Fluorescent dyes, their synthesis and use

Abstract

The present invention relates to dye compounds and their use as fluorescent markers, as well as to methods for preparing the compounds, nucleotides or oligonucleotides labeled with the compounds, and methods for determining nucleic acid sequences.

Description

[Technical Field]

[0001] This invention relates to the fields of organic chemistry, fluorescent dyes, and gene sequencing. In particular, this invention relates to dye compounds and their use as fluorescent markers, as well as methods for preparing the compounds, nucleotides or oligonucleotides labeled with the compounds, and nucleic acid sequencing methods. [Background technology]

[0002] Next-generation sequencing, also known as high-throughput sequencing, achieves sequencing-by-synthesis through the introduction of reversible terminators. DNA sequencing is determined by capturing fluorescent dye labels bound to newly added bases during DNA replication. Currently, most commercially available sequencers rely on next-generation sequencing based on sequencing-by-synthesis. In the sequencing process, base identification is achieved by detecting fluorescent dyes modified on the bases. The dyes used to modify four types of bases typically have emission wavelengths in two regions: green and red light. This results in a partial overlap of emission wavelengths between the two base dyes, and the insufficient discriminability between these dyes is a significant factor leading to increased sequencing error rates or reduced sequencing quality. Therefore, the development of novel dyes with emission wavelengths in other visible light regions for use in gene sequencing would help improve sequencing accuracy. [Invention Summary]

[0003] Acridine (azaanthracene) is a macrocyclic conjugated system with a robust tricyclic planar structure and strong fluorescence. Acridine functions as a good fluorescent marker and can be applied to fields such as in vitro diagnostics, immunoassays, and molecular labeling. Generally, dyes with acridine as the basic parent are synthesized using aniline derivatives as starting materials, and acridine derivatives are synthesized through multiple reaction steps.

[0004] During the structural modification of the substituents of pigment AF532, the inventors found that under ammonia / methanol conditions, AF532 undergoes a substitution reaction under the influence of its own substituents (1,8-disulfonic acid groups), and the nucleophile changes from the original xanthene to an acridine structure.

[0005] The present invention uses substituted xanthene as a raw material for synthesizing acridine dyes under ammonia / methanol reaction conditions. The corresponding maximum excitation wavelength and maximum emission wavelength of the synthesized dyes are subject to a blue shift; under the maximum excitation wavelength of blue light, the emission wavelengths of these dyes fall into the cyan light region. Therefore, dyes that can emit cyan light can be obtained.

[0006] This application provides the following inventions.

[0007] Pigment compounds In one aspect, this application relates to formula (I)

Chemical formula

[0008] In a particular embodiment, R 1 and R 2 In a particular embodiment, R 3 and R 4 In a particular embodiment, R 1 , R 2 , R 3 and R 4 They are the same.

[0009] In a particular embodiment, R 1 , R 2 , R 3 and R 4 These are H.

[0010] In a particular embodiment, R 1 , R 2 , R 3 and R 4 These are C1-C6 alkyl groups (for example, methyl and ethyl).

[0011] In a particular embodiment, R 1 , R 2 , R 3 and R 4 These are each a halo-C1~C6 alkyl group (for example, trifluoromethyl, trifluoroethyl).

[0012] In a particular embodiment, R 1 and R 3 In a particular embodiment, R 2 and R 4 They are the same.

[0013] In a particular embodiment, R 1 and R 3 They are the same, and each is H, R 2 and R 4 These are the same, and each is a halo-C1~C6 alkyl group (e.g., trifluoromethyl, trifluoroethyl).

[0014] In a particular embodiment, R 5 and R 6 They are the same.

[0015] In a particular embodiment, R 5 and R 6 These are H.

[0016] In a particular embodiment, R 5 and R 6 These are each halogens (for example, F).

[0017] In a particular embodiment, R 7 It is a carboxyl.

[0018] In a particular embodiment, R 8 H is H.

[0019] In a particular embodiment, R 8 It is a carboxyl.

[0020] In a particular embodiment, -NR 1 R 2 However, together with the benzene ring to which it is bonded, it forms a benzo-5-6 member nitrogen-containing heterocyclic group, and the 5-6 member nitrogen-containing heterocyclic group is optionally substituted with one or more methyl groups.

[0021] In a particular embodiment, -NR 3 R 4 However, together with the benzene ring to which it is bonded, it forms a benzo-5-6 member nitrogen-containing heterocyclic group, and the 5-6 member nitrogen-containing heterocyclic group is optionally substituted with one or more methyl groups.

[0022] In a particular embodiment, R 7 or R 8 The carboxyl is a cleavable linker, for example: [ka] It is connected to a severable linker having the structure shown.

[0023] In certain embodiments, the compound of the present invention is of formula (II) [ka] (In the formula, R 7 (As defined above) It has a structure represented by the following:

[0024] In certain embodiments, the compound of the present invention is of formula (III) [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 It has a structure represented as (as defined above).

[0025] In certain embodiments, the compound of the present invention is of formula (IV) [ka] (In the formula, R1 , R 2 , R 3 , R 4 , R 5 and R 6 (As defined above) It has a structure represented by the following:

[0026] The compounds of the present invention may have structures selected from the following. [ka]

[0027] In one embodiment, this application is based on formula (I') [ka] (In the formula, R a , R b , R c and R d These are either the same or different from each other, H, C1-C6 alkyl, halo-C1-C6 alkyl, -NR 1’ R 2’ , hydroxy, hydroxy-substituted C1-C6 alkyl, and halogen are independently selected; R 1’ and R 2’ They are either the same or different from each other, and are independently selected from H, C1-C6 alkyl and halo-C1-C6 alkyl. R e and R f They are the same or different, H, -COOH and -C(O)NR 3’ R 4’ Each is independently selected from R e and R f It is not H at the same time, R 3’ and R 4’ These are either the same or different from each other, and are independently selected from H and C1-C6 alkyl groups, wherein the C1-C6 alkyl groups are optionally substituted with carboxyl, -C(O)NH2, or sulfonic acid groups. Optionally, R ais -NR 1’ R 2’ when it is, -NR 1’ R 2’ together with the benzene ring to which it is attached forms a benzo 5 - 6 membered nitrogen - containing heterocyclic group, and the 5 - 6 membered nitrogen - containing heterocyclic group is optionally substituted with one or more groups selected from C1 - C6 alkyl, halogen, and halo - C1 - C6 alkyl; optionally, R b is -NR 1’ R 2’ when it is, -NR 1’ R 2’ together with the benzene ring to which it is attached forms a benzo 5 - 6 membered nitrogen - containing heterocyclic group, and the 5 - 6 membered nitrogen - containing heterocyclic group is optionally substituted with one or more groups selected from C1 - C6 alkyl, halogen, and halo - C1 - C6 alkyl) Provide a compound represented by, its ester, or its salt.

[0028] In certain embodiments, R a and R b are the same.

[0029] In certain embodiments, R a and R b are the same, and each is -NR 1’ R 2’ or each is hydroxy.

[0030] In certain embodiments, R c and R d are the same.

[0031] In certain embodiments, R c and R d are the same, and each is H or each is halogen.

[0032] In certain embodiments, R 1’ and R 2’ are each H.

[0033] In a particular embodiment, R 1’ and R 2’ One of the atoms is H, and the other is selected from C1-C6 alkyl groups (e.g., methyl, ethyl).

[0034] In a particular embodiment, R e and R f They differ, and each is H, -COOH, and -C(O)NR 3’ R 4’ It is selected independently of the others.

[0035] In a particular embodiment, R e and R f These differ, and each is independently selected from H and -COOH.

[0036] In a particular embodiment, R 3’ and R 4’ However, they are either the same or different from each other, and are independently selected from H and C1-C6 alkyl groups, and the C1-C6 alkyl groups are optionally substituted with carboxyl or sulfonic acid groups.

[0037] In a particular embodiment, R a ga-NR 1’ R 2’ and;-NR 1’ R 2’ However, together with the benzene ring to which it is bonded, it forms a benzo-5-6 member nitrogen-containing heterocyclic group, and the 5-6 member nitrogen-containing heterocyclic group is optionally substituted with one or more methyl groups.

[0038] In a particular embodiment, R b ga-NR 1’ R 2’ and;-NR 1’ R 2’ However, together with the benzene ring to which it is bonded, it forms a benzo-5-6 member nitrogen-containing heterocyclic group, and the 5-6 member nitrogen-containing heterocyclic group is optionally substituted with one or more methyl groups.

[0039] In certain embodiments, the compounds of the present invention have the structure represented by formula (II’) [Chemical Formula] (wherein R e and R f are as defined above) and have the structure represented by

[0040] In certain embodiments, the compounds of the present invention have the structure represented by formula (III’) [Chemical Formula] (wherein R a , R e and R f are as defined above) and have the structure represented by

[0041] The compounds of the present invention may have a structure selected from the following [Chemical Formula] .

[0042] The compounds of the present invention can be conjugated to nucleotides or oligonucleotides as fluorescent dyes. Thus, in certain embodiments, the compound represented by formula (I) of the present invention is covalently bonded to a nucleotide or oligonucleotide via R 7 , and 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 covalently bonded to a nucleotide or oligonucleotide via R e or R f .

[0043] In certain embodiments, a compound represented by formula (I) or formula (I') of the present invention can be covalently bonded to a nucleotide or oligonucleotide via a cleavable linker. Therefore, the present invention also includes compounds represented by formula (I) or formula (I'), esters thereof, or salts thereof, of formula (i) [ka] (In the formula, w is a carboxyl or ester group, and the dye is the dye compound mentioned above.) The present invention provides a compound represented by R. In a particular embodiment, the dye is R e or R f Through the remaining part ( [ka] It is a compound represented by formula (I') that is connected to ).

[0044] The esters described in the present invention are preferably carboxyl group activated esters. As used herein, the term "activated ester" refers to a carboxyl group derivative that can react, for example, with an amino group-containing compound under mild conditions. Non-limiting examples of activated esters include p-nitrophenyl esters, pentafluorophenyl esters, and succinimidyl esters. [ka] ) are some examples, but are not limited to these.

[0045] The salts of the compounds of the present invention are preferably salts formed by a sulfonic acid group on an acridine ring, for example, salts formed by a sulfonic acid group with an alkali metal ion, an alkaline earth metal ion, or an ammonium ion.

[0046] In a particular embodiment, the compound represented by formula (i) has the following structure [Chemical formula] (wherein, R a , R b , R c , R d and R e are as defined above). has

[0047] In certain embodiments, the compound represented by formula (i) has the following structure [Chemical formula] (wherein, R a , R b , R c , R d and R e are as defined above). has

[0048] In certain embodiments, the compound represented by formula (i) has the following structure [Chemical formula] (wherein, R a , R b , R c , R d and R e are as defined above). has

[0049] ​​​​​​​As used herein, the term "C1-C6 alkyl" refers to a group obtained by removing one hydrogen atom from a linear or branched alkane containing 1 to 6 carbon atoms. Specific examples of C1-C6 alkyls include, but are not limited to, methyl, ethyl, propyl, n-butyl, isobutyl, isopropyl, tert-butyl, n-pentyl, and n-hexyl. In the present invention, preferred C1-C6 alkyls are C1-C4 alkyls.

[0051] As used herein, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0052] As used herein, the term "halo" means that one or more hydrogen atoms of a group or compound are substituted with one or more halogen atoms, and includes perhalation and partialhalation.

[0053] As used herein, the term “heterocyclic group” refers to a group obtained by removing one hydrogen atom from a saturated or partially saturated monocyclic or fused ring compound containing 3 to 14 ring atoms and at least one heteroatom (e.g., 1, 2, 3, 4, or 5 heteroatoms). The term “5-6 membered nitrogen-containing heterocyclic group” refers to a heterocyclic group containing 5 or 6 ring atoms, of which 1, 2, or 3 ring atoms are nitrogen atoms; optionally, the heterocyclic group further contains 1 or 2 oxygen or sulfur atoms.

[0054] As used herein, the term "salt" means (i) alkali metal salts, e.g., sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, e.g., calcium salts, magnesium salts, etc.; other metal salts, e.g., aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, e.g., ammonium salts; organic base salts, e.g., tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts This refers to salts formed by an acidic functional group (e.g., -COOH) present in a compound provided by the present invention and a suitable inorganic or organic cation (base), including but not limited to guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzylphenethylamine salts, piperazine salts, tetramethylamine salts, and tris(hydroxymethyl)aminomethane salt. Furthermore, (ii) refers to salts formed by a basic functional group (e.g., -NH2) present in the compounds provided by the present invention and a suitable inorganic or organic anion (acid), including, but not limited to, hydrohalides such as hydrofluoric acid, hydrochloride, hydrobromide, hydroiodide, etc.; inorganic salts such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkanesulfonates such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; arylsulfonates such as benzenesulfonates, p-toluenesulfonates, etc.; organic salts such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; and amino acid salts such as glycine salts, trimethylglycine salts, arginine salts, ornithine salts, glutamates, aspartates, etc.

[0055] As used herein, the term "ester" refers to an ester formed by a -COOH group present in a compound provided by the present invention with a suitable alcohol, or an ester formed by a -OH group present in a compound provided by the present invention with a suitable acid (e.g., a carboxylic acid or an oxygen-containing inorganic acid). Suitable ester groups include formates, acetates, propionates, butyrates, acrylates, and ethyl succinates. Examples include, but are not limited to, esters, stearates, or palmitates. Esters can undergo hydrolysis in the presence of an acid or base to form the corresponding acid or alcohol.

[0056] Labeled nucleotides The dye compounds of the present invention are suitable for conjugation to substrate moieties. The substrate moiety can, in fact, be any molecule or substance to which the fluorescent dyes described herein can be conjugated, and, as an example not limited to, the dye can be conjugated to the substrate. The substrate moiety can include nucleosides, nucleotides, polynucleotides, carbohydrates, proteins, antibodies, ligands, particles or solid surfaces, organic polymers and inorganic polymers, and combinations or aggregates thereof, such as chromosomes, cell nuclei, and living cells. In some cases, such labeled nucleotides are also called "modified nucleotides."

[0057] A particularly useful application of the fluorescent dye of the present invention is for labeling biomolecules such as nucleotides or oligonucleotides. Therefore, in one embodiment, this application relates to nucleotides or oligonucleotides labeled with the fluorescent compound of the present invention.

[0058] The binding to biomolecules is -C(=O)R of the compound of formula (I). 7 This can be achieved through a part. In certain embodiments, R is a substituted alkoxy group that can be used to bond to the amino group of a biomolecule. In one embodiment, -C(=O)R 7 This portion may be the most suitable activated ester residue for the formation of additional amide / peptide bonds.

[0059] In certain embodiments, a dye compound can be covalently bonded to an oligonucleotide or nucleotide via a nucleoside base. For example, a labeled nucleotide or oligonucleotide may have a label bonded to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base via a linker moiety. The labeled nucleotide or oligonucleotide may also have a 3'OH protecting group covalently bonded to the ribose or deoxyribose of the nucleotide.

[0060] Nucleosides and nucleotides can be labeled at a site on a sugar or nucleic acid base. As understood by those skilled in the art, a “nucleotide” consists of a nitrogen-containing base, a sugar, and one or more phosphate groups. In RNA, the sugar is ribose, and in DNA, the sugar is deoxyribose, which is a sugar lacking the hydroxyl group present in ribose. The nitrogen-containing base is a derivative of a purine or pyrimidine. Purines are adenine (A) and guanine (G), and pyrimidines are cytosine (C) and thymine (T), or, in the context of RNA, uracil (U). The C-1 atom of deoxyribose is bonded to N-1 of the pyrimidine or N-9 of the purine. Nucleosides are also phosphate esters of nucleosides, and esterification occurs at a hydroxyl group bonded to C-3 or C-5 of the sugar. Nucleosides are typically monophosphates, diphosphates, or triphosphatates.

[0061] A "nucleoside" is structurally similar to a nucleotide but lacks a phosphate group. Examples of nucleoside analogs are those in which the label is attached to a base and there is no phosphate group attached to the sugar molecule.

[0062] Bases are generally called purines or pyrimidines, but those skilled in the art will understand that derivatives and analogues are available that do not alter the ability of a nucleotide or nucleoside to undergo Watson-Crick base pairing. “Derivative” or “analog” means a compound or molecule whose core structure is the same as or very similar to that of the parent compound, but which has chemical or physical modifications, such as different or additional side groups, that allow the derived nucleotide or nucleoside to be linked to another molecule. For example, the base is a deazapurine. Derivatives may undergo Watson-Crick pairing. "Derivatives" and "analogs" also mean synthetic nucleotide or nucleoside derivatives having a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are discussed, for example, in Scheit, Nucleotide analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analogs may also contain modified phosphodiester bonds, including phosphorothioate bonds, dithiophosphorate bonds, alkylphosphonate bonds, phosphoranilide bonds, phosphoramidate bonds, etc.

[0063] The dye can be attached to any position on the nucleoside base via a linker, provided that Watson-Crick base pairing can still occur. Specific nucleic acid base labeling sites include the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base. As described above, the dye can be covalently bonded to a nucleoside or nucleotide using a linker group.

[0064] In certain embodiments, the labeled nucleoside or nucleotide may be enzymatically embeddable and enzymatically elongable. Therefore, the linker portion may be long enough to connect the nucleotide to the compound so that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by the nucleic acid replicase. Thus, the linker may also contain spacer units. For example, the spacers keep the nucleoside base away from the cleavage site or label.

[0065] A nucleoside or nucleotide labeled with the dye compound of the present invention has the following structure [ka] (In the formula, the dye is a dye compound, B is a nucleic acid base, such as uracil, thymine, cytosine, adenine, or guanine, and L is an optional linker group that may or may not be present.) It may have the following: R' may be H, a monophosphate, a diphosphate, a triphosphate, a phosphorothioate, a phosphate analog, an -O- bonded to a reactive phosphorus-containing group, or an -O- protected by a protecting group. R'' may be H, an OH, a phosphoramidite, or a 3'-OH protecting group, where R''' is H or an OH; R'' is a phosphoramidite, and R is an acid-cleavable hydroxyl protecting group, allowing for subsequent monomer coupling under automated synthetic conditions.

[0066] This application also relates to polynucleotides incorporating the dye compounds of the present invention. Such polynucleotides may be DNA or RNA, each containing deoxyribonucleotides or ribonucleotides linked by phosphodiester bonds. The polynucleotides may include naturally occurring nucleotides, naturally occurring (or modified) nucleotides other than the labeled nucleotides described herein, or any combination thereof, provided that at least one nucleotide is labeled with the dye compounds of this application. The polynucleotides may also include non-natural skeletal linkages and / or non-nucleotide chemical modifications. Chimeric structures containing mixtures of ribonucleotides and deoxyribonucleotides, each containing at least one labeled nucleotide, are also conceivable.

[0067] In certain embodiments, the labeled nucleotide described in the present invention is of formula (1) [ka] (In the formula, the dye is a dye compound referred to above, which includes a compound represented by formula (I) or formula (I'), or a salt thereof.) It has a structure represented by R. In a particular embodiment, the dye is R e or R f via the remaining part ( [ka] It is a compound represented by formula (I') that is connected to ).

[0068] In a particular embodiment, the labeled nucleotide represented by formula (1) has the following structure [ka] (In the formula, R a , R b , R c , R d and R e (As defined above) It holds.

[0069] In a particular embodiment, the labeled nucleotide represented by formula (1) has the following structure [ka] (In the formula, R a , R b , R c , R d and R e (As defined above) It holds.

[0070] In a particular embodiment, the labeled nucleotide represented by formula (1) has the following structure [ka] (In the formula, R a , R b , R c , R d and R e (As defined above) It holds.

[0071] The nucleotide in the above formula can be selected from dATP, dGTP, dCTP, and dTTP. An exemplary labeled dATP is MGI471-V1-dATP. [ka] These include, but are not limited to, the following:

[0072] An exemplary labeled dATP is one obtained by replacing MGI471 with another dye compound of the present invention.

[0073] An example of labeled dGTP is MGI471-V1-dGTP. [ka] These include, but are not limited to, the following:

[0074] An exemplary labeled dGTP is one obtained by replacing MGI471 with another dye compound of the present invention.

[0075] An example of labeled dCTP is MGI471-V1-dCTP. [ka] These include, but are not limited to, the following:

[0076] An exemplary labeled dCTP is a labeled dCTP obtained by replacing MGI471 with another dye compound of the present invention.

[0077] An example of a labeled dTTP is MGI471-V1-dTTP. [ka] These include, but are not limited to, the following:

[0078] An exemplary labeled dTTP is a labeled dTTP obtained by replacing MGI471 with another dye compound of the present invention.

[0079] The labeled nucleotide of the present invention is [ka] (In the formula, dNTP has the following exemplary structure: [ka] (Selected from dATP, dGTP, dCTP, and dTTP) This can also be mentioned.

[0080] Sequence determination method The nucleotides (or nucleosides) containing the fluorescent dyes of the present invention can be used alone, incorporated into a larger molecular structure or conjugate, or associated with a larger molecular structure or conjugate, in any analytical method requiring the detection of a fluorescent label bound to a nucleotide or nucleoside. Certain embodiments of this application relate to a sequencing method comprising the steps of (a) incorporating at least one labeled nucleotide described herein into a polynucleotide, and (b) detecting the labeled nucleotide incorporated into the polynucleotide by detecting a fluorescent signal from a novel fluorescent dye bound to the modified nucleotide.

[0081] In certain embodiments, during the synthesis step, at least one labeled nucleotide is incorporated into the polynucleotide by the action of polymerase. However, other methods of incorporating labeled nucleotides into polynucleotides, such as chemical oligonucleotide synthesis or ligation of labeled oligonucleotides into unlabeled oligonucleotides, are not excluded. Thus, the term "incorporating" a nucleotide into a polynucleotide encompasses polynucleotide synthesis by both chemical and enzymatic methods.

[0082] In specific, non-limiting embodiments, modified nucleotides or nucleosides labeled with the fluorescent dyes of the present invention may be used in methods for any other applications involving nucleic acid sequencing, rearrangement, whole-genome analysis, single nucleotide polymorphism scoring, detection of modified nucleotides or nucleosides upon incorporation into polynucleotides, or for any other applications requiring the use of polynucleotides labeled with modified nucleotides containing the fluorescent dyes of the present invention.

[0083] In certain embodiments, this application provides the use of modified nucleotides containing the dye compounds of the present invention in a "sequencing bisynthesis" reaction of polynucleotides. Sequencing bisynthesis typically involves sequentially adding one or more nucleotides or oligonucleotides to a polynucleotide chain growing in the 5'→3' direction using a polymerase or ligase to form an elongated polynucleotide chain complementary to the template nucleic acid to be sequenced. The identity of the (one or more) bases present in the added nucleotides is determined in a detection step or “imaging” step. The identity of the added bases may be determined after each nucleotide integration step. The template sequence can then be estimated using the conventional Watson-Crick base pairing rule. The use of dye-labeled modified nucleotides according to this disclosure for determining single-base identity may be useful, for example, in single-nucleotide polymorphism scoring, and such single-base elongation reactions are within the scope of this application.

[0084] In one embodiment, the sequence of a 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, by detecting a fluorescent label bound to the incorporated nucleotides. The nucleic acid template to be sequenced can be DNA or RNA, or even a hybrid molecule containing both deoxynucleotides and ribonucleotides. The nucleic acid template can contain naturally occurring nucleotides and / or non-naturally occurring nucleotides, as well as natural or non-natural skeletal linkages, provided that these do not interfere with the replication of the template in the sequencing reaction.

[0085] One application of the modified nucleotides described herein is in sequencing bisynthesis reactions, but the usefulness of such labeled nucleotides is not limited to such methods. In fact, nucleotides can be advantageously used in any sequencing method requiring the detection of a fluorescent label attached to a nucleotide incorporated into a polynucleotide.

[0086] In a particular embodiment, the present invention provides a method for determining the sequence of a target single-stranded polynucleotide, comprising the following steps: (a) A step of preparing a double strand, nucleotides, polymerase, and a removal reagent, wherein the double strand comprises a growing nucleic acid strand and a nucleic acid molecule to be sequenced. (b) Steps (i), (ii), and (iii): Step (i): Using polymerase, incorporate nucleotides into a growing nucleic acid chain to form a nucleic acid intermediate containing a protecting group and a detectable label. Step (ii): A step of detecting a detectable label of the nucleic acid intermediate, Step (iii): Remove the protecting group of the nucleic acid intermediate using a removal reagent. The steps include carrying out a reaction cycle that includes and This provides a method that includes [something].

[0087] In a particular embodiment, the reaction cycle further includes step (iv): removing a detectable label from the nucleic acid intermediate using a removal reagent.

[0088] In the present invention, nucleic acids may include nucleotides or nucleotide analogs. Nucleotides typically include sugars, nucleic acid bases, 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), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxy Examples include denosine 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 containing modified nucleic acid bases may also be used in the methods described herein. Exemplary modified nucleic acid bases that can be included in polynucleotides, whether with a natural or similar 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-propyluracil. Examples include ropinylcytosine, 6-azouracil, 6-azocytosine, 6-azothimine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-aminoadenine or guanine, 8-thioadenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyadenine or guanine, 5-halogenated 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, such as adenosine 5'-phosphosulfate, cannot be included in polynucleotides.

[0089] 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 may be at least 10 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1000 bp, or at least 2000 bp. In certain preferred embodiments, the length of the nucleic acid molecule to be sequenced may be 10-20 bp, 20-30 bp, 30-40 bp, 40-50 bp, 50-100 bp, 100-200 bp, 200-300 bp, 300-400 bp, 400-500 bp, 500-1000 bp, 1000-2000 bp, or greater than 2000 bp. In certain preferred embodiments, the nucleic acid molecule to be sequenced may have a length of 10-1000 bp to facilitate high-throughput sequencing.

[0090] In certain preferred embodiments, nucleic acid molecules may be pre-treated before being immobilized on a support. Such pre-treatments include, but are not limited to, fragmentation, end blunting, adapter ligation, tagging, amplification, isolation and purification of nucleic acid molecules, and any combination thereof.

[0091] In certain embodiments, the surface of a solid support may have reactive functional groups that react with complementary functional groups of polynucleotide molecules to form covalent bonds in the same manner as, for example, techniques used to bind cDNA to microarrays. See, for example, Smirnov et al. (2004), Genes, Chromosomes & Cancer, 40:72-77 and Beaucage (2001), Current, both incorporated herein by reference. See Medicinal Chemistry, 8:1213-1244. DNB can also be effectively bonded to hydrophobic surfaces, such as clean glass surfaces with low concentrations of various reactive functional groups (e.g., -OH groups). Bonding via covalent bonds formed between polynucleotide molecules and reactive functional groups on the surface is also referred to herein as "chemical bonds".

[0092] In other embodiments, polynucleotide molecules may be adsorbed onto a surface. In such embodiments, the polynucleotides are immobilized through nonspecific interactions with the surface or through non-covalent interactions, such as hydrogen bonds or van der Waals forces.

[0093] In other embodiments, the nucleic acid library may consist of double-stranded nucleic acid fragments, which are immobilized on the surface of a solid support by a ligation reaction with oligonucleotides immobilized on the surface of the solid support, and a sequencing library is subsequently prepared by rolling circle amplification.

[0094] kit In another embodiment, this application provides a kit comprising nucleosides and / or nucleotides labeled with the dye compounds of the present invention. In certain embodiments, the kit comprises one or more nucleotides, wherein at least one nucleotide is labeled with the dye compounds of the present invention. In certain embodiments, the kit may comprise two or more labeled nucleotides. The fluorescent dye compounds, labeled nucleotides, or kits of the present invention may be used for sequencing, expression analysis, hybridization analysis, gene analysis, RNA analysis, or protein binding assays. Such use may be carried out with an automated sequencer. The sequencer may be equipped with two lasers operating at different wavelengths.

[0095] If the kit contains multiple nucleotides, particularly two or four nucleotides, labeled with dye compounds, then different nucleotides may be labeled with the same or different dye compounds, or one nucleotide may not be labeled with any of the dye compounds. If different nucleotides are labeled with the same or different dye compounds, the kit is characterized in that the nucleotides labeled with the dye compounds can be distinguished by fluorescence spectroscopy and algorithms. If two nucleotides labeled with fluorescent dye compounds are provided in the form of a kit, in a particular embodiment, spectrally distinguishable fluorescent dyes may be excited at the same wavelength (e.g., by the same laser). If four nucleotides labeled with fluorescent dye compounds are provided in the form of a kit, in a particular embodiment, two of the spectrally distinguishable fluorescent dyes may be excited together at one wavelength, and the other two spectrally distinguishable dyes may be excited together at different wavelengths.

[0096] The dye compounds of the present invention can be excited by blue light and have an emission wavelength that falls into the cyan light 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 (having an emission wavelength in the cyan light region) and two spectrally distinct dyes that are excited at different wavelengths.

[0097] In the present invention, blue light refers to light having a wavelength in the range of approximately 450 nm to 480 nm, and cyan light refers to light having a wavelength in the range of approximately 480 nm to 490 nm. In certain embodiments, the kit of the present invention may further include reagents for immobilizing nucleic acid molecules to be sequenced onto a support (e.g., immobilization via covalent or non-covalent bonds); primers for initiating a nucleotide polymerization reaction; polymerases for carrying out the nucleotide polymerization reaction; one or more buffers; one or more washing solutions; or any combination thereof.

[0098] In certain embodiments, the kit of the present invention may further include reagents and / or apparatus for extracting nucleic acid molecules from a sample. Methods for extracting nucleic acid molecules from a sample are well known in the art. Therefore, reagents for cell lysis, reagents for DNA precipitation, reagents for DNA washing, reagents for DNA lysis, reagents for RNA precipitation, reagents for RNA washing, reagents for RNA lysis, reagents for protein removal, reagents for DNA removal (for example, when the target nucleic acid molecule is RNA) Various reagents and / or apparatus for extracting nucleic acid molecules, such as reagents for RNA removal (for example, when the target nucleic acid molecule is DNA), and any combination thereof, may be included in the kit of the present invention as needed.

[0099] In certain embodiments, the kit of the present invention further comprises reagents for pretreatment of nucleic acid molecules. In the kit of the present invention, the reagents for pretreatment of nucleic acid molecules are not further limited and can be selected as required by the actual need. Examples of reagents for pretreatment of nucleic acid molecules include reagents for nucleic acid molecule fragmentation (e.g., DNase I), reagents for blunting the ends of nucleic acid molecules (e.g., DNA polymerases such as T4 DNA polymerase, Pfu DNA polymerase, Klenow DNA polymerase), adapter molecules, tag molecules, reagents for ligating adapter molecules to target nucleic acid molecules (e.g., ligases such as T4 DNA ligase), reagents for nucleic acid end repair (e.g., DNA polymerase lacking 3'-5' exonuclease activity but exhibiting 5'-3' exonuclease activity), reagents for amplifying nucleic acid molecules (e.g., DNA polymerase, primers, dNTPs), reagents for isolating and purifying nucleic acid molecules (e.g., chromatography columns), and any combination thereof.

[0100] In certain embodiments, the kit of the present invention further includes a support for immobilizing nucleic acid molecules to be sequenced. Typically, the support for immobilizing nucleic acid molecules to be sequenced is solid-phase for ease of handling. Thus, in this disclosure, “support” is sometimes also referred to as “solid support” or “solid-phase support.” However, it should be understood that “support” as referred to herein is not limited to solids and may be semi-solid (e.g., gels).

[0101] As used herein, the terms “loading,” “immobilization,” and “binding,” when referring to nucleic acids, mean direct or indirect binding to a solid support via covalent or non-covalent bonds. In certain embodiments of this disclosure, the method of the present invention includes the step of immobilizing a nucleic acid onto a solid support via covalent bonds. However, generally, under conditions where a solid support is intended to be used (e.g., in applications requiring nucleic acid amplification and / or sequencing), it is merely required that the nucleic acid remains immobilized or bound to the solid support. In certain embodiments, immobilization of a nucleic acid onto a solid support may involve immobilizing an oligonucleotide intended to be used as a capture primer or amplification primer onto the solid support such that its 3' end is available for enzymatic extension and at least a portion of the primer sequence can hybridize to a complementary nucleic acid sequence; the immobilized nucleic acid is then hybridized to the oligonucleotide, in which case the immobilized oligonucleotide or polynucleotide may be oriented 3'-5'. In certain embodiments, immobilization of nucleic acids onto a solid support may involve binding nucleic acid-binding proteins to the solid support via amination modification and capturing nucleic acid molecules via the nucleic acid-binding proteins. Alternatively, immobilization may be carried out by means other than base-pairing hybridization, such as the covalent bonding described above. Non-limiting examples of methods for binding nucleic acids to a solid support include nucleic acid hybridization, biotin-streptavidin binding, thiol binding, photoactivation binding, covalent bonding, antibody-antigen binding, and physical restraint via hydrogels or other porous polymers.Various exemplary methods for immobilizing nucleic acids on solid supports can be found, for example, in 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 No. 5,639603, U.S. Patent No. 5,641658, U.S. Patent Application Publication No. 2010248991; International Publication No. 2001062982, International Publication No. 2001012862, International Publication No. 2007111937, International Publication No. 0006770. All for the purposes of this work, and. For all teachings relating in particular to the preparation of solid supports on which nucleic acids are immobilized, the above-mentioned literature is incorporated herein by reference in its entirety.

[0102] In the present invention, the support can be made from a variety of suitable materials. Such materials include, for example, inorganic materials, 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, silica, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, polystyrene crosslinked with divinylbenzene, etc. (see, for example, Merrifield, Biochemistry 1964, 3, 1385-1390), polyacrylamide, latex, dextran, rubber, silicone, plastics, natural sponges, metal-plastics, crosslinked dextran (e.g., Sephadex®), agarose gel (Sepharose®), and other supports known to those skilled in the art.

[0103] In certain preferred embodiments, the support for immobilizing nucleic acid molecules to be sequenced may be a solid support comprising an inert substrate or matrix (e.g., slide, polymer beads, etc.) functionalized by applying an intermediate material containing reactive groups that enable covalent bonding 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 International Publication No. 2005 / 065814 and U.S. Patent Application Publication No. 2008 / 0280773, the entire contents of which are incorporated herein by reference. In such embodiments, biomolecules (e.g., polynucleotides) can be directly covalently bonded to an intermediate material (e.g., hydrogel) that can itself non-covalently bond to a substrate or matrix (e.g., a glass substrate). In certain preferred embodiments, the support is a slide glass or silicon wafer whose surface is modified with a layer of chemical groups such as avidin, amino, acrylamide, silane, or aldehyde.

[0104] In the present invention, the support or solid support is not limited by its size, shape, or structure. 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 may be in the form of a planar layer.

[0105] In a particular preferred embodiment, the support for immobilizing nucleic acid molecules to be sequenced is an array (also called a chip) of beads or wells. The array can be prepared using any of the materials outlined herein for preparing a solid support, 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 be 10 to 10 2 pieces, 10 2 ~10 3 pieces, 10 3 ~10 4 pieces, 10 4~10 5 pieces, 10 5 ~10 6 pieces, 10 6 ~10 7 pieces, 10 7 ~10 8 pieces, 10 8 ~10 9 pieces, 10 10 ~10 11 pieces, 10 11 ~10 12 It may contain 10 or more beads or wells. In certain exemplary embodiments, the surface of each bead or well may be immobilized with one or more nucleic acid molecules. Correspondingly, each array may contain 10 to 10 2 pieces, 10 2 ~10 3 pieces, 10 3 ~10 4 pieces, 10 4 ~10 5 pieces, 10 5 ~10 6 pieces, 10 6 ~10 7 pieces, 10 7 ~10 8 pieces, 10 8 ~10 9 pieces, 10 10 ~10 11 pieces, 10 11 ~10 12 Such arrays can immobilize one or more nucleic acid molecules. Therefore, they can be used particularly advantageously for high-throughput sequencing of nucleic acid molecules.

[0106] In a particular preferred embodiment, the kit of the present invention supports nucleic acid molecules to be sequenced. The material further comprises reagents for immobilization on a body (e.g., immobilization via covalent or non-covalent bonds). Such reagents include, for example, reagents for activating or modifying nucleic acid molecules (e.g., at their 5' end), e.g., phosphates, thiols, amines, carboxylic acids, or aldehydes; reagents for activating or modifying the surface of a support, e.g., aminoalkoxysilanes (e.g., aminopropyltrimethoxysilane, aminopropyltriethoxysilane, 4-aminobutyltriethoxysilane, etc.); crosslinking agents, e.g., succinic anhydride, phenyl diisothiocyanate (Guo et al., 1994), maleic anhydride (Yang et al., 1998), 1-ethyl Examples include 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), m-maleimidobenzoyl-N-hydroxysuccinimide (MBS), N-succinimidyl[4-iodoacetyl]aminobenzoic acid (SIAB), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-γ-maleimidobutyryloxysuccinimide (GMBS), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB); and any combination thereof.

[0107] In certain preferred embodiments, the kit of the present invention further comprises primers for initiating a nucleotide polymerization reaction. In the present invention, the primers are not further limited as long as they can specifically anneal to a region of the target nucleic acid molecule. In some exemplary embodiments, the length of the primers may be 5 to 50 bp, e.g., 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 bp. In some exemplary embodiments, the primers may contain naturally occurring nucleotides or nucleotides that do not exist naturally. In some exemplary embodiments, the primers contain or consist of naturally occurring nucleotides. In some exemplary embodiments, the primers contain modified nucleotides such as loc nucleic acid (LNA). In certain preferred embodiments, the primers contain a universal primer sequence.

[0108] In certain preferred embodiments, the kit of the present invention further comprises a polymerase for carrying out a nucleotide polymerization reaction. In the present invention, a variety of suitable polymerases can be used for the polymerization reaction. In some exemplary embodiments, a polymerase (e.g., DNA polymerase) can synthesize a new DNA strand using DNA as a template. In some exemplary embodiments, a polymerase (e.g., reverse transcriptase) can synthesize a new DNA strand using RNA as a template. In some exemplary embodiments, a polymerase (e.g., RNA polymerase) can synthesize a new RNA strand using DNA or RNA as a template. Thus, in certain preferred embodiments, the polymerase is selected from DNA polymerase, RNA polymerase, and reverse transcriptase.

[0109] In certain preferred embodiments, the kit of the present invention further comprises one or more removal reagents. In certain embodiments, the removal reagents are selected from endonuclease IV and alkaline phosphatase.

[0110] In certain preferred embodiments, the kit of the present invention further comprises one or more buffers. Such buffers include, but are not limited to, buffers for DNase I, buffers for DNA polymerase, buffers for ligase, buffers for eluting nucleic acid molecules, buffers for dissolving nucleic acid molecules, buffers for carrying out nucleotide polymerization reactions (e.g., PCR), and buffers for carrying out ligation reactions. The kit of the present invention may comprise any one or more of the above buffers.

[0111] In a particular embodiment, the buffer for DNA polymerase is a monovalent salt ion (for example, The buffer contains sodium ions, chloride ions, and / or divalent salt ions (e.g., magnesium ions, sulfate ions, manganese ions). In certain embodiments, the concentration of monovalent or divalent salt ions in the buffer is 10 μM to 200 mM, for example, 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.

[0112] In certain embodiments, the buffer for DNA polymerase contains tris(hydroxymethyl)aminomethane (tris). In certain embodiments, the concentration of tris in the buffer is 10 mM to 200 mM, for example, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, or 200 mM.

[0113] In certain embodiments, the buffer for DNA polymerase contains an organic solvent such as DMSO or glycerol. In certain embodiments, the mass content of the organic solvent in the buffer is 0.01% to 10%, for example, 0.01%, 0.02%, 0.05%, 1%, 2%, 5%, or 10%.

[0114] In a particular embodiment, the pH of the buffer for DNA polymerase is 7.0 to 9.0, for example, 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.

[0115] In certain embodiments, the buffer 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 is 8.8.

[0116] In certain preferred embodiments, the kit of the present invention further comprises one or more cleaning solutions. Examples of such cleaning solutions include, but are not limited to, phosphate buffer, citrate buffer, Tris-HCl buffer, acetate buffer, and carbonate buffer. The kit of the present invention may comprise one or more of the above cleaning solutions.

[0117] In another aspect, the present application provides the use of a fluorescent dye, labeled nucleotide or oligonucleotide, or kit of the present invention for determining the sequence of a target polynucleotide.

[0118] The present invention also provides the use of fluorescent dyes in the fields of sequencing, expression analysis, hybridization analysis, gene analysis, RNA analysis, protein binding assays, in vitro diagnostics, immunoassays, and molecular labeling. In certain embodiments, molecular labeling is used for cell imaging, tissue imaging, or in vivo biological imaging.

[0119] The present invention further provides the use of fluorescent dyes in the fluorescent labeling, quantification, or detection of proteins, enzymes, or nucleic acids.

[0120] Method for preparing dye compounds This application also provides a method for preparing the dye compounds of the present invention, comprising the step of subjecting a xanthene structure to a substitution reaction in the presence of ammonia and methanol to convert it to an acridine structure.

[0121] The reaction pathway for synthesizing the compound of formula (I) is as follows: [ka] (R 1 ~R 7 (This is defined as above).

[0122] The reaction pathway for synthesizing the compound of formula (I') is as follows: [ka] (R a ~R f (This is defined as above).

[0123] This method can be carried out at room temperature or under heating (for example, 50-60°C).

[0124] Method for preparing dye-labeled nucleotides This application also provides a method for preparing the labeled nucleotides of the present invention, which can be carried out according to any one of Method A, Method B, or Method C.

[0125] Method A: [ka] (In the formula, dye 2 is the dye compound of the present invention and has an acridine structure, while dye 1 is a precursor of dye 2 and has a xanthene structure). Method B: [ka] (In the formula, the dye is the dye compound of the present invention.) Method C: [ka] (In the formula, the dye is the dye compound of the present invention.) Method D: [ka] (In the formula, dye 2 is the dye compound of the present invention and has an acridine structure, while dye 1 is a precursor of dye 2 and has a xanthene structure). In the above method, the nucleotide may have a structure selected from the following: [ka]

[0126] [Advantageous effect] This invention uses substituted xanthenes as raw materials for synthesizing acridine dyes under ammonia / methanol reaction conditions. The corresponding maximum excitation wavelength and maximum emission wavelength of the synthesized dyes undergo a blue shift; under blue light excitation, their emission wavelengths fall into the cyan light region.

[0127] The present invention develops novel dyes in the blue light region or cyan light region, and uses these dyes for base labeling, thereby helping to improve sequencing accuracy in the field of gene sequencing.

[0128] The present invention provides a method for preparing pigments that has the advantages of simple steps and mild conditions.

[0129] Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are used only to illustrate the present invention and are not intended to limit its scope. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. [Brief explanation of the drawing]

[0130] [Figure 1] This figure shows the 1H NMR spectrum of MGI450b. [Figure 2A] This figure shows the 1H NMR spectrum of MGI443. [Figure 2B] This figure shows the F NMR spectrum of MGI443. [Figure 3] This figure shows the 1H NMR spectrum of MGI447. [Figure 4] This figure shows the 1H NMR spectrum of MGI477. [Figure 5] This figure shows the 1H NMR spectrum of MGI456. [Figure 6] This figure shows the 1H NMR spectrum of MGI485. [Figure 7A] This figure shows the 31P NMR spectrum of MGI501-V1-dATP. [Figure 7B] This figure shows the 31P NMR spectrum of MGI501-V1-dCTP. [Figure 8A] This figure shows the 31P NMR spectrum of MGI495-V1-dATP. [Figure 8B] This figure shows the 31P NMR spectrum of MGI495-V1-dCTP. [Figure 9A] This figure shows the 31P NMR spectrum of MGI450c-V1-dATP. [Figure 9B] This figure shows the 31P NMR spectrum of MGI450c-V1-dCTP. [Figure 9C] This figure shows the 31P NMR spectrum of MGI450c-V1-dGTP. [Figure 10A] This figure shows the 31P NMR spectrum of MGI450b-V1-dATP. [Figure 10B] This figure shows the 31P NMR spectrum of MGI450b-V1-dTTP. [Figure 10C] This figure shows the 31P NMR spectrum of MGI450b-V1-dGTP. [Figure 11A] This figure shows the 31P NMR spectrum of MGI443-V1-dATP. [Figure 11B] This figure shows the 31P NMR spectrum of MGI443-V1-dCTP. [Figure 11C] This figure shows the 31P NMR spectrum of MGI443-V1-dTTP. [Figure 11D] This figure shows the 31P NMR spectrum of MGI443-V1-dGTP. [Figure 12A] This figure shows the 31P NMR spectrum of MGI488-V1-dATP. [Figure 12B]This figure shows the 31P NMR spectrum of MGI488-V1-dCTP. [Figure 13A] This figure shows the 31P NMR spectrum of MGI447-V1-dATP. [Figure 13B] This figure shows the 31P NMR spectrum of MGI447-V1-dCTP. [Figure 13C] This figure shows the 31P NMR spectrum of MGI447-V1-dTTP. [Figure 13D] This figure shows the 31P NMR spectrum of MGI447-V1-dGTP. [Figure 14A] This figure shows the 19F NMR spectrum of MGI456-V1-dATP. [Figure 14B] This figure shows the 31P NMR spectrum of MGI456-V1-dATP. [Figure 14C] This figure shows the 19F NMR spectrum of MGI456-V1-dCTP. [Figure 14D] This figure shows the 31P NMR spectrum of MGI456-V1-dCTP. [Figure 14E] This figure shows the 19F NMR spectrum of MGI456-V1-dTTP. [Figure 14F] This figure shows the 19F NMR spectrum of MGI456-V1-dGTP. [Figure 14G] This figure shows the 31P NMR spectrum of MGI456-V1-dGTP. [Figure 15A] This figure shows the 31P NMR spectrum of MGI485-V1-dATP. [Figure 15B] This figure shows the 31P NMR spectrum of MGI485-V1-dCTP. [Figure 16] This figure shows the 31P NMR spectrum of MGI450d-V1-dATP. [Figure 17A] This figure shows the 1H NMR spectrum of MGI471-V1-dATP. [Figure 17B]This figure shows the 31P NMR spectrum of MGI471-V1-dATP. [Figure 17C] This figure shows the 31P NMR spectrum of MGI471-V1-dCTP. [Figure 17D] This figure shows the 31P NMR spectrum of MGI471-V1-dTTP. [Figure 17E] This figure shows the 31P NMR spectrum of MGI471-V1-dGTP. [Figure 18A] This figure shows the excitation spectrum and emission spectrum of MGI471-V1. [Figure 18B] This figure shows the excitation spectrum and emission spectrum of MGI471. [Figure 18C] This figure shows the excitation and emission spectra of MGI471-V1-dATP. [Figure 18D] This figure shows the excitation and emission spectra of MGI471-V1-dGTP. [Figure 18E] This figure shows the excitation spectrum and emission spectrum of MGI471-V1-dCTP. [Figure 18F] This figure shows the excitation and emission spectra of MGI471-V1-dTTP. [Figure 18G] This figure shows the excitation and emission spectra of MGI495-V1-dATP. [Figure 18H] This figure shows the excitation spectrum and emission spectrum of MGI495-V1-dCTP. [Figure 18I] This figure shows the excitation spectrum and emission spectrum of MGI501-V1-dATP. [Figure 18J] This figure shows the excitation spectrum and emission spectrum of MGI501-V1-dCTP. [Figure 18K] This figure shows the excitation spectrum and emission spectrum of MGI447. [Figure 18L] This figure shows the excitation spectrum and emission spectrum of MGI450b. [Figure 18M]This figure shows the excitation spectrum and emission spectrum of MGI456. [Figure 18N] This figure shows the excitation spectrum and emission spectrum of MGI477. [Figure 18O] This figure shows the excitation spectrum and emission spectrum of MGI485. [Figure 18P] This figure shows the excitation spectrum and emission spectrum of MGI450d. [Figure 18Q] This figure shows the excitation and emission spectra of MGI450d-V1-dATP. [Figure 18R] This figure shows the excitation spectrum and emission spectrum of MGI450d-V1-dCTP. [Figure 18S] This figure shows the excitation spectrum and emission spectrum of MGI450d-V1-dTTP. [Figure 18T] This figure shows the excitation and emission spectra of MGI485-V1-dATP. [Figure 18U] This figure shows the excitation spectrum and emission spectrum of MGI485-V1-dCTP. [Figure 18V] This figure shows the excitation and emission spectra of MGI447-V1-dATP. [Figure 18W] This figure shows the excitation spectrum and emission spectrum of MGI447-V1-dCTP. [Figure 18X] This figure shows the excitation spectrum and emission spectrum of MGI447-V1-dTTP. [Figure 18Y] This figure shows the excitation and emission spectra of MGI447-V1-dGTP. [Figure 18Z] This figure shows the excitation spectrum and emission spectrum of MGI453. [Figure 18a] This figure shows the excitation and emission spectra of MGI456-V1-dATP. [Figure 18b] This figure shows the excitation spectrum and emission spectrum of MGI456-V1-dCTP. [Figure 18c] This figure shows the excitation spectrum and emission spectrum of MGI456-V1-dGTP. [Figure 18d] This figure shows the excitation spectrum and emission spectrum of MGI456-V1-dTTP. [Figure 18e] This figure shows the excitation spectrum and emission spectrum of MGI450a. [Figure 18f] This figure shows the excitation and emission spectra of MGI450b-V1-dATP. [Figure 18g] This figure shows the excitation spectrum and emission spectrum of MGI450b-V1-dCTP. [Figure 18h] This figure shows the excitation spectrum and emission spectrum of MGI450b-V1-dGTP. [Figure 18i] This figure shows the excitation spectrum and emission spectrum of MGI450b-V1-dTTP. [Figure 18j] This figure shows the excitation and emission spectra of MGI488-V1-dATP. [Figure 18k] This figure shows the excitation spectrum and emission spectrum of MGI488-V1-dCTP. [Figure 18l] This figure shows the excitation and emission spectra of MGI443-V1-dATP. [Figure 18m] This figure shows the excitation spectrum and emission spectrum of MGI443-V1-dCTP. [Figure 18n] This figure shows the excitation spectrum and emission spectrum of MGI443-V1-dGTP. [Figure 18o] This figure shows the excitation spectrum and emission spectrum of MGI443-V1-dTTP. [Figure 18p] This figure shows the excitation spectrum and emission spectrum of MGI450c. [Figure 18q] This figure shows the excitation and emission spectra of MGI450c-V1-dATP. [Figure 18r]This figure shows the excitation spectrum and emission spectrum of MGI450c-V1-dCTP. [Figure 18s] This figure shows the excitation spectrum and emission spectrum of MGI450c-V1-dGTP. [Figure 18t] This figure shows the excitation spectrum and emission spectrum of MGI450c-V1-dTTP. [Figure 18u] This figure shows the excitation spectrum and emission spectrum of MGI455. [Figure 18v] This figure shows the excitation spectrum and emission spectrum of MGI488. [Figure 18w] This figure shows the excitation spectrum and emission spectrum of MGI443. [Figure 18x] This figure shows the excitation spectrum and emission spectrum of MGI453. [Figure 18y] This figure shows the excitation spectrum and emission spectrum of MGI450a. [Figure 19] This figure shows the Q30 data of the MGI471 dNTP on the sequencer. Detailed description of specific embodiments

[0131] Embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are used only to illustrate the present invention and should not be considered to limit its scope. Where otherwise specified in the examples, experiments are carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or equipment whose manufacturers are not indicated are conventional products available on the market.

[0132] As used herein, common organic abbreviations are defined as follows: Boc:tert-butoxycarbonyl ℃: Celsius dATP: Deoxyadenosine triphosphate dTTP: Deoxythymidine triphosphate dCTP: Deoxycytidine triphosphate dGTP: Deoxyguanosine triphosphate DIPEA: N,N-diisopropylethylamine DMAP: 4-dimethylaminopyridine DMF: N,N-dimethylformamide DMSO-d6: Deuterated Dimethyl Sulfoxide D2O: heavy water DSC:N,N'-Discuscinimidyl carbonate FA: Formic acid g: grams LCMS: Liquid Chromatography-Mass Spectrometry mg: milligrams mL: milliliter MeOH: methanol m / z: mass-to-charge ratio NH3·H2O: Ammonia TEAB: Triethylamine-Bicarbonate Buffer Solution THF: Tetrahydrofuran [ka]

[0133] Example 1: Synthesis of MGI453 [ka] (1) Synthesis of 3',6'-dihydroxy-3-carbonyl-3H-spiro[isobenzofuran-1,9'-xanthene]-4',5'-disulfonic acid (MGI453-1) [ka] In a 100 mL flask, 30% fuming sulfuric acid (30 mL) was added, followed by fluorescein (3 g, 9.03 mmol). The mixture was heated to 90°C and magnetically stirred for 2 hours. The reaction solution was slowly added dropwise to crushed ice to quench the reaction, followed by filtration. The filtrate was purified by flash preparative liquid chromatography (water / acetonitrile), and the prepared solution was concentrated under reduced pressure to obtain MGI453-1. LCMS: C 20 H 12 O 11 S2[M+H] + Calculated value: 492.98. Measured value, m / z, [M+H] + :493.10.

[0134] (2) Synthesis of 2-(3,6-dihydroxy-4,5-disulfacrylidine-9-yl)benzoic acid (MGI453-2) [ka] In a 100 mL flask, MGI453-1 (200 mg, 406.15 μmol) was dissolved in ammonia solution (25%-28%, 20 mL) and methanol (20 mL), heated to 50°C, and magnetically stirred for 15 hours. The reaction solution was concentrated under reduced pressure to obtain the crude product, which was purified by flash preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile). The prepared solution was concentrated under reduced pressure and lyophilized to obtain MGI453-2. LCMS: C 20 H 13 NO 10 S2[M+H] + Calculated value: 492.00. Measured value, m / z, [M+H] + :492.00. 1 H NMR(600 MHz, 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).

[0135] (3) Synthesis of 4-(2-(3,6-dihydroxy-4,5-disulfacrylidine-9-yl)-N-methylbenzamide)butyric acid (MGI453) [ka] In a 15 mL sample vial, MGI453-2 (50 mg, 101.74 μmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), then N,N'-disuccinimidyl carbonate (52 mg, 202.99 μmol) and 4-dimethylaminopyridine (13 mg, 106.41 μmol) were added, and the mixture was subsequently magnetically stirred at 20°C for 3 hours. Next, 4-methylaminobutyrate (78 mg, 507.79 μmol) and triethylamine (103 mg, 1.02 mmol) were added, and the mixture was further magnetically stirred at 20°C for 15 hours. The reaction solution was filtered, and the filtrate was first purified by flash preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile). The prepared solution was concentrated under reduced pressure and lyophilized to obtain a yellow solid. After further purification by preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile), the prepared solution was concentrated under reduced pressure and lyophilized to obtain MGI453. LCMS: C 25 H 22 N2O 11 S2[M+H] + Calculated value: 590.07. Measured value, m / z, [M+H] + : 591.04. 1 H NMR(600 MHz, 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, 1H), 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).

[0136] Example 2: Synthesis of MGI450a [ka] (1) Synthesis of 2-(6-amino-3-imino-3H-xanthene-9-yl)benzoic acid (MGI450a-1) [ka] To a 40 mL sample vial, 3-aminophenol (2.95 g, 27.01 mmol), methanesulfonic acid (15 mL), and then phthalic anhydride (2 g, 13.50 mmol) were added and heated to 180°C, followed by magnetic stirring for 6 hours. The reaction solution was slowly poured into ice water to quench the reaction, followed by filtration. The filtration cake was washed with a small amount of water to obtain a red solid. The red solid was slurryed with dichloromethane / methanol = 10 / 1 (50 mL), filtered, and dried to obtain MGI450a-1. LCMS: C 20 H 14 N2O3[M+H] + Calculated value: 331.10. Measured value, m / z, [M+H] + :331.18.

[0137] (2) Synthesis of 2-(3,6-diamino-4,5-disulfacrydin-9-yl)benzoic acid (MGI450a-2) [ka] In a 250 mL flask, 100 mL of 50% fuming sulfuric acid was added for dissolution, then 2.2 g, 6.66 mmol of MGI450a-1 were added in portions, and the mixture was heated to 60°C and magnetically stirred for 15 hours. The reaction solution was slowly added dropwise to ice water to quench the reaction, and then sodium carbonate was added to neutralize the solution. Equivolutes of methanol and ammonia solution (25%-28%, 100 mL) were added, and the mixture was subsequently magnetically stirred at 22°C for 15 hours. The reaction solution was concentrated under reduced pressure to remove most of the solvent, followed by filtration, and the filtrate was subjected to flash preparative liquid chromatography (0.1% trifluoroacetic acid / acetate). The solution was purified using tonitrile, and the prepared solution was concentrated under reduced pressure to obtain MGI450a-2. LCMS: C 20 H 15 N3O8S2[M+H] + Calculated value: 490.03. Measured value, m / z, [M+H] + : 490.13.

[0138] (3) Synthesis of 4-(2-(3,6-diamino-4,5-disulfacrylidine-9-yl)-N-methylbenzamide)butyric acid (MGI450a) [ka] In a 50 mL flask, MGI450a-2 (200 mg, 408.60 μmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL), then N,N'-disuccinimidyl carbonate (209 mg, 815.86 μmol) and 4-dimethylaminopyridine (50 mg, 409.26 μmol) were added, and the mixture was subsequently magnetically stirred at 22°C for 15 hours. Next, 4-methylaminobutyric acid (96 mg, 819.48 μmol) and triethylamine (165 mg, 1.63 mmol) were added, and the mixture was further magnetically stirred at 20°C for 15 hours. The reaction solution was filtered, and the filtrate was first purified by flash preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile), and then purified again by preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile). The prepared solution was concentrated under reduced pressure to obtain MGI450a. LCMS: C 25 H 24 N4O9S2[M+H] + Calculated value: 589.10. Measured value, m / z, [M+H] + : 589.28. 1 H NMR(600 MHz, 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).

[0139] Example 3: Synthesis of MGI450b [ka] (1) Synthesis of methyl 4-(6-amino-3-imino-3H-xanthene-9-yl)benzoate (MGI450b-1) [ka] To a 40 mL sample vial, 3-aminophenol (2.91 g, 26.64 mmol), methanesulfonic acid (20 mL), and then 4-formylbenzoic acid (2 g, 13.32 mmol) were added. The mixture was heated to 180°C and magnetically stirred for 5 hours. The reaction solution was transferred to a 250 mL flask, and methanol (50 mL) and dichloromethane (50 mL), and then 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (3.02 g, 13.32 mmol) were added. The mixture was then magnetically stirred at 18°C ​​for 2 hours. The reaction solution was concentrated under reduced pressure to remove methanol and dichloromethane. The concentrated solution was then poured into water (500 mL), stirred, and filtered to obtain the crude product. The crude product was purified by column chromatography to obtain MGI450b-1. LCMS: C 21 H 16 N2O3[M+H] + Calculated value: 345.12. Measured value, m / z, [M+H] + :345.17.

[0140] (2) Synthesis of 4-(6-amino-3-imino-3H-xanthene-9-yl)benzoic acid (MGI450b-2) [ka] In a 100 mL flask, MGI450b-1 (3.9 g, 11.33 mmol) was dissolved in methanol (90 mL) and an aqueous solution of sodium hydroxide (906 mg, 22.65 mmol) (30 mL), and the mixture was magnetically stirred at 19°C for 5 hours. The reaction solution was concentrated under reduced pressure to remove methanol, the pH was adjusted to 5-6 with 1 M hydrochloric acid aqueous solution, filtered, washed with a small amount of water, and dried to obtain MGI450b-2.

[0141] (3) Synthesis of 4-(6-amino-3-imino-4,5-disulfo-3H-xanthene-9-yl)benzoic acid (MGI450b-3) [ka] In a 250 mL flask, 100 mL of 50% fuming sulfuric acid was added for dissolution, then MGI450b-2 (2 g, 6.05 mmol) was added, and the mixture was heated to 60°C and magnetically stirred for 20 hours. The reaction solution was slowly added dropwise to crushed ice to quench the reaction, followed by filtration. The filtrate was purified by flash preparative liquid chromatography (water / acetonitrile), and the prepared solution was concentrated under reduced pressure to obtain MGI450b-3. LCMS: C 20 H 14 N2O9S2[MH] - Calculated value: 489.01. Measured value, m / z, [MH] - : 489.11.

[0142] (4) Synthesis of 4-(3,6-diamino-4,5-disulfacrydin-9-yl)benzoic acid (MGI450b) [ka] In a 40 mL sample vial, MGI450b-3 (100 mg, 203.89 μmol) was dissolved in ammonia solution (25%-28%, 5 mL) and methanol (5 mL), and the mixture was magnetically stirred at 25°C for 15 hours. The reaction solution was concentrated under reduced pressure to obtain the crude product, which was purified by preparative liquid chromatography (0.1 M TEAB / acetonitrile). The prepared solution was concentrated under reduced pressure and lyophilized to obtain MGI450b. LCMS: C 20 H 15 N3O8S2[MH] - Calculated value: 488.03. Measured value, m / z, [MH] - : 488.19. 1 H NMR(600 MHz, 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).

[0143] Example 4 The fluorescent dye MGI450c was synthesized according to the synthesis method for MGI450b. LCMS: C 21 H 15 N3O 10 S2[MH] - Calculated value: 532.02. Measured value, m / z, [MH] - : 532.18.

[0144] Example 5 The fluorescent dye MGI450d was synthesized according to the synthesis method for MGI450b. LCMS: C 24 H 22 N4O 12 S3[MH] - Calculated value: 653.04. Measured value, m / z, [MH] - : 653.11.

[0145] Example 6 The fluorescent dye MGI443 was synthesized according to the synthesis method for MGI450b. LCMS: C 20 H 13 F2N3O8S2[MH] - Calculated value: 524.01. Measured value, m / z, [MH] - : 524.17. 1 H NMR(400 MHz, DMSO) δ = 8.16 (d, J=8.2, 2H), 7.51 (d,J=8.1, 2H), 6.95 (d, J=11.6, 2H).

[0146] Example 7 The fluorescent dye MGI455 was synthesized according to the synthesis method for MGI450b. LCMS: C 20 H 13 Cl2N3O8S2[M+H] + Calculated value: 557.95. Measured value, m / z, [M+H] + : 558.90. 1H NMR(600 MHz, 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).

[0147] Example 8 The fluorescent dye MGI447 was synthesized according to the synthesis method for MGI450b. LCMS: C 22 H 19 N3O8S2[MH] - Calculated value: 516.06. Measured value, m / z, [MH] - : 516.12. 1 H NMR(600 MHz, DMSO) δ = 8.12 (d, J=7.6, 2H), 7.36 (d,J=7.7, 2H), 6.98 (s, 2H), 2.11 (s, 6H).

[0148] Example 9 The fluorescent dye MGI477 was synthesized according to the synthesis method for MGI450b. LCMS: C 24 H 23 N3O8S2[MH] - Calculated value: 544.09. Measured value, m / z, [MH] - : 544.18. 1 H NMR(600 MHz, 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).

[0149] Example 10 The fluorescent dye MGI456 was synthesized according to the synthesis method for MGI450b. LCMS: C 24 H 17 F6N3O8S2[M+H] +Calculated value: 654.04. Measured value, m / z, [M+H] + : 654.20. 1 H NMR(600 MHz, 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).

[0150] Example 11 The fluorescent dye MGI471 was synthesized according to the synthesis method for MGI450b. LCMS: C 30 H 31 N3O8S2[MH] - Calculated value: 624.16. Measured value, m / z, [MH] - : 624.23. 1 H NMR(600 MHz, DMSO-d6): δ 14.37 (s, 1H), 8.22 -8.18 (m, 2H), 7.83 (s, 2H), 7.52 (dt, J = 8.6, 4.5 Hz, 2H), 6.76 (s, 2H), 3.77- 3.71 (m, 2H), 1.18 - 1.14 (m, 12H), 0.99 (d, J = 4.3 Hz, 6H).

[0151] Example 12 The fluorescent dye MGI485 was synthesized according to the synthesis method for MGI450b. LCMS: C 32 H 35 N3O8S2[MH] - Calculated value: 652.19. Measured value, m / z, [MH] - : 652.45. 1H NMR(600 MHz, 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).

[0152] Example 13 The fluorescent dye MGI488 was synthesized according to the synthesis method for MGI450b. LCMS: C 29 H 32 N4O9S2[MH] - Calculated value: 643.16. Measured value, m / z, [MH] - : 643.22. 1 H NMR(400 MHz, 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), 3.15 - 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).

[0153] Example 14 Synthesis of MGI501-modified nucleotide [ka] (1) Synthesis of MGI501-1-V1-dATP or MGI501-1-V1-dCTP [ka] In a 4 mL sample vial, AF546 NHS (10 mg, 9.45 μmol) was dissolved in N,N-dimethylformamide (1 mL), and then V1-dATP (18 mg, 18.99 μmol) and N,N-diisopropylaminoethylamine (12 mg, 92.85 μmol) were added. The mixture was then magnetically stirred at 22°C for 15 hours. The reaction solution was filtered, and the filtrate was purified by flash preparative chromatography (0.1 M TEAB / acetonitrile). The prepared solution was concentrated under reduced pressure to obtain MGI501-1-V1-dATP. LCMS: C 70 H 82 Cl3N 16 O 28 P3S3[MH] - Calculated value: 1889.29. Measured value, m / z, [(M-2) / 2] - : 944.45. MGI501-1-V1-dCTP was synthesized according to the same method as described above. LCMS: C 68 H 80 Cl3N 14 O 30 P3S3[MH] - Calculated value: 1865.26. Measured value, m / z, [(M-2) / 2] - : 932.87.

[0154] (2) Synthesis of MGI501-V1-dATP or MGI501-V1-dCTP [ka] In a 15 mL sample vial, MGI501-1-V1-dATP (10 mg, 5.29 μmol of the compound was dissolved in a methanol solution of ammonia (7M, 2 mL) and magnetically stirred at 23°C for 15 hours. The reaction solution was filtered, and the filtrate was purified by preparative liquid chromatography (0.1% ammonia / acetonitrile). The prepared solution was concentrated under reduced pressure and lyophilized to obtain MGI501-V1-dATP. LCMS: C 70 H 83 Cl3N 17 O 27P3S3[MH] - Calculated value: 1888.31. Measured value, m / z, [(M-2) / 2] - : 943.87. 1 H NMR(400 MHz, DMSO-d6) δ 9.24 (t, J = 6.1 Hz,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, 2H), 7.08 (d, J = 14.6 Hz, 3H), 6.99 (d, J = 15.8 Hz, 2H), 6.49 (dd, J = 8.9, 5.8 Hz, 1H), 5.14 (s, 1H), 4.93 (d, J = 9.0 Hz, 1H), 4.86(d, J = 8.8 Hz, 1H), 4.57 (s, 1H), 4.25 - 4.20 (m, 1H), 4.14 (d, J = 5.5 Hz,4H), 3.98 (s, 7H), 3.68 (s, 2H), 2.86 (d, J = 62.4 Hz, 16H), 2.01 (s, 2H), 1.82(d, J = 12.8 Hz, 2H), 1.43 (s, 2H), 1.31 (s, 5H), 1.25 (d, J = 5.5 Hz, 3H), 1.20 (t, J = 6.3 Hz, 3H), 1.12 (dd, J = 6.5, 3.4 Hz, 2H), 1.07 - 1.00 (m, 3H). 31 P NMR (162 MHz, dmso) δ = -9.98 (d, J=18.5), -13.44, -22.65. MGI501-V1-dCTP was synthesized using the same method as described above. LCMS: C 68 H 81 Cl3N 15 O 29 P3S3[MH] - Calculated value: 1864.28. Measured value, m / z, [MH]- : [(M-2) / 2] - : 932.30. 1 H NMR(400 MHz, dmso) δ 9.24 (d, J = 5.9 Hz, 2H), 8.67 (s,2H), 8.03 (s, 3H), 7.75 (s, 1H), 7.45 (d, J = 7.6 Hz, 2H), 7.35 - 7.29 (m, 1H),7.11 (s, 2H), 7.07 (s, 1H), 7.00 (d, J = 15.7 Hz, 2H), 6.88 (s, 1H), 6.15 -6.07 (m, 1H), 5.16 (s, 1H), 4.92 (d, J = 9.1 Hz, 1H), 4.81 (d, J = 8.9 Hz, 1H), 4.41 (s, 1H), 4.27 - 4.21 (m, 2H), 4.15 (dd, J = 17.8, 8.4 Hz, 4H), 4.01 - 3.90(m, 5H), 3.89 - 3.82 (m, 2H), 3.70 - 3.65 (m, 2H), 3.49 (d, J = 8.2 Hz, 2H), 3.19 (s, 2H), 2.93 (d, J = 6.6 Hz, 3H), 2.85 (s, 2H), 2.71 (d, J = 17.9 Hz, 1H), 2.31 - 2.30 (m, 1H), 2.15 (s, 2H), 2.01 (s, 2H), 1.82 (d, J = 12.4 Hz, 2H), 1.42 (d, J = 8.2 Hz, 3H), 1.36 - 1.23 (m, 12H), 1.22 - 1.11 (m, 6H). 31 P NMR (162 MHz, dmso) δ 74.99 (s), -10.17 - -10.42 (m), -12.28 - -12.91 (m), -22.73 (s).

[0155] Example 15 Synthesis of MGI495 modified ヌクレオチドの

change

[0156] (2) Synthesis of MGI495-V1-dATP and MGI495-V1-dCTP [ka] In a 15 mL sample vial, MGI495-1-V1-dATP (25 mg, 14.29 μmol) was dissolved in methanol (2 mL), then ammonia (2 mL) was added, and the mixture was magnetically stirred at 25°C for 15 hours. The reaction solution was concentrated under reduced pressure to obtain the crude product, which was purified by preparative HPLC (0.1% NH3·H2O / acetonitrile). The prepared solution was concentrated under reduced pressure and lyophilized to obtain the compound MGI495-V1-dATP. LCMS: C 66 H 80 N 17 O 28 P3S3[MH] - Calculated value: 1746.37 Measured value, m / z, [(M-2) / 2] - : 837.09. 1 H NMR(400 MHz, dmso) δ 9.26 (d, J = 7.8 Hz, 2H), 9.04 (d, J= 42.3 Hz, 2H), 8.76 (s, 1H), 8.43 (t, J = 5.5 Hz, 1H), 8.24 (t, J = 11.7 Hz,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 = 8.3 Hz, 2H), 6.94 (d, J = 5.3 Hz, 2H),6.49 (dd, J = 8.7, 5.9 Hz, 1H), 5.15 (t, J = 4.6 Hz, 1H), 4.90 (dd, J = 31.3,8.9 Hz, 2H), 4.56 (d, J = 4.9 Hz, 1H), 4.25 (dd, J = 10.4, 4.0 Hz, 1H), 4.16(dd, J = 16.6, 5.3 Hz, 3H), 3.99 - 3.83 (m, 5H), 3.72 - 3.67 (m, 2H), 3.31 (s,3H), 3.06 (dd, J = 15.8, 8.6 Hz, 1H), 2.99 - 2.73 (m, 3H), 2.65 - 2.57 (m, 2H),2.42 - 2.37 (m, 1H), 1.81 (d, J = 13.6 Hz, 2H), 1.45 - 1.12 (m, 12H), 1.04 (dd,J = 6.0, 2.6 Hz, 6H). 31 P NMR (162 MHz, dmso) δ -10.51 (d, J = 18.9 Hz), -12.94 (d, J= 24.0 Hz), -23.00 - -23.49 (m). MGI495-V1-dCTP was synthesized using the same method as described above. LCMS: C 64 H 77 N 15 O 30 P3S3[MH] - Calculated value: 1724.34 Measured value, m / z, [(M-2) / 2] - : 861.35. 1 H NMR(400 MHz, dmso) δ 9.26 (d, J = 10.1 Hz, 2H), 9.06 (d, J= 32.6 Hz, 2H), 8.83 - 8.75 (m, 1H), 8.58 (dd, J = 12.2, 7.8 Hz, 1H), 8.24 (t,J = 11.5 Hz, 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 = 8.2 Hz, 2H), 6.94 (d, J = 5.2 Hz,2H), 6.89 (s, 1H), 6.11 (dd, J = 7.8, 6.0 Hz, 1H), 5.19 - 5.13 (m, 1H), 4.91(d, J = 8.8 Hz, 1H), 4.80 (d, J = 8.9 Hz, 1H), 4.44 - 4.39 (m, 1H), 4.26 (dd, J= 10.4, 4.3 Hz, 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, 6.9 Hz, 1H), 2.83 (dd, J = 28.6,23.2 Hz, 3H), 2.58 (t, J = 5.4 Hz, 2H), 2.40 (s, 1H), 2.32 (d, J = 8.0 Hz, 1H), 2.17 (dd, J = 14.7, 7.6 Hz, 1H), 1.81 (d, J = 12.1 Hz, 2H), 1.45 - 1.12 (m,12H), 1.05 (d, J = 6.4 Hz, 6H). 31 P NMR (162 MHz, dmso) δ -10.44 (d, J = 18.9 Hz), -12.86 (d, J= 27.2 Hz), -23.09 (t, J = 20.3 Hz).

[0157] Example 16 Synthesis of MGI450c modified ヌクレオチドの (1) The following compounds are synthesized according to the method described in Example 15: MGI450c-1-V1-dATP LCMS: C 51 H 52 N 15 O 27 P3S2[MH] - Calculated value: 1462.18. Measured value, m / z, [(M-2) / 2] - 730.68. MGI450c-1-V1-dCTP LCMS: C 49 H 50 N 13 O 29 P3S2[MH] - Calculated value: 1440.15. Measured value, m / z, [(M-2) / 2] - : 719.20. MGI450c-1-V1-dTTP LCMS: C 49 H 51 N 14 O 28 P3S2[MH] - Calculated value: 1439.17. Measured value, m / z, [(M-2) / 2] - : 719.73. MGI450c-1-V1-dGTP LCMS: C 51 H 52 N 15 O 28 P3S2[MH] - Calculated value: 1478.18. Measured value, m / z, [(M-2) / 2] - : 738.80.

[0158] (2) The following compounds were synthesized according to the method described in Example 15: MGI450c-V1-dATP LCMS: C 51 H 53 N 16 O 26 P3S2[MH] - Calculated value: 1461.20. Measured value, m / z, [(M-2) / 2] - : 730.27. 11H NMR (400 MHz, dmso) δ 9.07 (s, 1H), 8.76 (s, 1H), 8.67 (d, J = 1.4 Hz, 1H), 8.43 (t, J = 5.4 Hz, 1H), 8.20 (d, J = 8.2 Hz, 1H), 8.10 (s, 1H), 7.85 (s, 3H), 7.70 (s, 1H), 7.51 (d, J = 8.2 Hz, 2H), 7.42 - 7.34 (m, 2H), 7.12 (d, J = 8.3 Hz, 1H), 6.94 (dd, J = 18.2, 9.3 Hz, 3H), 6.49 (dd, J = 8.8, 5.8 Hz, 1H), 5.15 (t, J = 4.8 Hz, 1H), 4.89 (dd, J = 29.8, 8.9 Hz, 2H), 4.56 (d, J = 4.6 Hz, 1H), 4.25 (dd, J = 10.5, 4.4 Hz, 1H), 4.16 (dd, J = 17.2, 4.8 Hz, 3H), 3.98 (s, 1H), 3.96 - 3.81 (m, 4H), 3.68 (d, J = 6.1 Hz, 3H), 2.95 (dd, J = 14.4, 7.1 Hz, 2H), 2.85 (d, J = 7.2 Hz, 1H), 2.69 - 2.58 (m, 1H), 2.39 (dd, J = 13.3, 5.9 Hz, 1H). 31 31P NMR (162 MHz, dmso) δ -10.47 (d, J = 19.4 Hz), -12.76 (d, J = 20.9 Hz), -22.81 - -23.33 (m). MGI450c-V1-dCTP LCMS: C 49 H 51 N 14 O 28 P3S2[M-H] - Calculated value: 1439.17. Measured value, m / z, [(M - 2) / 2] - : 718.74. 1H NMR(400 MHz, dmso) δ 9.09 (s, 1H), 8.80 (s, 1H), 8.68 (d,J = 0.8 Hz, 1H), 8.22 (d, J = 7.4 Hz, 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 = 8.9 Hz, 1H), 6.92(dt, J = 16.5, 8.3 Hz, 3H), 6.17 - 6.06 (m, 1H), 5.17 (dd, J = 6.0, 4.3 Hz,1H), 4.90 (d, J = 8.8 Hz, 1H), 4.79 (d, J = 8.9 Hz, 1H), 4.41 (s, 1H), 4.27 (dd, J = 9.6, 6.0 Hz, 1H), 4.22 - 4.06 (m, 3H), 4.03 - 3.82 (m, 4H), 3.67 (dd,J = 7.9, 2.9 Hz, 2H), 3.52 (s, 2H), 2.88 (s, 3H), 2.31- 2.29 (m, 1H), 2.19 -2.12 (m, 1H). 31 P NMR(162 MHz, dmso) δ -9.64 - -10.54 (m), -12.89 - -13.79(m), -22.30 - -23.25 (m). MGI450c-V1-dTTP LCMS: C 49 H 52 N 15 O 27 P3S2[MH] - Calculated value: 1438.18. Measured value, m / z, [(M-2) / 2] - : 719.24. MGI450c-V1-dGTP LCMS: C 51 H 53 N 16 O 27 P3S2[MH] - Calculated value: 1477.19. Measured value, m / z, [(M-2) / 2]- : 738.15. 1 H NMR(400 MHz, dmso) δ 9.07 (s, 1H), 8.82 (s, 1H), 8.65 (s,1H), 8.35 - 8.25 (m, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.80 (s, 3H), 7.51 (d, J =8.0 Hz, 1H), 7.43 - 7.36 (m, 1H), 7.25 (d, J = 8.1 Hz, 1H), 7.19 (s, 1H), 7.12(d, J = 8.2 Hz, 1H), 6.97 (d, J = 9.3 Hz, 1H), 6.89 (d, J = 9.3 Hz, 1H), 6.54(s, 1H), 6.20 (dd, J = 8.8, 5.8 Hz, 1H), 5.21 - 5.14 (m, 1H), 4.87 (dd, J =25.6, 8.8 Hz, 2H), 4.53 (d, J = 4.0 Hz, 1H), 4.27 (dd, J = 10.3, 4.4 Hz, 1H), 4.20 (dd, J = 10.1, 5.8 Hz, 1H), 4.12 (d, J = 5.1 Hz, 1H), 4.06 (d, J = 4.7 Hz,1H), 3.98 - 3.83 (m, 4H), 3.68 (d, J = 8.8 Hz, 2H), 3.49 (s, 2H), 2.73 (d, J =7.0 Hz, 1H), 2.64 - 2.56 (m, 1H), 2.30 - 2.23 (m, 1H). 31 P NMR (162 MHz, dmso) δ -10.50 (d, J = 20.0 Hz), -11.96 --12.59 (m), -22.86 (dd, J = 24.2, 20.9 Hz).

[0159] Example 17 Synthesis of MGI450b modified ヌクレオチドの (1) Synthesis of MGI450b-V1

change

[0160] (2) Synthesis of MGI450b-V1-dATP [ka] In a 15 mL sample vial, MGI450b-V1 (10 mg, 11.92 μmol) was dissolved in anhydrous N,N-dimethylformamide (1 mL), and then N,N'-disuccinimidyl carbonate (6 mg, 23.42 μmol) and 4-dimethylaminopyridine (3 mg, 24.56 μmol) were added, followed by magnetic stirring at 25°C for 2 hours. Next, dATP (14 mg, 23.40 μmol) and triethylamine (6 mg, 59.29 μmol) were added, followed by further magnetic stirring at 25°C for 4 hours. The reaction solution was filtered, and the filtrate was first purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The prepared solution was concentrated under reduced pressure to obtain the crude product. Next, the crude product was purified by preparative liquid chromatography (0.1 M TEAB / acetonitrile), the prepared solution was concentrated under reduced pressure, and lyophilized to obtain MGI450b-V1-dATP. LCMS: C 50 H 53 N 16 O 24 P3S2[MH] - Calculated value: 1417.21. Measured value, m / z, [(M-2) / 2] - : 708.36. 1 H NMR(400 MHz, dmso) δ 8.97 (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 = 1.6 Hz, 1H), 7.50(t, J = 8.3 Hz, 2H), 7.42 - 7.35 (m, 1H), 7.30 - 7.24 (m, 1H), 7.16 - 7.05 (m,2H), 6.98 (d, J = 9.4 Hz, 1H), 6.65 (d, J = 4.7 Hz, 1H), 6.49 (dd, J = 8.7, 5.8Hz, 1H), 5.18 - 5.13 (m, 1H), 4.92 (d, J = 8.8 Hz, 1H), 4.85 (d, J = 8.9 Hz,1H), 4.56 (d, J = 5.1 Hz, 1H), 4.25 (dd, J = 10.6, 4.4 Hz, 1H), 4.22 - 4.11 (m,2H), 3.98 (s, 1H), 3.96 - 3.82 (m, 3H), 3.68 (dd, J = 6.5, 4.2 Hz, 2H), 3.51(s, 4H), 2.95 (s, 10H), 2.70 - 2.58 (m, 1H), 2.39 (dd, J = 14.0, 5.9 Hz, 1H). 31 P NMR (162 MHz, dmso) δ -10.28 (d, J = 18.7 Hz), -13.08 (d, J= 25.2 Hz), -22.76 (dd, J = 25.0, 18.9 Hz). The following compounds can be synthesized by the same method as described above: MGI450b-V1-dCTP LCMS: C 48 H 52 N 15 O 25 P3S2[MH] - Calculated value: 1394.19. Measured value, m / z, [(M-2) / 2] - : 696.72. 1 H NMR(400 MHz, dmso) δ 9.10 (s, 1H), 8.96 (s, 1H), 8.78 (d,J = 23.3 Hz, 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, 4.0 Hz, 1H), 6.90 (d, J = 8.3Hz, 1H), 6.12 (d, J = 7.4 Hz, 1H), 5.20 (d, J = 19.1 Hz, 1H), 4.94 - 4.75 (m,2H), 4.43 (s, 1H), 4.19 (dd, J = 49.6, 15.5 Hz, 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). 31 P NMR(162 MHz, dmso) δ -10.09 - -10.84 (m), -12.05 - -13.06(m), -22.42 - -23.22 (m). MGI450b-V1-dTTP LCMS: C 48 H 51 N 14 O 26 P3S2[MH] - Calculated value: 1395.18. Measured value, m / z, [(M-2) / 2] - : 697.42. 1H NMR(400 MHz, dmso) δ 9.14 (s, 1H), 9.01 (s, 1H), 8.96 -8.89 (m, 1H), 8.15 (dd, J = 8.5, 5.9 Hz, 2H), 7.96 (s, 3H), 7.61 (s, 1H), 7.49(t, J = 9.0 Hz, 2H), 7.38 (t, J = 7.9 Hz, 1H), 7.15 - 7.06 (m, 2H), 7.02 - 6.98(m, 1H), 6.10 (t, J = 6.8 Hz, 1H), 5.22 - 5.14 (m, 1H), 4.91 - 4.85 (m, 1H),4.80 (d, J = 8.9 Hz, 1H), 4.47 (s, 1H), 4.26 (ddd, J = 15.7, 10.7, 4.6 Hz, 2H),4.11 - 4.00 (m, 4H), 3.97 (d, J = 2.4 Hz, 1H), 3.92 - 3.83 (m, 4H), 3.67 (s,8H), 2.31 (dd, J = 11.1, 6.8 Hz, 2H). 31 P NMR (162 MHz, dmso) δ -10.84 (d, J = 21.3 Hz), -12.60 (d, J= 23.6 Hz), -23.29 (t, J = 22.4 Hz). MGI450b-V1-dGTP LCMS: C 50 H 53 N 16 O 25 P3S2[MH] - Calculated value: 1433.20. Measured value, m / z, [(M-2) / 2] - : 716.34. 1H NMR(400 MHz, dmso) δ 10.46 (s, 1H), 8.96 (s, 1H), 8.79 (s,1H), 8.29 (s, 1H), 8.13 (d, J = 8.2 Hz, 1H), 7.96 (s, 3H), 7.53 - 7.46 (m, 2H),7.41 - 7.35 (m, 1H), 7.19 (s, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.09 (d, J = 9.4Hz, 1H), 6.98 (d, J = 9.4 Hz, 1H), 6.39 (s, 1H), 6.24 - 6.18 (m, 1H), 5.19 -5.14 (m, 1H), 4.87 (dd, J = 26.2, 8.8 Hz, 2H), 4.52 (s, 1H), 4.27 (dd, J =10.6, 4.0 Hz, 1H), 4.21 - 4.15 (m, 1H), 4.09 (dd, J = 15.8, 5.3 Hz, 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). 31 P NMR(162 MHz, dmso) δ -10.18 (d, J = 18.8 Hz), -13.20 (dd, J = 15.3, 13.5 Hz), -22.61 - -22.73 (m).

[0161] Example 18 Synthesis of MGI443-modified nucleotide The following compounds were synthesized according to the method described in Example 17: MGI443-V1 LCMS: C 35 H 32 F2N8O 13 S2[MH] - Calculated value: 873.15. Measured value, m / z, [MH] - : 873.30. MGI443-V1-dATP LCMS: C50 H 51 F2N 16 O 24 P3S2[M-H] - Calculated value: 1453.19. Measured value, m / z, [(M-2) / 2] - : 726.60. 1 H NMR(400 MHz, dmso) δ 9.01 (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 = 1.6 Hz,1H), 7.54 (dd, J = 15.5, 8.1 Hz, 2H), 7.42 - 7.35 (m, 1H), 7.12 (d, J = 7.7 Hz,1H), 6.95 (d, J = 11.6 Hz, 1H), 6.49 (dd, J = 8.8, 5.9 Hz, 1H), 5.15 (t, J =4.8 Hz, 1H), 4.89 (dd, J = 25.8, 8.9 Hz, 2H), 4.57 (d, J = 4.9 Hz, 1H), 4.25(dd, J = 10.5, 4.5 Hz, 1H), 4.20 - 4.12 (m, 2H), 4.01 - 3.83 (m, 4H), 3.68 (dd,J = 6.7, 4.1 Hz, 2H), 2.94 (d, J = 6.0 Hz, 10H), 2.69 - 2.59 (m, 1H), 2.39 (dd,J = 13.8, 6.2 Hz, 1H). 31 P NMR(162 MHz, dmso) δ -10.49 (d, J = 19.5 Hz), -12.86 (d, J= 24.3 Hz), -22.97 (dd, J = 24.9, 19.7 Hz). MGI443-V1-dCTP LCMS: C 48 H 50 F2N 15 O 25 P3S2[M-H] -Calculated value: 1430.17. Measured value, m / z, [(M-2) / 2] - 714.95. 1 H NMR(400 MHz, dmso) δ 8.97 (s, 1H), 8.79 (s, 1H), 8.65 -8.58 (m, 1H), 8.15 (d, J = 8.0 Hz, 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, 1.7 Hz, 1H), 6.96 (d, J = 11.6Hz, 1H), 6.88 (s, 1H), 6.76 (d, J = 4.0 Hz, 1H), 6.14 - 6.09 (m, 1H), 5.19 -5.13 (m, 1H), 4.84 (dd, J = 40.5, 8.8 Hz, 2H), 4.44 - 4.38 (m, 1H), 4.22 (ddd,J = 15.4, 10.4, 4.6 Hz, 2H), 4.12 (d, J = 4.9 Hz, 1H), 4.04 - 3.83 (m, 4H),3.72 - 3.63 (m, 2H), 3.51 (s, 2H), 3.04 (d, J = 6.9 Hz, 6H), 2.91 (d, J = 6.9Hz, 2H), 2.31 (ddd, J = 8.1, 6.3, 2.6 Hz, 1H), 2.19 - 2.13 (m, 1H). 31 P NMR (162 MHz, dmso) δ -10.38 (d, J = 18.7 Hz), -13.29 (d, J= 25.4 Hz), -22.94 (dd, J = 25.0, 18.7 Hz). MGI443-V1-dTTP LCMS: C 48 H 49 F2N 14 O 26 P3S2[MH] - Calculated value: 1431.16. Measured value, m / z, [(M-2) / 2] -: 715.23. 1 1H NMR (400 MHz, dmso) δ 8.98 (s, 1H), 8.82 (s, 1H), 8.77 - 8.70 (m, 1H), 8.16 (d, J = 8.2 Hz, 1H), 8.08 (s, 1H), 7.97 - 7.77 (m, 3H), 7.56 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.41 - 7.36 (m, 1H), 7.14 (d, J = 8.2 Hz, 1H), 6.96 (d, J = 11.7 Hz, 1H), 6.13 - 6.07 (m, 1H), 5.20 - 5.15 (m, 1H), 4.90 (d, J = 8.9 Hz, 1H), 4.80 (d, J = 8.9 Hz, 1H), 4.44 (d, J = 2.5 Hz, 1H), 4.27 (dd, J = 10.6, 4.1 Hz, 1H), 4.19 (dd, J = 10.6, 5.2 Hz, 1H), 4.13 - 4.04 (m, 2H), 4.02 - 3.83 (m, 4H), 3.67 (dd, J = 7.0, 5.4 Hz, 2H), 3.51 (s, 2H), 2.97 (dd, J = 45.2, 6.5 Hz, 8H), 2.34 - 2.28 (m, 1H). 31 31P NMR (162 MHz, dmso) δ -10.35 (d, J = 18.5 Hz), -13.41 (d, J = 25.1 Hz), -22.70 - -23.05 (m). MGI443-V1-dGTP LCMS: C 50 H 51 F2N 16 O 25 P3S2[M-H] - Calculated value: 1469.18. Measured value, m / z, [(M - 2) / 2] - : 734.27. 1H NMR(400 MHz, dmso) δ 10.45 (s, 1H), 9.00 (s, 1H), 8.80 (d,J = 4.2 Hz, 1H), 8.26 (dd, J = 12.0, 6.5 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 7.97 - 7.67 (m, 3H), 7.56 - 7.45 (m, 2H), 7.35 (dd, J = 13.7, 5.7 Hz, 1H), 7.17(d, J = 3.4 Hz, 1H), 7.13 - 7.04 (m, 1H), 6.93 (d, J = 11.6 Hz, 1H), 6.37 (s,2H), 6.19 (dd, J = 8.9, 5.8 Hz, 1H), 5.16 - 5.10 (m, 1H), 4.84 (dd, J = 23.9,8.8 Hz, 2H), 4.50 (d, J = 4.8 Hz, 1H), 4.25 (dd, J = 10.4, 4.4 Hz, 1H), 4.16 (dd, J = 8.7, 4.9 Hz, 1H), 4.12 - 4.04 (m, 2H), 3.94 - 3.83 (m, 4H), 3.66 (dd,J = 8.4, 3.6 Hz, 2H), 3.16 (d, J = 5.4 Hz, 2H), 2.93 (d, J = 5.5 Hz, 10H), 2.65- 2.53 (m, 1H), 2.33 - 2.21 (m, 1H). 31 P NMR (162 MHz, dmso) δ -10.59 (t, J = 21.7 Hz), -12.93 (d, J= 25.7 Hz), -22.85 - -23.20 (m).

[0162] Example 19 Synthesis of MGI488 modified ヌクレオチドの The following compounds are synthesized according to the method described in Example 17: MGI488-V1 LCMS: C 44 H 51 N9O 14 S2[MH] -Calculated value: 992.30. Measured value, m / z, [MH] - : 992.49. MGI488-V1-dATP LCMS: C 59 H 70 N 17 O 25 P3S2[MH] - Calculated value: 1572.34. Measured value, m / z, [(M-2) / 2] - : 785.91. 1 H NMR (400 MHz, dmso) δ 8.95 - 8.87 (m, 1H), 8.60 (s, 1H), 8.45 (dd, J = 15.6, 11.0 Hz, 2H), 8.09 (s, 1H), 8.03 (s, 1H), 7.67 (dd, J =10.7, 5.9 Hz, 2H), 7.59 (dd, J = 14.4, 8.6 Hz, 1H), 7.44 (d, J = 9.0 Hz, 2H),7.35 (dd, J = 13.9, 6.4 Hz, 1H), 7.17 (d, J = 9.4 Hz, 1H), 7.09 (dd, J = 16.1,9.0 Hz, 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 = 5.1 Hz, 1H), 4.24 (dd, J = 10.0, 4.6 Hz, 1H), 4.18 - 4.11(m, 2H), 3.98 (s, 1H), 3.95 - 3. 84 (m, 2H), 3.69 (d, J = 4.2 Hz, 1H), 3.49 (s,2H), 3.17 (s, 3H), 2.97 (s, 8H), 2.62 (d, J = 8.8 Hz, 1H), 2.39 (dd, J = 12.7,6.3 Hz, 1H), 2.04 - 1.97 (m, 1H), 1.66 (dd, J = 22.4, 12.5 Hz, 2H), 1.28 - 1.14(m, 6H). 31 P NMR (162 MHz, dmso) δ -10.05 (d, J = 18.2 Hz), -13.38 (dd,J = 25.9, 2.8 Hz), -22.38 - -22.61 (m). MGI488-V1-dCTP LCMS: C 57 H 69 N 16 O 26 P3S2[MH] - Calculated value: 1549.32. Measured value, m / z, [(M-2) / 2] - 774.30. 1 H NMR(400 MHz, dmso) δ 8.93 - 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, 5.8 Hz, 3H), 7.17 (d, J = 9.4 Hz, 1H), 7.13(d, J = 7.8 Hz, 1H), 7.08 (d, J = 9.6 Hz, 1H), 6.89 (s, 1H), 6.15 - 6.10 (m,1H), 5.16 (d, J = 4.2 Hz, 1H), 4.91 (d, J = 8.8 Hz, 1H), 4.80 (d, J = 8.8 Hz,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 = 7.4 Hz, 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, 5.4 Hz, 3H), 1.16 (s, 3H). 31P NMR(162 MHz, dmso) δ -10.15 (d, J = 18.6 Hz), -13.31 (d, J= 24.6 Hz), -22.68 (dd, J = 23.5, 18.8 Hz).

[0163] Example 20 Synthesis of MGI447-modified nucleotides (1) Synthesis of MGI447-V1-dATP [ka] In a 4 mL sample vial, MGI447 (5 mg, 9.66 μmol) was dissolved in anhydrous N,N-dimethylformamide (1 mL), then N,N'-disuccinimidyl carbonate (5 mg, 19.52 μmol) and 4-dimethylaminopyridine (2 mg, 16.37 μmol) were added, and the mixture was subsequently magnetically stirred at 25°C for 2 hours. Next, V1-dATP (18 mg, 18.99 μmol) and triethylamine (5 mg, 49.41 μmol) were added, and the mixture was further magnetically stirred at 25°C for 4 hours. The reaction solution was filtered, and the filtrate was first purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The prepared solution was concentrated under reduced pressure to obtain the crude product. Next, the crude product was purified by preparative liquid chromatography (0.1 M TEAB / acetonitrile), the prepared solution was concentrated under reduced pressure, and lyophilized to obtain MGI447-V1-dATP. LCMS: C 52 H 57 N 16 O 24 P3S2[MH] - Calculated value: 1445.24. Measured value, m / z, [(M-2) / 2] - : 722.32. 1H NMR(400 MHz, dmso) δ 9.02 (s, 1H), 8.82 (s, 1H), 8.45 -8.38 (m, 1H), 8.15 (d, J = 8.2 Hz, 1H), 8.09 (s, 1H), 7.71 (d, J = 1.7 Hz, 1H),7.50 (dd, J = 16.2, 8.2 Hz, 2H), 7.42 - 7.35 (m, 1H), 7.20 (s, 1H), 7.12 (d, J= 9.0 Hz, 1H), 6.97 (s, 1H), 6.65 (s, 1H), 6.49 (dd, J = 8.8, 5.8 Hz, 1H), 5.35- 5.30 (m, 1H), 5.16 (t, J = 4.8 Hz, 1H), 4.89 (dd, J = 26.3, 8.9 Hz, 2H), 4.56(s, 1H), 4.26 (dd, J = 1 0.4, 4.4 Hz, 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 = 6.1 Hz, 10H), 2.63 (dd, J = 18.3,9.6 Hz, 1H), 2.39 (dd, J = 12.5, 6.1 Hz, 1H), 2.12 (s, 3H), 2.03 - 1.95 (m,1H), 1.23 (s, 3H). 31 P NMR (162 MHz, dmso) δ -10.49 (d, J = 19.2 Hz), -12.96 (d, J= 24.9 Hz), -22.98 (dd, J = 25.1, 19.5 Hz). The following compounds can be synthesized by the same method as described above: MGI447-V1-dCTP LCMS: C 50 H 55 N 14 O 26 P3S2[MH] - Calculated value: 1423.21. Measured value, m / z, [(M-2) / 2] - : 711.21. 1 1H NMR (400 MHz, DMSO) δ 9.03 (s, 1H), 8.86 (s, 1H), 8.71 - 8.62 (m, 1H), 8.15 (d, J = 8.1 Hz, 1H), 8.02 (s, 1H), 7.72 (s, 1H), 7.54 (d, J = 12.9 Hz, 1H), 7.50 - 7.46 (m, 1H), 7.42 - 7.35 (m, 1H), 7.14 (d, J = 6.5 Hz, 1H), 6.97 (s, 1H), 6.88 (s, 1H), 6.16 - 6.08 (m, 1H), 5.17 (t, J = 4.6 Hz, 1H), 4.89 (d, J = 8.8 Hz, 1H), 4.80 (d, J = 8.9 Hz, 1H), 4.40 (s, 1H), 4.27 (dd, J = 10.6, 4.0 Hz, 1H), 4.19 (dd, J = 10.7, 5.0 Hz, 1H), 4.11 (d, J = 4.8 Hz, 1H), 3.91 (dt, J = 11.6, 8.1 Hz, 4H), 3.67 (d, J = 9.0 Hz, 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). 31 31P NMR (162 MHz, DMSO) δ -10.33 (d, J = 19.1 Hz), -13.20 (d, J = 25.7 Hz), -22.83 (dd, J = 23.8, 18.4 Hz). MGI447-V1-dTTP LCMS: C 50 H 56 N 15 O 25 P3S2 [M - H] - Calculated value: 1422.22. Measured value, m / z, [(M - 2) / 2] - : 711.18. 11H NMR (400 MHz, dmso) δ 9.03 (s, 1H), 8.88 (s, 1H), 8.79 (s, 1H), 8.15 (d, J = 8.2 Hz, 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 = 8.2 Hz, 1H), 6.97 (s, 1H), 6.14 - 6.07 (m, 1H), 5.18 (d, J = 4.3 Hz, 1H), 4.90 (d, J = 8.9 Hz, 1H), 4.80 (d, J = 8.9 Hz, 1H), 4.45 (s, 1H), 4.31 - 4.26 (m, 1H), 4.20 (dd, J = 10.5, 5.4 Hz, 1H), 4.09 (d, J = 18.0 Hz, 2H), 4.01 - 3.82 (m, 4H), 3.67 (s, 2H), 3.52 (s, 2H), 2.94 (s, 10H), 2.30 (d, J = 8.0 Hz, 1H), 2.12 (s, 3H), 2.00 (d, J = 8.0 Hz, 1H), 1.23 (s, 3H). 31 31P NMR (162 MHz, dmso) δ -10.30 (d, J = 18.7 Hz), -13.32 (d, J = 24.8 Hz), -22.78 (dd, J = 24.8, 18.8 Hz). MGI447-V1-dGTP LCMS: C 52 H 57 N 16 O 25 P3S2[M-H] - Calculated value: 1461.23. Measured value, m / z, [(M - 2) / 2] - : 730.29. 1 1H NMR (400 MHz, dmso) δ 10.47 (s, 1H), 9.01 (s, 1H), 8.82 (d, J = 4.4 Hz, 1H), 8.33 - 8.25 (m, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.50 (dd, J =13.9, 7.3 Hz, 2H), 7.38 (t, J = 8.1 Hz, 1H), 7.19 (s, 1H), 7.13 (d, J = 6.7 Hz,1H), 6.97 (s, 1H), 6.65 (s, 1H), 6.39 (s, 1H), 6.21 (dd, J = 9.1, 5.6 Hz, 1H),5.17 (t, J = 4.8 Hz, 1H), 4.87 (dd, J = 25.1, 8.8 Hz, 2H), 4.52 (d, J = 5.2 Hz,1H), 4.27 (dd, J = 10.4, 4.4 Hz, 1H), 4.19 (dd, J = 9.6, 5.8 Hz, 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). 31 P NMR(162 MHz, dmso) δ -10.38 (d, J = 18.9 Hz), -13.06 (d, J= 26.4 Hz), -22.90 (dd, J = 25.2, 18.8 Hz).

[0164] Example 21: Synthesis of MGI456-modified nucleotides The following compounds were synthesized according to the method described in Example 20: MGI456-V1-dATP LCMS: C 54 H 55 F6N 16 O 24 P3S2[MH] - Calculated value: 1581.21. Measured value, m / z, [(M-2) / 2] - : 790.71. 1H NMR(400 MHz, dmso) δ 9.41 (t, J = 6.6 Hz, 1H), 9.07 (s,1H), 8.86 (s, 1H), 8.45 - 8.36 (m, 1H), 8.17 (d, J = 8.1 Hz, 1H), 8.09 (s, 1H),7.71 (d, J = 2.3 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.53 (d, J = 8.5 Hz, 1H),7.39 (dd, J = 17.0, 8.6 Hz, 2H), 7.28 (d, J = 9.6 Hz, 1H), 7.11 (d, J = 8.5 Hz,1H), 6.49 (dd, J = 8.5, 5.7 Hz, 1H), 5.16 (t, J = 4.8 Hz, 1H), 4.88 (dd, J =26.5, 8.9 Hz, 2H), 4.57 (d, J = 6.0 Hz, 1H), 4.53 - 4.42 (m, 2H), 4.26 (dd, J =10.4, 4.7 Hz, 1H), 4.21 - 4.11 (m, 2H), 4.00 - 3.80 (m, 4H), 3.68 (dd, J = 6.6,4.4 Hz, 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, 6.6 Hz, 1H). 19 F NMR(376 MHz, dmso) δ -70.45 (t, J = 9.4 Hz). 31 P NMR(162 MHz, dmso) δ -10.48 (d, J = 19.5 Hz), -12.83 (d, J= 25.1 Hz), -22.90 (dd, J = 25.0, 19.5 Hz). MGI456-V1-dCTP LCMS: C 52 H 53 F6N 14 O 26 P3S2[M-H] -Calculated value: 1559.18. Measured value, m / z, [(M - 2) / 2] - : 778.96. 1 H NMR(400 MHz, dmso) δ 9.43 - 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 = 8.0 Hz, 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 = 9.5 Hz, 1H), 7.15 (d, J = 13.7 Hz, 1H), 6.87 - 6.83 (m, 1H), 6.65 - 6.59 (m, 1H), 6.12 - 6.07 (m, 1H), 5.30 (t, J = 4.0 Hz, 1H), 5.17 - 5.13 (m, 1H), 4.87 (d, J = 8.8 Hz, 1H), 4.78 (d, J = 8.8 Hz, 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). 19 F NMR(376 MHz, dmso) δ -70.45 (t, J = 9.4 Hz). 31 P NMR(162 MHz, dmso) δ -9.67 - -10.31 (m), -13.02 - -13.53 (m), -22.26 - -22.81 (m). MGI456-V1-dTTP LCMS: C 52 H 54 F6N 15 O 25 P3S2[M - H][[ID=2,6]] -Calculated value: 1558.20. Measured value, m / z, [(M-2) / 2] - 779.04. 1 H NMR(400 MHz, dmso) δ 9.40-9.36 (m, 1H), 9.03 - 8.94 (m,1H), 8.85 - 8.79 (m, 1H), 8.77 - 8.70 (m, 1H), 8.14 (d, J = 8.4 Hz, 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 = 9.1 Hz, 1H), 4.77(d, J = 9.1 Hz, 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). 19 F NMR (376 MHz, dmso) δ -70.45 (t, J = 9.3 Hz). 31 P NMR(162 MHz, dmso) δ -9.82 - -10.46 (m), -12.89 - -13.69(m), -22.07 - -22.74 (m). MGI456-V1-dGTP LCMS: C 54 H 55 F6N 16 O 25 P3S2[MH] - Calculated value: 1597.21. Measured value, m / z, [(M-2) / 2] - : 798.71. 1H NMR(400 MHz, dmso) δ 10.48 (s, 1H), 9.40 (t, J = 6.9 Hz,1H), 9.04 (s, 1H), 8.85 (s, 1H), 8.33 - 8.23 ​​(m, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 6.4 Hz, 1H), 7.39 (dd, J = 18.0, 9.0 Hz,2H), 7.28 (d, J = 9.6 Hz, 1H), 7.19 (s, 1H), 7.13 (d, J = 7.4 Hz, 1H), 6.40 (s,1H), 6.21 (dd, J = 9.2, 5.7 Hz, 1H), 5.17 (t, J = 4.7 Hz, 1H), 4.87 (dd, J =24.2, 8.8 Hz (2H), 4.55 - 4.43 (m, 2H), 4.27 (dd, J = 10.4, 4.0 Hz, 1H), 4.19 (dd, J = 10.2, 4.5 Hz, 1H), 4.14 - 4.03 (m, 2H), 4.00 - 3.79 (m, 4H), 3.68 (dd,J = 8.5, 3.4 Hz, 2H), 3.52 (s, 2H), 2.93 (s, 10H), 2.66 - 2.56 (m, 1H), 2.28 (dd, J = 13.1, 6.3 Hz, 1H), 2.04 - 1.94 (m, 1H). 19 F NMR (376 MHz, dmso) δ -70.45 (t, J = 9.4 Hz). 31 P NMR (162 MHz, dmso) δ -10.48 (d, J = 19.0 Hz), -12.88 (d, J= 26.0 Hz), -22.76 - -23.21 (m).

[0165] Example 22 Synthesis of MGI485 modified ヌクレオチドの The following compounds are synthesized according to the method described in Example 20: MGI485-V1-dATP LCMS: C 62 H 73 N 16 O 24 P3S2[M-H] - Calculated value: 1581.36. Measured value, m / z, [(M-2) / 2] - : 790.38. 1 H NMR(400 MHz, dmso) δ 9.29 (s, 1H), 8.98 (s, 1H), 8.78 (s,1H), 8.46 - 8.39 (m, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.09 (s, 1H), 7.70 (s, 1H),7.51 (d, J = 8.2 Hz, 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, 8.8 Hz, 2H), 4.55 (s, 1H), 4.25 (dd, J = 10.3, 4.2 Hz, 1H),4.16 (dd, J = 18.4, 5.3 Hz, 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 = 8.0 Hz, 1H), 1.52 - 1.12 (m, 12H), 1.12 - 0.93 (m, 6H). 31 P NMR(162 MHz, dmso) δ -10.07 (d, J = 18.0 Hz), -13.31 (d, J= 26.4 Hz), -22.57 (dd, J = 25.7, 18.1 Hz). MGI485-V1-dCTP LCMS: C 60 H 71 N 14O 26 P3S2[MH] - Calculated value: 1559.33. Measured value, m / z, [(M-2) / 2] - 778.84. 1 H NMR(400 MHz, dmso) δ 9.27 (s, 1H), 8.97 (s, 1H), 8.79 (s,1H), 8.62 (s, 1H), 8.14 (dd, J = 7.8, 4.8 Hz, 1H), 7.99 (s, 1H), 7.70 (s, 1H),7.52 - 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, 9.1 Hz, 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), 2.96 (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, 34.1 Hz, 12H), 1.07 - 0.67 (m, 6H). 31 P NMR (162 MHz, dmso) δ -9.96 (d, J = 18.0 Hz), -13.41 (d, J= 4.1 Hz), -22.31 (d, J = 2.8 Hz).

[0166] Example 23 Synthesis of MGI450d modified ヌクレオチドの

change

[0167] (2) Synthesis of MGI450d-1-V1-dATP [ka] In a 4 mL sample vial, MGI450d-1-V1 (10 mg, 9.95 μmol) was dissolved in anhydrous N,N-dimethylformamide (1 mL), then N,N'-disuccinimidyl carbonate (5 mg, 19.52 μmol) and 4-dimethylaminopyridine (2 mg, 16.37 μmol) were added, and the mixture was magnetically stirred at 25°C for 2 hours. Next, dATP (12 mg, 20.06 μmol) and triethylamine (5 mg, 49.41 μmol) were added, and the mixture was further magnetically stirred at 25°C for 4 hours. The reaction solution was filtered, and the filtrate was purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The prepared solution was concentrated under reduced pressure to obtain MGI450d-1-V1-dATP. LCMS: C 54 H 59 N16 O 29 P3S3[MH] - Calculated value: 1583.20. Measured value, m / z, [(M-2) / 2] - : 791.25. The following compounds were synthesized according to the same method as described above: MGI450d-1-V1-dTTP LCMS: C 52 H 57 N 14 O 31 P3S3[MH] - Calculated value: 1561.17. Measured value, m / z, [(M-2) / 2] - : 780.23.MGI450d-1-V1-dCTP LCMS: C 52 H 58 N 15 O 30 P3S3[MH] - Calculated value: 1560.18. Measured value, m / z, [(M-2) / 2] - : 779.82.MGI450d-1-V1-dGTP LCMS: C 54 H 59 N 16 O 30 P3S3[MH] - Calculated value: 1599.20. Measured value, m / z, [(M-2) / 2] - : 799.65.

[0168] (3) Synthesis of MGI450d-V1-dATP [ka] In a 15 mL sample vial, compound MGI450d-1-V1-dATP (20 mg) was dissolved in methanol (2 mL), then ammonia (2 mL) was added, and the mixture was magnetically stirred at 25°C for 15 hours. The reaction solution was concentrated under reduced pressure to obtain the crude product, which was purified by preparative HPLC (0.1 M TEAB / acetonitrile). The prepared solution was concentrated under reduced pressure and lyophilized to obtain compound MGI450d-V1-dATP. LCMS: C 54 H 59 N 17 O 28 P3S3[MH] - Calculated value: 1581.21. Measured value, m / z, [M / 2-H] - : 790.78. 1 H NMR(400 MHz, dmso) δ 9.06 (s, 1H), 8.70 (s, 1H), 8.42 (s,1H), 8.23 ​​(s, 1H), 8.08 (s, 1H), 8.01 (d, J = 8.5 Hz, 1H), 7.97 - 7.70 (m, 3H),7.68 (s, 1H), 7.46 (dd, J = 21.3, 10.6 Hz, 2H), 7.35 (s, 1H), 7.22 - 7.14 (m,1H), 7.09 (s, 1H), 6.97 (dd, J = 20.9, 9.3 Hz, 2H), 6.63 (s, 1H), 6.48 (d, J =5.7 Hz, 1H), 5.30 (d, J = 4.6 Hz, 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 = 5.1 Hz, 2H), 3.96 (t, J =22.4 Hz, 3H), 3.66 (s, 1H), 3.49 (s, 2H), 2.95 (d, J = 14.4 Hz, 6H), 2.63 -2.56 (m, 1H), 2.38 (s, 1H), 1.97 (t, J = 7.0 Hz, 1H), 1.21 (s, 3H). 31 P NMR(162 MHz, DMSO) δ 74.99 (s), -10.12 (d, J = 18.1 Hz), -13.35 (d, J = 26.3 Hz), -22.66 (dd, J = 26.2, 18.2 Hz).

[0169] The following compounds were synthesized according to the same method as described above: MGI450d-V1-dTTP LCMS: C 52 H 57 N 15 O 30 P3S3[MH] - Calculated value: 1559.18. Measured value, m / z, [M / 2-H] - : 779.47. MGI450d-V1-dCTP LCMS: C 52 H 58 N 16 O 29 P3S3[MH] - Calculated value: 1558.19. Measured value, m / z, [M / 2-H] - : 778.76. MGI450d-V1-dGTP LCMS: C 54 H 59 N 17 O 29 P3S3[MH] - Calculated value: 1598.21. Measured value, m / z, [M / 2-H] - : 798.49.

[0170] Example 24 Synthesis of MGI471-modified nucleotide (1) Synthesis of MGI471-V1-dATP [ka] In a 4 mL sample vial, compound AF532-V1-dATP (10 mg, 15.96 μmol) was dissolved in methanol (0.5 mL), then ammonia (0.5 mL) was added, and the mixture was magnetically stirred at 22°C for 15 hours. The reaction solution was concentrated under reduced pressure to obtain the crude product, which was purified by preparative HPLC (0.1% ammonia / acetonitrile). The prepared solution was concentrated under reduced pressure and lyophilized to obtain compound MGI471-V1-dATP. 1 H NMR(600 MHz, D2O): δ 7.92 (s, 1H), 7.73-7.68(m, 2H), 7.43 (d, J = 12.2 Hz, 1H), 7.04-6.93 (m, 4H), 6.87 (d, J = 13.7 Hz,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 = 7.3 Hz, 2H), 2.36-2.27 (m, 2H), 1.11(t, J = 7.3 Hz, 3H), 0.94-0.91 (m,6H), 0.79-0.76 (m, 6H), 0.66 - 0.62 (m, 6H). 31 P NMR(243 MHz, D2O) δ -10.77 (d, J = 19.2 Hz), -11.36 (d, J= 19.5 Hz), -23.13 (t, J = 19.4 Hz). LCMS: C 60 H 69 N 16 O 24 P3S2[MH] - Calculated value: 1553.33. Measured value, m / z, [M-2 / 2] - : 776.31.

[0171] (2) Synthesis of MGI471-V1-dGTP [ka] (2-1) Synthesis of MGI471-1-V1-dGTP [ka] In a 15 mL sample vial, compound V1-dGTP (40 mg, 41.51 μmol) was dissolved in anhydrous N,N-dimethylformamide (4 mL). Then, 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 added, and the mixture was magnetically stirred at 25 °C for 15 hours. The reaction solution was filtered, and the filtrate was 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-dGTP. LCMS: C 60 H 68 N 15 O 26 P3S2[MH] - Calculated value: 1570.31. Measured value, m / z, [MH] ] - 1570.24.

[0172] (2-2) Synthesis of MGI471-V1-dGTP [ka] In a 15 mL sample vial, compound MGI471-1-V1-dGTP (20 mg, 12.72 μmol) was dissolved in methanol (3 mL), then ammonia (3 mL) was added, and the mixture was magnetically stirred at 24 °C for 15 hours. The reaction solution was concentrated under reduced pressure to obtain the crude product, which was purified by preparative HPLC (0.1% ammonia / acetonitrile). The prepared solution was concentrated under reduced pressure and lyophilized to obtain compound MGI471-V1-dGTP. 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). 31 P NMR (243 MHz, D2O) δ -10.14, -11.41 (d, J = 20.2Hz), -22.93 (t, J = 19.4 Hz). LCMS: C 60 H 69 N 16 O 25 P3S2[MH] - Calculated value: 1569.33. Measured value, m / z, [M-2 / 2] - : 785.10.

[0173] (3) Synthesis of MGI471-V1-dCTP

change

change

[0174] (3-2) Synthesis of MGI471-V1-dCTP [ka] In a 15 mL sample vial, compound MGI471-1-V1-dCTP (20 mg, 13.04 μmol) was dissolved in methanol (3 mL), then ammonia (3 mL) was added, and the mixture was magnetically stirred at 24 °C for 15 hours. The reaction solution was concentrated under reduced pressure to obtain the crude product, which was purified by preparative HPLC (0.1% ammonia / acetonitrile). The prepared solution was concentrated under reduced pressure and lyophilized to obtain compound MGI471-1-V1-dCTP. 1H 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). 31 P 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). LCMS: C 58 H 68 N 15 O 25 P3S2[MH] - Calculated value: 1530.32. Measured value, m / z, [M-2 / 2] - 764.85.

[0175] (4) Synthesis of MGI471-V1-dTTP

change

[0176] (4-2) Synthesis of MGI471-V1-dTTP [ka] In a 15 mL sample vial, compound MGI471-1-V1-dTTP (20 mg, 13.04 μmol) was dissolved in methanol (3 mL), then ammonia (3 mL) was added, and the mixture was subsequently magnetically stirred at 24°C for 15 hours. The reaction solution was concentrated under reduced pressure to obtain a crude solution. The product was obtained, the crude product was purified by preparative HPLC (0.1% ammonia / acetonitrile), the prepared solution was concentrated under reduced pressure, and lyophilized to obtain compound MGI471-V1-dTTP. 1 H NMR(600 MHz, D2O): δ 7.88-7.81 (m, 2H),7.74-7.71 (m, 1H), 7.27 (d, J = 7.3 Hz, 2H), 7.23-7.14 (m, 2H), 7.01-6.95 (m,1H), 6.88 (d, J = 8.1 Hz, 1H), 6.73 (d, J = 3.1 Hz, 2H), 5.80 (dt, J = 28.9,7.1 Hz, 1H), 4.78 (d, J = 5.0 Hz, 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 = 7.3 Hz, 1H), 2.35-2.26 (m, 1H),2.13-2.05 (m, 1H), 1.12 (t, J = 7.3 Hz, 2H), 1.02 (d, J = 6.7 Hz, 6H),0.96-0.93 (m, 6H), 0.83-0.79 (m, 6H). 31 P NMR(243 MHz, D2O) δ -10.44, -11.54 (d, J = 19.1Hz), -22.87. LCMS: C 58 H 67 N 14 O 26 P3S2[MH] - Calculated value: 1531.30. Measured value, m / z, [M-2 / 2] - : 765.26.

[0177] Example 25 Synthesis of MGI471-V2 [ka] (1) Synthesis of tert-butyl(2-(trifluoroacetamido)ethyl)carbamate [ka] In a 100 mL flask, N-tert-butoxycarbonyl-1,2-ethanediamine (5 g, 31.21 mmol) was dissolved in tetrahydrofuran (30 mL). Ethyl trifluoroacetate (4.88 g, 34.33 mmol) was added under ice water bath conditions at 0-5°C. After the addition was complete, the reaction solution was heated to 24°C and magnetically stirred for 4 hours. The reaction solution was concentrated under reduced pressure to obtain tert-butyl(2-(trifluoroacetamido)ethyl)carbamate.

[0178] (2) Synthesis of N-trifluoroacetyl-1,2-ethylenediamine [ka] In a 100 mL flask, tert-butyl(2-(trifluoroacetamido)ethyl)carbamate (8 g, 31.21 mmol) was dissolved in formic acid (50 mL) and magnetically stirred at 24 °C for 15 hours. The reaction solution was concentrated under reduced pressure, then acetonitrile (50 mL x 2) was added, and subsequently concentrated to dryness under reduced pressure to obtain N-trifluoroacetyl-1,2-ethanediamine.

[0179] (3) Synthesis of MGI471-1-V1 [ka] In a 15 mL sample vial, AF532 NHS (100 mg, 138.17 μmol) was dissolved in anhydrous N,N-dimethylformamide (3 mL), and then V1 (102 mg, 277.66 μmol) and N,N-diisopropylethylamine (89 mg, 688.60 μmol) were added. The mixture was then magnetically stirred at 22 °C for 15 hours. The reaction solution was filtered, and the filtrate was 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. LCMS: C 45 H 49 N7O 14 S2[MH] -Calculated value: 974.28. Measured value, m / z, [MH] - : 974.34.

[0180] (4) Synthesis of MGI471-1-V2-1 [ka] In a 50 mL flask, MGI471-1-V1 (90 mg, 92.21 μmol) was dissolved in anhydrous N,N-dimethylformamide (5 mL), then N,N'-disuccinimidyl carbonate (36 mg, 140.53 μmol) and 4-dimethylaminopyridine (2 mg, 16.37 μmol) were added, and the mixture was magnetically stirred at 24°C for 3 hours. Next, N-trifluoroacetyl-1,2-ethanediamine (58 mg, 371.54 μmol) and N,N-diisopropylethylamine (60 mg, 464.23 μmol) were added, and the mixture was further magnetically stirred at 24°C for 15 hours. The reaction solution was filtered, and the filtrate was purified by flash preparative liquid chromatography (0.1 M TEAB / acetonitrile). The prepared solution was concentrated under reduced pressure to obtain MGI471-1-V2-1. LCMS: C 49 H 54 F3N9O 14 S2[MH] - Calculated value: 1112.32. Measured value, m / z, [MH] - : 1112.49.

[0181] (5) Synthesis of MGI471-V2 [ka] In a 15 mL sample vial, MGI471-1-V2-1 (60 mg, 53.85 μmol) was dissolved in methanol (5 mL), then ammonia (5 mL) was added, and the mixture was magnetically stirred at 25°C for 3 hours. The reaction solution was concentrated under reduced pressure to obtain the crude product, which was purified by preparative HPLC (0.1 M TEAB / acetonitrile). The prepared solution was concentrated under reduced pressure and lyophilized to obtain MGI471-V2. 1 H NMR(600 MHz, DMSO-d6): δ 14.34 (s, 1H),8.88-8.84 (m, 1H), 8.67 (t, J = 5.2 Hz, 1H), 8.14 - 8.11 (m, 2H), 7.91 (t, J =5.9 Hz, 1H), 7.87-7.82 (m, 2H), 7.51 - 7.47 (m, 4H), 7.39 (t, J = 7.9 Hz, 1H),7.14 - 7.11 (m, 1H), 6.77 (s, 2H), 5.10 (t, J = 5.0 Hz, 1H), 4.21 (dd, J =10.3, 4.4 Hz, 1H), 4.14 (dd, J = 10.4, 5.5 Hz, 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, 2.4 Hz, 2H), 2.94 - 2.89(m, 2H), 1.17 - 1.13 (m, 12H), 0.98 (d, J = 3.9 Hz, 6H). 13C NMR(151 MHz, DMSO-d6): δ 170.40, 166.69,166.61, 158.11, 152.32, 151.48, 143.66, 138.11, 138.02, 136.62, 135.48, 130.04,129.95, 128.22, 120.69, 120.64, 118.12, 116.85,113.47, 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. LCMS: C 47 H 56 N 10 O 12 S2[MH] - Calculated value: 1015.35. Measured value, m / z, [MH] - : 1015.51. The nuclear magnetic resonance (NMR) characterization spectra of some of the above compounds are shown in Figures 1 to 17.

[0182] Example 26: Testing of the photochemical properties of fluorescent dyes or modified nucleotides The photochemical properties of fluorescent dyes or modified nucleotides were tested, and the results are shown in Table 1.

[0183] [Table 1] TIFF2026517479000080.tif58149

[0184] The excitation and emission spectra of the above compound are shown in Figure 18.

[0185] Example 27: Biochemical Experiment Experimental objective: To verify the performance of modified nucleotides in a sequencer. Experimental protocol: Using dNTPs from the MGI471 series, two-color hot dNTP mixes were prepared according to the following criteria: double-labeled T bases (MGI471-dTTP & AF532-dTTP), AF532-dATP, MGI471-dCTP, and G non-luminescent type.

[0186] PE100+10 was tested on-machine using a modified DNBSEQ-200RS sequencer (equipped with an H-light channel modified for 465 nm excitation), and the results were compared with those of PE100+10 tested on-machine using an unmodified DNBSEQ-200RS sequencing kit.

[0187] Experimental results The PE100 off-machine report shows that while the total number of on-machine reads using MGI471 dNTPs is slightly lower than that using conventional two-color reagents, the data volume can exceed 650M, which is equally excellent.

[0188] For PE100, the Q30%, partition rate, and mapping rate are comparable between the two types of dNTPs. The average error rate (N) of the MGI471 dNTP monomer is slightly lower than that of conventional reagents.

[0189] Regarding runon / lag, the overall runon of the MGI471 dNTP monomer is slightly high, indicating the need for further improvement in monomer purity. The lag shows little difference from conventional reagents, demonstrating the superior polymerization efficiency of MGI471 dNTP.

[0190] The drawbacks 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, damage to the DNB is much greater than under conventional conditions, affecting the recovery of the second chain to some extent. Therefore, optimization of the monomer structure and sequencing reagents is needed to reduce photodamage.

[0191] Overall, MGI471 dNTPs can, to some extent, meet the requirements for gene sequencing applications.

[0192] Table 2 shows the performance of the MGI471 dNTP in the sequencer. Figure 19 shows the Q30 data for the MGI471 dNTP in the sequencer.

[0193] [Table 2]

[0194] Interpretation of important biochemical indicators: Q30(%): Percentage of bases in base call results where the estimated error rate is less than 0.001 (i.e., accuracy is greater than 99.9%).

[0195] Run-on / lag: Run-on can be understood as the proportion of copies of reactions that proceed in a DNB, while lag can be understood as the proportion of copies of reactions that are delayed in a DNB.

[0196] Total number of reads (M): The total number of reads contained in the fq file generated off-machine by Zebracall without splitting.

[0197] Partition Rate (%): By classifying and partitioning the barcode portion of the sequences being sequenced, the number of sequences corresponding to the barcode list is counted, and the percentage of this number relative to the total number of fastq sequences is the partition rate. The denominator for calculating the partition rate is the number of reads in all off-machine fastq files (i.e., reads that have been filtered out), and currently, the total number of reads is the numerator for calculating the partition rate.

[0198] Mapping rate (%): The ratio of the number of mapped reads to the total number of reads.

[0199] Average Error Rate N(%): Represents the average error rate of the remaining mismatch types after removing some of the mismatches caused by Call N.

[0200] The above embodiments are used merely to illustrate the technical solutions of the present invention and do not limit the invention. Any modifications or equivalent substitutions made to the technical solutions of the present invention shall all be covered within the scope of the invention without departing from the object and scope of the technical solutions of the present invention.

Claims

1. Equation (I) 【Chemistry 1】 (In the formula, R １ , R ２ , R ３ and R ４ H, C are either the same or different from each other. １ ~C ６ Alkyl and Halo-C １ ~C ６ Each alkyl group is independently selected, R ５ and R ６ are the same or different and are each independently selected from H, C １ ~C ６ alkyl, halogen and halo-C １ ~C ６ alkyl, respectively R ７ and R ８ They are the same or different, H, -COOH, -C(O)NH-(C １ ~C ６ Alkyl) and -C(O)NH ２ Each is independently selected from, R ７ and R ８ It is not H at the same time, Optional: -NR １ R ２ It, together with the benzene ring to which it is bonded, forms a benzo 5-6 member nitrogen-containing heterocyclic group, and the 5-6 member nitrogen-containing heterocyclic group is optionally C １ ~C ６ Alkyl, halogen, and halo-C １ ~C ６ Substituted with one or more groups selected from alkyl groups, Optional: -NR ３ R ４ It, together with the benzene ring to which it is bonded, forms a benzo 5-6 member nitrogen-containing heterocyclic group, and the 5-6 member nitrogen-containing heterocyclic group is optionally C １ ~C ６ Alkyl, halogen, and halo-C １ ~C ６ (Substituted with one or more groups selected from alkyl groups) A compound represented by, its ester, or its salt.

2. R １ , R ２ , R ３ , R ４ Each of them is H, or R １ , R ２ , R ３ and R ４ Each is C １ ~C ６ Alkyl (e.g., methyl, ethyl), or R １ , R ２ , R ３ and R ４ Each of them is Hello C １ ~C ６ Alkyl (e.g., trifluoromethyl, trifluoroethyl), or R １ and R ３ The same, and each is H; R ２ and R ４ They are the same, and each is Halo C １ ~C ６ Alkyl (e.g., trifluoromethyl, trifluoroethyl), The compound according to claim 1, its ester, or its salt.

3. R ５ and R ６ They are the same, Preferably, R ５ and R ６ Either H or R ５ and R ６ Each of these is a halogen (for example, F). The compound according to claim 1 or 2, its ester, or its salt.

4. R ７ A compound according to any one of claims 1 to 3, an ester thereof, or a salt thereof, wherein the compound is a carboxyl molecule.

5. R ８ A compound according to any one of claims 1 to 4, an ester thereof, or a salt thereof, wherein is H or carboxyl.

6. -NR １ R ２ However, together with the benzene ring to which it is bonded, it forms a benzo 5-6 member nitrogen-containing heterocyclic group, and the 5-6 member nitrogen-containing heterocyclic group is optionally substituted with one or more methyl groups. Preferably, -NR ３ R ４ However, together with the benzene ring to which it is bonded, it forms a benzo 5-6 member nitrogen-containing heterocyclic group, and the 5-6 member nitrogen-containing heterocyclic group is optionally substituted with one or more methyl groups. A compound, an ester thereof, or a salt thereof, according to any one of claims 1 to 5.

7. R ７ or R ８ The carboxyl is a cleavable linker, for example: 【Chemistry 2】 A compound, ester thereof, or salt thereof according to any one of claims 1 to 6, connected to a severable linker having the structure shown in [figure name].

8. The aforementioned compound is of formula (II) 【Transformation 3】 (In the formula, R ７ (This is as defined in claim 1 or 4.) Having a structure represented by, The aforementioned compound is of formula (III) 【Chemistry 4】 (In the formula, R １ , R ２ , R ３ , R ４ , R ５ , R ６ and R ７ (This is as defined in any one of claims 1 to 4 and 6.) Having a structure represented by, The aforementioned compound is of formula (IV) 【Transformation 5】 (In the formula, R １ , R ２ , R ３ , R ４ , R ５ and R ６ (This is as defined in any one of claims 1 to 3 and 6.) Having a structure represented by, A compound, an ester thereof, or a salt thereof, according to any one of claims 1 to 7.

9. The aforementioned compound is as follows: 【Transformation 6】 A compound, ester, or salt thereof according to any one of claims 1 to 8, having a structure selected from the above.

10. Equation (I') 【Transformation 7】 (wherein, R ａ , R ｂ , R ｃ and R ｄ are the same or different from each other, and are each independently selected from H, C １ - C ６ alkyl, halo - C １ - C ６ alkyl, - NR １’ R ２’ , hydroxy, hydroxy - substituted C １ - C ６ alkyl, and halogen; R １’ and R ２’ are the same or different from each other, and are each independently selected from H, C １ - C ６ alkyl and halo - C １ - C ６ alkyl, respectively, R ｅ and R ｆ They are the same or different, H, -COOH and -C(O)NR ３’ R ４’ Each is independently selected from, R ｅ and R ｆ It is not H at the same time, R ３’ and R ４’ H and C are either the same or different from each other. １ ~C ６ Each alkyl is independently selected, and the C １ ~C ６ Alkyl can be optionally carboxyl, -C(O)NH ２ Alternatively, it is substituted with a sulfonic acid group. Optional, R ａ ga-NR １’ R ２’ If so, -NR １’ R ２’ It, together with the benzene ring to which it is bonded, forms a benzo 5-6 member nitrogen-containing heterocyclic group, and the 5-6 member nitrogen-containing heterocyclic group is optionally C １ ~C ６ Alkyl, halogen, and halo-C １ ~C ６ Substituted with one or more groups selected from alkyl groups, Optional, R ｂ ga-NR １’ R ２’ If so, -NR １’ R ２’ It, together with the benzene ring to which it is bonded, forms a benzo 5-6 member nitrogen-containing heterocyclic group, and the 5-6 member nitrogen-containing heterocyclic group is optionally C １ ~C ６ Alkyl, halogen, and halo-C １ ~C ６ (Substituted with one or more groups selected from alkyl groups) A compound represented by, its ester, or its salt.

11. R ａ and R ｂ The same, preferably each is -NR １’ R ２’ or each is hydroxyl, and / or R ｃ and R ｄ They are the same, preferably each is H or each is a halogen, Preferably, R １’ and R ２’ These are H, Preferably, R １’ and R ２’ One of them is H, and the other is C １ ~C ６ Selected from alkyl (e.g., methyl, ethyl), The compound according to claim 10, its ester, or its salt.

12. R ｅ and R ｆ They differ, and each is H, -COOH, and -C(O)NR ３’ R ４’ Selected independently from, Preferably, R ３’ and R ４’ However, H and C are either the same or different from each other. １ ~C ６ Each alkyl is independently selected, and the C １ ~C ６ The alkyl group is optionally substituted with a carboxyl or sulfonic acid group. The compound according to claim 10 or 11, its ester, or its salt.

13. R ａ ga-NR １’ R ２’ and; -NR １’ R ２’ However, together with the benzene ring to which it is bonded, it forms a benzo 5-6 member nitrogen-containing heterocyclic group, and the 5-6 member nitrogen-containing heterocyclic group is optionally substituted with one or more methyl groups, and / or R ｂ ga-NR １’ R ２’ and; -NR １’ R ２’ However, together with the benzene ring to which it is bonded, it forms a benzo 5-6 member nitrogen-containing heterocyclic group, and the 5-6 member nitrogen-containing heterocyclic group is optionally substituted with one or more methyl groups. A compound, an ester thereof, or a salt thereof according to any one of claims 10 to 12.

14. The aforementioned compound is of formula (II'): 【Transformation 8】 (In the formula, R ｅ and R ｆ (This is as defined in claim 10 or 12.) A compound according to any one of claims 10 to 13, an ester thereof, or a salt thereof, having the structure represented by [the formula shown].

15. The aforementioned compound is of formula (III'): 【Chemistry 9】 (In the formula, R ａ , R ｅ and R ｆ (This is as defined in any one of claims 10 to 12.) A compound, ester, or salt thereof according to any one of claims 10 to 14, having the structure represented by [the formula shown].

16. The aforementioned compound is as follows: 【Chemistry 10】 A compound, ester, or salt thereof according to any one of claims 10 to 15, having a structure selected from the above.

17. Equation (i): 【Chemistry 11】 (wherein w is a carboxyl or ester group, and the dye is a compound or salt thereof as described in any one of claims 1 to 16.) A compound represented by the formula.

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

19. The salt of the above compound is a salt formed by a sulfonic acid group on the acridine ring, for example, A compound according to any one of claims 1 to 17, an ester thereof, or a salt thereof, which is a salt formed by a ruhonic acid group with an alkali metal ion, an alkaline earth metal ion, or an ammonium ion.

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

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

22. Formula (1) 【Chemistry 12】 (In the formula, the dye is a compound, an ester thereof, or a salt thereof, as described in any one of claims 1 to 16.) A labeled nucleotide according to claim 20 or 21, having a structure represented by the given name.

23. The following structure: 【Chemistry 13】 (In the formula, R ａ , R ｂ , R ｃ , R ｄ and R ｅ (This is as defined in any one of claims 10 to 13.) The labeled nucleotide according to claim 22, having the characteristics of the label.

24. The following structure: 【Chemistry 14】 (In the formula, R ａ , R ｂ , R ｃ , R ｄ and R ｅ (This is as defined in any one of claims 10 to 13.) A labeled nucleotide according to claim 22 or 23, having the characteristics of the label.

25. The following structure: 【Chemistry 15】 (In the formula, R ａ , R ｂ , R ｃ , R ｄ and R ｅ (This is as defined in any one of claims 10 to 13.) A labeled nucleotide according to any one of claims 22 to 24, having the characteristics of the nucleotide.

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

27. A sequencing method comprising the step of incorporating a labeled nucleotide according to any one of claims 20 to 26 into a sequencing assay, Preferably, the method further includes the step of detecting the labeled nucleotide, Preferably, the sequencing assay is performed using an automated sequencer, and the automated sequencer comprises two light sources operating at different wavelengths. Sequence determination method.

28. (a) the step of incorporating at least one labeled nucleotide according to any one of claims 20 to 26 into a polynucleotide, (b) A step of detecting the labeled nucleotide incorporated into the polynucleotide by detecting a fluorescent signal from a novel fluorescent dye bound to the modified nucleotide. The sequence determination method according to claim 27, including the method described in claim 27.

29. A kit comprising one or more nucleosides, wherein at least one nucleoside is a labeled nucleotide according to any one of claims 20 to 26, Preferably, the kit contains two or more labeled nucleotides. kit.

30. The use of a compound, an ester thereof, or a 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 assays, in vitro diagnostics, immunoassays, and molecular labeling, Preferably, the molecular label is used for cell imaging, tissue imaging, or in vivo biological imaging. use.

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

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

33. A method for preparing a compound, an ester thereof, or a salt thereof according to any one of claims 1 to 16, comprising the step of converting a xanthene structure to an acridine structure by subjecting it to a substitution reaction in the presence of ammonia and methanol.

34. The reaction pathway for synthesizing the compound of formula (I) is as follows: 【Chemistry 16】 (R １ ~R ７ (This is as defined in any one of claims 1 to 6.) The method according to claim 33.

35. The reaction pathway for synthesizing the compound of formula (I') is as follows: 【Chemistry 17】 (R ａ ~R ｆ (This is as defined in any one of claims 10 to 13.) The method according to claim 33.

36. A method for preparing labeled nucleotides, comprising one of method A, method B, or method C: Method A: [Chemistry 18] (In the formula, dye 2 is a compound according to any one of claims 1 to 16 having an acridine structure, an ester thereof, or a salt thereof, and dye 1 is a precursor of dye 2 and has a xanthene structure); Method B: 【Chemistry 19】 (In the formula, the dye is a compound, an ester thereof, or a salt thereof, as described in any one of claims 1 to 16); Method C: 【Chemistry 20】 (In the formula, the dye is a compound, an ester thereof, or a salt thereof, as described in any one of claims 1 to 16.) Method D: 【Chemistry 21】 (In the formula, dye 2 is a compound according to any one of claims 1 to 16 having an acridine structure, an ester thereof, or a salt thereof, and dye 1 is a precursor of dye 2 and has a xanthene structure.)

37. The aforementioned nucleotides are as follows: 【Chemistry 22】 The method according to claim 36, having a structure selected from the following.

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