker may also include spacer units. Spacers, for example, move nucleotide bases away from cleavage sites or labels.

[0147] The nucleosides or nucleotides labeled with the dyes described herein may have the following formula:

[0148] [ka]

[0149] In the formula, the dye is the chromenoquinoline dye compound (labeled) moiety described herein (after covalent bonding between the functional group of the dye and the functional group of linker "L"), B is a nucleic acid base such as uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, or 7-deazaguanine, L is any linker which may or may not be present, R' may be H, or -OR' may be monophosphate, diphosphate, triphosphate, thiophosphate, phosphate ester analog, -O- bonded to a reactive phosphorus-containing group, or -O- protected by a blocking group, R'' may be H or OH, R''' may be H, a 3'OH blocking group as described herein, or -OR''' may form a phosphoramidite. If -OR''' is a phosphoramidite, R' is an acid-cleavable hydroxyl protecting group that allows subsequent monomer coupling under autosynthetic conditions. In some further embodiments, B is

[0150] [ka] or include optionally substituted derivatives thereof and analogs thereof. In some further embodiments, the labeled nucleic acid base is structure

[0151] [ka] Includes.

[0152] In certain embodiments, the blocking group is separate from the dye compound, i.e., it is not bound to the dye compound. Alternatively, the dye may contain all or some of the 3'-OH blocking groups. Therefore, R''' can be a 3'-OH blocking group that may or may not contain the dye compound.

[0153] In yet another alternative embodiment, there is no blocking group on the 3' carbon of the pentose sugar, and, for example, the base-bound dye (or dye and linker structure) may be large or structurally large enough to act as a block to further nucleotide incorporation. Thus, whether the dye is bound to the 3' position of the sugar or not, the block may be due to steric interference or to a combination of size, charge, and structure.

[0154] In yet another alternative embodiment, the blocking group may be located on the 2' or 4' carbon of the pentose sugar and may be of a size or structure sufficient to act as a block for the incorporation of further nucleotides.

[0155] The use of blocking groups allows for the control of polymerization, for example, by stopping elongation when a labeled nucleotide is incorporated. If the blocking effect is reversible, for example by changing chemical conditions or removing the chemical block (but not limited to these examples), elongation can be temporarily stopped at a specific point and then allowed to continue.

[0156] In certain embodiments, both linkers (between the dye and the nucleotide) and blocking groups are present and are separate parts from each other. In certain embodiments, both the linker and the blocking group are cleavable under the same conditions, or substantially similar conditions. Therefore, the deprotection and deblocking processes 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 similar conditions, rather than being cleavable individually under different conditions.

[0157] This disclosure also includes polynucleotides incorporating dye compounds. Such polynucleotides may be DNA or RNA consisting of deoxyribonucleotides or ribonucleotides joined by phosphate diester links, respectively. Polynucleotides may 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 this disclosure may also include non-naturally occurring skeletal linkages and / or non-nucleotide chemical modifications. Chimeric structures consisting of mixtures of ribonucleotides and deoxyribonucleotides containing at least one labeled nucleotide can also be conceived.

[0158] Examples of non-exclusive, exemplary labeled nucleotides described herein include:

[0159] [ka]

[0160] [ka] In the formula, L represents a linker, and R represents the ribose or deoxyribose moiety described above, or a ribose or deoxyribose moiety in which the 5' position is substituted with monophosphate, diphosphate, or triphosphate.

[0161] In some embodiments, non-limiting exemplary fluorescent dye conjugates are shown below.

[0162] [ka]

[0163] [ka]

[0164] [ka] In the formula, 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.

[0165] [ka] This refers to a linkage point of the dye having a cleavable linker as a result of a reaction between the amino group of the linker moiety and the carboxyl group of the dye. In any embodiment of the labeled nucleotides described herein, the nucleotide is a nucleotide triphosphate.

[0166] Additional embodiments of this disclosure relate to oligonucleotides comprising labeled nucleotides as described herein. In some embodiments, the oligonucleotide is 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 multiple 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 are directly aligned on each CMOS photodiode (pixel).

[0167] kit This specification provides a kit comprising a first nucleotide labeled with a chromenoquinoline compound of the Disclosure (i.e., first labeling). In some embodiments, the kit also comprises a second labeled nucleotide labeled with a second compound different from the chromenoquinoline compound in the first labeled nucleotide (i.e., second labeling). 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 the overlapping region of the spectrum causes both labels to fluoresce. In some further embodiments, the kit may comprise a third nucleotide, the third nucleotide being labeled with a third compound different from the first and second labels (i.e., third labeling). 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 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 different compound from the first, second, and third nucleotides, and each label has a distinct absorbance value that is distinguishable from the other labels. In yet another embodiment, 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 an alternative embodiment, the first light source has an excitation wavelength of approximately 400 nm to approximately 480 nm, approximately 420 nm to approximately 470 nm, or 450 nm to approximately 460 nm (e.g., 450 nm).The second excitation light source has a wavelength of approximately 500 nm to approximately 550 nm, approximately 510 to approximately 540 nm, or approximately 520 to approximately 530 nm (e.g., 520 nm). The second light source has an excitation wavelength of approximately 400 nm to approximately 480 nm, approximately 420 nm to approximately 470 nm, or 450 nm to approximately 460 nm (e.g., 450 nm). In further embodiments, each of the first label, the second label, and the third label has an emission spectrum that can be collected in a single emission collection filter or channel.

[0168] In some embodiments, the kit may comprise four labeled nucleotides (A, C, G, and T or U), where the first of the four nucleotides is labeled with a compound disclosed herein. In such a kit, each of the four nucleotides may be labeled with the same or different compounds as the labels on the other three nucleotides. Alternatively, the first of the four nucleotides may be a labeled nucleotide as described herein, the second of the four nucleotides may have a second label, the third nucleotide may have a third label, and the fourth nucleotide may be unlabeled (dark). Another example is the first of the four nucleotides may be a labeled nucleotide as described herein, the second of the four nucleotides may have a second label, the third nucleotide may carry a mixture of the two labels, and the fourth nucleotide may be unlabeled (dark). Thus, one or more of the labeled compounds may have different absorbance and / or emission values ​​so that the compound is distinguishable from the other compounds. For example, each compound may have different absorbance and / or emission maximums so that each compound is spectroscopically distinguishable from the other three compounds (or two compounds if the fourth nucleotide is not labeled). The portions of the absorbance and / or emission spectra other than the maximums may differ, and it will be understood that these differences may be used to distinguish the compounds. A kit may be such that two or more compounds have different absorbance maximums. The chromenoquinoline dyes disclosed herein typically absorb light in the region below 500 nm. For example, these chromenoquinoline dyes may have absorption wavelengths of approximately 450 nm to 530 nm, approximately 460 nm to 520 nm, approximately 475 nm to 510 nm, or approximately 490 nm to 500 nm.

[0169] The compounds, nucleotides, or kits described herein may be used to detect, measure, or identify biological systems (e.g., processes or their components). Exemplary techniques in which the compounds, nucleotides, or kits may be used include sequencing, expression analysis, hybridization analysis, gene analysis, RNA analysis, cell assays (e.g., cell binding or cell function analysis), or protein assays (e.g., protein binding assays or protein activity assays). Their use may also be in automated equipment for performing specific techniques, such as automated sequencing instruments. A sequencing instrument may include two light sources operating at different wavelengths.

[0170] In certain embodiments, the labeled nucleotides described herein may be supplied in combination with unlabeled or natural nucleotides, or any combination thereof. Nucleotide combinations may be supplied as separate individual components (e.g., one nucleotide type per container or tube) or as nucleotide mixtures (e.g., two or more nucleotides mixed in the same container or tube).

[0171] If the kit contains multiple nucleotides, particularly two, three, or more specifically four, different nucleotides may be labeled with different dye compounds or may be dark in color, without any dye compounds. A key feature of the kit is that, when different nucleotides are labeled with different dye compounds, the dye compounds are spectrally distinct fluorescent dyes. As used herein, the term “spectrometrically distinct fluorescent dye” refers to a fluorescent dye that emits fluorescence energy at wavelengths that can be distinguished by a fluorescence detector (e.g., a commercially available capillary-based DNA sequencing platform) when two or more dyes are present in a single sample. In some embodiments, when two nucleotides labeled with fluorescent dye compounds are supplied in kit form, the spectrally distinct fluorescent dyes may be excited at the same wavelength, for example, by the same light source. In some embodiments, when four nucleotides labeled with fluorescent dye compounds are supplied in kit form, two of the spectrally distinct fluorescent dyes may be excited at one wavelength, while the other two spectrally distinct dyes may both be excited at different wavelengths. The specific excitation wavelengths of the dyes are 450-460 nm, 490-500 nm, or 520 nm or higher (e.g., 532 nm).

[0172] In some embodiments, the kit comprises a first nucleotide labeled with the chromenoquinoline dye of the Disclosure and a second nucleotide labeled with the second dye, the dyes having a difference in maximum absorbance of at least 10 nm, particularly 20 nm to 50 nm or 30 nm to 40 nm. More specifically, the first label may have a Stokes shift of greater than 50 nm to greater than 60 nm, greater than 70 nm, or greater than 80 nm. The second label may have a Stokes shift of about greater than 80 nm, greater than 90 nm, or greater than 100 nm (where "Stokes shift" is the distance between the peak absorption wavelength and the peak emission wavelength). Furthermore, the first label may have a maximum absorbance 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 a maximum absorbance 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, the third label having an absorption maximum above 520 nm. The third label may have a Stokes shift above 20 nm, above 30 nm, or above 40 nm, or a Stokes shift between 20 and 40 nm. The kit may further include an unlabeled fourth nucleotide. In further embodiments, each of the first, second, and third labels has an emission maximum above 540 nm, above 550 nm, above 560 nm, above 570 nm, above 580 nm, above 590 nm, or above 600 nm. In some embodiments, the emission spectra of the first, second, and third labels 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).

[0173] In alternative embodiments, the kit of the present disclosure may contain nucleotides in which the same base is labeled with two different compounds. The first nucleotide may be labeled with the compound of the present disclosure. The second nucleotide may be labeled with a spectrally different compound, for example, a “green” dye absorbed below 600 nm. The third nucleotide may be labeled as a mixture of the compound of the present disclosure and a spectrally different compound, and the fourth nucleotide may be “dark” and may not contain any labeling. Thus, simply put, nucleotides 1-4 can be labeled as “blue”, “green”, “blue / green”, and dark. To further simplify the instrument, the four nucleotides can be labeled with two dyes excited by a single light source, and thus the labeling of nucleotides 1-4 may be “blue 1”, “blue 2”, “blue 1 / blue 2”, and dark.

[0174] While kits are illustrated herein with respect to configurations having different nucleotides labeled with different dye compounds, it will be understood that kits may contain two, three, four or more different nucleotides having the same dye compound.

[0175] In addition to the labeled nucleotides, the kit may include at least one additional component. The further component may be one or more of the components identified in the methods described herein or in the following examples section. Some non-limiting examples of components that can be combined in the kit of this disclosure are listed 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. Additional buffer compositions may include reagents that can be used to cleave the 3' blocking group and / or cleavable linker. For example, a water-soluble phosphine or water-soluble transition metal catalyst may be formed from a transition metal such as a palladium complex and at least partially water-soluble ligands. Various components of the kit may be provided in concentrated forms that are 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 kit of this disclosure. In any embodiment of the nucleotide or labeled nucleotide described herein, the nucleotide contains a 3' hydroxyl blocking group.

[0176] Sequence determination method The nucleotides comprising the dye compounds according to this disclosure may be used, whether by themselves, incorporated into or associated with a larger molecular structure or conjugate, in any analytical method, such as a method that includes the detection of a fluorescent label attached to such nucleotide. In this context, the term “incorporated into a polynucleotide” may mean that a 5' phosphate is attached by a phosphate diester linkage to the 3' hydroxyl group of a second nucleotide, which itself may form part of a longer polynucleotide chain. The 3' end of the nucleotides described herein may or may not be attached by a phosphate diester linkage to the 5' phosphate of a further nucleotide. Accordingly, in one non-limiting embodiment, the disclosure provides a method for detecting a labeled nucleotide incorporated into a polynucleotide, comprising: (a) incorporating at least one labeled nucleotide of the disclosure into a polynucleotide; and (b) determining the identity of the nucleotide incorporated into the polynucleotide by detecting a fluorescent signal from a dye compound attached to the nucleotide.

[0177] 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 one or more labeled nucleotides incorporated into the polynucleotide are detected by detecting or quantitatively measuring their fluorescence.

[0178] Some embodiments of this application are methods for determining the sequence of a target polynucleotide (e.g., a single-stranded target polynucleotide), comprising: (a) contacting a primer polynucleotide with one or more labeled nucleotides (nucleoside triphosphates A, G, C, and T), wherein at least one of the labeled nucleotides is a labeled nucleotide as 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; 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 the labeled moiety and 3' hydroxy-blocking group from the nucleotide incorporated into the primer polynucleotide. In some further embodiments, the method may also comprise (e) washing off the removed labeled moiety and 3'-blocking group from the primer polynucleotide chain. In some embodiments, steps (a)–(d) or (a)–(e) are repeated until the sequence of at least a portion of the target polynucleotide chain is determined. In some cases, steps (a)–(d) or (a)–(e) are repeated for at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, or 300 cycles. In some embodiments, the labeled portion and 3' blocking group from the nucleotides incorporated into the primer polynucleotide chain are removed in a single chemical reaction. In some further embodiments, the method is carried out using an automated sequencing instrument, which includes two light sources operating at different wavelengths. In some embodiments, sequencing is performed after the completion of a repeating cycle of the sequencing steps described herein.

[0179] 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), wherein (a) a primer polynucleotide is contacted with an incorporated mixture comprising one or more of four different types of nucleotide conjugates, wherein the first type of nucleotide conjugate comprises a first label, the second type of nucleotide conjugate comprises a second label, the third type of nucleotide conjugate comprises a third label, and each of the first, second, and third labels is spectrally distinct from the other, and the primer nucleotide is at least a portion of the target polynucleotide. The method comprises (b) contacting the mixture, which is complementary to the first imaging event, and (c) incorporating a single nucleotide conjugate into a primer polynucleotide from the mixture to produce an extended primer polynucleotide, and (d) performing a first imaging event using a first excitation light source and detecting a first emission signal from the extended polynucleotide, wherein the first and second excitation light sources have different wavelengths, and the first and second emission signals are detected or collected by a single emission detection channel. In some embodiments, the chromenoquinoline dyes described herein may be used as any one of the first, second, or third labels described herein. In some embodiments, the method does not involve 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 integration mixture further comprises a fourth type of nucleotide, which is labeled with an unlabeled fluorescent moiety that does not emit a signal from either the first or second imaging event. In this sequencing method, the identity of each integrated nucleotide conjugate is determined based on the detection patterns of the first and second imaging events. For example, the integration of the first type of nucleotide conjugate is determined by the signal state in the first imaging event and the dark state in the second imaging event.The incorporation of a second type of nucleotide conjugate is determined by the dark state in the first imaging event and the signal state in the second imaging event. The incorporation of a third type of nucleotide conjugate is determined by the signal state in both the first and second imaging events. The incorporation of a fourth type of nucleotide conjugate is determined by the dark state in both the first and second imaging events. In further embodiments, steps (a) to (d) are carried out in repeated cycles (e.g., at least 30, 50, 100, 150, 200, 250, 300, 400, or 500 times), and the method further includes sequentially determining the sequence of at least a portion of a single-stranded target polynucleotide based on the identity of each sequentially 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 approximately 520 nm. The second excitation light source has a wavelength of approximately 400 nm to approximately 480 nm, approximately 420 nm to approximately 470 nm, or approximately 450 nm to approximately 460 nm (i.e., "blue light"). In one embodiment, the second excitation light source has a wavelength of approximately 450 nm.

[0180] In some embodiments, at least one nucleotide is incorporated into a polynucleotide (such as the single-stranded primer polynucleotide described herein) in the synthesis process by the action of a polymerase enzyme. However, other methods of linking nucleotides to polynucleotides may be used, such as chemical oligonucleotide synthesis or ligation of labeled oligonucleotides to unlabeled oligonucleotides. Thus, when the term “incorporate” is used with respect to nucleotides and polynucleotides, it can encompass both chemical and enzymatic polynucleotide synthesis.

[0181] In certain embodiments, the synthesis step may be carried out and optionally include incubating a template or target polynucleotide chain with a reaction mixture containing the fluorescently labeled nucleotides of the Disclosure. The polymerase may also be provided under conditions that allow for the formation of phosphate diester links between the free 3' hydroxyl group on the polynucleotide chain annealed to the template or target polynucleotide chain and the 5' phosphate group on the labeled nucleotide. Thus, the synthesis step may include the formation of a polynucleotide chain directed by complementary base pairing of the nucleotides to the template / target chain.

[0182] In all embodiments of this method, the detection step may be performed while the polynucleotide chain into which the labeled nucleotide is incorporated is annealed to the template / target chain, or after the denaturation step in which the two chains are separated. Further 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 chain into which the labeled nucleotide is incorporated may be isolated or purified and then further processed or used for subsequent analysis. For example, a polynucleotide into which the labeled nucleotide described herein is incorporated in the synthesis step may subsequently be used as a labeled probe or primer. In other embodiments, the product of the synthesis step described herein may be subjected to further reaction steps, or, if desired, purified or isolated products of these subsequent steps.

[0183] The conditions suitable for the synthesis process are well known to those familiar with standard molecular biological techniques. In one embodiment, the synthesis process may be analogous to a standard primer extension reaction using a nucleotide precursor containing the labeled nucleotide described herein to form an extended target chain (primer polynucleotide) complementary to the template / target chain in the presence of a suitable polymerase enzyme. In other embodiments, the synthesis process may itself form part of an amplification reaction that produces a labeled double-stranded amplification product consisting of an annealed complementary chain derived from copies of the primer and template polynucleotide chains. Other exemplary synthesis processes include nick translation, strand substitution polymerization, and random priming DNA labeling. Polymerase enzymes particularly useful for the synthesis process are those capable of catalyzing the incorporation of the labeled nucleotide described herein. A variety of naturally occurring or mutated / modified polymerases may be used. For example, thermostable polymerases can be used in synthesis reactions carried out using thermal cycling conditions, although thermostable polymerases may be undesirable for isothermal primer extension reactions. Suitable thermostable polymerases capable of incorporating the labeled nucleotides according to this disclosure include those described in International Publication No. 2005 / 024010 or International Publication No. 06 / 120433, each of which is incorporated herein by reference. In synthetic reactions carried out at low temperatures such as 37°C, the polymerase enzyme does not necessarily have to be a thermostable polymerase, and therefore the selection of the polymerase depends on several factors such as reaction temperature, pH, and chain displacement activity.

[0184] In certain non-limiting embodiments, the Disclosure includes methods for any other applications involving nucleic acid sequencing, rearrangement, whole genome sequencing, single nucleotide polymorphism scoring, and detection of modified nucleotides or nucleosides labeled with the dyes described herein when incorporated into polynucleotides.

[0185] Certain embodiments of this disclosure provide the use of labeled nucleotides containing the dye moieties of this disclosure in a sequencing reaction of polynucleotides by synthesis. Sequencing by synthesis generally involves sequentially adding one or more nucleotides or oligonucleotides in the 5' to 3' direction to a growing polynucleotide chain using a polymerase or ligase to form an elongated polynucleotide chain complementary to the template / target nucleic acid to be sequenced. The identity of the bases present in one or more of the added nucleotides may be determined in a detection or “imaging” step. The identity of the added bases may be determined after each nucleotide incorporation step. The sequence of the template may then be inferred using conventional Watson-Crick base pairing rules. The use of dye-labeled nucleotides as described herein for determining single-base identity may be useful, for example, in single-nucleotide polymorphism scoring, and such single-base elongation reactions are within the scope of this disclosure.

[0186] 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 chain complementary to the template polynucleotide, which is sequenced via detection of a fluorescent label bound to the incorporated nucleotides. Sequence determination of the template polynucleotide can be performed by priming with a suitable primer (or a primer prepared as a hairpin structure containing the primer as part of a hairpin), and the nascent chain is extended stepwise by adding nucleotides to the 3' end of the primer in a polymer-catalyzed reaction.

[0187] In certain embodiments, each of the different nucleotide triphosphates (A, T, G, and C) may be labeled with a specific fluorophore and may also contain a blocking group at the 3' position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides may be unlabeled (dark). A polymerase enzyme incorporates the nucleotides into a nascent chain complementary to the template / target polynucleotide, and the blocking group prevents further incorporation of nucleotides. 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 a suitable emission filter. The 3' blocking group and fluorescent dye compound can then be removed (deprotected) (simultaneously or sequentially) to expose the nascent chain for further nucleotide incorporation. Typically, the identity of the incorporated nucleotides is determined after each incorporation step, although this is not strictly required. Similarly, U.S. Patent No. 5,302,509 (incorporated herein by reference) discloses a method for sequencing polynucleotides immobilized on a solid support.

[0188] The method exemplified above involves incorporating fluorescently labeled 3'-blocked nucleotides A, G, C, and T into a growth chain complementary to an immobilized polynucleotide in the presence of 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 is then determined, and the blocking group may subsequently be removed by chemical cleavage to allow further polymerization. The nucleic acid template to be sequenced in the synthetic sequencing reaction may be any polynucleotide to be sequenced. The nucleic acid template for the sequencing reaction typically includes a double-stranded region having a free 3'-hydroxyl group that functions as a primer or starting point for further nucleotide addition in the sequencing reaction. The region of the sequencing template overhangs this free 3'-hydroxyl group onto the complementary chain. The overhang region of the sequencing template may be single-stranded, but may also be double-stranded, insofar as a "break exists" on a chain complementary to the sequencing template chain, providing a free 3'OH group to initiate the sequencing reaction. In such embodiments, sequencing may proceed by chain substitution. In certain embodiments, a primer having a free 3' hydroxyl group may be added as a separate component (e.g., a short oligonucleotide) that hybridizes to the single-stranded region of the sequencing template. Alternatively, the sequencing primer and template chain may each form a portion of a partially self-complementary nucleic acid chain that can form an intramolecular double helix, such as a hairpin loop structure. Hairpin polynucleotides and methods for binding them to a solid support are disclosed in PCT International Publications 0157248 and 2005 / 047301, each of which is incorporated herein by reference. Nucleotides can be sequentially added to a growing primer to synthesize a polynucleotide chain in the 5'-3' direction. The properties of the added bases may be determined after each nucleotide addition, but this is not necessarily required. This determination provides sequence information about the nucleic acid template.Therefore, nucleotides are incorporated into nucleic acid chains (or polynucleotides) by attaching them to the free 3' hydroxyl group of the nucleic acid chain via the formation of a phosphate diester linkage with the 5' phosphate group of the nucleotide.

[0189] The nucleic acid template for sequencing may be DNA or RNA, or even a hybrid molecule consisting of deoxynucleotides and ribonucleotides. The nucleic acid template may include naturally occurring nucleotides and / or non-naturally occurring nucleotides and naturally occurring or non-naturally occurring skeletal links, as long as this does not prevent the template from being copied in the sequencing reaction.

[0190] In certain embodiments, the sequenced nucleic acid template may be bound to a solid support by any preferred linking method known in the art, such as covalent bonding. In certain embodiments, the template polynucleotide may be directly bound to a solid support (e.g., a silica-based support). However, in other embodiments of the present disclosure, the surface of the solid support may be modified in any way to allow either direct covalent bonding of the template polynucleotide or immobilization of the template polynucleotide via a hydrogel or polyelectrolyte multilayer that can itself be non-covalently bonded to the solid support.

[0191] An array in which polynucleotides are directly bound to a support (for example, a silica-based support such as those disclosed in International Publication No. 2000 / 06770 (incorporated herein by reference)), wherein the polynucleotides are immobilized on the glass support by a reaction between pendant epoxide groups on the glass and internal amino groups on the polynucleotides. In addition, the polynucleotides are solidified with a sulfur-based nucleophile, as described, for example, in International Publication No. 2005 / 047301 (incorporated herein by reference). They can be bound to a solid support by reaction with the support. Further examples of solid-supported template polynucleotides include cases where the template polynucleotide is bound to a hydrogel supported on a silica-based or other solid support, as described, for example, in International Publication Nos. 2000 / 31148, 2001 / 01143, 2002 / 12566, 2003 / 014392, U.S. Patent No. 6,465,178, and 2000 / 53812, each of which is incorporated herein by reference.

[0192] Specific surfaces on which template polynucleotides can be immobilized include polyacrylamide hydrogels. Polyacrylamide hydrogels are described in the above references and International Publication No. 2005 / 065814, which are incorporated herein by reference. Specific hydrogels that may 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)).

[0193] 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 may be useful for sequencing applications. Bead libraries can be prepared, each containing a different DNA sequence. Exemplary libraries and methods for their preparation are described in Nature, 437, 376-380 (2005) and Science, 309, 5741, 1728-1732 (2005), each of which is incorporated herein by reference. Sequencing of arrays of such beads using the nucleotides described herein is within the scope of this disclosure.

[0194] The sequencing template may form part of an "array" on a solid support, in which case the array can take any convenient form. Therefore, the methods of this 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 this disclosure can be used to sequence templates on virtually any type of array, including but not limited to those formed by the immobilization of nucleic acid molecules on a solid support.

[0195] However, nucleotides labeled with the dye compounds of this disclosure are particularly advantageous in the context of sequencing clustered arrays. In a clustered array, distinct regions on the array (often referred to as sites or feature regions) contain multiple polynucleotide template molecules. Generally, multiple polynucleotide molecules are not individually degradable by optical means, but rather detected as an ensemble. Depending on how the array is formed, each site on the array may contain multiple copies of a single individual polynucleotide molecule (e.g., the site is homogeneous with respect to a particular 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. For example, International Publication No. 98 / 44151 and International Publication No. 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 an array consisting of clusters or “colonies” of immobilized nucleic acid molecules. The nucleic acid molecules present on the clustered arrays prepared according to these methods are suitable templates for sequencing using nucleotides labeled with the dye compounds of this disclosure.

[0196] Nucleotides labeled with the dye compounds of this 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 collection of polynucleotide molecules distributed (or arranged) on a solid support, such that the separation distance of any individual polynucleotide from all other polynucleotides in the collection allows for the individual identification of each polynucleotide molecule. Thus, in some embodiments, target nucleic acid molecules immobilized on the surface of a solid support can be degraded by optical means. This means that one or more distinct signals, each representing a single polynucleotide, are generated within an area that can be distinguished by the particular imaging device used.

[0197] Single-molecule detection can be achieved when the spacing between adjacent polynucleotide molecules on the array is at least 100 nm, more specifically at least 250 nm, even more specifically at least 300 nm, and even more specifically at least 350 nm. Thus, each molecule is individually degradable and detectable, detectable as a single-molecule fluorescence site, and fluorescence from this single-molecule fluorescence site also exhibits single-step photobleaching.

[0198] The terms “individually decomposed” and “individual resolution” are used herein to specify that, when visualized, it is possible to distinguish one molecule on an array from its adjacent molecules. The separation between individual molecules on an array is determined in part by specific techniques used to decompose the individual molecules. The general characteristics of single-molecule arrays will be understood by referring to International Publication Nos. 00 / 06770 and International Publication Nos. 01 / 57248, respectively, which are incorporated herein by reference. One use of labeled nucleotides in this disclosure is in synthetic sequencing reactions, but the usefulness of such nucleotides is not limited to such methods. In fact, labeled nucleotides described herein may be advantageously used in any sequencing method that requires the detection of a fluorescent label attached to a nucleotide incorporated into a polynucleotide.

[0199] In particular, modified nucleotides labeled with the dye compounds of this disclosure can be used in automated fluorescence sequencing protocols, especially in fluorescent dye-terminator cycle sequencing based on the Sanger and collaborators' chain arrest sequencing method. Such methods generally use enzymes and cycle sequencing to incorporate fluorescently labeled dideoxynucleotides in a primer extension sequencing reaction. The so-called Sanger sequencing method and related protocols (Sanger-type) utilize chain ends randomized with labeled dideoxynucleotides.

[0200] Therefore, this disclosure also includes nucleotides labeled with dye compounds that are dideoxynucleotides lacking hydroxyl groups at both the 3' and 2' positions, and such modified deoxynucleotides are suitable for use in Sanger sequencing and the like.

[0201] Since the same effect achieved by using a dideoxy base can be achieved by using a nucleotide having a 3'-OH blocking group (both can prevent the incorporation of subsequent nucleotides), it will be recognized that nucleotides labeled with the dye compounds of the present disclosure incorporating a 3'-blocking group may be useful in Sanger sequencing and related protocols. When using a nucleotide of the present disclosure having a 3'-blocking group in a Sanger sequencing method, it will be understood that in each instance in which the labeled nucleotide of the present disclosure is incorporated, it is not necessary for a subsequent nucleotide to be incorporated, and therefore, it is not necessary to remove the label from the nucleotide, and thus the dye compound or detectable label attached to the nucleotide does not need to be linked via a cleavable linker.

[0202] Alternatively, the sequencing method described herein may also be carried out using affinity reagents containing unlabeled nucleotides and fluorescent dyes described herein. For example, one, two, three, or each of the four different types of nucleotides in the incorporation mixture of step (a) (e.g., dATP, dCTP, dGTP, and dTTP or dUTP) may not be labeled. 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 polymerase. After the incorporation of the unlabeled nucleotides in step (b), the remaining unincorporated nucleotides are washed away. Then, an affinity reagent is introduced that specifically recognizes and binds to the incorporated dNTPs, providing a labeled extension product containing the incorporated dNTPs. The use of unlabeled nucleotides and affinity reagents in synthetic sequencing is disclosed in International Publication 2018 / 129214 and International Publication 2020 / 097607. A modified sequencing method of this disclosure using unlabeled nucleotides may include the following steps: (a') Contacting a primer polynucleotide / target polynucleotide complex with one or more unlabeled nucleotides (e.g., dATP, dCTP, dGTP, and dTTP or dUTP), wherein the primer polynucleotide is complementary to at least a portion of the target polynucleotide. (b') Incorporating the nucleotide into the primer polynucleotide to generate an extended primer polynucleotide, (c') Contacting an extended primer polynucleotide with a set of affinity reagents, wherein one affinity reagent specifically binds to an incorporated unlabeled nucleotide to provide a labeled extended primer polynucleotide / target polynucleotide complex. (d') Perform one or more fluorescence measurements on the labeled extended primer polynucleotide / target polynucleotide complex to determine the identity of the incorporated nucleotides.

[0203] In some embodiments of the modified sequencing method described herein, each unlabeled nucleotide in the integration mixture contains a 3' hydroxyl blocking group. In further embodiments, the 3' hydroxyl blocking group of the integrated nucleotide is removed before the next integration cycle. In further embodiments, the method further includes removing an affinity reagent from the integrated nucleotide. In 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 may include a first affinity reagent that specifically binds to a first type of nucleotide, a second affinity reagent that specifically binds to a second type of nucleotide, and a third affinity reagent that specifically binds to a third type of nucleotide. In some further embodiments, each of the first, second, and third affinity reagents includes a spectroscopically distinguishable, detectable label. In some embodiments, the affinity reagent may include a protein tag, an antibody (including, but not limited to, antibody conjugate fragments, single-chain antibodies, bispecific antibodies, etc.), an aptamer, a Nottin, an affimer, or any other known agent that binds to the integrated nucleotide with appropriate specificity and affinity. In one embodiment, at least one affinity reagent is an antibody or protein tag. In another embodiment, at least one of the first, second, and third type affinity reagents is an antibody or protein tag comprising one or more detectable labels (e.g., multiple copies of the same detectable label), wherein the detectable label is or comprises the chromenoquinoline pigment moiety described herein. [Examples]

[0204] Additional embodiments are disclosed in more detail in the following embodiments, but these embodiments are not intended to limit the scope of the claims. Example 1. Synthesis of chromenoquinoline pigment Synthesis of chrome quinoline pigment intermediates

[0205] [ka]

[0206] γ-(7-hydroxy-1,2,3,4-tetrahydroquinoline-1-yl) butyrate tert-butyl ester (1) (3.94 g, 13.5 mmol) was co-evaporated with anhydrous N,N'-dimethylformamide (DMF) (3 × 10 mL). Phosphorus oxychloride (1.9 mL, 20 mmol) was added to 30 mL of anhydrous DMF at 0°C. After 15 minutes, the solution turned pale pink, and the solution for 1 in 10 mL of DMF was added. The reaction mixture was stirred at room temperature for 3 hours, and then quenched with 10 mL of saturated NaHCO3 aqueous solution. After 5 minutes, volatile substances were removed under reduced pressure, leaving a deep green oil. 300 mL of saturated NaHCO3 aqueous solution was added, and the solution was extracted with 5 × 200 mL of ethyl acetate. The pooled organic phase was dried over MgSO4, filtered, and evaporated under reduced pressure to dryness. The residue was purified by flash chromatography on silica gel to obtain compound 2. Yield: 3.656 g, green oil (11.4 mmol, 84%). LC-MS (ES and CI): (positive ion) m / z 320 (M+H + ), (negative ions) m / z 318 (MH + ).

[0207] γ-(6-formyl-7-hydroxy-1,2,3,4-tetrahydroquinoline-1-yl) butyrate tert-butyl ester (2) (2.46 g, 7.72 mmol) was dissolved in anhydrous DMF, and then K2CO3 (2.13 g, 15.4 mmol), tetrabutylammonium iodide (258 mg, 0.70 mmol), and propargyl bromide (80 wt% in toluene, 1.28 mL, 11.6 mmol) were added. The reaction mixture was stirred overnight at room temperature, then quenched with 150 mL of water, and then extracted with 200 mL of ethyl acetate. The aqueous phase was separated and extracted with 2 × 100 mL of ethyl acetate. The combined organic phase was then extracted again with 2 × 100 mL of water. The organic phase was dried over MgSO4, filtered, and evaporated under reduced pressure to dryness. The residue was purified by flash chromatography on silica gel to obtain compound 3. Yield: 2.01 g, yellow solid (5.65 mmol, 73%). LC-MS (ESI): (positive ion) m / z 358 (M+H + ), 380(M+Na + ).

[0208] [ka]

[0209] Compound 5 was synthesized from γ-(1-dimethyl-3-methyl-6-formyl-7-hydroxy-1,2,3,4-tetrahydroquinol-1-yl)butyrate tert-butyl ester (4) as a grayish-white solid. Yield: 351 mg (0.86 mmol, 86%). LC-MS (ES and CI): (positive ion) m / z 400 (M+H + ).

[0210] [ka]

[0211] Compound 7 was synthesized from 4-(diethylamino)salicylaldehyde (6) as a pale pink solid, following the procedure described for compound 3. Yield: 2.263 mg (9.97 mmol, 95%). LC-MS (ES and CI): (positive ion) m / z 232 (M+H + ). Common synthesis of chrome quinoline pigment [ka]

[0212] Substituted 4-amino-2-propargyloxybenzaldehyde (0.336 mmol) and substituted aniline (0.37 mmol) were dissolved in anhydrous DMF under N2. Acetic acid (10 μL, 0.16 mmol) was then added, and the solution was heated at 60°C for 1 hour. TLC (petroleum ether / ethyl acetate 1:1) showed the formation of a yellow intermediate. CuI (13 mg, 0.067 mmol) was then added, and the mixture was stirred at 60-100°C until the reaction was complete. The reaction mixture was diluted with 30 mL of ethyl acetate, filtered through a silica gel plug, and then purified by flash chromatography.

[0213] Compound 8a: Starting from intermediate 3 and p-anisidine, compound 8a was obtained as a yellow solid in 31% yield. LC-MS (ESI): (positive ion) m / z 461 (M + H + ).

[0214] Compound 8b: Starting from intermediates 3 and 4-methylaniline, compound 8b was obtained as a yellow solid in 35% yield (89 mg, 0.20 mmol). LC-MS (ESI): (positive ion) m / z 445 (M+H + ).

[0215] Compound 8c: Starting from intermediates 3 and 3,4-dimethylaniline, compound 8c was obtained as a yellow solid in 21% yield as a 6:4 mixture of the two positional isomers (32 mg, 0.07 mmol). LC-MS (ESI): (positive ion) m / z 459 (M+H) + ).

[0216] Compound 8d: Starting from intermediates 3 and 3,4-dimethoxyaniline, compound 8d was obtained as a yellow solid in 48% yield (68 mg, 0.138 mmol). LC-MS (ESI): (positive ion) m / z 491 (M+H + ).

[0217] Compound 8e: Starting from intermediate 5 and p-anisidine, compound 8e was obtained as a yellow solid in 40% yield (76 mg, 0.151 mmol). LC-MS (ESI): (positive ion) m / z 503 (M+H) + ).

[0218] Compound 8f: Starting from intermediate 7 and methyl-(4-aminophenyl)-acetate, compound 8f was obtained as a yellow solid in 39% yield (321 mg, 0.85 mmol). LC-MS (ESI): (positive ion) m / z 377 (M+H) + ).

[0219] 8 g of compound: Starting from intermediates 3 and 3,4-(methylenedioxy)aniline, 8 g of compound was obtained as a yellow solid in 40% yield (65 mg, 0.137 mmol). LC-MS (ESI): (positive ion) m / z 475 (M+H + ).

[0220] Compound 8h: Starting from intermediates 3 and 3,4,5-trimethoxyaniline, compound 8h was obtained as a yellow solid in 35% yield (65 mg, 0.117 mmol). LC-MS (ESI): (positive ion) m / z 521 (M+H) + ).

[0221] Compound 8i: Starting from intermediates 3 and 5-amino-2,3-dihydrobenzofuran, compound 8i was obtained as a yellow solid in 31% yield (50 mg, 0.117 mmol). LC-MS (ESI): (positive ion) m / z 473 (M+H + ).

[0222] [ka]

[0223] Compound 8a (82 mg, 0.178 mmol) was dissolved in dry 1,4-dioxane (2 mL) in a pressure tube. Iodomethane (0.5 mL, 8 mmol) was added, and the reaction mixture was heated at 100°C. After 2 hours, it was cooled, volatile substances were removed under reduced pressure, and the crude product was purified by flash column chromatography. The intermediate was then dissolved in dry DCM (1 mL), 0.5 mL of trifluoroacetic acid was added, and the reaction mixture was stirred at room temperature for 1.5 hours. Volatile substances were then removed under reduced pressure, and the crude product was purified by flash column chromatography to obtain compound I-1 as a red solid. Yield: 28 mg (0.067, 37%). 1 H NMR (400MHz, MeOD): δ (ppm) 8.38 (s, 1H, Ar-CH), 7.64 (dd, J=9.5, 2.9Hz, 1H, Ar-CH) , 7.57(s, 1H, Ar-CH), 7.51(d, J=2.9Hz, 1H, Ar-CH), 6.52(s, 1H, Ar-CH), 5.18(s, 2H, CH2-O), 4.46(s, 3H, NCH3), 3.99(s, 3H, OCH3), 3.55-3.45(m, 4H, CH2-N), 2.86(t, J= 6.2Hz, 2H, CH2-Ar), 2.41(t, J=6.9Hz, 2H, CH2-COO), 2.07-1.90(m, 4H, CH2-CH2-N). LC-MS(ESI):(Positive ion) m / z419(M+H + ).

[0224] [ka]

[0225] Compound 8b (89 mg, 0.20 mmol) was dissolved in dry 1,2-dichloroethane (2 mL) in a pressure tube. Methyl trifluoromethylmethanesulfonate (0.45 mL, 4 mmol) and K2CO3 (138 mg, 1 mmol) were added, and the reaction mixture was heated at 110°C. After 18 hours, the reaction mixture was cooled, volatile substances were removed under reduced pressure, and the crude product was purified by flash column chromatography. The intermediate was then dissolved in methanol (10 mL), and 0.5 mL of a 4 M solution of NaOH in water was added. The reaction mixture was stirred at room temperature for 18 hours. The solution was then neutralized with 1 N HCl, volatile substances were removed under reduced pressure, and the crude product was purified by preparative scale RP-HPLC on a C18 column to obtain compound I-2 as a red solid. Yield: 30 μmol (15%). 1 H NMR (400MHz, MeOD): δ (ppm) 8.33 (s, 1H, Ar-CH), 8.10 (d, J=8.8Hz, 1H, Ar-CH), 7.92-7.8 4(m, 2H, Ar-CH), 7.57(s, 1H, Ar-CH), 6.53(s, 1H, Ar-CH), 5.16(s, 2H, CH2-O), 4.44(s, 3 H, NCH3), 3.56-3.52(m, 2H, CH2-N), 3.50-3.43(m, 2H, CH2-N), 2.85(t, J=6.2Hz, 2H, CH2 -Ar), 2.59(s, 3H, CH3), 2.25(t, J=7.2Hz, 2H, CH2-COO), 2.06-1.89(m, 4H, CH2-CH2-N). LC-MS(ESI):(Positive ion) m / z403(M+H + ).

[0226] [ka]

[0227] Compound 8c (30 mg, 0.065 mmol) was dissolved in dry 1,2-dichloroethane (1 mL) in a pressure tube. Methyl trifluoromethanesulfonate (1 mL) and diisopropylethylamine (0.5 mL) were added. After 1 hour at room temperature, the mixture was diluted with DCM (20 mL) and extracted with 20 mL of saturated NaHCO3. The organic phase was dried over MgSO4, filtered, and evaporated under reduced pressure to dryness. The red residue was purified by flash column chromatography. The intermediate was then dissolved in methanol (10 mL) and 0.15 mL of a 4 M solution of NaOH in water was added. The reaction mixture was stirred at room temperature for 18 hours. The solution was then neutralized with 1 N HCl (0.6 mL), volatile substances were removed under reduced pressure, and the crude product was purified by RP chromatography on a C18 column to obtain compound I-3 as a red solid in a 6:4 mixture of two positional isomers. Yield: 15 μmol (35%). 1 H NMR (400MHz, MeOD): δ (ppm) 8.71 (s, 0.4H, Ar-CH, isomer B), 8.32 (s, 0.6H, isomer A), 8.0 0(s, 0.6H, Ar-CH, isomer A), 7.96(d, J=9.0Hz, 0.4H, Ar-CH, isomer B), 7.85(d, J=9.0Hz, 0.4H, Ar-CH, isomer B), 7.82(s, 0.6H, Ar-CH, isomer A), 7.56(s, 1H, Ar-CH), 6.54(s, 0.4 H, Ar-CH, isomer B), 6.53(s, 1H, 0.6H, Ar-CH, isomer A), 5.21(s, 0.8H, CH2-O, isomer B), 5.15 (s, 1.2H, CH2-O, isomer A), 4.44(s, 3H, CH3-N), 3.71-3.57(m, TEAB), 3.57-3.51(m, 2 H, CH2-N), 3.50-3.43(m, 2H, CH2-N), 2.85(t, J=6.2Hz, 2H, Ar-CH2), 2.69(s, 1H, C H3 isomer B), 2.63 (s, 2H, CH3 isomer A), 2.56 (s, 1H, CH3 isomer B), 2.51 (s, 2H, CH3 isomer A), 2.2 7(t, J=7.2, 2H, CH2-COO), 2.06-1.91(m, 4H, CH2-CH2-N), 1.20(t, J=7.0Hz, TEAB). LC-MS(ESI):(Positive ion) m / z417(M+H + ).

[0228] [ka]

[0229] Compound 8d (65 mg, 0.132 mmol) was dissolved in dry dichloromethane (1 mL). Diisopropylethylamine (114 μL, 0.66 mmol) and methyl trifluoromethanesulfonate (300 μL, 2.65 mmol) were added under an N2 atmosphere. After 1 hour, volatile substances were removed under reduced pressure. The crude product was then dissolved in methanol (10 mL), and 0.5 mL of a 4 M solution of NaOH in water was added. The reaction mixture was stirred at room temperature for 18 hours. The solution was then neutralized with acetic acid (0.15 mL), and volatile substances were removed under reduced pressure. The residue was purified first by flash column chromatography and then by RP chromatography on a C18 column to obtain compound I-4 as a red solid. Yield: 92 μmol (69%). 1 H NMR (400MHz, MeOD / CDCl3): δ(ppm)8.26(s, 1H, Ar-CH), 7.71(s, 1H, Ar-CH), 7.43(s, 1H, Ar-CH), 7.42(s, 1H , Ar-CH), 6.48(s, 1H, Ar-CH), 5.12(s, 2H, CH2-O), 4.44(s, 3H, CH3-N), 4.15(s, 3H, CH3-O), 4.03(s, 3H, CH3 -O), 3.70-3.55(m, 1H, TEAB), 3.50(d, J=5.6Hz, 2H, CH2-N), 3.44(d, J=7.7Hz, 2H, CH2-N), 2.82(t, J=6.2Hz , 2H, Ar-CH2), 2.25(t, J=7.2Hz, 2H, CH2-COO), 2.06-1.85(m, 4H, CH2-CH2-N), 1.17(t, J=7.0Hz, 1H, TEAB). LC-MS(ESI):(Positive ion) m / z449(M+H + ).

[0230] [ka]

[0231] Compound 8e (80 mg, 0.159 mmol) was dissolved in dry 1,2-dichloroethane (2 mL) in a pressure tube. Methyl trifluoromethanesulfonate (360 μL, 3.18 mmol) and K2CO3 (109 mg, 0.795 mmol) were added under an N2 atmosphere, and the reaction mixture was heated at 110 °C for 24 hours. Volatile substances were removed under reduced pressure, and the red residue was purified by flash column chromatography. The intermediate was then dissolved in methanol (5 mL), and 0.35 mL of a 4 M solution of NaOH in water was added. The reaction mixture was stirred at room temperature for 18 hours. The solution was then neutralized with 1 N HCl (0.15 mL). Volatile substances were removed under reduced pressure, and the crude product was first purified by flash column chromatography, and then by RP chromatography on a C18 column to obtain compound I-5 as a red solid. Yield: 69 μmol (43%). 1 H NMR (400MHz, MeOD / CDCl3): δ(ppm)8.36(s, 1H, Ar-CH), 8.16(d, J=9.5Hz, 1H, Ar-CH), 7.69-7.63(m, 2H, Ar-CH), 7.51(d, J=2.9Hz, 1H, Ar-CH), 6.54(s, 1H, Ar-CH), 5.19(d, J=13.7Hz, 1H, O-CHH), 5.12(d, J=13.7Hz, 1H, O-CHH), 4.47(s, 3H, CH3-N), 4.00(s, 3H) , CH3-O), 3.63(q, J=7.1Hz, 1H, TEAB), 3.58-3.47(m, 1H, CHH-N), 3.36-3.29(m, CHH-N (under MeOH), 3.03-2.89 (m, 1H, Ar-CH), 2.27 (d, J=7.1Hz, 2H, CH2-COO), 2.04-1.84 (m, 4H, CH2-CH2- N), 1.47(s, 3H, CH3), 1.46(d, J=6.5Hz, 3H, CH3), 1.35(s, 3H, CH3), 1.20(t, J=7.0Hz, 2H, TEAB). LC-MS(ESI):(Positive ion) m / z461(M+H + ).

[0232] [ka]

[0233] Compound 8f (310 mg, 0.182 mmol) was dissolved in dry 1,4-dioxane (2 mL) in a pressure tube. Iodomethane (0.5 mL, 8 mmol) was added, and the reaction mixture was heated in the dark at 100 °C. After 2 days, volatile substances were removed under reduced pressure, and the intermediate was then dissolved in methanol (5 mL), to which 1 mL of a 4 M solution of NaOH in water was added. The reaction mixture was stirred at room temperature for 18 hours. The solution was then neutralized with 1 N HCl (0.15 mL). Volatile substances were removed under reduced pressure, and the crude product was purified by preparative scale RP-HPLC on a C18 column to obtain compound I-6 as a red solid. Yield: 28 μmol (15%). 1 H NMR (400MHz, MeOD): δ (ppm) 8.47 (s, 1H, Ar-CH), 8.15 (d, J=9.0Hz, 1H, Ar-CH), 8.02 (dd, J=9.0, 2.0Hz, 1 H, Ar-CH), 7.98(d, J=2.0Hz, 1H, Ar-CH), 7.91(d, J=9.4Hz, 1H, Ar-CH), 6.80(dd, J=9.4, 2.6Hz, 1H, Ar-CH ), 6.51(d, J=2.7Hz, 1H, Ar-CH), 5.25(s, 2H, CH2-O), 4.49(s, 3H, NCH3), 3.73(s, 3H, OCH3), 3.59(q, J=7. 1Hz, 4H, CH2-N), 2.65 (q, J=7.2Hz, 6H, TEAB), 1.29 (t, J=7.1Hz, 6H, CH3Et), 1.10 (t, J=7.2Hz, 9H, TEAB). LC-MS(ESI):(Positive ion) m / z377(M+H + ).

[0234] [ka]

[0235] 8 g (65 mg, 0.137 mmol) of the compound was dissolved in 2 mL of dry dichloromethane. Methyl trifluoromethanesulfonate (77 μL, 0.68 mmol) and N,N-diisopropylethylamine (118 μL, 0.68 mmol) were added under an N2 atmosphere at 0°C, and the reaction mixture was stirred at room temperature for 4 hours. The remaining methyl trifluoromethanesulfonate (77 μL, 0.68 mmol) and N,N-diisopropylethylamine (118 μL, 0.68 mmol) and 2 mL of dry DCM were added. After a further 2 hours, volatile substances were removed under reduced pressure. The red residue was purified by flash column chromatography. The intermediate was then dissolved in methanol (10 mL), and 0.375 mL of a 4 M solution of NaOH in water was added. The reaction mixture was stirred at 60°C for 2 hours. The solution was then neutralized with acetic acid (0.09 mL). Volatile substances were removed under reduced pressure, and the crude product was purified by RP chromatography on a C18 column to obtain compound I-7 as a red solid. Yield: 9.8 μmol (7%). 1 H NMR (400MHz, MeOD): δ (ppm) 8.24 (s, 1H, Ar-CH), 7.62 (s, 1H, Ar-CH), 7.47 (s, 1H, Ar-CH), 7 .34(s, 1H, Ar-CH), 6.48(s, 1H, Ar-CH), 6.29(s, 2H, O-CH2-O), 5.08(s, 2H, CH2-O), 4.38-4. 31(m, 3H, CH3-N), 3.50(t, J=5.4Hz, 2H, CH2-N), 3.44(t, J=7.8Hz, 2H, CH2-N), 2.81(m, 2H, A r-CH2), 2.25(m, 2H, CH2-COO), 2.02-1.87(m, 4H, CH2-CH2-N), 1.20(t, J=7.0Hz, 2H, TEAB). LC-MS(ESI):(Positive ion) m / z433(M+H + ).

[0236] [ka]

[0237] Compound 8h (65 mg, 0.117 mmol) was dissolved in dry dichloromethane (2 mL). Methyl trifluoromethanesulfonate (27 μL, 0.234 mmol) and N,N-diisopropylethylamine (41 μL, 0.234 mmol) were added under an N2 atmosphere at 0°C, and the reaction mixture was then stirred at room temperature for 2 hours. Next, methyl trifluoromethanesulfonate (54 μL, 0.46 mmol) and N,N-diisopropylethylamine (82 μL, 0.46 mmol) were added, and the reaction mixture was stirred at room temperature for 2 hours. Next, methyl trifluoromethanesulfonate (100 μL, 0.88 mmol) and N,N-diisopropylethylamine (82 μL, 0.46 mmol) were added, and the reaction mixture was stirred at room temperature for 1 hour, after which volatile substances were removed under reduced pressure. The intermediate was dissolved in methanol (2 mL), and 0.5 mL of a 4 M solution of NaOH in water was added. The reaction mixture was stirred overnight at room temperature. The solution was then neutralized with acetic acid (0.24 mL). Volatile substances were removed under reduced pressure, and the crude product was purified by RP chromatography on a C18 column to obtain compound I-8 as a red solid. Yield: 70 μmol (60%). 1 H NMR (400MHz, MeOD / CDCl3): δ(ppm)8.49(s, 1H, Ar-CH), 7.51(s, 1H, Ar-CH), 7.23(s, 1H, Ar- CH), 6.49(s, 1H, Ar-CH), 5.09(s, 2H, CH2-O), 4.40(s, 3H, CH3-N), 4.16(s, 3H, CH3-O), 4.15 (s, 3H, CH3-O), 3.98(s, 3H, CH3-O), 3.54-3.50(m, 2H, CH2-N), 3.47-3.41(m, 2H, CH2-N), 2. 84(t, J=6.2Hz, 2H, Ar-CH2), 2.24(t, J=7.2Hz, 2H, CH2-COO), 2.04-1.89(m, 4H, CH2-CH2-N). LC-MS(ESI):(Positive ion) m / z479(M+H + ).

[0238] [ka]

[0239] Compound 8i (50 mg, 0.1 mmol) was dissolved in dry dichloromethane (1 mL). N,N-diisopropylethylamine (208 μL, 1.2 mmol) and methyl trifluoromethanesulfonate (600 μL, 5 mmol) were added under an N2 atmosphere at 0°C, and the reaction mixture was stirred at room temperature for 2 hours. Then, methyl trifluoromethanesulfonate (600 μL, 5 mmol) and N,N-diisopropylethylamine (208 μL, 1.2 mmol) were added, and the reaction mixture was stirred at room temperature for 2 hours. Then, volatile substances were removed under reduced pressure. The red residue was purified by flash column chromatography. The intermediate was dissolved in methanol (5 mL), and 0.25 mL of a 4 M solution of NaOH in water was added. The reaction mixture was stirred at room temperature overnight. Then, the solution was neutralized with acetic acid (0.06 mL). Volatile substances were removed under reduced pressure, and the crude product was purified by RP chromatography on a C18 column to obtain compound I-9 as a red solid. Yield: 4.1 μmol (4%) as a red solid. 1 H NMR (400MHz, MeOD / CDCl3): δ(ppm)8.30(s, 1H, Ar-CH), 8.13(s, 1H, Ar-CH), 7.54(s, 1H, A r-CH), 7.28(s, 1H, Ar-CH), 6.54(s, 1H, Ar-CH), 5.17-5.14(m, 2H, CH2-O), 4.80(t, J=8.4H z, 2H, CH2-CH2-O), 4.49-4.40(m, 3H, CH3-N), 3.70-3.44(m, 6H, CH2-N, CH2-CH2-O), 2.85 (t, J=6.2Hz, 2H, Ar-CH2), 2.27(t, J=7.2Hz, 2H, CH2-COO), 2.04-1.93(m, 4H, CH2-CH2-N). LC-MS(ESI):(Positive ion) m / z431(M+H + ).

[0240] Example 2. General synthesis of nucleotides labeled with chromenoquinoline dye. 0.020 mmol of chromenoquinoline dye of formula (I) was co-evaporated with 2 × 2 mL of anhydrous N,N'-dimethylformamide (DMF), and then dissolved in 2 mL of anhydrous N,N'-dimethylacetamide (DMA). 28.4 μL, 0.163 mmol of N,N-diisopropylethylamine was added, followed by the addition of N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate as a 0.1 M solution in anhydrous DMA (TSTU, 232 μL, 0.023 mmol). The reaction mixture was stirred under nitrogen at room temperature for 30 minutes. During this time, an aqueous solution of 2'-deoxyadenosine triphosphate linker (0.01 mmol) was evaporated under reduced pressure to dryness and resuspended in a 0.1 M solution of triethylammonium bicarbonate (TEAB) in 200 μL of water. The activated chromenoquinoline dye solution was added to the triphate, and the reaction was stirred at room temperature for 18 hours. The crude product was first purified by ion-exchange chromatography on DEAE-Sephadex A25 (25 g). The fraction containing the triphate was pooled, and the solvent was evaporated under reduced pressure to dryness. The crude material was further purified by preparative scale RP-HPLC using a YMC-Pack-Pro C18 column. The final compound was characterized by LC-MS, analytical RP-HPLC, and UV-Vis spectroscopy.

[0241] [ka]

[0242] Compound ffA-LN3-I-1: Yield: 2.7 μmol (54%). LC-MS(ES): (Negative ion) m / z 1346 (MH) + ), 673 (M-2H + ). UV-visible λ 最大 =503nm. Fluorescence emission λ 最大 = 590nm.

[0243] [ka]

[0244] Compound ffA-LN3-I-2: Yield: 6.8 μmol (68%). LC-MS (ES): (negative ion) m / z 1330 (M-H + ), 665 (M-2H + ). UV-visible λ 最大 = 500 nm. Fluorescence emission λ 最大 = 582 nm.

[0245]

Chem.

[0246] Compound ffA-sPA-LN3-I-3: Yield: 6.3 μmol (63%). LC-MS (ES): (negative ion) m / z 1343 (M-H + ), 671 (M-2H + ). UV-visible λ 最大 = 500 nm. Fluorescence emission λ 最大 = 578 nm.

[0247]

Chem.

[0248] <重要说明:这里的“

化

Chem.

[0249]

Chem.

[0250] Compound ffA-LN3-I-5: Yield: 7.3 μmol (73%). LC-MS (ES): (negative ion) m / z 1388 (M-H + ), 694 (M-2H + ). UV-visible λ 最大= 501 nm. Fluorescent emission λ 最大 = 585 nm.

[0251]

Chemical formula

[0252] Compound ffA-LN3-I-6: Yield: 3.1 μmol (31%). LC-MS (ES): (negative ion) m / z 1304 (M-H + ), 652 (M - 2H + ). UV-visible λ 最大 = 495 nm. Fluorescent emission λ 最大 = 574 nm.

[0253]

Chemical formula

[0254] Compound ffA-sPA-LN3-I-8: Yield: 6.9 μmol (69%). LC-MS (ES): (negative ion) m / z 1405 (M-H + ), 702 (M - 2H + ). UV-visible λ 最大 [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Compound ffA-(DB)-AOM-AOL-I-4: Yield: 2.1 μmol (21%). LC-MS(ES):(Negative ion) m / z1407(MH + ), 703 (M-2H + ). UV-visible λ 最大 =500nm. Fluorescence emission λ 最大 = 578 nm.

[0259] Example 3. Spectral characteristics of ffA nucleotides conjugated with chromenoquinoline dye. In this example, the spectral characteristics of several fully functionalized A nucleotides (ffA) conjugated with the chrome quinoline dyes described herein were characterized. Figure 1 shows the fluorescence absorption spectra of ffA nucleotides conjugated with chrome quinoline dyes I-1 to I-6 and I-8 as a 2 μM solution in a universal scan mixture (USM, 1 M Tris pH 7.5, 0.05% Tween, 20 mM sodium ascorbate, 10 mM ethyl gallate). Figures 2A to 2D show the fluorescence emission spectra of ffA nucleotides conjugated with chrome quinoline dyes I-1 to I-6 and I-8, acquired in USM using either 450 nm or 520 nm as the excitation wavelength. The spectra were acquired using an Agilent Cary100 UV-Vis spectrophotometer and a CaryEclipse fluorescence spectrophotometer, using quartz or plastic cuvettes. The chromenoquinoline dyes described herein were observed to be excited by both blue and green light sources and to exhibit strong fluorescence emission.

[0260] Example 4. Stability of ffA labeled with chromenoquinoline dye. The stability of compounds ffA-sPA-I-3 and ffA-sPA-I-4 in high-pH aqueous solutions was evaluated by incubating a 10 μM solution of a given compound in two buffers: 1) 50 mM ethanolamine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.02% CHAPS, 4 mM MgSO4, and 2) 50 mM glycine buffer, pH 9.8, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO4, in the dark at 45°C for 6 days. At the start of the incubation period, the UV-visible absorption and fluorescence emission of the solutions were measured using a quartz cuvette with an Agilent Cary 100 UV-visible spectrophotometer and a Cary Eclipse fluorescence spectrophotometer. In addition, aliquots of the solutions were taken and analyzed by analytical HPLC. Figures 3A and 3B show that the fluorescence emission of ffA-sPA-I-3 and ffA-sPA-I-4 decreased very slowly over time, indicating that the chromoquinoline dyes I-3 and I-4 are stable under these conditions compared to ffC labeled with bright coumarin reference dye A under the same conditions. Coumarin reference dye A is disclosed in U.S. Patent Application Publication No. 2018 / 0094140 and, when conjugated with ffC, has a structural component

[0261] [ka] It has.

[0262] In addition, the stability of compounds ffA-sPA-I-3 and ffA-sPA-I-4 in buffers suitable for nucleotide storage was evaluated by incubating a 10 μM solution in two buffers: 1) 10 mM Tris buffer, pH 8.0, and 2) 100 mM MOPS buffer, pH 7.0, at 40°C in the dark for 14 days. At the start of the incubation period, the UV-visible absorption and fluorescence emission of the solution were measured using a quartz cuvette with an Agilent Cary 100 UV-visible spectrophotometer and a Cary Eclipse fluorescence spectrophotometer. In addition, aliquots of the solution were taken and analyzed by analytical HPLC. Figures 3C and 3D show that, compared to coumarin reference dye A under the same conditions, ffA-sPA-I-3 and ffA-sPA-I-4 retained >90% of their fluorescence after 14 days in Tris pH 8.0 and >75% of their fluorescence after 14 days in MOPS pH 7.0.

[0263] Example 5. Sequence determination experiment using the Illumina iSeq(trademark) 100 instrument. The ffA labeled with the chromenoquinoline dye described herein was tested using an Illumina iSeq® 100 instrument set up to acquire a first image at green excitation light (approximately 520 nm) and a second image at blue excitation light (approximately 450 nm). The sequencing strategy was modified to perform a standard 1 × 300 cycle SBS cycle (integration, followed by imaging, followed by dehiscation). The incorporation mixtures used in each of these experiments contained the following four ffNs, ffA labeled with the chrome quinoline dye of formula (I), ffC excitable with 450 nm blue light (e.g., ffC-linker-coumarin reference dye A), ffT excitable with green light (e.g., ffT-linker-NR550s0), and unlabeled ffG (dark ffG), in 50 mM ethanolamine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO4, and DNA polymerase. Figures 4A to 4E show scattering plots obtained at 26 cycles for each incorporation mixture with ffA nucleotides labeled with chrome quinoline dyes I-1, I-2, I-3, I-4, and I-5. Figure 4F shows phasing and pre-phasing metricing at 1 × 300 cycles. All ffA samples tested showed excellent quality scattering plots and sequencing metric results.

[0264] Table 1 shows the phasing, prephasing, PhiX error rate, and Q30% metric for 2 × 300 cycles performed on an Illumina iSeq™ 100 instrument with an acquisition mixture containing ffA-sPA-LN3-I-4. The instrument was set up to acquire a first image with green excitation light and a second image with blue excitation light, and its strategy was modified to perform a standard 2 × 300 cycle SBS cycle (acquisition, followed by imaging, followed by dehiscation). The incorporated mixtures used in these experiments contained nucleotides ffA-sPA-LN3-I-4, ffC (e.g., ffC-linker-coumarin reference dye A) excitable with 450 nm blue light, ffT (e.g., ffT-linker-NR550s0) excitable with green light, and dark ffG in 50 mM ethanolamine buffer, pH 9.6, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO4, and DNA polymerase.

[0265] [Table 2]

[0266] Table 2 shows the phasing, prephasing, PhiX error rate, and Q30% metric for 2 × 150 cycles and 2 × 300 cycle runs on the IlluminaiSeq(trademark) 100 instrument, performed with an acquisition mixture containing ffA-AOM-AOL-I-4. The instrument was set up to acquire a first image with green excitation light and a second image with blue excitation light, and its strategy was modified to perform a standard 2 × 300 cycle SBS cycle (acquisition, followed by imaging, followed by dehiscation). The incorporated mixtures used in these experiments contained nucleotides ffA-AOM-aOL-I-4, excitable to 450 nm blue light and cleavable with a palladium complex (ffC), excitable to 520 nm green light and cleavable with a palladium complex (ffT), and dark ffG in 50 mM glycine buffer, pH 9.8, 50 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 4 mM MgSO4, and DNA polymerase. The cleavage solution used in these experiments contained 100 mM diethylethanolamine buffer, pH 9.5, 100 mM tris(hydroxypropyl)phosphine, 10 mM [AllylPdCl]2, 10 mM sodium ascorbate, 1 M NaCl, and 0.1% Tween20.

[0267] [Table 3]

Claims

1. A compound of formula (I), a salt or a mesomeric form thereof, [Formula 1] In the formula, R １ , R ４ , R ５ , R ７ , R ８ , R ９ , R １０ , R １１ , R １２ａ and R １２ｂ each independently represents 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; R 2 is C 1 -C 6 alkyl or 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-), -C(O)OR 15 , and -C(O)NR 16 R 17 ; R 15 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; each of R 16 and R 17 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; R3 is H, C １ -C ６ Alkyl or substituted C １ -C ６ is alkyl, R ６ But, C １ -C ６ Alkyl or substituted C １ -C ６ is alkyl, Alternatively, R ８ and R ９ together with the atoms to which they are attached, optionally substituted C ６ -C １０ aryl, optionally substituted 3- to 10-membered carbocyclyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 3- to 10-membered heterocyclyl; However, R ２ and R ３ is ethyl, then R １ , R ４ , R ５ , R ７ , R ８ , R １０ , R １１ , R １２ａ and R １２ｂ is H, and R ６ is methyl, R ９ is a substituted C containing a carboxyl group １ -C ６ The compound, its salt or mesomeric form, provided that it is alkyl.

2. R ２ and R ３ Each of the groups independently represents C １ -C ６ The compound of claim 1 , wherein the aryl group is an alkyl group.

3. The compound of formula (I) is represented by formula (Ia), a salt or a mesomeric form thereof, [Case 2] In the formula, R １３ａ , R １３ｂ , R １４ａ and R １４ｂ each independently represents 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 [C3] is selected from the group consisting of a single bond and a double bond, with the proviso that [C4] When is a double bond, R １４ｂ The compound of claim 1 , provided that: is not present.

4. Solid and dashed lines [C5] is a double bond, Optionally, R 14a is H or C 1 -C 6 alkyl; Further optionally, R 14a is methyl; The compound according to claim 3.

5. Solid and dashed lines [C6] is a single bond, Optionally, R 14a is H and R 14b is C 1 -C 6 alkyl; or Each of R 14a and R 14b is H; The compound according to claim 3.

6. (i) R １３ａ and R １３ｂ is H, or (ii) each of R 13a and R 13b is C 1 -C 6 alkyl; or optionally each of R 13a and R 13b is methyl; The compound according to any one of claims 3 to 5.

7. R ２ is carboxyl or -C(O)NR １６ R １７ C substituted with １ -C ６ alkyl, and each R １６ and R １７ are independently carboxyl, carboxylate, -C(O)OR １５ , C substituted with sulfo or sulfonate １ -C ６ The compound according to any one of claims 1 to 6, which is alkyl.

8. (i) R １ is H, and / or (ii) each R 5 and R 11 is H; and / or (iii) R 6 is C 1 -C 6 alkyl or C 1 -C 6 alkyl substituted with carboxyl or -C(O)NR 16 R 17 ; each R 16 and R 17 is independently C 1 -C 6 alkyl substituted with carboxyl, carboxylate, —C(O)OR 15 , sulfo, or sulfonate; A compound according to any one of claims 1 to 7.

9. (i) R ７ , R ８ , R ９ and R １０ At least one of the groups is independently selected from the group consisting of C １ -C ６ Alkyl or C １ -C ６ and / or (ii) at least one of R 7 , R 8 , R 9 and R 10 is C 1 -C 6 alkyl substituted with carboxyl, carboxylate, sulfo, sulfonate, -C(O)OR 15 or -C(O)NR 16 R 17 , and each R 16 and R 17 is independently H or C 1 -C 6 alkyl substituted with carboxyl, carboxylate, -C(O)OR 15 , sulfo or sulfonate; A compound according to any one of claims 1 to 8.

10. R ８ and R ９ together with the atom to which they are attached form an optionally substituted 5- or 6-membered heterocyclyl, Optionally, said optionally substituted 5- or 6-membered heterocyclyl is [C7] The compound according to any one of claims 1 to 9,

11. R １２ａ and R １２ｂ The compound of any one of claims 1 to 10, wherein each of is H.

12.

8. 2. The compound of claim 1, a salt or mesomeric form thereof, selected from the group consisting of:

13. A nucleotide or oligonucleotide labelled with a compound according to any one of claims 1 to 12, which is attached to the C5 position of a pyrimidine base or to the C7 position of a 7-deazapurine base.

14. The compound is (i) R in formula (I) ２ Or R ６ or via the carboxyl group of (ii) via a carboxyl group of one of R 7 , R 8 , R 9 and R 10 of formula (I); Attached to the nucleotide or oligonucleotide, A labeled nucleotide or oligonucleotide according to claim 13.

15. 15. The labeled nucleotide or oligonucleotide of claim 13 or 14, further comprising a 3'OH blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide.

16. the nucleotide or oligonucleotide is an oligonucleotide hybridized to at least a portion of a target polynucleotide; Optionally, the target polynucleotide is immobilized on a solid support; Optionally, the solid support comprises an array of a plurality of immobilized target polynucleotides. The oligonucleotide according to any one of claims 13 to 15.

17. A kit comprising a first nucleotide labelled with a first compound according to any one of claims 13 to 16, and optionally further comprising a DNA polymerase and one or more buffer compositions.

18. a second nucleotide, said second nucleotide being labeled with a second compound 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, further comprising 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, further comprising a fourth nucleotide, said fourth nucleotide being unlabeled (dark); The kit of claim 17.

19. The kit of claim 17, wherein the first labeled nucleotide has an emission spectrum that is detectable in a single detection channel; or the kit of claim 18, wherein 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.

20. 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, at least one of which is a nucleotide according to any one of claims 13 to 16, and wherein the primer polynucleotide is 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; (c) performing one or more fluorescence measurements of the extended primer polynucleotide to determine the identity of the incorporated nucleotide.

21. 21. The method of claim 20, wherein the primer polynucleotide / target polynucleotide complex is formed by contacting the target polynucleotide with a primer polynucleotide that is complementary to at least a portion of the target polynucleotide.

22. (d) removing a label and a 3′ blocking group from the nucleotide incorporated into the primer polynucleotide; Optionally, (e) further comprising washing the removed label and the 3' blocking group from the primer polynucleotide; Optionally, repeating steps (a) through (e) until the sequence of at least a portion of the template polynucleotide strand is determined; Optionally, steps (a) to (e) are repeated at least 50 times.

22. The method according to claim 20 or 21. (i) the label and the 3' blocking group from the nucleotides incorporated into the primer polynucleotide are removed in a single chemical reaction; and / or (ii) The method according to any one of claims 20 to 22, wherein the method is carried out on an automated sequencing instrument, the automated sequencing instrument comprising two light sources operating at different wavelengths.

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