Fluorescent dye as well as synthesis and application thereof

Abstract

Relates to dye compounds and uses thereof as fluorescent markers, and also relates to a method for preparing the compounds, nucleotides or oligonucleotides labeled by the compounds, and a nucleic acid sequencing method.

Description

A fluorescent dye, its synthesis and application Technical Field

[0001] The present invention relates to the fields of organic chemistry, fluorescent dyes, and gene sequencing. In particular, the present invention relates to a dye compound and its use as a fluorescent marker, a method for preparing the compound, nucleotides or oligonucleotides labeled with the compound, and a nucleic acid sequencing method. Background Art

[0002] Second-generation sequencing, also known as high-throughput sequencing, introduces reversible terminators to achieve sequencing-by-synthesis. During the DNA replication process, the DNA sequence is determined by capturing the fluorescent dye labels on the newly added bases. Currently, commercially available sequencers are mainly second-generation sequencing that uses sequencing-by-synthesis. During the sequencing process, the recognition of bases comes from the recognition of fluorescent dyes modified on the bases. The dyes that modify the four bases generally use emission wavelengths in the green and red regions, which makes the emission wavelengths of the dyes of the two bases partially overlap. The lack of distinction is the key reason for the increase in sequencing error rate or the decrease in sequencing quality. Therefore, the development of new dyes in other visible light regions and their application in gene sequencing will help improve sequencing accuracy.

[0003] Summary of the Invention

[0004] Acridine (azaanthracene) is a macrocyclic conjugated system with a rigid, three-ring planar structure and strong fluorescence, making it an excellent fluorescent marker for applications in in vitro diagnostics, immunoassays, and molecular labeling. Dyes based on acridine are typically synthesized using aniline derivatives as starting materials, followed by a multi-step reaction to create acridine derivatives.

[0005] During the process of structural modification of the substituents of the dye AF532, the inventors discovered that AF532 would undergo a substitution reaction under the conditions of ammonia / methanol under the influence of its own substituents (1,8-disulfonic acid group), and the parent core would be transformed from the original xanthene to an acridine structure.

[0006] The present invention uses substituted xanthenes as raw materials and synthesizes acridine dyes through an ammonia / methanol reaction. The corresponding maximum excitation wavelength and emission wavelength are blue-shifted, with the emission wavelength falling into the cyan region at the maximum excitation wavelength of blue light, thus producing a dye that emits cyan light.

[0007] This application provides the following inventions:

[0008] Dye compounds

[0009] In one aspect, the present application provides a compound as shown in formula (I), an ester thereof or a salt thereof,

[0010] Among them, R 1 、R 2 、R 3 、R 4 Each is the same or different and is independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl;

[0011] R 5 、R 6 are the same or different and are each independently selected from hydrogen, C1-C6 alkyl, halogen, and halogenated C1-C6 alkyl; R 7 、R 8 are the same or different and are each independently selected from hydrogen, -COOH, -C(O)NH-(C1-C6 alkyl) and -C(O)NH2, and R 7 and R 8 Not simultaneously hydrogen;

[0012] Optionally, -NR 1 R 2 Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, wherein the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more groups selected from C1-C6 alkyl, halogen, and halogenated C1-C6 alkyl;

[0013] Optionally, -NR 3 R 4 Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, and the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more groups selected from C1-C6 alkyl, halogen, and halogenated C1-C6 alkyl.

[0014] In certain embodiments, R 1 With R 2 In certain embodiments, R 3 With R 4 In certain embodiments, R 1 、R 2 、R 3 、R 4 same.

[0015] In certain embodiments, R 1 、R 2 、R 3 、R 4 Each is hydrogen.

[0016] In certain embodiments, R 1 、R 2 、R 3 、R 4 Each is a C1-C6 alkyl group (eg, methyl, ethyl).

[0017] In certain embodiments, R 1 、R 2 、R 3 、R 4 Each is a halogenated C1-C6 alkyl group (eg, trifluoromethyl, trifluoroethyl).

[0018] In certain embodiments, R 1 With R 3 In certain embodiments, R 2 With R 4 same.

[0019] In certain embodiments, R 1 With R 3 The same, each is hydrogen, R 2 With R 4 The same, each is a halogenated C1-C6 alkyl group (eg, trifluoromethyl, trifluoroethyl).

[0020] In certain embodiments, R 5 、R 6 same.

[0021] In certain embodiments, R 5 、R 6 Each is hydrogen.

[0022] In certain embodiments, R 5 、R 6 Each is a halogen (eg, fluorine).

[0023] In certain embodiments, R 7 It is a carboxyl group.

[0024] In certain embodiments, R 8 For hydrogen.

[0025] In certain embodiments, R 8 It is a carboxyl group.

[0026] In certain embodiments, -NR 1 R 2 Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, and the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more methyl groups.

[0027] In certain embodiments, -NR 3 R 4 Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, and the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more methyl groups.

[0028] In certain embodiments, R 7 or R 8is a carboxyl group, and the carboxyl group is connected to a cleavable linker, for example, a cleavable linker having the structure shown below:

[0029] In certain embodiments, the compounds of the present invention have a structure as shown in Formula (II):

[0030] Among them, R 7 As defined above.

[0031] In certain embodiments, the compounds of the present invention have a structure as shown in Formula (III):

[0032] Among them, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 As defined above.

[0033] In certain embodiments, the compounds of the present invention have a structure as shown in Formula (IV):

[0034] Among them, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 As defined above.

[0035] The compounds of the present invention may have a structure selected from the group consisting of:

[0036] In one aspect, the present application provides a compound as shown in formula (I'), an ester thereof or a salt thereof,

[0037] Among them, R a 、R b 、R c 、R d Each is the same or different and is independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, -NR 1’ R 2’ , hydroxyl, hydroxyl-substituted C1-C6 alkyl, halogen; R 1’ 、R 2’ Each is the same or different and is independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl;

[0038] R e 、Rf are the same or different and are each independently selected from hydrogen, -COOH, -C(O)NR 3’ R 4’ , and R e and R f Not simultaneously hydrogen;

[0039] R 3’ 、R 4’ are each the same or different and are each independently selected from hydrogen, C1-C6 alkyl, the C1-C6 alkyl being optionally substituted with carboxyl, -C(O)NH2 or sulfonic acid;

[0040] Optionally, when R a -NR 1’ R 2’ When -NR 1’ R 2’ Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, wherein the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more groups selected from C1-C6 alkyl, halogen, and halogenated C1-C6 alkyl;

[0041] Optionally, when R b -NR 1’ R 2’ When -NR 1’ R 2’ Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, and the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more groups selected from C1-C6 alkyl, halogen, and halogenated C1-C6 alkyl.

[0042] In certain embodiments, R a With R b same.

[0043] In certain embodiments, R a With R b Same, and both are -NR 1’ R 2’ Or both are hydroxyl groups.

[0044] In certain embodiments, R c With R d same.

[0045] In certain embodiments, R c With R d are the same, and are both hydrogen or both halogen.

[0046] In certain embodiments, R 1’ 、R 2’ Each is hydrogen.

[0047] In certain embodiments, R 1’ 、R 2’ One of them is hydrogen and the other is selected from C1-C6 alkyl (such as methyl, ethyl).

[0048] In certain embodiments, R e 、R f Different, and each independently selected from hydrogen, -COOH, -C(O)NR 3’ R 4’ .

[0049] In certain embodiments, R e 、R f Different from each other and are each independently selected from hydrogen, -COOH.

[0050] In certain embodiments, R 3’ 、R 4’ are the same or different and are each independently selected from hydrogen, C1-C6 alkyl, said C1-C6 alkyl being optionally substituted with carboxyl or sulfonic acid.

[0051] In certain embodiments, R a -NR 1’ R 2’ , -NR 1’ R 2’ Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, and the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more methyl groups.

[0052] In certain embodiments, R b -NR 1’ R 2’ , -NR 1’ R 2’ Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, and the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more methyl groups.

[0053] In certain embodiments, the compounds of the present invention have a structure as shown in Formula (II'):

[0054] Among them, R e 、R f As defined above.

[0055] In certain embodiments, the compounds of the present invention have a structure as shown in Formula (III'):

[0056] Among them, R a 、R e 、R f As defined above.

[0057] The compounds of the present invention may have a structure selected from the group consisting of:

[0058] The compounds of the present invention can be conjugated to nucleotides or oligonucleotides as fluorescent dyes. 7 is covalently attached to a nucleotide or oligonucleotide, R 7 is selected from -COOH, -C(O)NH-(C1-C6 alkyl) and -C(O)NH2. In certain embodiments, the compound represented by formula (I') of the present invention is e or R f Covalently attached to a nucleotide or oligonucleotide.

[0059] In certain embodiments, the compound represented by formula (I) or formula (I') of the present invention can be covalently attached to a nucleotide or oligonucleotide via a cleavable linker. Therefore, the present invention also provides a compound represented by formula (i):

[0060] Wherein, w is a carboxyl group or an ester group, and the dye is a dye compound including the compound represented by formula (I) or formula (I'), an ester thereof or a salt thereof. In certain embodiments, the dye is a compound represented by formula (I'), which is e or R f With the rest connected.

[0061] The esters of the present invention are preferably activated esters of carboxyl groups. As used herein, the term "activated ester" refers to a derivative of a carboxyl group that is capable of reacting 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 esters, pentafluorophenyl esters, and succinimidyl esters.

[0062] The salt of the compound of the present invention is preferably a salt formed by the sulfonate group on the acridinium ring, such as a salt formed by the sulfonate group and an alkali metal ion, an alkaline earth metal particle or an ammonium ion.

[0063] In certain embodiments, the compound represented by formula (i) has the following structure:

[0064] Among them, R a 、R b 、R c 、R d 、R e As defined above.

[0065] In certain embodiments, the compound represented by formula (i) has the following structure:

[0066] Among them, R a 、R b 、R c 、R d 、R e As defined above.

[0067] In certain embodiments, the compound represented by formula (i) has the following structure:

[0068] Among them, R a 、R b 、R c 、R d 、R e As defined above.

[0069] The compound represented by formula (i) can be used as an intermediate for synthesizing the dye-labeled nucleotide or oligonucleotide of the present invention.

[0070] the term

[0071] As used herein, the term "C1-C6 alkyl" refers to a group obtained by removing a hydrogen atom from a straight-chain or branched alkane containing 1 to 6 carbon atoms, specific examples of which include but are not limited to: methyl, ethyl, propyl, n-butyl, isobutyl, isopropyl, tert-butyl, n-pentyl, and n-hexyl. In the present invention, the preferred C1-C6 alkyl group is a C1-C4 alkyl group.

[0072] As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine.

[0073] As used herein, the term "halogenated" refers to a group or compound in which hydrogen is replaced by one or more halogen atoms, including full halogenation and partial halogenation.

[0074] As used herein, the term "heterocyclyl" refers to a radical derived from a saturated or partially saturated monocyclic or condensed ring compound containing 3 to 14 ring atoms and at least one heteroatom (e.g., 1, 2, 3, 4, or 5 heteroatoms) by removing one hydrogen atom. The term "5-6 membered nitrogen-containing heterocyclyl" refers to a heterocyclyl containing 5 or 6 ring atoms, wherein 1, 2, or 3 of the ring atoms are nitrogen atoms, and optionally, the heterocyclyl further contains 1 or 2 oxygen atoms or sulfur atoms.

[0075] As used herein, the term "salt" refers to (i) a salt formed by an acidic functional group (e.g., -COOH) present in the compounds provided herein with a suitable inorganic or organic cation (base), and includes, but is not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, tris(hydroxymethyl)aminomethane salts. and (ii) salts formed by basic functional groups (e.g., -NH2) present in the compounds provided by the present invention and appropriate inorganic or organic anions (acids), including but not limited to hydrohalides, such as hydrofluorides, hydrochlorides, hydrobromides, hydroiodides, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkanesulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; arylsulfonates, such as benzenesulfonates, p-toluenesulfonates, etc.; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts, such as glycine, trimethylglycine, arginine, ornithine, glutamate, aspartate, etc.

[0076] As used herein, term " ester " refers to the ester that the-COOH existing in the compound provided by the present invention forms with suitable alcohol, or the ester that the-OH existing in the compound provided by the present invention forms with suitable acid (for example, carboxylic acid or oxygen-containing inorganic acid). Suitable ester groups include but are not limited to formates, acetates, propionates, butyrates, acrylates, ethyl succinates, stearic acid esters or palmitates. In the presence of acid or alkali, esters can undergo hydrolysis reactions to generate corresponding acids or alcohols.

[0077] Labeled nucleotides

[0078] Dye compounds of the present invention are suitable for being attached to substrate moiety.Substrate moiety can actually be any molecule or material that fluorescent dye described herein can be conjugated to, and dyestuff can be attached to substrate by way of non-limiting examples, and substrate moiety can include nucleosides, nucleotides, polynucleotides, carbohydrates, proteins, antibodies, parts, particles or solid surfaces, organic polymers and inorganic polymers and combinations thereof or assembly (assemblage), such as chromosome, nucleus, living cells and analogues.In some cases, the nucleotide of such labeling is also referred to as " modified nucleotide (modified nucleotide) ".

[0079] A particularly useful application of the fluorescent dyes of the invention is for labeling biomolecules, such as nucleotides or oligonucleotides. Thus, in one aspect, the present application relates to nucleotides or oligonucleotides labeled with a fluorescent compound of the invention.

[0080] Attachment to biomolecules can be via -C(=O)R 7 In certain embodiments, R is a substituted alkoxy group, which can be used to attach to an amino group of a biomolecule. In one embodiment, -C(=O)R 7 The moiety may be an activated ester residue best suited for further amide / peptide bond formation.

[0081] In certain embodiments, the dye compound can be covalently attached to an oligonucleotide or nucleotide via a nucleotide base. For example, the labeled nucleotide or oligonucleotide can have a label that is attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base by a linker moiety. The labeled nucleotide or oligonucleotide can also have a 3'OH blocking group that is covalently attached to the ribose or deoxyribose of the nucleotide.

[0082] Nucleoside and nucleotide can be marked at the site on sugar or core base.As understood by those of ordinary skill in the art, " nucleotide " is made up of nitrogenous base, sugar and one or more phosphate groups.In RNA, sugar is ribose and in DNA, sugar is deoxyribose, i.e. lacks the sugar of the hydroxyl group present in ribose. Nitrogenous base is the derivative of purine or pyrimidine. Purine is adenine (A) and guanine (G), and pyrimidine is cytosine (C) and thymine (T) or is uracil (U) in the context of RNA. The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine. Nucleotide is also the phosphate ester of nucleoside, wherein esterification occurs on the hydroxyl group of the C-3 or C-5 being attached to sugar. Nucleotide is typically monophosphate, diphosphate or triphosphate.

[0083] A "nucleoside" is structurally similar to a nucleotide but without the phosphate moiety. An example of a nucleoside analog would be one in which a label is linked to the base and there is no phosphate group attached to the sugar molecule.

[0084] " derivative " or " analogue " means that its core structure is identical or very similar to the core structure of the parent compound but it has a chemically modified or physically modified compound or molecule, such as a different or additional side group, which allows the nucleotide or nucleoside to be connected to another molecule. For example, the base can be a deazapurine. Derivatives should be able to undergo Watson-Crick pairing. " Derivative " and " analogue " also mean nucleotide derivatives or nucleoside derivatives with the synthesis of a modified base moiety and / or a modified sugar moiety. Such derivatives and analogues are discussed in, for example, Scheit, Nucleotide analogs (John Wiley & Son, 1980) and Uhlman et al. Chemical Reviews 90: 543-584, 1990. Nucleotide analogs can also contain modified phosphodiester linkages, including phosphorothioate linkages, phosphorodithioate linkages, alkylphosphonate linkages, phosphoranilidate linkages, phosphoramidate linkages, and the like.

[0085] Dye can be attached to any position on the nucleotide base by connecting base, and condition is that Watson-Crick base pairing can still be carried out.Specific core base labeling site includes the C5 position of pyrimidine base or the C7 position of 7-deazapurine base.As described above, connecting base group can be used to dye is covalently attached to nucleoside or nucleotide.

[0086] In a specific embodiment, the nucleoside or nucleotide of the label can be enzymatically incorporable and enzymatically extendable. Therefore, the linker moiety can have sufficient length to connect the nucleotide to the compound so that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by the nucleic acid replicator. Therefore, the linker can also include a spacer unit. For example, the spacer keeps the nucleotide base away from the cleavage site or the label.

[0087] The nucleoside or nucleotide labeled with the dye compound of the present invention may have the following structure:

[0088] Wherein the dye is a dye compound, B is a nucleobase such as, for example, uracil, thymine, cytosine, adenine, guanine, and the like, and L is an optional linker group that may or may not be present. R' can be H, a monophosphate, a diphosphate, a triphosphate, a phosphorothioate, a phosphate analog, -O- attached to a reactive phosphorus-containing group, or -O- protected by a blocking group. R" can be H, OH, a phosphoramidite, or a 3'-OH blocking group, and R'" is H or OH; wherein R" is a phosphoramidite and R is an acid-cleavable hydroxyl protecting group that allows subsequent monomer coupling under automated synthesis conditions.

[0089] The application also relates to and encompasses the polynucleotides that are incorporated into the dye compound of the present invention.Such polynucleotides can be DNA or RNA comprising deoxyribonucleotides or ribonucleotides connected with phosphodiester bonds respectively.Polynucleotides can comprise naturally occurring nucleotides, nucleotides that are different from the non-natural existence (or modification) of the nucleotides of mark described herein or its any combination, provided that there is at least one mark according to the application that has the nucleotides of dye compound.Polynucleotides can also comprise the chemical modification of non-natural backbone connection and / or non-nucleotide.Also contemplate the chimeric structure of the mixture of ribonucleotides and deoxyribonucleotides comprising the nucleotides of at least one mark.

[0090] In certain embodiments, the labeled nucleotides of the present invention have a structure as shown in formula (1):

[0091] Wherein, the dye is a dye compound or a salt thereof including the compound represented by formula (I) or formula (I') above. In certain embodiments, the dye is a compound represented by formula (I'), which is e or R f With the rest connected.

[0092] In certain embodiments, the labeled nucleotide represented by formula (1) has the following structure:

[0093] Among them, R a 、R b 、R c 、R d 、R e As defined above.

[0094] In certain embodiments, the labeled nucleotide represented by formula (1) has the following structure:

[0095] Among them, R a 、R b 、R c 、Rd 、R e As defined above.

[0096] In certain embodiments, the labeled nucleotide represented by formula (1) has the following structure:

[0097] Among them, R a 、R b 、R c 、R d 、R e As defined above.

[0098] The nucleotides in the above formulas can be selected from dATP, dGTP, dCTP, and dTTP. Exemplary labeled dATPs include but are not limited to MGI471-V1-dATP

[0099] Also included are labeled dATP obtained by replacing MGI471 with other dye compounds of the present invention.

[0100] Exemplary labeled dGTPs include, but are not limited to, MGI471-V1-dGTP

[0101] Also included are labeled dGTPs obtained by replacing MGI471 with other dye compounds of the present invention.

[0102] Exemplary labeled dCTPs include, but are not limited to, MGI471-V1-dCTP

[0103] Also included are labeled dCTPs obtained by replacing MGI471 with other dye compounds of the present invention.

[0104] Exemplary labeled dTTPs include, but are not limited to, MGI471-V1-dTTP

[0105] Also included are labeled dTTPs obtained by replacing MGI471 with other dye compounds of the present invention.

[0106] The labeled nucleotides of the present invention also include:

[0107] Wherein, dNTP is selected from dATP, dGTP, dCTP, dTTP, and an exemplary structure is as follows:

[0108] Sequencing methods

[0109] Nucleotides (or nucleosides) comprising fluorescent dyes of the present invention can be used in any analytical method requiring detection of fluorescent markers attached to nucleotides or nucleosides, whether by themselves or incorporated into or associated with larger molecular structures or conjugates. Certain embodiments of the present application relate to methods for sequencing, comprising: (a) incorporating at least one labeled nucleotide as described herein into a polynucleotide; and (b) detecting the labeled nucleotides incorporated into the polynucleotide by detecting a fluorescent signal from a new fluorescent dye attached to the modified nucleotide.

[0110] In certain embodiments, in the synthesis step, at least one labeled nucleotide is incorporated into the polynucleotide by the action of a polymerase. However, other methods for incorporating labeled nucleotides into polynucleotides are not excluded, such as chemical oligonucleotide synthesis or the connection of labeled oligonucleotides to unlabeled oligonucleotides. Therefore, the term "incorporating" nucleotides into a polynucleotide encompasses polynucleotide synthesis by chemical methods as well as enzymatic methods.

[0111] In specific non-limiting embodiments, modified nucleotides or nucleosides labeled with fluorescent dyes according to the present invention can be used in methods of nucleic acid sequencing, resequencing, whole genome sequencing, single nucleotide polymorphism scoring, any other application involving the detection of modified nucleotides or nucleosides when incorporated into polynucleotides, or any other application requiring the use of polynucleotides labeled with modified nucleotides comprising a fluorescent dye of the present invention.

[0112] In specific embodiments, the application provides the purposes of the nucleotide of the modification that comprises dye compound of the present invention in polynucleotide " by synthesis sequencing " reaction.By synthesis sequencing generally relate to and use polymerase or ligase on 5 ' to 3 ' direction one or more nucleotide or oligonucleotide are added to the polynucleotide chain of growth in succession, so that form the polynucleotide chain of the extension complementary with template nucleic acid to be sequenced.The identity (identity) of the base that is present in one or more of the nucleotide of interpolation is determined in detection step or " imaging " step.The identity of the base of interpolation can be determined after each nucleotide is incorporated into step.Then, the sequence of template can use conventional Watson-Crick base pairing rule to infer.Use the nucleotide that is marked with the modification of the dyestuff according to the disclosure to be used to determine that the identity of single base can be useful for example in the scoring of single nucleotide polymorphism, and such single base extension reaction is within the scope of the application.

[0113] In an embodiment, the sequence of the template polynucleotide is determined by detecting the incorporation of one or more nucleotides into a nascent chain complementary to the template polynucleotide to be sequenced via detection of a fluorescent marker attached to the incorporated nucleotide. The nucleic acid template to be sequenced can be DNA or RNA, or even a hybrid molecule comprising deoxynucleotides and ribonucleotides. The nucleic acid template can comprise naturally occurring nucleotides and / or non-naturally occurring nucleotides and natural or non-natural backbone linkages, provided that these do not prevent replication of the template in the sequencing reaction.

[0114] While one application of the modified nucleotides of the present disclosure is in sequencing by synthesis reactions, the utility of such labeled nucleotides is not limited to such methods. In fact, the nucleotides can be advantageously used in any sequencing method requiring detection of fluorescent labels attached to nucleotides incorporated into a polynucleotide.

[0115] In certain embodiments, the present invention provides a method for determining the sequence of a target single-stranded polynucleotide comprising the steps of:

[0116] (a) providing a duplex, nucleotides, a polymerase, and an excision reagent; the duplex comprises a growing nucleic acid chain and a nucleic acid molecule to be sequenced;

[0117] (b) performing a reaction cycle comprising the following steps (i), (ii) and (iii):

[0118] Step (i): using a polymerase to incorporate nucleotides into the growing nucleic acid chain to form a nucleic acid intermediate comprising a blocking group and a detectable label;

[0119] Step (ii): detecting the detectable label on the nucleic acid intermediate;

[0120] Step (iii): using a cleavage reagent to remove the blocking group on the nucleic acid intermediate.

[0121] In certain embodiments, the reaction cycle further comprises step (iv): removing the detectable label on the nucleic acid intermediate using a cleavage reagent.

[0122] In the present invention, nucleic acids may include nucleotides or nucleotide analogs. Nucleotides generally contain a sugar, a nucleobase, and at least one phosphate group. Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include, for example, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidylic acid (CMP), cytidylic acid diphosphate (CDP), cytidylic acid triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), Deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP) and deoxyuridine triphosphate (dUTP). Nucleotide analogs comprising modified nucleobases can also be used in the methods described herein. Exemplary modified nucleobases that can be included in polynucleotides, whether having a natural backbone or an analogous structure, include, for example, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynyl Cytosine, 6-azouracil, 6-azocytosine, 6-azothymidine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-aminoadenine or guanine, 8-thioadenine or guanine, 8-sulfanyladenine or guanine, 8-hydroxyadenine or guanine, 5-halosubstituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, etc. As is known in the art, certain nucleotide analogs cannot be incorporated into polynucleotides, for example, nucleotide analogs such as adenosine 5'-phosphosulfate.

[0123] In the method of the present invention, the nucleic acid molecule to be sequenced is not limited by its length. In certain preferred embodiments, the length of the nucleic acid molecule to be sequenced can be at least 10bp, at least 20bp, at least 30bp, at least 40bp, at least 50bp, at least 100bp, at least 200bp, at least 300bp, at least 400bp, at least 500bp, at least 1000bp, or at least 2000bp. In certain preferred embodiments, the length of the nucleic acid molecule to be sequenced can be 10-20bp, 20-30bp, 30-40bp, 40-50bp, 50-100bp, 100-200bp, 200-300bp, 300-400bp, 400-500bp, 500-1000bp, 1000-2000bp, or more than 2000bp. In certain preferred embodiments, the nucleic acid molecule to be sequenced can have a length of 10-1000bp, to facilitate high-throughput sequencing.

[0124] In certain preferred embodiments, the nucleic acid molecules may be pretreated before being fixed to a support. Such pretreatments include, but are not limited to, fragmentation of the nucleic acid molecules, end-padding, addition of adapters, addition of tags, amplification of the nucleic acid molecules, separation and purification of the nucleic acid molecules, and any combination thereof.

[0125] In certain embodiments, the solid support surface may carry reactive functional groups that react with complementary functional groups on the polynucleotide molecules to form covalent bonds, for example, using the same method as that used for attaching cDNA to a microarray, for example, see Smirnov et al. (2004), Genes, Chromosomes & Cancer, 40:72-77 and Beaucage (2001), Current Medicinal Chemistry, 8:1213-1244, both of which are incorporated herein by reference. DNB can also effectively attach to hydrophobic surfaces, for example, clean glass surfaces with low concentrations of various reactive functional groups (e.g., -OH groups). Attachment via the covalent bonds formed between the polynucleotide molecules and the reactive functional groups on the surface is also referred to as "chemical attachment" in this article. In other embodiments, the polynucleotide molecules can be adsorbed onto the surface. In this embodiment, the polynucleotides are fixed by non-specific interactions with the surface, or by non-covalent interactions such as hydrogen bonds, van der Waals forces, etc.

[0126] In other embodiments, the nucleic acid library can be double-stranded nucleic acid fragments, which are immobilized on the surface of a solid support by ligation reaction with oligonucleic acids immobilized on the surface of a solid support, and then subjected to rolling circle amplification reaction to prepare a sequencing library.

[0127] Reagent test kit

[0128] In another aspect, the application provides a kind of test kit, it comprises the nucleosides and / or nucleotides that are marked with the dye compound of the present invention.In certain embodiments, the test kit comprises one or more nucleotides, wherein at least one nucleotide is the nucleotide that is marked with the dye compound of the present invention.In certain embodiments, the test kit can comprise two or more labeled nucleotides.The fluorescent dye compound of the present invention, the labeled nucleotide or the test kit can be used for order-checking, expression analysis, hybridization analysis, gene analysis, RNA analysis or protein binding assay.This purposes can be carried out on an automatic sequencing instrument.Sequencing instrument can include two lasers operating at different wavelengths.

[0129] In the case that test kit comprises the multiple Nucleotide, especially two Nucleotide and four kinds of Nucleotide that are marked with dye compounds, different Nucleotide can be marked with identical or different dye compounds, or a kind of Nucleotide can not mark dye compounds.In the case that different Nucleotide marks have identical or different dye compounds, the feature of test kit is that the Nucleotide of described dye compound mark can be distinguished by fluorescence spectrum and algorithm.When two kinds of Nucleotide that are marked with fluorescent dye compounds are supplied with test kit form, in certain embodiments, spectrally distinguishable fluorescent dye can be excited at identical wavelength (such as for example by identical laser).When four kinds of Nucleotide that are marked with fluorescent dye compounds are supplied with test kit form, in certain embodiments, spectrally distinguishable two kinds can both be excited at a wavelength, and other two kinds of spectrally distinguishable dye can both be excited at another wavelength.

[0130] The dye compounds of the present invention can be excited by blue light and have an emission wavelength falling in the cyan region. Therefore, in certain embodiments, the kit of the present invention may include at least one fluorescent dye that can be excited by blue light and has an emission wavelength falling in the cyan region, and further includes two spectrally distinguishable dyes that are excited by another wavelength.

[0131] In the present invention, blue light refers to light with a wavelength in the range of approximately 450 nm to 480 nm, and cyan light refers to light with a wavelength in the range of approximately 480 nm to 490 nm. In certain embodiments, the kit of the present invention may further comprise: a reagent for fixing the nucleic acid molecule to be sequenced to a support (e.g., by covalent or non-covalent attachment); a primer for initiating nucleotide polymerization; a polymerase for carrying out nucleotide polymerization; one or more buffer solutions; one or more washing solutions; or any combination thereof.

[0132] In certain embodiments, the test kit of the present invention may also include reagents and / or devices for extracting nucleic acid molecules from a sample. Methods for extracting nucleic acid molecules from a sample are well known in the art. Therefore, various reagents and / or devices for extracting nucleic acid molecules may be configured as needed in the test kit of the present invention, such as reagents for crushing cells, reagents for precipitating DNA, reagents for washing DNA, reagents for dissolving DNA, reagents for precipitating RNA, reagents for washing RNA, reagents for dissolving RNA, reagents for removing protein, reagents for removing DNA (for example, when the target nucleic acid molecule is RNA), reagents for removing RNA (for example, when the target nucleic acid molecule is DNA), and any combination thereof.

[0133] In certain embodiments, kit of the present invention also comprises, for the reagent of pre-treatment nucleic acid molecule.In kit of the present invention, for the reagent of pre-treatment nucleic acid molecule, not subject to additional restriction, and can be selected according to actual needs.Described reagent for pre-treatment nucleic acid molecule comprises for example, for the reagent (for example DNA enzyme I) of nucleic acid molecule fragmentation, for the reagent (for example DNA polymerase, for example T4 DNA polymerase, Pfu DNA polymerase, Klenow DNA polymerase) of polishing nucleic acid molecule end, joint molecule, label molecule, for the reagent (for example ligase, for example T4 DNA ligase) that joint molecule is connected with target nucleic acid molecule, for example, for the reagent (for example, losing 3'-5' exonuclease activity but showing 5'-3' exonuclease activity) of repairing nucleic acid end, for example, for the reagent (for example, DNA polymerase, primer, dNTP) of amplifying nucleic acid molecule, for the reagent (for example chromatography column) of separating and purifying nucleic acid molecule, and its any combination.

[0134] In certain embodiments, the kit of the present invention further comprises a support for fixing the nucleic acid molecules to be sequenced. Typically, the support for fixing the nucleic acid molecules to be sequenced is in a solid phase for ease of operation. Therefore, in this disclosure, "support" is sometimes also referred to as "solid support" or "solid phase support". However, it should be understood that the "support" mentioned herein is not limited to solid, and it can also be a semi-solid (e.g., gel).

[0135] As used herein, the terms "loaded", "fixed" and "attached" when used in reference to nucleic acids, mean attached directly or indirectly to a solid support via covalent or non-covalent bonds. In certain embodiments of the present disclosure, the methods of the present invention include immobilizing nucleic acids on a solid support via covalent attachment. However, generally, it is only necessary that the nucleic acids remain fixed or attached to the solid support under conditions where the solid support is desired to be used (e.g., in applications where nucleic acid amplification and / or sequencing is required). In certain embodiments, immobilizing nucleic acids on a solid support can include immobilizing an oligonucleotide to be used as a capture primer or an amplification primer on a solid support such that the 3' end is available for enzymatic extension and at least a portion of the primer sequence is capable of hybridizing to a complementary nucleic acid sequence; the nucleic acid to be immobilized is then hybridized to the oligonucleotide, in which case the fixed oligonucleotide or polynucleotide can be in a 3'-5' direction. In certain embodiments, immobilizing nucleic acids on a solid support can include binding a nucleic acid binding protein to the solid support by amino modification, and capturing nucleic acid molecules by the nucleic acid binding protein. Alternatively, loading can occur by other means besides base pair hybridization, such as covalent attachment as described above. Non-limiting examples of nucleic acid attachment to a solid support include nucleic acid hybridization, biotin-streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, physical confinement via a hydrogel or other porous polymer, and the like. Various exemplary methods for immobilizing nucleic acids on solid supports can be found in, for example, G. Steinberg-Tatman et al., Bioconjugate Chemistry 2006, 17, 841-848; Xu X. et al. Journal of the American Chemical Society 128 (2006) 9286-9287; U.S. patent applications US 5639603, US 5641658, US2010248991; international patent applications WO 2001062982, WO 2001012862, WO 2007111937, WO0006770, all of which are incorporated herein by reference in their entirety for all purposes, in particular for all teachings relating to the preparation of solid supports having nucleic acids immobilized thereon.

[0136] In the present invention, the support can be made of various suitable materials. Such materials include, for example, inorganic substances, natural polymers, synthetic polymers, and any combination thereof. Specific examples include, but are not limited to, cellulose, cellulose derivatives (e.g., nitrocellulose), acrylic resins, glass, silica gel, silicon dioxide, polystyrene, gelatin, polyvinyl pyrrolidone, copolymers of vinyl and acrylamide, cross-linked polystyrenes such as divinylbenzene (see, for example, Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamide, latex, dextran, rubber, silicon, plastics, natural sponges, metal plastics, cross-linked dextran (e.g., Sephadex TM ), agarose gel (Sepharose TM ), and other supports known to those skilled in the art.

[0137] In certain preferred embodiments, the support for immobilizing the nucleic acid molecules to be sequenced can be a solid support comprising an inert substrate or matrix (e.g., a glass slide, polymer beads, etc.) that has been functionalized, for example, by applying an intermediate material containing reactive groups that allow for covalent attachment of biomolecules such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, particularly the polyacrylamide hydrogels described in WO 2005 / 065814 and US 2008 / 0280773, the contents of which are incorporated herein by reference in their entirety. In such embodiments, the biomolecules (e.g., polynucleotides) can be directly covalently attached to the intermediate material (e.g., hydrogel), while the intermediate material itself can be non-covalently attached to the substrate or matrix (e.g., a glass substrate). In certain preferred embodiments, the support is a glass or silicon wafer whose surface is modified with a layer of avidin, amino, acrylamide silane, or aldehyde chemical groups.

[0138] In the present invention, the support or solid support is not limited to its size, shape and configuration. In some embodiments, the support or solid support is a planar structure, such as a slide, chip, microchip and / or array. The surface of such a support can be in the form of a planar layer.

[0139] In certain preferred embodiments, the support for immobilizing the nucleic acid molecules to be sequenced is an array of beads or wells (also referred to as a chip). The array can be prepared using any of the materials outlined herein for preparing solid supports, and preferably, the surface of the beads or wells on the array is functionalized to facilitate the immobilization of nucleic acid molecules. The number of beads or wells on the array is not limited. For example, each array may contain 10-10 2 , 10 2-10 3 , 10 3 -10 4 , 10 4 -10 5 , 10 5 -10 6 , 10 6 -10 7 , 10 7 -10 8 , 10 8 -10 9 , 10 10 -10 11 , 10 11 -10 12 In certain exemplary embodiments, the surface of each bead or hole can be fixed with one or more nucleic acid molecules. Accordingly, each array can be fixed with 10-10 2 , 10 2 -10 3 , 10 3 -10 4 , 10 4 -10 5 , 10 5 -10 6 , 10 6 -10 7 , 10 7 -10 8 , 10 8 -10 9 , 10 10 -10 11 , 10 11 -10 12 or more nucleic acid molecules. Therefore, such arrays can be particularly advantageously used for high-throughput sequencing of nucleic acid molecules.

[0140] In certain preferred embodiments, the kit of the present invention further comprises a reagent for fixing the nucleic acid molecule to be sequenced to the support (e.g., by covalent or non-covalent attachment). Such reagents include, for example, reagents for activating or modifying nucleic acid molecules (e.g., their 5' ends), such as phosphoric acid, thiol, amine, carboxylic acid, or aldehyde; reagents for activating or modifying the surface of the support, such as amino-alkoxysilanes (e.g., aminopropyltrimethoxysilane, aminopropyltriethoxysilane, 4-aminobutyltriethoxysilane, etc.); cross-linking agents, such as succinic anhydride, phenyl diisocyanate (Guo et al., 1994), maleic anhydride (Yang et al., 1998), 1-ethyl-3-(3-dimethoxysilane)-1-ol; methylaminopropyl)-carbodiimide hydrochloride (EDC), m-maleimidobenzoic acid-N-hydroxysuccinimide ester (MBS), N-succinimidyl[4-iodoacetyl]aminobenzoic acid (SIAB), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), N-γ-maleimidobutyryloxy-succinimide ester (GMBS), 4-(p-maleimidophenyl)butyric acid succinimide (SMPB); and any combination thereof.

[0141] In certain preferred embodiments, the test kit of the present invention also includes a primer for initiating nucleotide polymerization. In the present invention, the primer is not subject to additional restrictions, as long as it can specifically anneal to a region of the target nucleic acid molecule. In some exemplary embodiments, the length of the primer can be 5-50bp, such as 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50bp. In some exemplary embodiments, the primer can include naturally occurring or non-naturally occurring nucleotides. In some exemplary embodiments, the primer includes naturally occurring nucleotides or is composed of naturally occurring nucleotides. In some exemplary embodiments, the primer includes modified nucleotides, such as locked nucleic acid (LNA). In certain preferred embodiments, the primer includes a universal primer sequence.

[0142] In some preferred embodiments, test kit of the present invention also comprises the polymerase for carrying out nucleotide polymerization reaction.In the present invention, various suitable polymerases can be used to carry out polyreaction.In some exemplary embodiments, described polymerase can be the new DNA chain (such as DNA polymerase) synthesized with DNA as a template.In some exemplary embodiments, described polymerase can be the new DNA chain (such as reverse transcriptase) synthesized with RNA as a template.In some exemplary embodiments, described polymerase can be the new RNA chain (such as RNA polymerase) synthesized with DNA or RNA as a template.Therefore, in some preferred embodiments, described polymerase is selected from DNA polymerase, RNA polymerase, and reverse transcriptase.

[0143] In certain preferred embodiments, the kit of the present invention further comprises one or more excision reagents. In certain embodiments, the excision reagent is selected from endonuclease IV and alkaline phosphatase.

[0144] In certain preferred embodiments, the kit of the present invention further comprises one or more buffer solutions. Such buffer solutions include, but are not limited to, buffer solutions for DNA enzyme I, buffer solutions for DNA polymerase, buffer solutions for ligase, buffer solutions for eluting nucleic acid molecules, buffer solutions for dissolving nucleic acid molecules, buffer solutions for carrying out nucleotide polymerization reactions (e.g., PCR), and buffer solutions for carrying out ligation reactions. The kit of the present invention may comprise any one or more of the above-mentioned buffer solutions.

[0145] In certain embodiments, the buffer solution for DNA polymerase comprises monovalent salt ions (e.g., sodium ions, chloride ions) and / or divalent salt ions (e.g., magnesium ions, sulfate ions, manganese ions). In certain embodiments, the concentration of the monovalent salt ions or divalent salt ions in the buffer solution is 10 μM-200 mM, e.g., 10 μM, 50 μM, 100 μM, 200 μM, 500 μM, 1 mM, 3 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, or 200 mM.

[0146] In certain embodiments, the buffer solution for DNA polymerase comprises tris (hydroxymethylaminomethane) (Tris).In certain embodiments, the concentration of Tris in the buffer solution is 10mM-200mM, such as 10mM, 20mM, 50mM, 100mM, 150mM or 200mM.

[0147] In certain embodiments, the buffer solution for DNA polymerase comprises an organic solvent, such as DMSO or glycerol (glycerol). In certain embodiments, the mass content of the organic solvent in the buffer solution is 0.01%-10%, such as 0.01%, 0.02%, 0.05%, 1%, 2%, 5% or 10%.

[0148] In certain embodiments, the pH of the buffer solution for DNA polymerase is 7.0-9.0, such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

[0149] In certain embodiments, the buffer solution for DNA polymerase comprises: monovalent salt ions (e.g., sodium ions, chloride ions), divalent salt ions (e.g., magnesium ions, sulfate ions, manganese ions), Tris, and an organic solvent (e.g., DMSO or glycerol). In certain embodiments, the pH of the buffer solution phase is 8.8.

[0150] In certain preferred embodiments, the kit of the present invention further comprises one or more washing solutions. Examples of such washing solutions include, but are not limited to, phosphate buffer, citrate buffer, Tris-HCl buffer, acetate buffer, carbonate buffer, and the like. The kit of the present invention may comprise any one or more of the above-mentioned washing solutions.

[0151] In another aspect, the present application provides use of the fluorescent dye, labeled nucleotide or oligonucleotide, or kit of the present invention for determining the sequence of a target polynucleotide.

[0152] The present invention also provides uses of the fluorescent dye in sequencing, expression analysis, hybridization analysis, gene analysis, RNA analysis, protein binding assays, in vitro diagnosis, immunoassays, and molecular markers. In certain embodiments, the molecular markers are used for cell imaging, tissue imaging, or in vivo imaging.

[0153] The present invention also provides use of the fluorescent dye in fluorescent labeling, quantification or detection of proteins, enzymes or nucleic acids.

[0154] Preparation method of dye compound

[0155] The present application also provides a method for preparing the dye compound of the present invention, which comprises: in the presence of ammonia water and methanol, causing a xanthene structure to undergo a substitution reaction to convert it into an acridine structure.

[0156] The reaction route for synthesizing the compound of formula (I) is as follows:

[0157] R 1 -R 7 As defined above.

[0158] The reaction route for synthesizing the compound of formula (I') is as follows:

[0159] R a -R f As defined above.

[0160] The method can be carried out at room temperature or under heating (eg, 50-60°C).

[0161] Preparation method of dye-labeled nucleotides

[0162] The present application also provides a method for preparing the labeled nucleotides of the present invention, which can be performed according to any one of method A, method B, and method C.

[0163] Method A:

[0164] Wherein, dye 2 is the dye compound of the present invention, having an acridine structure, and dye 1 is the precursor of dye 2, having a xanthene structure.

[0165] Method B:

[0166] The dye is the dye compound of the present invention.

[0167] Method C:

[0168] The dye is the dye compound of the present invention.

[0169] Method D:

[0170] Wherein, dye 2 is the dye compound of the present invention, having an acridine structure, and dye 1 is the precursor of dye 2, having a xanthene structure.

[0171] In each of the above methods, the nucleotide may have a structure selected from the following: Beneficial effects

[0172] The present invention adopts substituted xanthene as raw material and synthesizes acridine dyes under ammonia / methanol reaction conditions. The corresponding maximum excitation wavelength and emission wavelength are blue-shifted, and the emission wavelength falls into the cyan region after being excited by blue light.

[0173] The present invention develops new dyes in the blue light region or the cyan light region and uses them to label bases, which helps to improve sequencing accuracy in the field of gene sequencing.

[0174] The preparation method of the dye of the present invention has the advantages of simple steps and mild conditions.

[0175] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples. However, it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0176] Figure 1MGI450b 1 H NMR spectrum.

[0177] Figure 2A MGI443 1 H NMR spectrum.

[0178] FIG2B is the F NMR of MGI443.

[0179] Figure 3 shows the MGI447 1 H NMR spectrum.

[0180] Figure 4 shows the MGI477 1 H NMR spectrum.

[0181] Figure 5 shows the MGI456 1 H NMR spectrum.

[0182] Figure 6 shows the MGI485 1 H NMR spectrum.

[0183] Figure 7A shows the MGI501-V1-dATP 31 P NMR spectrum.

[0184] Figure 7B shows the MGI501-V1-dCTP 31 P NMR spectrum.

[0185] Figure 8A shows the MGI495-V1-dATP 31 P NMR spectrum.

[0186] Figure 8B shows the MGI495-V1-dCTP 31 P NMR spectrum.

[0187] Figure 9A shows the MGI450c-V1-dATP 31 P NMR spectrum.

[0188] Figure 9B shows the MGI450c-V1-dCTP 31 P NMR spectrum.

[0189] Figure 9C shows the MGI450c-V1-dGTP 31 P NMR spectrum.

[0190] Figure 10A shows the MGI450b-V1-dATP 31 P NMR spectrum.

[0191] Figure 10B shows the MGI450b-V1-dTTP 31 P NMR spectrum.

[0192] Figure 10C shows MGI450b-V1-dGTP 31 P NMR spectrum.

[0193] Figure 11A shows the MGI443-V1-dATP 31 P NMR spectrum.

[0194] Figure 11B shows the MGI443-V1-dCTP 31 P NMR spectrum.

[0195] Figure 11C shows the MGI443-V1-dTTP 31 P NMR spectrum.

[0196] Figure 11D shows the MGI443-V1-dGTP 31 P NMR spectrum.

[0197] Figure 12A shows the MGI488-V1-dATP 31 P NMR spectrum.

[0198] Figure 12B shows the MGI488-V1-dCTP 31 P NMR spectrum.

[0199] Figure 13A shows the MGI447-V1-dATP 31 P NMR spectrum.

[0200] Figure 13B shows the MGI447-V1-dCTP 31 P NMR spectrum.

[0201] Figure 13C shows the MGI447-V1-dTTP 31 P NMR spectrum.

[0202] Figure 13D shows MGI447-V1-dGTP 31 P NMR spectrum.

[0203] Figure 14A shows the MGI456-V1-dATP 19F NMR spectrum.

[0204] Figure 14B shows the MGI456-V1-dATP 31 P NMR spectrum.

[0205] Figure 14C shows the MGI456-V1-dCTP 19 F NMR spectrum.

[0206] Figure 14D shows the MGI456-V1-dCTP 31 P NMR spectrum.

[0207] Figure 14E shows the MGI456-V1-dTTP 19 F NMR spectrum.

[0208] Figure 14F shows the MGI456-V1-dGTP 19 F NMR spectrum.

[0209] Figure 14G shows the MGI456-V1-dGTP 31 P NMR spectrum.

[0210] Figure 15A shows the MGI485-V1-dATP 31 P NMR spectrum.

[0211] Figure 15B shows the MGI485-V1-dCTP 31 P NMR spectrum.

[0212] Figure 16 shows the MGI450d-V1-dATP 31 P NMR spectrum.

[0213] FIG17A shows the MGI471-V1-dATP 1 H NMR spectrum.

[0214] Figure 17B shows the MGI471-V1-dATP 31 P NMR spectrum.

[0215] Figure 17C shows the MGI471-V1-dCTP 31 P NMR spectrum.

[0216] Figure 17D shows the MGI471-V1-dTTP 31 P NMR spectrum.

[0217] Figure 17E shows the MGI471-V1-dGTP 31 P NMR spectrum.

[0218] FIG18A shows the excitation and emission spectra of MGI471-V1.

[0219] FIG18B shows the excitation and emission spectra of MGI471.

[0220] FIG18C shows the excitation and emission spectra of MGI471-V1-dATP.

[0221] FIG18D shows the excitation and emission spectra of MGI471-V1-dGTP.

[0222] FIG18E shows the excitation and emission spectra of MGI471-V1-dCTP.

[0223] FIG18F shows the excitation and emission spectra of MGI471-V1-dTTP.

[0224] FIG18G shows the excitation and emission spectra of MGI495-V1-dATP.

[0225] FIG18H shows the excitation and emission spectra of MGI495-V1-dCTP.

[0226] FIG18I shows the excitation and emission spectra of MGI501-V1-dATP.

[0227] FIG18J shows the excitation and emission spectra of MGI501-V1-dCTP.

[0228] Figure 18K shows the excitation and emission spectra of MGI447.

[0229] FIG18L shows the excitation and emission spectra of MGI450b.

[0230] FIG18M shows the excitation and emission spectra of MGI456.

[0231] FIG18N shows the excitation and emission spectra of MGI477.

[0232] FIG18O shows the excitation and emission spectra of MGI485.

[0233] FIG18P shows the excitation and emission spectra of MGI450d.

[0234] FIG18Q shows the excitation and emission spectra of MGI450d-V1-dATP.

[0235] FIG18R shows the excitation and emission spectra of MGI450d-V1-dCTP.

[0236] FIG18S shows the excitation and emission spectra of MGI450d-V1-dTTP.

[0237] FIG18T shows the excitation and emission spectra of MGI485-V1-dATP.

[0238] FIG18U shows the excitation and emission spectra of MGI485-V1-dCTP.

[0239] FIG18V shows the excitation and emission spectra of MGI447-V1-dATP.

[0240] FIG18W shows the excitation and emission spectra of MGI447-V1-dCTP.

[0241] FIG18X shows the excitation and emission spectra of MGI447-V1-dTTP.

[0242] FIG18Y shows the excitation and emission spectra of MGI447-V1-dGTP.

[0243] FIG18Z shows the excitation and emission spectra of MGI453.

[0244] FIG18 a shows the excitation and emission spectra of MGI456-V1-dATP.

[0245] FIG18 b shows the excitation and emission spectra of MGI456-V1-dCTP.

[0246] FIG18 c shows the excitation and emission spectra of MGI456-V1-dGTP.

[0247] FIG18 d shows the excitation and emission spectra of MGI456-V1-dTTP.

[0248] Figure 18e shows the excitation and emission spectra of MGI450a.

[0249] FIG18 f shows the excitation and emission spectra of MGI450b-V1-dATP.

[0250] Figure 18g shows the excitation and emission spectra of MGI450b-V1-dCTP.

[0251] Figure 18h shows the excitation and emission spectra of MGI450b-V1-dGTP.

[0252] FIG18i shows the excitation and emission spectra of MGI450b-V1-dTTP.

[0253] FIG18j shows the excitation and emission spectra of MGI488-V1-dATP.

[0254] Figure 18k shows the excitation and emission spectra of MGI488-V1-dCTP.

[0255] FIG181 shows the excitation and emission spectra of MGI443-V1-dATP.

[0256] Figure 18m shows the excitation and emission spectra of MGI443-V1-dCTP.

[0257] FIG18n shows the excitation and emission spectra of MGI443-V1-dGTP.

[0258] FIG18o shows the excitation and emission spectra of MGI443-V1-dTTP.

[0259] Figure 18p shows the excitation and emission spectra of MGI450c.

[0260] Figure 18q shows the excitation and emission spectra of MGI450c-V1-dATP.

[0261] Figure 18r shows the excitation and emission spectra of MGI450c-V1-dCTP.

[0262] Figure 18s shows the excitation and emission spectra of MGI450c-V1-dGTP.

[0263] FIG18t shows the excitation and emission spectra of MGI450c-V1-dTTP.

[0264] Figure 18u shows the excitation and emission spectra of MGI455.

[0265] Figure 18v shows the excitation and emission spectra of MGI488.

[0266] Figure 18w shows the excitation and emission spectra of MGI443.

[0267] Figure 18x shows the excitation and emission spectra of MGI453.

[0268] Figure 18y shows the excitation and emission spectra of MGI450a.

[0269] FIG19 shows the Q30 data of MGI471 dNTP on the sequencer. DETAILED DESCRIPTION

[0270] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0271] As used herein, common organic abbreviations are defined as follows: Boc tert-butyloxycarbonyl °C degrees Celsius dATP deoxyadenosine triphosphate dTTP deoxythymidine triphosphate dCTP deoxycytidine triphosphate dGTP deoxyguanosine triphosphate DIPEA N'N-diisopropylethylamine acetonitrile DMAP 4-dimethylaminopyridine DMF N'N-dimethylformamide DMSO-d6 deuterated dimethyl sulfoxide D2O deuterated water DSC N,N'-disuccinimidyl carbonate FA formic acid g gram LCMS liquid chromatography mass spectrometry mg milligram mL milliliter MeOH methanol m / z mass-to-charge ratio NH3·H2O ammonia TEAB triethylamine-carbonic acid buffer THF tetrahydrofuran

[0272] Example 1 Synthesis of MGI453

[0273] (1) Synthesis of 3',6'-dihydroxy-3-carbonyl-3H-spiro[isobenzofuran-1,9'-xanthen]-4',5'-disulfonic acid (MGI453-1)

[0274] To a 100 mL flask, add 30% fuming sulfuric acid (30 mL) and then fluorescein (3 g, 9.03 mmol). Heat to 90°C and stir for 2 hours. Slowly add the reaction solution dropwise to crushed ice to quench the reaction. Filter and purify the filtrate by flash preparative liquid chromatography (water / acetonitrile). The preparative solution is concentrated under reduced pressure to obtain MGI453-1. LCMS: calculated for C 20 H 12 O 11 S2[M+H] + :492.98.Found,m / z,[M+H] + :493.10.

[0275] (2) Synthesis of 2-(3,6-dihydroxy-4,5-disulfonic acid acridin-9-yl)benzoic acid (MGI453-2)

[0276] In a 100 mL flask, weigh MGI453-1 (200 mg, 406.15 μmol) and dissolve it in aqueous ammonia (25-28%, 20 mL) and methanol (20 mL). Heat to 50°C and stir under electromagnetic field for 15 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product, which was purified by flash preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain MGI453-2. LCMS: calculated for C 20 H 13 NO10 S2[M+H] + :492.00.Found,m / z,[M+H] + :492.00.

[0277] 1 H NMR (600MHz, dmso) δ = 12.70 (s, 1H), 8.28 (d, J = 7.8, 1H), 7.86 (dt, J = 30.8, 7.6, 2H), 7.51 (d, J = 7.5, 1H), 7.43 (d, J = 9.4, 2H), 7.30 (d, J = 9.4, 2H).

[0278] (3) Synthesis of 4-(2-(3,6-dihydroxy-4,5-disulfonic acid acridin-9-yl)-N-methylbenzamido)butyric acid (MGI453)

[0279] In a 15 mL vial, MGI453-2 (50 mg, 101.74 μmol) was weighed and dissolved in anhydrous N'N-dimethylformamide (2 mL). N,N'-disuccinimidyl carbonate (52 mg, 202.99 μmol) and 4-dimethylaminopyridine (13 mg, 106.41 μmol) were then added. The mixture was stirred at 20°C for 3 hours. 4-Methylaminobutyric acid hydrochloride (78 mg, 507.79 μmol) and triethylamine (103 mg, 1.02 mmol) were then added. The mixture was stirred at 20°C for 15 hours. The filtrate was purified by flash preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain a yellow solid. This was then purified by preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain MGI453. LCMS: calculated for C 25 H 22 N2O 11 S2[M+H] + :590.07.Found,m / z,[M+H] + :591.04.

[0280] 1H NMR(600MHz,dmso)δ=12.73(s,1H),7.78-7.67(m,3H),7.61-7.51(m,3H),7.31(dd,J=18.5,9.4,2H),3.21-3.13(m,1 H),2.95(t,J=7.2,1H),2.90(s,3H),2.16(t,J=7.0,1H),1.89(t,J=7.1,1H),1.63-1.57(m,1H),1.25(p,J=7.1,1H).

[0281] Example 2 Synthesis of MGI450a

[0282] (1) Synthesis of 2-(6-amino-3-imino-3H-xanthen-9-yl)benzoic acid (MGI450a-1)

[0283] In a 40 mL sample bottle, weigh 3-aminophenol (2.95 g, 27.01 mmol), add methanesulfonic acid (15 mL), then phthalic anhydride (2 g, 13.50 mmol), heat to 180°C, and stir for 6 hours. The reaction mixture was slowly poured into ice water to quench the reaction, filtered, and the filter cake was washed with a small amount of water to obtain a red solid. This solid was slurried in dichloromethane / methanol = 10 / 1 (50 mL), filtered, and dried to obtain MGI450a-1. LCMS: calculated for C 20 H 14 N2O3[M+H] + :331.10.Found,m / z,[M+H] + :331.18.

[0284] (2) Synthesis of 2-(3,6-diamino-4,5-disulfonic acid acridin-9-yl)benzoic acid (MGI450a-2)

[0285] Take a 250mL flask, add 50% fuming sulfuric acid (100mL) to dissolve, then add MGI450a-1 (2.2g, 6.66mmol) in batches, heat to 60℃, and stir under electromagnetic field for 15 hours. The reaction solution is slowly added dropwise to ice water to quench the reaction, and then sodium carbonate is added to adjust the pH to neutral. Equal volumes of methanol and ammonia water (25-28%, 100mL) are added and stirred under electromagnetic field for 15 hours at 22℃. The reaction solution is concentrated under reduced pressure to remove most of the solvent, filtered, and the filtrate is purified by flash preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile). The preparative solution is concentrated under reduced pressure to obtain MGI450a-2. LCMS: calculated for C 20 H 15N3O8S2[M+H] + :490.03.Found,m / z,[M+H] + :490.13.

[0286] (3) Synthesis of 4-(2-(3,6-diamino-4,5-disulfonic acid acridin-9-yl)-N-methylbenzamido)butyric acid (MGI450a)

[0287] MGI450a-2 (200 mg, 408.60 μmol) was weighed into a 50 mL flask and dissolved in anhydrous N'N-dimethylformamide (10 mL). N,N'-disuccinimidyl carbonate (209 mg, 815.86 μmol) and 4-dimethylaminopyridine (50 mg, 409.26 μmol) were then added. The mixture was stirred at 22°C for 15 hours. 4-Methylaminobutyric acid (96 mg, 819.48 μmol) and triethylamine (165 mg, 1.63 mmol) were then added. The mixture was stirred at 20°C for another 15 hours. The filtrate was purified by flash preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile) and then by preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile). The preparative solution was concentrated under reduced pressure to obtain MGI450a. LCMS: calculated for C 25 H 24 N4O9S2[M+H] + :589.10.Found,m / z,[M+H] + :589.28.

[0288] 1 H NMR (600MHz, DMSO) δ = 14.62 (s, 1H), 8.13 (d, J = 7.3, 1H), 7.67 (d, J = 7.0, 2H), 7.23 (d, J = 6.3, 1H), 6.98 (d, J = 9.3, 2H), 6.91 (d, J = 9.3, 2H).

[0289] Example 3 Synthesis of MGI450b

[0290] (1) Synthesis of methyl 4-(6-amino-3-imino-3H-xanthen-9-yl)benzoate (MGI450b-1)

[0291] Take a 40mL sample bottle, weigh 3-aminophenol (2.91g, 26.64mmol), add methanesulfonic acid (20mL), then add p-formylbenzoic acid (2g, 13.32mmol), heat to 180℃, and stir under electromagnetic field for 5 hours. The reaction solution is transferred to a 250mL flask, methanol (50mL) and dichloromethane (50mL) are added, and then 2,3-dichloro-5,6-dicyano-p-benzoquinone (3.02g, 13.32mmol) is added. Stir under electromagnetic field for 2 hours at 18℃. The reaction solution is concentrated under reduced pressure to remove methanol and dichloromethane, and then the concentrate is poured into water (500mL), stirred, and filtered to obtain the crude product. It is purified by column chromatography to obtain MGI450b-1. LCMS: calcd for C 21 H 16 N2O3[M+H] + :345.12.Found,m / z,[M+H] + :345.17.

[0292] (2) Synthesis of 4-(6-amino-3-imino-3H-xanthen-9-yl)benzoic acid (MGI450b-2)

[0293] In a 100 mL flask, weigh MGI450b-1 (3.9 g, 11.33 mmol), add methanol (90 mL), and then dissolve in an aqueous solution (30 mL) of sodium hydroxide (906 mg, 22.65 mmol). Stir under electromagnetic field at 19°C for 5 hours. The reaction mixture is concentrated under reduced pressure to remove the methanol, adjusted to pH 5-6 with 1 M aqueous hydrochloric acid, filtered, washed with a small amount of water, and dried to obtain MGI450b-2.

[0294] (3) Synthesis of 4-(6-amino-3-imino-4,5-disulfonic-3H-xanthen-9-yl)benzoic acid (MGI450b-3)

[0295] To a 250 mL flask, add 50% fuming sulfuric acid (100 mL) to dissolve the product. Then, add MGI450b-2 (2 g, 6.05 mmol), heat to 60°C, and stir under electromagnetic stirring for 20 hours. The reaction solution was slowly added dropwise to crushed ice to quench the reaction. The filtrate was filtered and purified by flash preparative liquid chromatography (water / acetonitrile). The preparative solution was concentrated under reduced pressure to obtain MGI450b-3. LCMS: calculated for C 20 H 14 N2O9S2[MH] - :489.01.Found,m / z,[MH] - :489.11.

[0296] (4) Synthesis of 4-(3,6-diamino-4,5-disulfonic acid acridin-9-yl)benzoic acid (MGI450b)

[0297] Weigh MGI450b-3 (100 mg, 203.89 μmol) in a 40 mL sample vial and dissolve in 5 mL of ammonia solution (25-28%) and methanol. Stir under electromagnetic field at 25°C for 15 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product, which was purified by preparative liquid chromatography (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain MGI450b. LCMS: calculated for C 20 H 15 N3O8S2[MH] - :488.03.Found,m / z,[MH] - :488.19.

[0298] 1 H NMR (600MHz, dmso) δ = 8.14 (d, J = 8.2, 2H), 7.47 (d, J = 8.0, 2H), 7.07 (d, J = 9.3, 2H), 6.96 (d, J = 9.4, 2H).

[0299] Example 4

[0300] The fluorescent dye MGI450c was synthesized according to the method of synthesizing MGI450b.

[0301] LCMS:calcd for C 21 H 15 N3O 10 S2[MH] - :532.02.Found,m / z,[MH] - :532.18.

[0302] Example 5

[0303] The fluorescent dye MGI450d was synthesized according to the method of synthesizing MGI450b.

[0304] LCMS:calcd for C 24 H 22 N4O 12 S3[MH] - :653.04.Found,m / z,[MH] - :653.11.

[0305] Example 6

[0306] The fluorescent dye MGI443 was synthesized according to the method of synthesizing MGI450b.

[0307] LCMS:calcd for C 20 H 13 F2N3O8S2[MH] - :524.01.Found,m / z,[MH] - :524.17.

[0308] 1 H NMR (400MHz, dmso) δ = 8.16 (d, J = 8.2, 2H), 7.51 (d, J = 8.1, 2H), 6.95 (d, J = 11.6, 2H).

[0309] Example 7

[0310] The fluorescent dye MGI455 was synthesized according to the method of synthesizing MGI450b.

[0311] LCMS:calcd for C 20 H 13 Cl2N3O8S2[M+H] + :557.95.Found,m / z,[M+H] + :558.90.

[0312] 1 H NMR (600MHz, DMSO) δ = 14.97 (s, 1H), 8.73 (s, 2H), 8.21-8.16 (m, 2H), 7.56 (d, J = 8.1, 2H), 7.28 (s, 2H).

[0313] Example 8

[0314] The fluorescent dye MGI447 was synthesized according to the method of synthesizing MGI450b.

[0315] LCMS:calcd for C 22 H 19 N3O8S2[MH] - :516.06.Found,m / z,[MH] - :516.12.

[0316] 1 H NMR (600MHz, dmso) δ = 8.12 (d, J = 7.6, 2H), 7.36 (d, J = 7.7, 2H), 6.98 (s, 2H), 2.11 (s, 6H).

[0317] Example 9

[0318] The fluorescent dye MGI477 was synthesized according to the method of synthesizing MGI450b.

[0319] LCMS:calcd for C 24 H 23 N3O8S2[MH] - :544.09.Found,m / z,[MH] - :544.18.

[0320] 1 H NMR (600MHz, dmso) δ = 8.94 (s, 2H), 8.16 (d, J = 7.4, 2H), 7.51 (d, J = 7.3, 2H), 7. 17(d,J=9.5,2H),7.09(d,J=9.6,2H),3.41-3.35(m,4H),1.21(t,J=7.1,6H).

[0321] Example 10

[0322] The fluorescent dye MGI456 was synthesized according to the method of synthesizing MGI450b.

[0323] LCMS:calcd for C 24 H 17 F6N3O8S2[M+H] + :654.04.Found,m / z,[M+H] + :654.20.

[0324] 1 H NMR (600MHz, dmso) δ = 9.39 (t, J = 6.7, 2H), 8.17 ( d, J = 7.5, 2H), 7.55 ( d, J = 7.0, 2H), 7.40 ( d, J = 9.7, 2H), 7.26 ( d, J = 9.6, 2H), 4.47 ( p, J = 8.9, 4H).

[0325] Example 11

[0326] The fluorescent dye MGI471 was synthesized according to the method of synthesizing MGI450b.

[0327] LCMS:calcd for C 30 H 31 N3O8S2[MH] - :624.16.Found,m / z,[MH] - :624.23.

[0328] 1H NMR (600MHz, DMSO-d6): δ14.37(s,1H),8.22-8.18(m,2H),7.83(s,2H),7.52(dt,J=8.6 ,4.5Hz,2H),6.76(s,2H),3.77-3.71(m,2H),1.18-1.14(m,12H),0.99(d,J=4.3Hz,6H).

[0329] Example 12

[0330] The fluorescent dye MGI485 was synthesized according to the method of synthesizing MGI450b.

[0331] LCMS:calcd for C 32 H 35 N3O8S2[MH] - :652.19.Found,m / z,[MH] - :652.45.

[0332] 1 H NMR (600MHz, dmso) δ = 9.28 (s, 2H), 8.17 (d, J = 6.4, 2H), 7.49 (d, J = 5.4, 2H), 6.98 (s, 2H), 1.81 (d, J = 9.0, 2H), 1.30 -1.24(m,16H),1.04(dd,J=14.6,8.5,6H).

[0333] Example 13

[0334] The fluorescent dye MGI488 was synthesized according to the method of synthesizing MGI450b.

[0335] LCMS:calcd for C 29 H 32 N4O9S2[MH] - :643.16.Found,m / z,[MH] - :643.22.

[0336] 1H NMR (400MHz, dmso) δ = 8.88 (t, J = 5.2, 2H), 7.72-7.63 (m, 2H), 7.62-7.53 (m ,1H),7.43-7.31(m,1H),7.21-7.00(m,4H),3.35(dt,J=12.5,6.8,4H),3.1 5-3.06(m,1H),3.05-2.98(m,1H),2.83(s,3H),2.12(t,J=6.9,1H),1.95( t,J=7.1,1H),1.56(q,J=7.3,1H),1.34(q,J=7.3,1H),1.21(t,J=7.2,6H).

[0337] Example 14 Synthesis of MGI501-modified nucleotides

[0338] (1) Synthesis of MGI501-1-V1-dATP or MGI501-1-V1-dCTP

[0339] In a 4 mL vial, weigh AF546 NHS (10 mg, 9.45 μmol) and dissolve in N,N-dimethylformamide (1 mL). Add V1-dATP (18 mg, 18.99 μmol) and N,N-diisopropylaminoethylamine (12 mg, 92.85 μmol). Stir at 22°C for 15 hours. Filter the reaction mixture and purify by flash preparative chromatography (0.1 M TEAB / acetonitrile). Concentrate the preparative solution under reduced pressure to obtain MGI501-1-V1-dATP. LCMS: calculated for C 70 H 82 Cl3N 16 O 28 P3S3[MH] - :1889.29.Found,m / z,[(M-2) / 2] - :944.45.

[0340] MGI501-1-V1-dCTP was synthesized by the same method as above.

[0341] LCMS:calcd for C 68 H 80 Cl3N 14 O 30 P3S3[MH] - :1865.26.Found,m / z,[(M-2) / 2] - :932.87.

[0342] (2) Synthesis of MGI501-V1-dATP or MGI501-V1-dCTP

[0343] Weigh MGI501-1-V1-dATP (10 mg, 5.29 μmol) in a 15 mL vial and dissolve in 7 M ammonia in methanol (2 mL). Stir at 23°C for 15 hours. Filter the reaction mixture and purify by preparative liquid chromatography (0.1% ammonia / acetonitrile). Concentrate the solution under reduced pressure and freeze-dry to obtain MGI501-V1-dATP. LCMS: calculated for C 70 H 83 Cl3N 17 O 27 P3S3[MH] - :1888.31.Found,m / z,[(M-2) / 2] - :943.87.

[0344] 1 H NMR (400MHz, DMSO-d6) δ9.24(t,J=6.1Hz,2H),8.44(s,1H),8.10(s,1H),8.01(s,2H),7.70(s,1H),7.48-7.41(m,2H),7.35-7.27(m,2 H),7.08(d,J=14.6Hz,3H),6.99(d,J=15.8Hz,2H),6.49(dd,J=8.9,5.8Hz,1H),5.14(s,1H),4.93(d,J=9.0Hz,1H),4.86(d,J=8.8Hz, 1H),4.57(s,1H),4.25-4.20(m,1H),4.14(d,J=5.5Hz,4H),3.98(s,7H),3.68(s,2H),2.86(d,J=62.4Hz,16H),2.01(s,2H),1.82(d,J =12.8Hz,2H),1.43(s,2H),1.31(s,5H),1.25(d,J=5.5Hz,3H),1.20(t,J=6.3Hz,3H),1.12(dd,J=6.5,3.4Hz,2H),1.07-1.00(m,3H).

[0345] 31 P NMR (162MHz, dmso) δ = -9.98 (d, J = 18.5), -13.44, -22.65.

[0346] MGI501-V1-dCTP was synthesized by the same method as above.

[0347] LCMS:calcd for C 68 H 81 Cl3N 15 O 29 P3S3[MH] - :1864.28.Found,m / z,[MH] - :[(M-2) / 2] - :932.30.

[0348] 1 H NMR(400MHz,dmso)δ9.24(d,J=5.9Hz,2H),8.67(s,2H),8.03(s,3H),7.75(s ,1H),7.45(d,J=7.6Hz,2H),7.35-7.29(m,1H),7.11(s,2H),7.07(s,1H),7. 00(d,J=15.7Hz,2H),6.88(s,1H),6.15-6.07(m,1H),5.16(s,1H),4.92(d,J =9.1Hz,1H),4.81(d,J=8.9Hz,1H),4.41(s,1H),4.27-4.21(m,2H),4.15(dd ,J=17.8,8.4Hz,4H),4.01-3.90(m,5H),3.89-3.82(m,2H),3.70-3.65(m,2H ),3.49(d,J=8.2Hz,2H),3.19(s,2H),2.93(d,J=6.6Hz,3H),2.85(s,2H),2. 71(d,J=17.9Hz,1H),2.31-2.30(m,1H),2.15(s,2H),2.01(s,2H),1.82(d,J =12.4Hz,2H),1.42(d,J=8.2Hz,3H),1.36-1.23(m,12H),1.22-1.11(m,6H).

[0349] 31 P NMR(162MHz,dmso)δ74.99(s),-10.17--10.42(m),-12.28--12.91(m),-22.73(s).

[0350] Example 15 Synthesis of MGI495-modified nucleotides

[0351] (1) Synthesis of MGI495-1-V1-dATP and MGI495-1-V1-dCTP

[0352] MB543 NHS (15 mg, 16.36 μmol) was weighed into a 15 mL vial and dissolved in anhydrous N'N-dimethylformamide (2 mL). V1-dATP (18 mg, 18.99 μmol) and N'N-diisopropylethylamine (19 mg, 139.27 μmol) were then added. The mixture was stirred at 22°C for 15 hours. The reaction mixture was filtered and purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure to obtain MGI495-1-V1-dATP. LCMS: calculated for C 66 H 79 N 16 O 29 P3S3[MH] - :1747.36Found,m / z,[(M-2) / 2] - :873.35.

[0353] MGI495-1-V1-dCTP was synthesized by the same method as above.

[0354] LCMS:calcd for C 64 H 77 N 14 O 31 P3S3[MH] - :1725.33Found,m / z,[(M-2) / 2] - :862.03.

[0355] (2) Synthesis of MGI495-V1-dATP and MGI495-V1-dCTP

[0356] MGI495-1-V1-dATP (25 mg, 14.29 μmol) was weighed into a 15 mL vial and dissolved in methanol (2 mL). Ammonia (2 mL) was then added and stirred at 25°C for 15 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product, which was then purified by preparative HPLC (0.1% NH3·H2O / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain compound MGI495-V1-dATP. LCMS: calculated for C 66 H 80 N 17 O 28 P3S3[MH] - :1746.37Found,m / z,[(M-2) / 2] - :837.09.

[0357] 1H NMR(400MHz,dmso)δ9.26(d,J=7.8Hz,2H),9.04(d,J=42.3Hz,2H),8.76(s,1H) ,8.43(t,J=5.5Hz,1H),8.24(t,J=11.7Hz,1H),8.16-8.08(m,1H),8.07-7.97(m ,1H),7.70(s,1H),7.57-7.47(m,2H),7.46-7.34(m,2H),7.12(d,J=8.3Hz,2H) ,6.94(d,J=5.3Hz,2H),6.49(dd,J=8.7,5.9Hz,1H),5.15(t,J=4.6Hz,1H),4.90 (dd,J=31.3,8.9Hz,2H),4.56(d,J=4.9Hz,1H),4.25(dd,J=10.4,4.0Hz,1H),4 .16(dd,J=16.6,5.3Hz,3H),3.99-3.83(m,5H),3.72-3.67(m,2H),3.31(s,3H), 3.06(dd,J=15.8,8.6Hz,1H),2.99-2.73(m,3H),2.65-2.57(m,2H),2.42-2.37( m,1H),1.81(d,J=13.6Hz,2H),1.45-1.12(m,12H),1.04(dd,J=6.0,2.6Hz,6H).

[0358] 31 P NMR(162MHz,dmso)δ-10.51(d,J=18.9Hz),-12.94(d,J=24.0Hz),-23.00--23.49(m).

[0359] MGI495-V1-dCTP was synthesized by the same method as above.

[0360] LCMS:calcd for C 64 H 77 N 15 O 30 P3S3[MH] - :1724.34Found,m / z,[(M-2) / 2] - :861.35.

[0361] 1H NMR(400MHz,dmso)δ9.26(d,J=10.1Hz,2H),9.06(d,J=32.6Hz,2H),8.83-8.75(m,1H),8 .58(dd,J=12.2,7.8Hz,1H),8.24(t,J=11.5Hz,1H),8.16-8.01(m,2H),7.98(s,1H),7.76 (s,1H),7.57-7.48(m,2H),7.45-7.35(m,2H),7.14(d,J=8.2Hz,2H),6.94(d,J=5.2Hz,2H ),6.89(s,1H),6.11(dd,J=7.8,6.0Hz,1H),5.19-5.13(m,1H),4.91(d,J=8.8Hz,1H),4.8 0(d,J=8.9Hz,1H),4.44-4.39(m,1H),4.26(dd,J=10.4,4.3Hz,1H),4.20-4.06(m,4H),3. 99-3.90(m,4H),3.86(d,J=4.5Hz,2H),3.68(s,2H),3.03(dd,J=14.2,6.9Hz,1H),2.83(d d,J=28.6,23.2Hz,3H),2.58(t,J=5.4Hz,2H),2.40(s,1H),2.32(d,J=8.0Hz,1H),2.17(d d,J=14.7,7.6Hz,1H),1.81(d,J=12.1Hz,2H),1.45-1.12(m,12H),1.05(d,J=6.4Hz,6H).

[0362] 31 P NMR (162MHz, dmso) δ -10.44 (d, J = 18.9Hz), -12.86 (d, J = 27.2Hz), -23.09 (t, J = 20.3Hz).

[0363] Example 16 Synthesis of MGI450c-modified nucleotides

[0364] (1) The following compounds were synthesized according to the method of Example 15:

[0365] MGI450c-1-V1-dATP

[0366] LCMS:calcd for C 51 H 52 N 15 O 27 P3S2[MH] - :1462.18.Found,m / z,[(M-2) / 2]- :730.68.

[0367] MGI450c-1-V1-dCTP

[0368] LCMS:calcd for C 49 H 50 N 13 O 29 P3S2[MH] - :1440.15.Found,m / z,[(M-2) / 2] - :719.20.

[0369] MGI450c-1-V1-dTTP

[0370] LCMS:calcd for C 49 H 51 N 14 O 28 P3S2[MH] - :1439.17.Found,m / z,[(M-2) / 2] - :719.73.

[0371] MGI450c-1-V1-dGTP

[0372] LCMS:calcd for C 51 H 52 N 15 O 28 P3S2[MH] - :1478.18.Found,m / z,[(M-2) / 2] - :738.80.

[0373] (2) The following compounds were synthesized according to the method of Example 15:

[0374] MGI450c-V1-dATP

[0375] LCMS:calcd for C 51 H 53 N 16 O 26 P3S2[MH] - :1461.20.Found,m / z,[(M-2) / 2] - :730.27.

[0376] 1H NMR(400MHz,dmso)δ9.07(s,1H),8.76(s,1H),8.67(d,J=1.4Hz,1H),8.43(t,J=5.4Hz,1H),8.20(d,J=8.2Hz,1H),8.10(s,1H),7.85(s,3H),7.70(s,1H),7.51(d,J=8.2Hz,2H),7.42-7.34(m,2H),7.12(d,J=8.3Hz,1H),6.94(dd,J=18.2,9.3Hz,3H),6.49(dd,J=8.8,5.8Hz,1H),5.15(t,J=4.8Hz,1H),4.89(dd,J=29.8,8.9Hz,2H),4.56(d,J=4.6Hz,1H),4.25(dd,J=10.5,4.4Hz,1H),4.16(dd,J=17.2,4.8Hz,3H),3.98(s,1H),3.96-3.81(m,4H),3.68(d,J=6.1Hz,3H),2.95(dd,J=14.4,7.1Hz,2H),2.85(d,J=7.2Hz,1H),2.69-2.58(m,1H),2.39(dd,J=13.3,5.9Hz,1H).

[0377] 31 P NMR(162MHz,dmso)δ-10.47(d,J=19.4Hz),-12.76(d,J=20.9Hz),-22.81--23.33(m).

[0378] MGI450c-V1-dCTP

[0379] LCMS:calcd for C 49 H 51 N 14 O 28 P3S2[M-H] - :1439.17.Found,m / z,[(M-2) / 2] - :718.74。

[0380] 1H NMR(400MHz,dmso)δ9.09(s,1H),8.80(s,1H),8.68(d,J=0.8Hz,1H),8.22(d,J=7.4Hz,1H),8.02(s,1H),7.85(s,3H),7.72(s,1H),7.58-7.45(m,1H),7.42-7.34(m,1H),7.14(d,J=8.9Hz,1H),6.92(dt,J=16.5,8.3Hz,3H),6.17-6.06(m,1H),5.17(dd,J=6.0,4.3Hz,1H),4.90(d,J=8.8Hz,1H),4.79(d,J=8.9Hz,1H),4.41(s,1H),4.27(dd,J=9.6,6.0Hz,1H),4.22-4.06(m,3H),4.03-3.82(m,4H),3.67(dd,J=7.9,2.9Hz,2H),3.52(s,2H),2.88(s,3H),2.31-2.29(m,1H),2.19-2.12(m,1H).

[0381] 31 P NMR(162MHz,dmso)δ-9.64--10.54(m),-12.89--13.79(m),-22.30--23.25(m).

[0382] MGI450c-V1-dTTP

[0383] LCMS:calcd for C 49 H 52 N 15 O 27 P3S2[M-H] - :1438.18.Found,m / z,[(M-2) / 2] - :719.24。

[0384] MGI450c-V1-dGTP

[0385] LCMS:calcd for C 51 H 53 N 16 O 27 P3S2[M-H] - :1477.19.Found,m / z,[(M-2) / 2] - :738.15。

[0386] 1H NMR(400MHz,dmso)δ9.07(s,1H),8.82(s,1H),8.65(s,1H),8.35-8.25(m, 1H),8.09(d,J=7.6Hz,1H),7.80(s,3H),7.51(d,J=8.0Hz,1H),7.43-7.36 (m,1H),7.25(d,J=8.1Hz,1H),7.19(s,1H),7.12(d,J=8.2Hz,1H),6.97(d ,J=9.3Hz,1H),6.89(d,J=9.3Hz,1H),6.54(s,1H),6.20(dd,J=8.8,5.8Hz, 1H),5.21-5.14(m,1H),4.87(dd,J=25.6,8.8Hz,2H),4.53(d,J=4.0Hz,1H ),4.27(dd,J=10.3,4.4Hz,1H),4.20(dd,J=10.1,5.8Hz,1H),4.12(d,J=5 .1Hz,1H),4.06(d,J=4.7Hz,1H),3.98-3.83(m,4H),3.68(d,J=8.8Hz,2H) ,3.49(s,2H),2.73(d,J=7.0Hz,1H),2.64-2.56(m,1H),2.30-2.23(m,1H).

[0387] 31 P NMR(162MHz,dmso)δ-10.50(d,J=20.0Hz),-11.96--12.59(m),-22.86(dd,J=24.2,20.9Hz).

[0388] Example 17 Synthesis of MGI450b-modified nucleotides

[0389] (1) Synthesis of MGI450b-V1

[0390] In a 15 mL vial, weigh MGI450b (20 mg, 40.86 μmol) and dissolve it in anhydrous N'N-dimethylformamide (2 mL). N,N'-disuccinimidyl carbonate (21 mg, 81.98 μmol) and 4-dimethylaminopyridine (5 mg, 40.93 μmol) were then added. Stir the mixture at 25°C for 2 hours. V1 (30 mg, 81.66 μmol) and triethylamine (21 mg, 207.52 μmol) were then added. Stir the mixture at 25°C for another 4 hours. The filtrate was purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure to obtain the crude product. The crude product was then purified by preparative liquid chromatography (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain MGI450b-V1. LCMS: calculated for C 35 H 34 N8O 13 S2[MH] - :837.17.Found,m / z,[MH] - :837.36.

[0391] (2) Synthesis of MGI450b-V1-dATP

[0392] In a 15 mL vial, weigh MGI450b-V1 (10 mg, 11.92 μmol) and dissolve it in anhydrous N'N-dimethylformamide (1 mL). N,N'-disuccinimidyl carbonate (6 mg, 23.42 μmol) and 4-dimethylaminopyridine (3 mg, 24.56 μmol) were then added. Stir the mixture at 25°C for 2 hours. dATP (14 mg, 23.40 μmol) and triethylamine (6 mg, 59.29 μmol) were then added. Stir the mixture at 25°C for another 4 hours. The filtrate was purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure to obtain the crude product. The product was then purified by preparative liquid chromatography (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain MGI450b-V1-dATP. LCMS: calculated for C 50 H 53 N 16 O 24 P3S2[MH] - :1417.21.Found,m / z,[(M-2) / 2] - :708.36.

[0393] 1H NMR(400MHz,dmso)δ8.97(s,1H),8.79(s,1H),8.45-8.38(m,1H),8.15-8.09(m,2H),7.95(s,3H),7.70(d,J=1.6Hz,1H),7.50(t,J=8.3H z,2H),7.42-7.35(m,1H),7.30-7.24(m,1H),7.16-7.05(m,2H),6.98(d,J=9.4Hz,1H),6.65(d,J=4.7Hz,1H),6.49(dd,J=8.7,5.8Hz,1H) ,5.18-5.13(m,1H),4.92(d,J=8.8Hz,1H),4.85(d,J=8.9Hz,1H),4.56(d,J=5.1Hz,1H),4.25(dd,J=10.6,4.4Hz,1H),4.22-4.11(m,2H), 3.98(s,1H),3.96-3.82(m,3H),3.68(dd,J=6.5,4.2Hz,2H),3.51(s,4H),2.95(s,10H),2.70-2.58(m,1H),2.39(dd,J=14.0,5.9Hz,1H).

[0394] 31 P NMR (162MHz, dmso) δ -10.28 (d, J = 18.7Hz), -13.08 (d, J = 25.2Hz), -22.76 (dd, J = 25.0, 18.9Hz).

[0395] The following compounds were synthesized using the same method as above:

[0396] MGI450b-V1-dCTP

[0397] LCMS:calcd for C 48 H 52 N 15 O 25 P3S2[MH] - :1394.19.Found,m / z,[(M-2) / 2] - :696.72.

[0398] 1H NMR(400MHz,dmso)δ9.10(s,1H),8.96(s,1H),8.78(d,J=23.3Hz,1H),8.52(s,1H),8.19-8.01(m,2H),7.95(s,1H),7.72(s,1H),7.58(s,1H),7.53-7.44(m,1H),7.42-7.34(m,1H),7.26(d,J=7.5Hz,1H),7.15-7.06(m,1H),6.98(dd,J=9.4,4.0Hz,1H),6.90(d,J=8.3Hz,1H),6.12(d,J=7.4Hz,1H),5.20(d,J=19.1Hz,1H),4.94-4.75(m,2H),4.43(s,1H),4.19(dd,J=49.6,15.5Hz,3H),4.04-3.75(m,4H),3.67(s,2H),3.07-2.95(m,1H), 2.90(s,2H),2.77(S,3H),2.31-2.28(m,1H),2.02-1.97(m,1H).

[0399] 31 P NMR(162MHz,dmso)δ-10.09--10.84(m),-12.05--13.06(m),-22.42--23.22(m).

[0400] MGI450b-V1-dTTP

[0401] LCMS:calcd for C 48 H 51 N 14 O 26 P3S2[M-H] - :1395.18.Found,m / z,[(M-2) / 2] - :697.42。

[0402] 1H NMR(400MHz,dmso)δ9.14(s,1H),9.01(s,1H),8.96-8.89(m,1H),8.15(dd,J=8.5,5.9Hz,2H),7.96(s,3H),7.61(s,1H),7.49(t,J=9.0Hz,2H),7.38(t,J=7.9Hz,1H),7.15-7.06(m,2H),7.02-6.98(m,1H),6.10(t,J=6.8Hz,1H),5.22-5.14(m,1H),4.91-4.85(m,1H),4.80(d,J=8.9Hz,1H),4.47(s,1H),4.26(ddd,J=15.7,10.7,4.6Hz,2H),4.11-4.00(m,4H),3.97(d,J=2.4Hz,1H),3.92-3.83(m,4H),3.67(s,8H),2.31(dd,J=11.1,6.8Hz,2H).

[0403] 31 P NMR(162MHz,dmso)δ-10.84(d,J=21.3Hz),-12.60(d,J=23.6Hz),-23.29(t,J=22.4Hz).

[0404] MGI450b-V1-dGTP

[0405] LCMS:calcd for C 50 H 53 N 16 O 25 P3S2[M-H] - :1433.20.Found,m / z,[(M-2) / 2] - :716.34。

[0406] 1H NMR(400MHz,dmso)δ10.46(s,1H),8.96(s,1H),8.79(s,1H),8.29(s,1H),8.13(d,J=8.2Hz,1H),7.96(s,3H),7.53-7.46(m,2 H),7.41-7.35(m,1H),7.19(s,1H),7.13(d,J=7.6Hz,1H),7.09(d,J=9.4Hz,1H),6.98(d,J=9.4Hz,1H),6.39(s,1H),6.24-6.1 8(m,1H),5.19-5.14(m,1H),4.87(dd,J=26.2,8.8Hz,2H),4.52(s,1H),4.27(dd,J=10.6,4.0Hz,1H),4.21-4.15(m,1H),4.09( dd,J=15.8,5.3Hz,2H),3.99-3.81(m,4H),3.72-3.63(m,2H),3.51(s,2H),2.90(s,8H),2.68-2.56(m,1H),2.35-2.22(m,1H).

[0407] 31 P NMR(162MHz,dmso)δ-10.18(d,J=18.8Hz),-13.20(dd,J=15.3,13.5Hz),-22.61--22.73(m).

[0408] Example 18 Synthesis of MGI443-modified nucleotides

[0409] The following compounds were synthesized according to the method of Example 17:

[0410] MGI443-V1

[0411] LCMS:calcd for C 35 H 32 F2N8O 13 S2[MH] - :873.15.Found,m / z,[MH] - :873.30.

[0412] MGI443-V1-dATP

[0413] LCMS:calcd for C 50 H 51 F2N 16 O 24 P3S2[MH] -:1453.19.Found,m / z,[(M-2) / 2] - :726.60。

[0414] 1 H NMR(400MHz,dmso)δ9.01(s,1H),8.82(s,1H),8.46-8.37(m,1H),8.20-8.07(m,2H),8.03-7.76(m,3H),7.71(d,J=1.6Hz,1H),7.54(dd,J=15.5,8.1Hz,2H),7.42- 7.35(m,1H),7.12(d,J=7.7Hz,1H),6.95(d,J=11.6Hz,1H),6.49(dd,J=8.8,5.9Hz,1H),5.15(t,J=4.8Hz,1H),4.89(dd,J=25.8,8.9Hz,2H),4.57(d,J=4.9Hz,1H),4.25(dd,J=10.5,4.5Hz,1H),4.20-4.12(m,2H),4.01-3.83(m,4H),3.68(dd,J=6.7,4.1Hz,2H),2.94(d,J=6.0Hz,10H),2.69-2.59(m,1H),2.39(dd,J=13.8,6.2Hz,1H).

[0415] 31 P NMR(162MHz,dmso)δ-10.49(d,J=19.5Hz),-12.86(d,J=24.3Hz),-22.97(dd,J=24.9,19.7Hz).

[0416] MGI443-V1-dCTP

[0417] LCMS:calcd for C 48 H 50 F2N 15 O 25 P3S2[M-H] - :1430.17.Found,m / z,[(M-2) / 2] - :714.95。

[0418] 1H NMR(400MHz,dmso)δ8.97(s,1H),8.79(s,1H),8.65-8.58(m,1H),8.15(d,J=8.0Hz,1H),8.00(s,1H),7.73(s,1H),7.58-7.48(m,2H),7.42-7.36(m,1H),7.14(dd,J=8.2,1.7Hz,1H),6.96(d,J=11.6Hz,1H),6.88(s,1H),6.76(d,J=4.0Hz,1H),6.14-6.09(m,1H),5.19-5.13(m,1H),4.84(dd,J=40.5,8.8Hz,2H),4.44-4.38(m,1H),4.22(ddd,J=15.4,10.4,4.6Hz,2H),4.12(d,J=4.9Hz,1H),4.04-3.83(m,4H),3.72-3.63(m,2H),3.51(s,2H),3.04(d,J=6.9Hz,6H),2.91(d,J=6.9Hz,2H),2.31(ddd,J=8.1,6.3,2.6Hz,1H),2.19-2.13(m,1H).

[0419] 31 P NMR(162MHz,dmso)δ-10.38(d,J=18.7Hz),-13.29(d,J=25.4Hz),-22.94(dd,J=25.0,18.7Hz).

[0420] MGI443-V1-dTTP

[0421] LCMS:calcd for C 48 H 49 F2N 14 O 26 P3S2[M-H] - :1431.16.Found,m / z,[(M-2) / 2] - :715.23。

[0422] 1H NMR(400MHz,dmso)δ8.98(s,1H),8.82(s,1H),8.77-8.70(m,1H),8.16(d,J=8.2Hz,1H),8.08(s,1H),7.97-7.77(m,3H),7.56(d,J=8.4Hz,1H),7.50(d,J=7.8Hz,1H),7.41-7.36(m,1H),7.14(d,J=8.2Hz,1H),6.96(d,J=11.7Hz,1H),6.13-6.07(m,1H),5.20-5.15(m,1H),4.90(d,J=8.9Hz,1H),4.80(d,J=8.9Hz,1H),4.44(d,J=2.5Hz,1H),4.27(dd,J=10.6,4.1Hz,1H),4.19(dd,J=10.6,5.2Hz,1H),4.13-4.04(m,2H),4.02-3.83(m,4H),3.67(dd,J=7.0,5.4Hz,2H),3.51(s,2H),2.97(dd,J=45.2,6.5Hz,8H),2.34-2.28(m,1H).

[0423] 31 P NMR(162MHz,dmso)δ-10.35(d,J=18.5Hz),-13.41(d,J=25.1Hz),-22.70--23.05(m).

[0424] MGI443-V1-dGTP

[0425] LCMS:calcd for C 50 H 51 F2N 16 O 25 P3S2[M-H] - :1469.18.Found,m / z,[(M-2) / 2] - :734.27。

[0426] 1H NMR (400MHz, dmso) δ10.45 (s, 1H), 9.00 (s, 1H), 8.80 (d, J = 4.2Hz, 1H), 8.26 (dd, J=12.0,6.5Hz,1H),8.14(d,J=8.1Hz,1H),7.97-7.67(m,3H),7.56-7.45(m,2H),7.35(dd,J=13.7,5.7Hz,1H),7.17(d,J=3. 4Hz,1H),7.13-7.04(m,1H),6.93(d,J=11.6Hz,1H),6.37(s,2H),6.19(dd,J=8.9,5.8Hz,1H),5.16-5.10(m,1H),4.84(dd,J= 23.9,8.8Hz,2H),4.50(d,J=4.8Hz,1H),4.25(dd,J=10.4,4.4Hz,1H),4.16(dd,J=8.7,4.9Hz,1H),4.12-4.04(m,2H),3.94-3 .83(m,4H),3.66(dd,J=8.4,3.6Hz,2H),3.16(d,J=5.4Hz,2H),2.93(d,J=5.5Hz,10H),2.65-2.53(m,1H),2.33-2.21(m,1H).

[0427] 31 P NMR(162MHz,dmso)δ-10.59(t,J=21.7Hz),-12.93(d,J=25.7Hz),-22.85--23.20(m).

[0428] Example 19 Synthesis of MGI488-modified nucleotides

[0429] The following compounds were synthesized according to the method of Example 17:

[0430] MGI488-V1

[0431] LCMS:calcd for C 44 H 51 N9O 14 S2[MH] - :992.30.Found,m / z,[MH] - :992.49.

[0432] MGI488-V1-dATP

[0433] LCMS:calcd for C 59 H 70 N 17 O25 P3S2[M-H] - :1572.34.Found,m / z,[(M-2) / 2] - :785.91。

[0434] 1 H NMR(400MHz,dmso)δ8.95-8.87(m,1H),8.60(s,1H),8.45(dd,J=15.6,11.0Hz,2H),8.09(s,1H),8.03(s,1H),7.67(dd,J=10.7,5.9Hz,2H),7.59(dd,J=14.4,8.6Hz,1H),7.44(d,J=9.0Hz,2H),7.35(dd,J=13.9,6.4Hz,1H),7.17(d,J=9.4Hz,1H),7.09(dd,J=16.1,9.0Hz,2H),6.52-6.46(m,1H),5.18-5.11(m,1H),4.89(dd,J=28.8,8.9Hz,2H),4.56(d,J=5.1Hz,1H),4.24(dd,J=10.0,4.6Hz,1H),4.18-4.11(m,2H),3.98(s,1H),3.95-3.84(m,2H),3.69(d,J=4.2Hz,1H),3.49(s,2H),3.17(s,3H),2.97(s,8H),2.62(d,J=8.8Hz,1H),2.39(dd,J=12.7,6.3Hz,1H),2.04-1.97(m,1H),1.66(dd,J=22.4,12.5Hz,2H),1.28-1.14(m,6H).

[0435] 31 P NMR(162MHz,dmso)δ-10.05(d,J=18.2Hz),-13.38(dd,J=25.9,2.8Hz),-22.38--22.61(m).

[0436] MGI488-V1-dCTP

[0437] LCMS:calcd for C 57 H 69 N 16 O 26 P3S2[M-H] - :1549.32.Found,m / z,[(M-2) / 2] - :774.30。

[0438] 1 H NMR(400MHz,dmso)δ8.93-8.87(m,1H),8.67-8.61(m,1H),8.56-8.48(m,1H),8.11-7 .97(m,2H),7.76-7.65(m,2H),7.63-7.51(m,2H),7.41(ddd,J=22.8,13.9,5.8Hz,3H ),7.17(d,J=9.4Hz,1H),7.13(d,J=7.8Hz,1H),7.08(d,J=9.6Hz,1H),6.89(s,1H),6 .15-6.10(m,1H),5.16(d,J=4.2Hz,1H),4.91(d,J=8.8Hz,1H),4.80(d,J=8.8Hz,1H), 4.44-4.37(m,1H),4.28-4.21(m,1H),4.20-4.09(m,2H),4.00-3.91(m,2H),3.89-3.82(m,1H),3.67(d,J=7.4Hz,1H),3.17(s,3H ),3.05-2.87(m,8H),2.79(s,1H),2.29(s,1H),2.01(s,1H),1.71(s,1H),1.62(s,1H),1.23(dd,J=8.9,5.4Hz,3H),1.16(s,3H).

[0439] 31 P NMR (162MHz, dmso) δ -10.15 (d, J = 18.6Hz), -13.31 (d, J = 24.6Hz), -22.68 (dd, J = 23.5, 18.8Hz).

[0440] Example 20 Synthesis of MGI447-modified nucleotides

[0441] (1) Synthesis of MGI447-V1-dATP

[0442] In a 4 mL vial, weigh MGI447 (5 mg, 9.66 μmol) and dissolve it in anhydrous N'N-dimethylformamide (1 mL). N,N'-disuccinimidyl carbonate (5 mg, 19.52 μmol) and 4-dimethylaminopyridine (2 mg, 16.37 μmol) were then added. Stir the mixture at 25°C for 2 hours. V1-dATP (18 mg, 18.99 μmol) and triethylamine (5 mg, 49.41 μmol) were then added. Stir the mixture at 25°C for another 4 hours. The filtrate was purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure to obtain the crude product. The product was then purified by preparative liquid chromatography (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain MGI447-V1-dATP. LCMS: calculated for C 52 H 57 N 16 O 24 P3S2[MH] - :1445.24.Found,m / z,[(M-2) / 2] - :722.32.

[0443] 1 H NMR(400MHz,dmso)δ9.02(s,1H),8.82(s,1H),8.45-8.38(m,1H),8.15(d,J=8.2Hz,1H),8.09(s,1H),7.71(d,J=1.7Hz,1H),7.50(dd,J=16.2, 8.2Hz,2H),7.42-7.35(m,1H),7.20(s,1H),7.12(d,J=9.0Hz,1H),6.97 (s,1H),6.65(s,1H),6.49(dd,J=8.8,5.8Hz,1H),5.35-5.30(m,1H),5. 16(t,J=4.8Hz,1H),4.89(dd,J=26.3,8.9Hz,2H),4.56(s,1H),4.26(dd,J=10.4,4.4Hz,1H),4.21-4.12(m,2H),3.98-3.84(m,4H),3.68(s,2H) ,3.51(s,2H),2.94(d,J=6.1Hz,10H),2.63(dd,J=18.3,9.6Hz,1H),2.39(dd,J=12.5,6.1Hz,1H),2.12(s,3H),2.03-1.95(m,1H),1.23(s,3H).

[0444] 31P NMR (162MHz, dmso) δ -10.49 (d, J = 19.2Hz), -12.96 (d, J = 24.9Hz), -22.98 (dd, J = 25.1, 19.5Hz).

[0445] The following compounds were synthesized using the same method as above:

[0446] MGI447-V1-dCTP

[0447] LCMS:calcd for C 50 H 55 N 14 O 26 P3S2[MH] - :1423.21.Found,m / z,[(M-2) / 2] - :711.21.

[0448] 1 H NMR(400MHz,dmso)δ9.03(s,1H),8.86(s,1H),8.71-8.62(m,1H),8.15(d,J=8.1Hz,1H),8.02(s,1H),7.72(s,1H),7.54(d,J=12.9Hz,1H) ,7.50-7.46(m,1H),7.42-7.35(m,1H),7.14(d,J=6.5Hz,1H),6.97(s,1H),6.88(s,1H),6.16-6.08(m,1H),5.17(t,J=4.6Hz,1H),4.89(d, J=8.8Hz,1H),4.80(d,J=8.9Hz,1H),4.40(s,1H),4.27(dd,J=10.6,4.0Hz,1H),4.19(dd,J=10.7,5.0Hz,1H),4.11(d,J=4.8Hz,1H),3.91( dt,J=11.6,8.1Hz,4H),3.67(d,J=9.0Hz,2H),3.52(s,2H),2.92(s,10H),2.31-2.29(m,1H),2.12(s,3H),2.04-1.95(m,1H),1.23(s,3H).

[0449] 31 P NMR (162MHz, dmso) δ -10.33 (d, J = 19.1Hz), -13.20 (d, J = 25.7Hz), -22.83 (dd, J = 23.8, 18.4Hz).

[0450] MGI447-V1-dTTP

[0451] LCMS:calcd for C 50 H 56 N 15 O 25 P3S2[M-H] - :1422.22.Found,m / z,[(M-2) / 2] - :711.18。

[0452] 1 H NMR(400MHz,dmso)δ9.03(s,1H),8.88(s,1H),8.79(s,1H),8.15(d,J=8.2Hz,1H),8.10(s,1H),7.56(s,1H),7.53-7.45(m,2H),7.42-7.36(m,1H),7.14(d,J=8.2Hz,1H),6.97(s,1H),6.14-6.07(m,1H),5.18(d,J=4.3Hz,1H),4.90(d,J=8.9Hz,1H),4.80(d,J=8.9Hz,1H),4.45(s,1H),4.31-4.26(m,1H),4.20(dd,J=10.5,5.4Hz,1H),4.09(d,J=18.0Hz,2H),4.01-3.82(m,4H),3.67(s,2H),3.52(s,2H),2.94(s,10H),2.30(d,J=8.0Hz,1H),2.12(s,3H),2.00(d,J=8.0Hz,1H),1.23(s,3H).

[0453] 31 P NMR(162MHz,dmso)δ-10.30(d,J=18.7Hz),-13.32(d,J=24.8Hz),-22.78(dd,J=24.8,18.8Hz).

[0454] MGI447-V1-dGTP

[0455] LCMS:calcd for C 52 H 57 N 16 O 25 P3S2[M-H] - :1461.23.Found,m / z,[(M-2) / 2] - :730.29。

[0456] 1H NMR(400MHz,dmso)δ10.47(s,1H),9.01(s,1H),8.82(d,J=4.4Hz,1H),8.33-8.25(m,1H),8.15(d,J=8.1Hz,1H),7.50(dd,J=13.9,7.3Hz,2H), 7.38(t,J=8.1Hz,1H),7.19(s,1H),7.13(d,J=6.7Hz,1H),6.97(s,1H),6.65(s,1H),6.39(s,1H),6.21(dd,J=9.1,5.6Hz,1H),5.17(t,J=4.8Hz ,1H),4.87(dd,J=25.1,8.8Hz,2H),4.52(d,J=5.2Hz,1H),4.27(dd,J=10.4,4.4Hz,1H),4.19(dd,J=9.6,5.8Hz,1H),4.15-4.03(m,2H),3.98- 3.81(m,4H),3.72-3.63(m,2H),3.52(s,2H),2.94(s,10H),2.64-2.57( m,1H),2.30-2.25(m,1H),2.12(s,3H),2.03-1.94(m,1H),1.23(s,3H).

[0457] 31 P NMR (162MHz, dmso) δ -10.38 (d, J = 18.9Hz), -13.06 (d, J = 26.4Hz), -22.90 (dd, J = 25.2, 18.8Hz).

[0458] Example 21 Synthesis of MGI456-modified nucleotides

[0459] The following compounds were synthesized according to the method of Example 20:

[0460] MGI456-V1-dATP

[0461] LCMS:calcd for C 54 H 55 F6N 16 O 24 P3S2[MH] - :1581.21.Found,m / z,[(M-2) / 2] - :790.71.

[0462] 1H NMR(400MHz,dmso)δ9.41(t,J=6.6Hz,1H),9.07(s,1H),8.86(s,1H),8.45-8.36(m,1H),8.17(d,J=8.1Hz,1H),8.09(s,1H),7.71(d,J=2.3Hz,1H),7.57(d,J=8.3Hz,1H),7.53(d,J=8.5Hz,1H),7.39(dd,J=17.0,8.6Hz,2H),7.28(d,J=9.6Hz,1H),7.11(d,J=8.5Hz,1H),6.49(dd,J=8.5,5.7Hz,1H),5.16(t,J=4.8Hz,1H),4.88(dd,J=26.5,8.9Hz,2H),4.57(d,J=6.0Hz,1H),4.53-4.42(m,2H),4.26(dd,J=10.4,4.7Hz,1H),4.21-4.11(m,2H),4.00-3.80(m,4H),3.68(dd,J=6.6,4.4Hz,2H),3.52(s,2H),2.85(s,8H),2.63-2.60(m,1H),2.41-2.36(m,1H),2.00(dd,J=15.2,6.6Hz,1H).

[0463] 19 F NMR(376MHz,dmso)δ-70.45(t,J=9.4Hz).

[0464] 31 P NMR(162MHz,dmso)δ-10.48(d,J=19.5Hz),-12.83(d,J=25.1Hz),-22.90(dd,J=25.0,19.5Hz).

[0465] MGI456-V1-dCTP

[0466] LCMS:calcd for C 52 H 53 F6N 14 O 26 P3S2[M-H] - :1559.18.Found,m / z,[(M-2) / 2] - :778.96。

[0467] 1H NMR(400MHz,dmso)δ9.43-9.35(m,1H),9.02-8.959(m,1H),8.84-8.792(m,1H),8.67-8.61(m,1H),8.14(d,J=8.0Hz,1H),7.99(s,1H),7.71-7.66(m,1H),7.56-7.47(m,2H),7.41-7.33(m,1H),7.26(d,J=9.5Hz,1H),7.15(d,J=13.7Hz,1H),6.87-6.83(m,1H),6.65-6.59(m,1H),6.12-6.07(m,1H),5.30(t,,J=4.0Hz,1H),5.17-5.13(m,1H),4.87(d,J=8.8Hz,1H),4.78(d,J=8.8Hz,1H),4.51-4.42(m,2H),4.27-4.17(m,1H),4.12-4.06(m,1H),3.99-3.81(s,3H),3.66-3.60(m,1H),3.54-3.45(m,1H),3.04-2.74(m,6H),1.96-1.89(m,1H),1.47-1.40(m,1H).

[0468] 19 F NMR(376MHz,dmso)δ-70.45(t,J=9.4Hz).

[0469] 31 P NMR(162MHz,dmso)δ-9.67--10.31(m),-13.02--13.53(m),-22.26--22.81(m).

[0470] MGI456-V1-dTTP

[0471] LCMS:calcd for C 52 H 54 F6N 15 O 25 P3S2[M-H] - :1558.20.Found,m / z,[(M-2) / 2] - :779.04。

[0472] 1H NMR(400MHz,dmso)δ9.40-9.36(m,1H),9.03-8.94(m,1H),8.85-8.79(m,1H), 8.77-8.70(m,1H),8.14(d,J=8.4Hz,1H),8.07(s,1H),7.56-7.46(m,2H),7.42-7.09(m,4H),6.66-6.57(m,1H),6.10-6.06(m,1H),5.31-5.29(m,1H),5.21-5.12(m,1H),4.88(d,J=9.1Hz,1H),4.77(d,J=9.1Hz,1H),,4.51-4.42(m,2H),4.5-3.84(m,6H),3.68-3.61(m,1H),3.53-3.47(m,1H),3.06-2.80(s,6H),1.97-1.93(m,1H),1.45-1.40(m,1H).

[0473] 19 F NMR(376MHz,dmso)δ-70.45(t,J=9.3Hz).

[0474] 31 P NMR(162MHz,dmso)δ-9.82--10.46(m),-12.89--13.69(m),-22.07--22.74(m).

[0475] MGI456-V1-dGTP

[0476] LCMS:calcd for C 54 H 55 F6N 16 O 25 P3S2[M-H] - :1597.21.Found,m / z,[(M-2) / 2] - :798.71。

[0477] 1H NMR(400MHz,dmso)δ10.48(s,1H),9.40(t,J=6.9Hz,1H),9.04(s,1H),8.8 5(s,1H),8.33-8.23(m,1H),8.16(d,J=8.1Hz,1H),7.57(d,J=8.3Hz,1H), 7.52(d,J=6.4Hz,1H),7.39(dd,J=18.0,9.0Hz,2H),7.28(d,J=9.6Hz,1H) ,7.19(s,1H),7.13(d,J=7.4Hz,1H),6.40(s,1H),6.21(dd,J=9.2,5.7Hz,1 H),5.17(t,J=4.7Hz,1H),4.87(dd,J=24.2,8.8Hz,2H),4.55-4.43(m,2H) ,4.27(dd,J=10.4,4.0Hz,1H),4.19(dd,J=10.2,4.5Hz,1H),4.14-4.03(m ,2H),4.00-3.79(m,4H),3.68(dd,J=8.5,3.4Hz,2H),3.52(s,2H),2.93(s ,10H),2.66-2.56(m,1H),2.28(dd,J=13.1,6.3Hz,1H),2.04-1.94(m,1H).

[0478] 19 F NMR (376MHz, dmso) δ-70.45 (t, J = 9.4Hz).

[0479] 31 P NMR(162MHz,dmso)δ-10.48(d,J=19.0Hz),-12.88(d,J=26.0Hz),-22.76--23.21(m).

[0480] Example 22 Synthesis of MGI485-modified nucleotides

[0481] The following compounds were synthesized according to the method of Example 20:

[0482] MGI485-V1-dATP

[0483] LCMS:calcd for C 62 H 73 N 16 O 24 P3S2[MH] - :1581.36.Found,m / z,[(M-2) / 2] - :790.38.

[0484] 1 H NMR(400MHz,dmso)δ9.29(s,1H),8.98(s,1H),8.78(s,1H),8.46-8.39(m,1H),8.15(d,J=8.3Hz,1H),8.09(s,1H),7.70(s,1H),7.51(d,J=8.2Hz,1H),7.41-7.33(m,1H),7.19(s,1H),7.12(d,J=7.9Hz,1H),7.01(s,1H),6.66(s,1H),6.54-6.46(m,1H),5.18-5.12(m,1H),4.89(dd,J=28.5,8.8Hz,2H),4.55(s,1H),4.25(dd,J=10.3,4.2Hz,1H),4.16(dd,J=18.4,5.3Hz,2H),4.02-3.83(m,3H),3.68(s,1H),3.51(s,2H),2.98(s,8H),2.64-2.59(m,1H),2.41-2.35(m,1H),2.03-1.95(m,1H),1.82(d,J=8.0Hz,1H),1.52-1.12(m,12H),1.12-0.93(m,6H).

[0485] 31 P NMR(162MHz,dmso)δ-10.07(d,J=18.0Hz),-13.31(d,J=26.4Hz),-22.57(dd, J=25.7,18.1Hz).

[0486] MGI485-V1-dCTP

[0487] LCMS:calcd for C 60 H 71 N 14 O 26 P3S2[M-H] - :1559.33.Found,m / z,[(M-2) / 2] - :778.84。

[0488] 1H NMR(400MHz,dmso)δ9.27(s,1H),8.97(s,1H),8.79(s,1H),8.62(s,1H),8.14(dd,J=7.8,4.8Hz,1H),7.99(s,1H),7.70(s,1H),7.5 2-7.45(m,1H),7.40-7.33(m,1H),7.16-7.08(m,1H),6.99(s,1H),6.84(s,1H),6.62(s,1H),6.14-6.05(m,1H),5.30(s,1H),5.15( s,1H),4.83(dd,J=42.4,9.1Hz,2H),4.41-4.35(m,1H),4.29-4.20(m,1H),4.08(s,2H),3.95(s,3H),3.66(s,1H),3.49(s,2H),2.9 6(s,6H),2.63-2.57(m,1H),2.29-2.23(m,1H),2.01-1.94(m,1H),1.79(s,1H),1.21(dd,J=41.8,34.1Hz,12H),1.07-0.67(m,6H).

[0489] 31 P NMR (162MHz, dmso) δ -9.96 (d, J = 18.0Hz), -13.41 (d, J = 4.1Hz), -22.31 (d, J = 2.8Hz).

[0490] Example 23 Synthesis of MGI450d-modified nucleotides

[0491] (1) Synthesis of MGI450d-1-V1

[0492] MB488 NHS (50 mg, 66.43 μmol) was weighed into a 15 mL vial and dissolved in anhydrous N'N-dimethylformamide (5 mL). V1 (49 mg, 133.38 μmol) and N'N-diisopropylethylamine (26 mg, 201.17 μmol) were then added. The mixture was stirred at 24°C for 15 hours. The reaction mixture was filtered and purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile) and then by flash preparative liquid chromatography (0.1% triethylamine / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain MGI450d-1-V1. LCMS: calculated for C 39 H 40 N8O 18 S3[MH] -:1003.16Found,m / z,[(M-2) / 2] - : 501.02.

[0493] (2) Synthesis of MGI450d-1-V1-dATP

[0494] In a 4 mL vial, weigh MGI450d-1-V1 (10 mg, 9.95 μmol) and dissolve it in anhydrous N'N-dimethylformamide (1 mL). N,N'-disuccinimidyl carbonate (5 mg, 19.52 μmol) and 4-dimethylaminopyridine (2 mg, 16.37 μmol) were then added. Stir the mixture at 25°C for 2 hours. dATP (12 mg, 20.06 μmol) and triethylamine (5 mg, 49.41 μmol) were then added. Stir the mixture at 25°C for another 4 hours. The filtrate was purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure to obtain MGI450d-1-V1-dATP. LCMS: calculated for C 54 H 59 N 16 O 29 P3S3[MH] - :1583.20.Found,m / z,[(M-2) / 2] - :791.25.

[0495] The following compounds were synthesized using the same method as above:

[0496] MGI450d-1-V1-dTTP

[0497] LCMS:calcd for C 52 H 57 N 14 O 31 P3S3[MH] - :1561.17.Found,m / z,[(M-2) / 2] - :780.23.

[0498] MGI450d-1-V1-dCTP

[0499] LCMS:calcd for C 52 H 58 N 15 O 30 P3S3[MH] - :1560.18.Found,m / z,[(M-2) / 2] - :779.82.

[0500] MGI450d-1-V1-dGTP

[0501] LCMS:calcd for C 54 H 59 N 16 O 30 P3S3[MH] - :1599.20.Found,m / z,[(M-2) / 2] - :799.65.

[0502] (3) Synthesis of MGI450d-V1-dATP

[0503] In a 15 mL vial, weigh compound MGI450d-1-V1-dATP (20 mg) and dissolve it in methanol (2 mL). Then, add ammonia (2 mL) and stir at 25°C for 15 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product, which was purified by preparative HPLC (0.1 M TEAB / acetonitrile). The prepared solution was concentrated under reduced pressure and freeze-dried to obtain compound MGI450d-V1-dATP. LCMS: calculated for C 54 H 59 N 17 O 28 P3S3[MH] - :1581.21.Found,m / z,[M / 2-H] - :790.78.

[0504] 1H NMR(400MHz,dmso)δ9.06(s,1H),8.70(s,1H),8.42(s,1H),8.23(s,1H),8.08(s,1H),8.01(d,J=8.5Hz,1H),7.97-7.70(m,3H),7.68(s ,1H),7.46(dd,J=21.3,10.6Hz,2H),7.35(s,1H),7.22-7.14(m,1H),7.09(s,1H),6.97(dd,J=20.9,9.3Hz,2H),6.63(s,1H),6.48(d,J =5.7Hz,1H),5.30(d,J=4.6Hz,1H),5.13(s,1H),4.87(dd,J=29.5,8.9Hz,2H),4.54(s,1H),4.22(s,1H),4.12(d,J=5.1Hz,2H),3.96(t ,J=22.4Hz,3H),3.66(s,1H),3.49(s,2H),2.95(d,J=14.4Hz,6H),2.63-2.56(m,1H),2.38(s,1H),1.97(t,J=7.0Hz,1H),1.21(s,3H).

[0505] 31 P NMR (162MHz, dmso) δ74.99 (s), -10.12 (d, J = 18.1Hz), -13.35 (d, J = 26.3Hz), -22.66 (dd, J = 26.2, 18.2Hz).

[0506] The following compounds were synthesized using the same method as above:

[0507] MGI450d-V1-dTTP

[0508] LCMS:calcd for C 52 H 57 N 15 O 30 P3S3[MH] - :1559.18.Found,m / z,[M / 2-H] - :779.47.

[0509] MGI450d-V1-dCTP

[0510] LCMS:calcd for C 52 H 58 N 16 O 29 P3S3[MH] -:1558.19.Found,m / z,[M / 2-H] - :778.76.

[0511] MGI450d-V1-dGTP

[0512] LCMS:calcd for C 54 H 59 N 17 O 29 P3S3[MH] - :1598.21.Found,m / z,[M / 2-H] - :798.49.

[0513] Example 24 Synthesis of MGI471-modified nucleotides

[0514] (1) Synthesis of MGI471-V1-dATP

[0515] Compound AF532-V1-dATP (10 mg, 15.96 μmol) was weighed into a 4 mL vial and dissolved in methanol (0.5 mL). Ammonia (0.5 mL) was then added and stirred at 22°C for 15 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product, which was then purified by preparative HPLC (0.1% ammonia / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain compound MGI471-V1-dATP.

[0516] 1 H NMR (600MHz, D2O): δ7.92 (s, 1H), 7.73-7.68 (m, 2H), 7.43 (d, J = 12.2Hz, 1H), 7.04-6.93 (m, 4H), 6.87(d,J=13.7Hz,1H),6.63-6.55(m,1H),6.50(s,2H),6.25-6.14(m,1H),4.47-4.42(m,1H),4 .17(s,1H),4.05-3.96(m,4H),3.94-3.68(m,6H),3.67-3.34(m,12H),3.05(q,J=7.3Hz,2H),2. 36-2.27(m,2H),1.11(t,J=7.3Hz,3H),0.94-0.91(m,6H),0.79-0.76(m,6H),0.66-0.62(m,6H).

[0517] 31P NMR(243MHz,D2O)δ-10.77(d,J=19.2Hz),-11.36(d,J=19.5Hz),-23.13(t,J=19.4Hz).

[0518] LCMS:calcd for C 60 H 69 N 16 O 24 P3S2[MH] - :1553.33.Found,m / z,[M-2 / 2] - :776.31.

[0519] (2) Synthesis of MGI471-V1-dGTP

[0520] (2-1) Synthesis of MGI471-1-V1-dGTP

[0521] Compound V1-dGTP (40 mg, 41.51 μmol) was weighed into a 15 mL vial and dissolved in anhydrous N'N-dimethylformamide (4 mL). Compound AF532 NHS (30 mg, 41.45 μmol), N'N-diisopropylethylamine (22 mg, 170.22 μmol), and 4-dimethylaminopyridine (11 mg, 81.85 μmol) were then added. The mixture was stirred at 25°C for 15 hours. The reaction mixture was filtered and purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure to obtain compound MGI471-1-V1-dGTP. LCMS: calculated for C 60 H 68 N 15 O 26 P3S2[MH] - :1570.31.Found,m / z,[MH] - :1570.24.

[0522] (2-2) Synthesis of MGI471-V1-dGTP

[0523] Compound MGI471-1-V1-dGTP (20 mg, 12.72 μmol) was weighed into a 15 mL sample vial and dissolved in methanol (3 mL). Ammonia (3 mL) was then added and stirred at 24°C for 15 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product, which was then purified by preparative HPLC (0.1% ammonia / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain compound MGI471-V1-dGTP.

[0524] 1 1H NMR (600 MHz, D2O) δ 7.81 (d, J = 7.7 Hz, 2H), 7.79 - 7.64 (m, 1H), 7.23 (d, J = 7.0 Hz, 2H), 7.18 - 7.10 (m, 2H), 6.97 - 6.94 (m, 1H), 6.90 (dt, J = 10.6, 2.9 Hz, 1H), 6.81 (d, J = 6.8 Hz, 1H), 6.69 (s, 2H), 6.57 - 6.44 (m, 1H), 5.92 - 5.81 (m, 1H), 4.84 - 4.71 (m, 1H), 4.37 (s, 1H), 4.12 - 4.07 m, 1H), 3.98 - 3.73 (m, 11H), 3.69 - 3.64 (m, 3H), 3.61 - 3.51 (m, 9H), 2.32 - 2.24 (m, 1H), 2.18 - 2.09 (m, 1H), 1.03 (d, J = 6.7 Hz, 6H), 0.94 - 0.91 (m, 6H), 0.80 (dd, J = 9.0, 5.0 Hz, 6H).

[0525] 31 31P NMR (243 MHz, D2O) δ -10.14, -11.41 (d, J = 20.2 Hz), -22.93 (t, J = 19.4 Hz).

[0526] LCMS: calcd for C 60 H 69 N 16 O 25 P3S2 [M - H] - : 1569.33. Found, m / z, [M - 2 / 2] - : 785.10。

[0527] (3) Synthesis of MGI471 - V1 - dCTP

[0528] (3 - 1) Synthesis of MGI471 - 1 - V1 - dCTP

[0529] Compound V1-dCTP (40 mg, 43.26 μmol) was weighed into a 15 mL vial and dissolved in anhydrous N'N-dimethylformamide (4 mL). Compound AF532 NHS (32 mg, 44.21 μmol), N'N-diisopropylethylamine (22 mg, 170.22 μmol), and 4-dimethylaminopyridine (11 mg, 90.04 μmol) were then added. The mixture was stirred at 25°C for 15 hours. The reaction mixture was filtered and purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The prepared solution was concentrated under reduced pressure to obtain compound MGI471-1-V1-dCTP. LCMS: calculated for C 58 H 67 N 14 O 26 P3S2[MH] - :1531.30.Found,m / z,[MH] - :1532.24.

[0530] (3-2) Synthesis of MGI471-V1-dCTP

[0531] Compound MGI471-1-V1-dCTP (20 mg, 13.04 μmol) was weighed into a 15 mL sample vial and dissolved in methanol (3 mL). Ammonia (3 mL) was then added and stirred at 24°C for 15 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product, which was then purified by preparative HPLC (0.1% ammonia / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain compound MGI471-1-V1-dCTP.

[0532] 11H NMR (600 MHz, D2O): δ 7.82 (d, J = 7.9 Hz, 2H), 7.75 - 7.69 (m, 1H), 7.31 (d, J = 7.8 Hz, 2H), 7.23 - 7.15 (m, 2H), 6.99 - 7.02 (m, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.75 (s, 2H), 5.81 (dt, J = 26.2, 6.0 Hz, 1H), 4.78 (t, J = 4.9 Hz, 1H), 4.33 (s, 1H), 4.12 (s, 1H), 4.03 - 3.91 (m, 6H), 3.88 - 3.72 (m, 5H), 3.70 - 3.66 (m, 2H), 3.63 - 3.50 (m, 8H), 3.48 - 3.34 (m, 2H), 3.04 (q, J = 7.3 Hz, 3H), 2.29 - 2.36 (m, 1H), 2.01 - 1.94 (m, 1H), 1.12 (t, J = 7.3 Hz, 5H), 1.03 (d, J = 6.7 Hz, 6H), 0.97 - 0.94 (m, 6H), 0.83 (d, J = 7.1 Hz, 6H).

[0533] 31 31P NMR (243 MHz, D2O) δ -10.84 (d, J = 20.0 Hz), -11.63 (d, J = 20.1 Hz), -23.22 (t, J = 19.6 Hz).

[0534] LCMS: calcd for C 58 H 68 N 15 O 25 P3S2 [M - H] - : 1530.32. Found, m / z, [M - ½] - : 764.85。

[0535] (4) Synthesis of MGI471 - V1 - dTTP

[0536] (4 - 1) Synthesis of MGI471 - 1 - V1 - dTTP

[0537] Compound V1-dTTP (40 mg, 43.22 μmol) was weighed into a 15 mL vial and dissolved in anhydrous N'N-dimethylformamide (4 mL). Compound AF532 NHS (32 mg, 22.21 μmol), N'N-diisopropylethylamine (22 mg, 170.22 μmol), and 4-dimethylaminopyridine (11 mg, 90.04 μmol) were then added. The mixture was stirred at 25°C for 15 hours. The reaction mixture was filtered and purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The prepared solution was concentrated under reduced pressure to obtain compound MGI471-1-V1-dTTP.

[0538] LCMS:calcd for C 58 H 66 N 13 O 27 P3S2[MH] - :1532.28.Found,m / z,[MH] - :1533.10.

[0539] (4-2) Synthesis of MGI471-V1-dTTP

[0540] Compound MGI471-1-V1-dTTP (20 mg, 13.04 μmol) was weighed into a 15 mL sample vial and dissolved in methanol (3 mL). Ammonia (3 mL) was then added and stirred at 24°C for 15 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product, which was then purified by preparative HPLC (0.1% ammonia / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain compound MGI471-V1-dTTP.

[0541] 1H NMR (600MHz, D2O): δ7.88-7.81(m,2H),7.74-7.71(m,1H),7.27(d,J=7.3Hz,2H),7.23-7.14(m,2H),7.01-6 .95(m,1H),6.88(d,J=8.1Hz,1H),6.73(d,J=3.1Hz,2H),5.80(dt,J=28.9,7.1Hz,1H),4.78(d,J=5.0Hz,1H) ,4.36(s,1H),4.13-4.06(m,1H),4.01-3.89(m,6H),3.88-3.31(m,18H),3.04(q,J=7.3Hz,1H),2.35-2.26(m ,1H),2.13-2.05(m,1H),1.12(t,J=7.3Hz,2H),1.02(d,J=6.7Hz,6H),0.96-0.93(m,6H),0.83-0.79(m,6H).

[0542] 31 P NMR (243MHz, D2O) δ -10.44, -11.54 (d, J = 19.1Hz), -22.87.

[0543] LCMS:calcd for C 58 H 67 N 14 O 26 P3S2[MH] - :1531.30.Found,m / z,[M-2 / 2] - :765.26.

[0544] Example 25 Synthesis of MGI471-V2

[0545] (1) Synthesis of tert-butyl (2-(trifluoroacetamide)ethyl)carbamate

[0546] In a 100 mL flask, weigh N-tert-butyloxycarbonyl-1,2-ethylenediamine (5 g, 31.21 mmol) and dissolve it in tetrahydrofuran (30 mL). Place the mixture in an ice-water bath at 0-5°C. Add ethyl trifluoroacetate (4.88 g, 34.33 mmol). After addition, warm to 24°C and stir under electromagnetic stirring for 4 hours. The reaction mixture is concentrated under reduced pressure to obtain tert-butyl (2-(trifluoroacetamido)ethyl)carbamate.

[0547] (2) Synthesis of N-trifluoroacetyl-1,2-ethylenediamine

[0548] In a 100 mL flask, weigh tert-butyl (2-(trifluoroacetamido)ethyl)carbamate (8 g, 31.21 mmol) and dissolve it in formic acid (50 mL). Stir the mixture at 24°C for 15 hours. Concentrate the reaction mixture under reduced pressure, then add acetonitrile (50 mL x 2) and concentrate under reduced pressure to dryness to obtain N-trifluoroacetyl-1,2-ethylenediamine.

[0549] (3) Synthesis of MGI471-1-V1

[0550] In a 15 mL vial, weigh AF532 NHS (100 mg, 138.17 μmol) and dissolve in anhydrous N'N-dimethylformamide (3 mL). V1 (102 mg, 277.66 μmol) and N'N-diisopropylethylamine (89 mg, 688.60 μmol) were then added. Stir for 15 hours at 22°C. The reaction mixture was filtered and purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure to obtain compound MGI471-1-V1. LCMS: calculated for C 45 H 49 N7O 14 S2[MH] - :974.28.Found,m / z,[MH] - :974.34.

[0551] (4) Synthesis of MGI471-1-V2-1

[0552] MGI471-1-V1 (90 mg, 92.21 μmol) was weighed into a 50 mL flask and dissolved in anhydrous N'N-dimethylformamide (5 mL). N,N'-disuccinimidyl carbonate (36 mg, 140.53 μmol) and 4-dimethylaminopyridine (2 mg, 16.37 μmol) were then added. The mixture was stirred at 24°C for 3 hours. N-trifluoroacetyl-1,2-ethylenediamine (58 mg, 371.54 μmol) and N'N-diisopropylethylamine (60 mg, 464.23 μmol) were then added. The mixture was stirred at 24°C for 15 hours. The filtrate was purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure to obtain MGI471-1-V2-1. LCMS: calculated for C 49 H 54 F3N9O 14 S2[MH] - :1112.32.Found,m / z,[MH] - :1112.49.

[0553] (5) Synthesis of MGI471-V2

[0554] MGI471-1-V2-1 (60 mg, 53.85 μmol) was weighed into a 15 mL vial and dissolved in methanol (5 mL). Ammonia (5 mL) was then added and stirred at 25°C for 3 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product, which was then purified by preparative HPLC (0.1 M TEAB / acetonitrile). The preparative solution was concentrated under reduced pressure and freeze-dried to obtain MGI471-V2.

[0555] 1 H NMR (600MHz, DMSO-d6): δ14.34(s,1H),8.88-8.84(m,1H),8.67(t,J=5.2Hz,1H),8.14-8.11(m,2H),7.91(t,J= 5.9Hz,1H),7.87-7.82(m,2H),7.51-7.47(m,4H),7.39(t,J=7.9Hz,1H),7.14-7.11(m,1H),6.77(s,2H),5.10(t ,J=5.0Hz,1H),4.21(dd,J=10.3,4.4Hz,1H),4.14(dd,J=10.4,5.5Hz,1H),3.89-3.83(m,3H),3.77-3.73(m,3H ),3.60-3.49(m,8H),3.06(qd,J=7.4,2.4Hz,2H),2.94-2.89(m,2H),1.17-1.13(m,12H),0.98(d,J=3.9Hz,6H).

[0556] 13 C NMR (151MHz, DMSO-d6): δ170.40,166.69,166.61,158.11,152.32,151.48,143.66,13 8.11,138.02,136.62,135.48,130.04,129.95,128.22,120.69,120.64,118.12,116.8 5,113.47,108.09,89.75,70.35,70.22,69.24,68.73,65.21,58.40,46.21,42.65,41.88,40.50,39.28,36.54,27.84,27.69,23.28,23.21,16.20,16.12,11.49,9.28,8.10.

[0557] LCMS:calcd for C47 H 56 N 10 O 12 S2[MH] - :1015.35.Found,m / z,[MH] - :1015.51.

[0558] The NMR characterization spectra of some of the above compounds are shown in Figures 1 to 17.

[0559] Example 26 Testing the Photochemical Properties of Fluorescent Dyes or Modified Nucleotides

[0560] The photochemical properties of fluorescent dyes or modified nucleotides were tested, and the results are shown in Table 1.

[0561] Table 1

[0562] The excitation and emission spectra of the above compounds are shown in FIG18 .

[0563] Example 27 Biochemical Experiment

[0564] Experimental purpose: To verify the performance of modified nucleotides on sequencers.

[0565] Experimental plan:

[0566] Use MGI471 series dNTPs and prepare a 2color Hot dNTP mix in the form of double-labeled T base (MGI471-dTTP & AF532-dTTP), AF532-dATP, MGI471-dCTP, and G non-luminescent.

[0567] PE100+10 was tested on the modified DNBSEQ-200RS sequencer (H light channel changed to 465nm excitation) and compared with the results of PE100+10 on the unmodified DNBSEQ-200RS sequencing kit.

[0568] Experimental results:

[0569] Judging from the PE100 off-machine report results, the TotalReads using MGI471 dNTP is slightly lower than that of conventional 2-color reagents, but the data volume can reach more than 650M, which is also a good level.

[0570] The Q30%, SplitRate, and MappingRate of the two dNTP PE100s are comparable, and the AvgErrorRate N (%) of the MGI471 dNTP monomer is slightly lower than that of conventional reagents.

[0571] In terms of Runon / Lag, the Runon of MGI471 dNTP monomer is slightly higher overall, indicating that the purity of the monomer needs to be improved. The Lag is not much different from that of conventional reagents, indicating that the polymerization efficiency of MGI471 dNTP is still very good.

[0572] The shortcomings of the MGI471 dNTP monomer lie in signal recovery and Q30% recovery. Due to the short wavelength and high laser energy of the MGI471 monomer, the damage to DNB is much stronger than under conventional conditions. Therefore, the recovery of the second chain is affected to some extent. It is necessary to optimize the monomer structure and sequencing reagents for photodamage.

[0573] Overall, MGI471 dNTP can meet the application of gene sequencing to a certain extent.

[0574] The performance of MGI471 dNTP on the sequencer is shown in Table 2. The Q30 data of MGI471 dNTP on the sequencer is shown in Figure 19.

[0575] Table 2

[0576] Interpretation of key biochemical indicators:

[0577] Q30(%): The proportion of bases with an estimated error rate lower than 0.001 (accuracy higher than 99.9%) in the basecall results.

[0578] Runon / Lag: Runon can be understood as the proportion of the number of copies of the leading reaction within the DNB, and Lag can be understood as the proportion of the number of copies of the lagging reaction within the DNB.

[0579] TotalReads(M): The total number of reads contained in the fq file generated by Zebracall without splitting.

[0580] SplitRate (%): The barcode portion of the sequencing sequence is classified and split, and the number of sequences that can be mapped to the barcode list is counted. The ratio of the number of reads in the total number of fastq sequences is the split rate (SplitRate). The denominator of the split rate is the number of reads in all the downloaded fastq files (i.e., the reads retained by filtering). In this case, TotalReads is the numerator of the split rate calculation.

[0581] MappingRate (%): The ratio of MappedReads to TotalReads.

[0582] AvgErrorRate! N (%): represents the average error rate of the remaining mismatch types after removing the mismatch caused by call N.

[0583] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. Any modification or equivalent replacement of the technical solutions of the present invention without departing from the purpose and scope of the technical solutions of the present invention should be included in the scope of protection of the present invention.

Claims

1. A compound represented by formula (I), an ester thereof or a salt thereof, in, R 1 , R 2 , R 3 , R 4 Each is the same or different, and each is independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl; R 5 , R 6 are the same or different and are each independently selected from hydrogen, C1-C6 alkyl, halogen, and halogenated C1-C6 alkyl; R 7 , R 8 are the same or different and are each independently selected from hydrogen, -COOH, -C(O)NH-(C1-C6 alkyl) and -C(O)NH2, and R 7 and R 8 Not at the same time hydrogen; Optionally, -NR 1 R 2 Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, wherein the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more groups selected from C1-C6 alkyl, halogen, and halogenated C1-C6 alkyl; Optionally, -NR 3 R 4 Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, and the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more groups selected from C1-C6 alkyl, halogen, and halogenated C1-C6 alkyl.

2. The compound, ester or salt thereof according to claim 1, wherein R 1 , R 2 , R 3 , R 4 are each hydrogen; or R 1 , R 2 , R 3 , R 4 Each is a C1-C6 alkyl group (e.g., methyl, ethyl); or R 1 , R 2 , R 3 , R 4 Each is a halogenated C1-C6 alkyl group (eg, trifluoromethyl, trifluoroethyl); or R 1 With R 3 The same, each is hydrogen, R 2 With R 4 The same is true for each of them, and each is a halogenated C1-C6 alkyl group (eg, trifluoromethyl, trifluoroethyl).

3. The compound, ester or salt thereof according to claim 1 or 2, wherein R 5 , R 6 same; Preferably, R 5 , R 6 are each hydrogen, or, R 5 , R 6 Each is a halogen (eg, fluorine).

4. The compound, ester or salt thereof according to any one of claims 1 to 3, wherein R 7 It is a carboxyl group.

5. The compound, ester or salt thereof according to any one of claims 1 to 4, wherein R 8 It is hydrogen or carboxyl.

6. The compound, ester or salt thereof according to any one of claims 1 to 5, wherein -NR 1 R 2 Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, wherein the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more methyl groups; Preferably, -NR 3 R 4 Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, and the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more methyl groups.

7. The compound, ester or salt thereof according to any one of claims 1 to 6, wherein R 7 or R 8 is a carboxyl group, and the carboxyl group is connected to a cleavable linker, for example, a cleavable linker having the structure shown below:

8. The compound, ester or salt thereof according to any one of claims 1 to 7, wherein the compound has a structure as shown in formula (II): in, R 7 as defined in claim 1 or 4; or The compound has a structure as shown in formula (III): Among them, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 As defined in any one of claims 1 to 4 or 6; or The compound has a structure as shown in formula (IV): Among them, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 As defined in any one of claims 1 to 3 and 6.

9. The compound, ester or salt thereof according to any one of claims 1 to 8, which has a structure selected from the group consisting of:

10. A compound represented by formula (I'), an ester thereof or a salt thereof, in, R a , R b , R c , R d are the same or different and are independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, -NR 1’ R 2’ , hydroxyl, hydroxyl-substituted C1-C6 alkyl, halogen; R 1’ , R 2’ Each is the same or different, and each is independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl; R e , R f are the same or different and are each independently selected from hydrogen, -COOH, -C(O)NR 3’ R 4’ , and R e and R f Not at the same time hydrogen; R 3’ , R 4’ are each the same or different and are each independently selected from hydrogen, C1-C6 alkyl, the C1-C6 alkyl being optionally substituted with carboxyl, -C(O)NH2 or sulfonic acid; Optionally, when R a For-NR 1’ R 2’ When -NR 1’ R 2’ Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, wherein the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more groups selected from C1-C6 alkyl, halogen, and halogenated C1-C6 alkyl; Optionally, when R b For-NR 1’ R 2’ When -NR 1’ R 2’ Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, and the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more groups selected from C1-C6 alkyl, halogen, and halogenated C1-C6 alkyl.

11. The compound, ester or salt thereof according to claim 10, wherein R a With R b Same, preferably -NR 1’ R 2’ or are all hydroxyl groups; and / or R c With R d The same, preferably both are hydrogen or both are halogen; Preferably, R 1’ , R 2’ Each is hydrogen; Preferably, R 1’ , R 2’ One of them is hydrogen and the other is selected from C1-C6 alkyl (e.g. methyl, ethyl).

12. The compound, ester or salt thereof according to claim 10 or 11, wherein R e , R f different and are each independently selected from hydrogen, -COOH, -C(O)NR 3’ R 4’ ; Preferably, R 3’ , R 4’ Each is the same or different and is independently selected from hydrogen, C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted with carboxyl or sulfonic acid.

13. The compound, ester or salt thereof according to any one of claims 10 to 12, wherein: R a For-NR 1’ R 2’ , -NR 1’ R 2’ Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, wherein the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more methyl groups; and / or R b For-NR 1’ R 2’ , -NR 1’ R 2’ Together with the benzene ring to which it is connected, it forms a benzo 5-6 membered nitrogen-containing heterocyclic group, and the 5-6 membered nitrogen-containing heterocyclic group is optionally substituted by one or more methyl groups.

14. The compound, ester or salt thereof according to any one of claims 10 to 13, wherein the compound has a structure as shown in formula (II'): in, R e , R f As defined in claim 10 or 12.

15. The compound, ester or salt thereof according to any one of claims 10 to 14, wherein the compound has a structure as shown in formula (III'): in, R a , R e , R f As defined in any one of claims 10 to 12.

16. The compound, ester or salt thereof according to any one of claims 10 to 15, wherein the compound has a structure selected from the following:

17. A compound as shown in formula (i): in, w is a carboxyl group or an ester group, and the dye is a compound or a salt thereof according to any one of claims 1 to 16.

18. The compound, its ester or its salt according to any one of claims 1 to 17, wherein the ester of the compound is an activated ester of a carboxyl group, such as a nitrophenyl ester, a pentafluorophenyl ester or a succinimidyl ester.

19. The compound, ester or salt thereof according to any one of claims 1 to 17, wherein the salt of the compound is a salt formed by a sulfonate group on an acridine ring, such as a salt formed by a sulfonate group and an alkali metal ion, an alkaline earth metal particle or an ammonium ion.

20. A labeled nucleotide or oligonucleotide, wherein the labeled nucleotide or oligonucleotide is labeled with the compound, ester or salt thereof according to any one of claims 1 to 19.

21. The labeled nucleotide or oligonucleotide of claim 20, wherein the compound is attached to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base of the nucleotide or oligonucleotide via a cleavable linker.

22. The labeled nucleotide according to claim 20 or 21, having a structure as shown in formula (1): in, The dye is a compound, an ester or a salt thereof according to any one of claims 1 to 16.

23. The labeled nucleotide of claim 22, having the following structure: in, R a , R b , R c , R d , R e As defined in any one of claims 10 to 13.

24. The labeled nucleotide of claim 22 or 23, having the following structure: in, R a , R b , R c , R d , R e As defined in any of 10-13.

25. The labeled nucleotide according to any one of claims 22 to 24, having the following structure: in, R a , R b , R c , R d , R e As defined in any of 10-13.

26. The labeled nucleotide according to any one of claims 22 to 25, wherein the nucleotide in each formula is selected from dATP, dGTP, dCTP, and dTTP.

27. A method of sequencing, comprising incorporating the labeled nucleotide of any one of claims 20-26 into a sequencing assay; Preferably, the method further comprises detecting the labeled nucleotide; Preferably, the sequencing assay is performed on an automated sequencing instrument, and wherein the automated sequencing instrument comprises two light sources operating at different wavelengths.

28. The sequencing method of claim 27, comprising: (a) incorporating at least one labeled nucleotide according to any one of claims 20 to 26 into a polynucleotide; as well as (b) detecting the labeled nucleotide incorporated into the polynucleotide by detecting the fluorescent signal from the new fluorescent dye attached to the modified nucleotide.

29. A kit comprising one or more nucleotides, wherein at least one nucleotide is a labeled nucleotide of any one of claims 20-26; Preferably, the kit comprises two or more labeled nucleotides.

30. Use of the compound, ester or salt thereof according to any one of claims 1 to 16 in the fields of sequencing, expression analysis, hybridization analysis, gene analysis, RNA analysis, protein binding assay, in vitro diagnosis, immunoassay, and molecular labeling; Preferably, the molecular marker is used for cell imaging, tissue imaging or in vivo imaging.

31. Use of the compound, ester or salt thereof according to any one of claims 1 to 16 in fluorescent labeling, quantification or detection of proteins, enzymes or nucleic acids.

32. Use of the labeled nucleotide or oligonucleotide according to any one of claims 20 to 26 or the kit according to claim 29 in sequencing.

33. A method for preparing a compound, an ester or a salt thereof according to any one of claims 1 to 16, the method comprising: In the presence of ammonia and methanol, the xanthene structure undergoes a substitution reaction and becomes an acridine structure.

34. The method of claim 33, wherein The reaction route for synthesizing the compound of formula (I) is as follows: R 1 -R 7 As defined in any one of claims 1 to 6.

35. The method of claim 33, wherein The reaction route for synthesizing the compound of formula (I') is as follows: R a -R f As defined in any one of claims 10 to 13.

36. A method for preparing labeled nucleotides, the method being carried out according to any one of method A, method B, and method C: Method A: in, Dye 2 is a compound, an ester or a salt thereof according to any one of claims 1 to 16, wherein the compound has an acridine structure, and dye 1 is a precursor of dye 2 and has a xanthene structure; Method B: Wherein, the dye is a compound, an ester or a salt thereof according to any one of claims 1 to 16; Method C: Wherein, the dye is a compound, an ester or a salt thereof according to any one of claims 1 to 16; Method D: Wherein, dye 2 is the compound, its ester or salt thereof according to any one of claims 1 to 16, the compound has an acridine structure, and dye 1 is a precursor of dye 2 and has a xanthene structure.

37. The method of claim 36, wherein The nucleotide has a structure selected from the group consisting of:

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