Red-region dye, synthesis method therefor and use thereof in gene sequencing

Abstract

The present invention relates to the fields of organic chemistry, fluorescent dyes and gene sequencing. In particular, the present invention relates to a red-region dye compound and the use thereof as a fluorescent marker, and further relates to a method for preparing the compound, a nucleotide or oligonucleotide labeled with the compound, and a nucleic acid sequencing method.

Description

A red light region dye, a synthesis method thereof and application thereof in gene sequencing TECHNICAL FIELD

[0001] The present application relates to the fields of organic chemistry, fluorescent dyes and gene sequencing. In particular, the present application relates to a red light region dye compound and its use as a fluorescent marker, to a method for preparing said compound, to a nucleotide or oligonucleotide labeled with said compound, and to a method for sequencing nucleic acids. BACKGROUND

[0002] DNA sequencing technologies include the first generation DNA sequencing technology represented by Sanger sequencing and the second generation DNA sequencing technology represented by Illumina Hiseq2500, Roche 454, ABI Solid, BGISEQ-500, etc. Sanger sequencing has the characteristics of simple experimental operation, intuitive and accurate results, and short experimental period, and is widely used in the fields of clinical gene mutation detection and genotyping, etc. which have high time effectiveness requirements for detection results. However, the disadvantages of Sanger sequencing are small throughput and high cost, which limit its application in large-scale gene sequencing.

[0003] Compared with the first generation DNA sequencing technology, the second generation DNA sequencing technology has the characteristics of large sequencing throughput, low cost, high automation degree and single molecule sequencing. For example, the sequencing technology of Hiseq2500V2 can produce 10-200G base data in one experimental process, and the average sequencing cost of each base is less than 1 / 1000 of the sequencing cost of Sanger sequencing, and the obtained sequencing results can be directly processed and analyzed by computer. Therefore, the second generation DNA sequencing technology is very suitable for large-scale sequencing.

[0004] The second generation DNA sequencing technologies developed at present mainly involve sequencing by ligation (SBL) technology and sequencing by synthesis (SBS) technology. Typical examples of these sequencing technologies include the SOLiD sequencing method developed by Applied Biosystems, the cPAL (combinatorial probe anchor ligation) method independently developed by Complete Genomics, the cPAS (combinatorial probe anchor synthesis) method developed by Huada Gene, the Illumina sequencing method developed by Illumina and Solexa technology, etc. Among these sequencing methods, Illumina and Complate Genomics use the method of detecting optical signals, and in order to realize the identification and differentiation of 4 bases (A, T / U, C and G), 4 fluorescent dyes are usually used to label the 4 bases respectively.

[0005] The accuracy of gene sequencing has always been the focus of the sequencing industry. There are many factors that cause high error rate in gene sequencing. When using four-color fluorescent sequencing principle for sequencing, the signal crosstalk between the fluorescent dyes labeled on the bases is the main source of the increase in error rate, mainly manifested as the mutual reading error between the bases. In the prior art, some sequencing products use Cy5 to label G base and IF700 to label C base, which is more prone to crosstalk.

[0006] SUMMARY

[0007] To solve the above problems, the distance between the emission wavelengths of the fluorescent dyes can be widened. The present application moves the emission wavelength of the red light region dye in the long wave direction by tens of nanometers through molecular structure design. The application of such dye to gene sequencing can significantly reduce the fluorescent crosstalk between C and G bases, thereby reducing the error rate between them.

[0008] The present application provides the following invention:

[0009] Dye compound

[0010] In one aspect, the present application provides a compound as shown in formula (I), an ester thereof or a salt thereof,

[0011] wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 are the same or different, and each is independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, hydroxyl, halogen, nitro, sulfonic acid group, phosphoric acid group;

[0012] R 7 , R 8 , R 9 , R 10 , R 11 are the same or different, and each is independently selected from hydrogen, C1-C6 alkyl, phenyl, substituted phenyl, nitrogen-containing heterocyclic group;

[0013] R 12 , R 13 are the same or different, and each is independently selected from alkyl having 1-18 carbons, carboxyalkyl having 1-18 carbons, amine alkyl having 1-18 carbons, alkyl sulfonate having 1-18 carbons, polyethylene glycol substituted alkyl;

[0014] R 14 , R 15 , R 16 , R 17each the same or different and each independently selected from the group consisting of hydrogen, C1-C6alkyl, haloC1-C6alkyl, hydroxyl, halogen, nitro, sulfonic acid group, phosphoric acid group;

[0015] R 18 selected from the group consisting of hydrogen, alkyl of 1-18 carbons, carboxyalkyl of 1-18 carbons, aminealkyl of 1-18 carbons, alkylsulfonate of 1-18 carbons, polyethylene glycol substituted alkyl;

[0016] each m is independently selected from an integer between 0-5 (e.g., 0, 1, 2, 3, 4, 5).

[0017] In some embodiments, the C1-C6alkyl is C1-C4alkyl.

[0018] In some embodiments, the C1-C6alkyl is selected from the group consisting of methyl, ethyl, propyl, n-butyl, i-butyl, i-propyl, t-butyl, n-pentyl, n-hexyl.

[0019] In some embodiments, the halogen is selected from the group consisting of fluorine, chlorine, bromine, and iodine.

[0020] In some embodiments, the haloC1-C6alkyl is fluoroC1-C6alkyl, e.g., trifluoromethyl.

[0021] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 each the same or different and each independently selected from the group consisting of hydrogen, C1-C6alkyl, hydroxyl, halogen, sulfonic acid group, phosphoric acid group.

[0022] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 each the same or different and each independently selected from the group consisting of hydrogen, C1-C6alkyl, hydroxyl, halogen.

[0023] In some embodiments, R 2 is C1-C6alkyl.

[0024] In some embodiments, R 3 is hydrogen, hydroxyl, or halogen.

[0025] In some embodiments, R 4 is hydrogen or hydroxyl.

[0026] In some embodiments, R5 is hydrogen or halogen.

[0027] In some embodiments, R 6 is hydrogen.

[0028] In some embodiments, R 7 , R 8 , R 9 , R 10 , R 11 are each independently selected from hydrogen, substituted phenyl.

[0029] In some embodiments, R 7 , R 8 , R 9 , R 10 , R 11 are each hydrogen.

[0030] In some embodiments, R 9 is substituted phenyl.

[0031] In some embodiments, R 12 , R 13 are each independently selected from C1-C6 alkyl, sulfonic acid substituted C1-C6 alkyl. In some embodiments, R 12 , R 13 are each methyl.

[0032] In some embodiments, R 12 , R 13 one is methyl and the other is sulfonic acid substituted C1-C6 alkyl.

[0033] In some embodiments, R 14 , R 15 , R 16 , R 17 are each the same or different and are each independently selected from hydrogen, sulfonic acid group.

[0034] In some embodiments, R 15 is a sulfonic acid group.

[0035] In some embodiments, R 18 is a carboxyalkyl group having 1-18 carbons.

[0036] In some embodiments, R 18 is -(CH2) 3～6 -COOH.

[0037] In some embodiments, the substituted phenyl means a phenyl group substituted with one or more (e.g., 2, 3, 4, or 5) substituents each independently selected from the group consisting of hydrogen, C1-C6alkyl, haloC1-C6alkyl, hydroxy, halogen, nitro, C1-C6alkoxy, cyano, carboxy, said C1-C6alkyl optionally substituted with carboxy, cyano, hydroxy, or nitro.

[0038] In some embodiments, the substituted phenyl has the following structure:

[0039] In the present application, the term "nitrogen-containing heterocycle" means a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic ring structure whose ring atoms are composed of carbon atoms and at least one (e.g., 1, 2, or 3) nitrogen atom. The heterocycle group can be connected to the rest of the molecule through any one of the ring atoms if the valence requirement is met. In some embodiments, the nitrogen-containing heterocycle is a 5-6 membered nitrogen-containing heterocycle having 5 to 6 ring atoms, wherein at least one (e.g., 1, 2, or 3) ring atom is a nitrogen atom.

[0040] In the present application, the "alkyl" preferably has 1-18 carbon atoms.

[0041] In the present application, the "1-18" can be 1-3, 3-6, 6-9, 9-12, 12-15, or 15-18, exemplarily 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. In some embodiments, the compound has a structure as shown in formula (II):

[0042] wherein R 2 , R 3 , R 4 , R 5 , R 9 , R 10 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 are as defined above.

[0043] In some embodiments, the compound has a structure as shown in formula (III):

[0044] wherein R 2 , R 3 , R 4 , R5 9 10 12 13 15 18 as defined above.

[0045] In some embodiments, the compound has a structure selected from the group consisting of:

[0046] The compounds of the present application can be conjugated to nucleotides or oligonucleotides as fluorescent dyes. Thus, in some embodiments, the compounds of the present application are covalently attached to nucleotides or oligonucleotides via the carboxyl group in the chemical structure above. In some embodiments, the carboxyl group on the compound is linked to a cleavable linker, for example, a cleavable linker having the structure shown below:

[0047] As used herein, the term "salt" refers to, (i) salts of acidic functional groups (e.g., -COOH) present in the compounds provided herein with appropriate inorganic or organic cations (bases), and includes, but is not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, and the like; alkaline earth metal salts, such as calcium salts, magnesium salts, and the like; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, and the like; inorganic base salts, such as ammonium salts; organic base salts, such as tertiary octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, tris(hydroxymethyl)aminomethane salts, and (ii) salts of basic functional groups (e.g., -NH2) present in the compounds provided herein with appropriate inorganic or organic anions (acids), and includes, but is not limited to, hydrogen halide salts, such as hydrofluoride salts, hydrochloride salts, hydrobromide salts, hydroiodide salts, and the like; inorganic acid salts, such as nitrate salts, perchlorate salts, sulfate salts, phosphate salts, and the like; lower alkyl sulfonic acid salts, such as methanesulfonate salts, trifluoromethanesulfonate salts, ethanesulfonate salts, and the like; aryl sulfonic acid salts, such as benzenesulfonate salts, p-toluenesulfonate salts, and the like; organic acid salts, such as acetate salts, malate salts, fumarate salts, succinate salts, citrate salts, tartrate salts, oxalate salts, maleate salts, and the like; amino acid salts, such as glycine salts, trimethylglycine salts, arginine salts, ornithine salts, glutamic acid salts, aspartic acid salts, and the like.

[0048] The salt of the compound of the present application is preferably a salt formed by a sulfonic acid group on the indole ring, for example, a salt of the sulfonic acid group with an alkali metal ion, an alkaline earth metal ion, or an ammonium ion.

[0049] ​​​​​​As used herein, the term "ester" refers to an ester formed from a -COOH present in a compound provided herein and a suitable alcohol, or an ester formed from a -OH present in a compound provided herein and a suitable acid (e.g., a carboxylic acid or an oxygen-containing inorganic acid). Suitable ester groups include, but are not limited to, formate, acetate, propionate, butyrate, acrylate, ethylsuccinate, stearyl or palmitate. Esters can undergo hydrolysis in the presence of an acid or a base to form the corresponding acid or alcohol.

[0050] Esters of the compounds of the present application are preferably activated esters of carboxylic acid groups. As used herein, the term "activated ester" refers to a derivative of a carboxylic acid group that is capable of reacting with, for example, a compound comprising an amino group under mild conditions. Non-limiting examples of activated esters include, but are not limited to, p-nitrophenyl ester, pentafluorophenyl ester, and succinimidyl ester.

[0051] Labeled nucleotides

[0052] The dye compounds of the present application are suitable for attachment to a substrate moiety. The substrate moiety can be virtually any molecule or substance to which the fluorescent dyes described herein can be conjugated, and the dyes can be attached to the substrate by way of non-limiting examples, the substrate moiety can include a nucleoside, a nucleotide, a polynucleotide, a carbohydrate, a protein, an antibody, a ligand, a particle or solid surface, an organic and inorganic polymer, and combinations or assemblages thereof, such as chromosomes, nuclei, living cells, and the like. In certain instances, such labeled nucleotides are also referred to as "modified nucleotides."

[0053] A particularly useful application of the fluorescent dyes of the present application is for labeling biomolecules, such as nucleotides or oligonucleotides. Accordingly, in one aspect, the present application relates to a nucleotide or oligonucleotide labeled with a fluorescent compound of the present application.

[0054] Attachment to a biomolecule can be via a carboxyl group on the compound of the present application, for example, to a biomolecule through a cleavable linker group as described above.

[0055] In certain embodiments, the dye compounds can be covalently attached to an oligonucleotide or nucleotide via a nucleotide base. For example, a labeled nucleotide or oligonucleotide can have a label attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base through a linker moiety. A labeled nucleotide or oligonucleotide can also have a 3' OH blocking group covalently attached to the ribose or deoxyribose of the nucleotide.

[0056] Nucleosides and nucleotides can be labeled at a site on the sugar or the nucleobase. As understood by one of ordinary skill in the art, a "nucleotide" consists of a nitrogenous base, a sugar, and one or more phosphate groups. In RNA, the sugar is ribose and in DNA the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present in ribose. The nitrogenous base is a derivative of a purine or a pyrimidine. The purines are adenine (A) and guanine (G), and the pyrimidines are cytosine (C) and thymine (T) or in the context of RNA, uracil (U). The C-1 atom of the deoxyribose sugar is bonded to the N-1 of a pyrimidine or the N-9 of a purine. Nucleotides are also phosphates of nucleosides, where esterification occurs on the hydroxyl group attached to C-3 or C-5 of the sugar. Nucleotides are typically mono-, di-, or tri-phosphates.

[0057] A "nucleoside" is structurally similar to a nucleotide but lacks the phosphate moiety. An example of a nucleoside analog would be a nucleoside in which a label is attached to the base and there is no phosphate group attached to the sugar molecule.

[0058] While the bases are generally referred to as purines or pyrimidines, the skilled artisan will appreciate that derivatives and analogs are available that do not change the ability of the nucleotide or nucleoside to undergo Watson-Crick base pairing. By "derivative" or "analog" is meant a compound or molecule whose core structure is the same or very similar to that of the parent compound but which has a chemical modification or physical modification, such as, for example, a different or additional pendant group, which allows the derivatized nucleotide or nucleoside to be attached to another molecule. For example, the base can be a deazapurine. The derivative should be able to undergo Watson-Crick pairing. By "derivative" and "analog" is also meant a synthetic nucleotide derivative or nucleoside derivative having a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are discussed in, for example, Scheit, Nucleotide analogs (John Wiley & Son, 1980) and Uhlman et al. Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also include modified phosphodiester linkages, including phosphorothioate linkages, phosphorodithioate linkages, alkylphosphonate linkages, phosphoranilidate linkages, phosphoramidate linkages, and the like.

[0059] The dye can be attached to any position on the nucleotide base through a linker, provided that Watson-Crick base pairing can still occur. Particular nucleobase labeling sites include the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base. As described above, linker groups can be used to covalently attach a dye to a nucleoside or nucleotide.

[0060] In particular embodiments, the labeled nucleoside or nucleotide can be enzymatically incorporable and enzymatically extendable. Thus, the linker moiety can have sufficient length to link the nucleotide to the compound such that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by nucleic acid replicase enzymes. Thus, the linker can also comprise a spacer unit. For example, the spacer distances the nucleotide base from the cleavage site or label.

[0061] The nucleoside or nucleotide labeled with the dye compound of the present application can have the following structure:

[0062] where B is a nucleobase such as, for example, uracil, thymine, cytosine, adenine, guanine, and the like, and L is an optional (i.e., can or can not be present) linking group. R' can be H, mono-, di-, tri-phosphate, phosphorothioate, phosphate analog, -O- attached to a reactive phosphorous-containing group, or -O- protected by a blocking group. R" can be H, OH, phosphoramidite, or a 3'-OH blocking group, and R'" is H or OH.

[0063] In some embodiments, the linking group is a cleavable linking group, for example, a linking group having a structure selected from:

[0064] In some embodiments, the nucleotide modified or labeled with the dye compound of the present application is a deoxyribonucleotide triphosphate (dNTP), such as dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dTTP (deoxythymidine triphosphate), or dCTP (deoxycytidine triphosphate).

[0065] In some embodiments, the nucleotide modified or labeled with the dye compound of the present application is a nucleoside triphosphate (NTP), such as ATP (adenosine triphosphate), GTP (guanosine triphosphate), CTP (cytidine triphosphate), and UTP (uridine triphosphate).

[0066] The present application also relates to polynucleotides encompassing incorporation of the dye compound of the present application. Such polynucleotides can be DNA or RNA comprising deoxyribonucleotides or ribonucleotides, respectively, linked by phosphodiester bonds. The polynucleotides can comprise naturally occurring nucleotides, non-naturally occurring (or modified) nucleotides different from the labeled nucleotides described herein, or any combination thereof, provided that there is at least one nucleotide labeled with the dye compound according to the present application. The polynucleotides can also include non-natural backbone linkages and / or non-nucleotide chemical modifications. Chimeric structures comprising a mixture of ribonucleotides and deoxyribonucleotides comprising at least one labeled nucleotide are also contemplated.

[0067] Exemplary labeled nucleotides as described herein include, but are not limited to:

[0068] Dye-labeled dNTPs, for example:

[0069] wherein each L is independently absent or a linker, for example, a cleavable linker as described above, and each dNTP is independently selected from dATP, dGTP, dTTP, or dCTP.

[0070] In some embodiments, the dye-labeled dCTP can have a structure selected from:

[0071] In some embodiments, the compound 32 and compound 33 labeled dATP can have a structure selected from:

[0072] In some embodiments, the compound 32 and compound 33 labeled dTTP can have a structure selected from:

[0073] In some embodiments, the compound 32 and compound 33 labeled dGTP can have a structure selected from:

[0074] Methods of sequencing

[0075] Nucleotides (or nucleosides) comprising the fluorescent dyes of the present application can be used in any analytical method requiring detection of a fluorescent label attached to a nucleotide or nucleoside, whether in its own right or incorporated into a larger molecular structure or conjugate or associated with a larger molecular structure or conjugate. The present application provides a method of sequencing comprising incorporating a labeled nucleotide of the present application into a sequencing assay, in some embodiments, the method further comprises detecting the labeled nucleotide. In some embodiments, the sequencing assay is performed on an automated sequencing instrument, and wherein the automated sequencing instrument comprises two light sources operating at different wavelengths.

[0076] Certain embodiments of the present application relate to a method of sequencing, the method of sequencing comprising: (a) incorporating at least one labeled nucleotide as described herein into a polynucleotide; and (b) detecting the labeled nucleotide incorporated into the polynucleotide by detecting a fluorescent signal from the new fluorescent dye attached to the modified nucleotide.

[0077] In certain embodiments, in the synthesis step, at least one labeled nucleotide is incorporated into the polynucleotide by the action of a polymerase. However, other methods of incorporating labeled nucleotides into polynucleotides are not excluded, such as chemical oligonucleotide synthesis or ligation of labeled oligonucleotides to unlabeled oligonucleotides. Thus, the term "incorporation" of nucleotides into polynucleotides encompasses polynucleotide synthesis by chemical methods as well as enzymatic methods.

[0078] In particular non-limiting embodiments, modified nucleotides or nucleosides labeled with a fluorescent dye according to the present application can be used in methods of nucleic acid sequencing, resequencing, whole genome sequencing, scoring of single nucleotide polymorphisms, any other application involving detection of modified nucleotides or nucleosides when incorporated into a polynucleotide, or any other application requiring the use of polynucleotides labeled with modified nucleotides comprising a fluorescent dye of the present application.

[0079] In particular embodiments, the present application provides the use of modified nucleotides comprising a dye compound of the present application in polynucleotide "sequencing by synthesis" reactions. Sequencing by synthesis generally involves the sequential addition of one or more nucleotides or oligonucleotides to a growing polynucleotide chain in the 5' to 3' direction using a polymerase or ligase enzyme, so as to form an extended polynucleotide chain complementary to a template nucleic acid to be sequenced. The identity of the bases present in the added nucleotide(s) is determined in a detection step or "imaging" step. The identity of the added bases can be determined after each nucleotide incorporation step. The sequence of the template can then be inferred using the conventional Watson-Crick base pairing rules. The use of modified nucleotides labeled with a dye according to the present disclosure for determining the identity of single bases can be useful, for example, in the scoring of single nucleotide polymorphisms, and such single base extension reactions are within the scope of the present application.

[0080] In embodiments, the sequence of a template polynucleotide is determined by detecting incorporation of one or more nucleotides into a nascent strand complementary to a template polynucleotide to be sequenced via detection of a fluorescent label attached to the incorporated nucleotide. The nucleic acid template to be sequenced can be DNA or RNA, or even a hybrid molecule comprising both deoxy- and ribonucleotides. The nucleic acid template can comprise naturally occurring nucleotides and / or non-naturally occurring nucleotides and natural or non-natural backbone linkages, provided that these do not prevent replication of the template in the sequencing reaction.

[0081] While one application of the modified nucleotides of the present disclosure is in sequencing-by-synthesis reactions, the utility of such labeled nucleotides is not limited to such methods. In fact, nucleotides can be advantageously used in any sequencing method that requires detection of fluorescent labels attached to nucleotides incorporated into a polynucleotide.

[0082] In certain embodiments, the present application provides a method of determining the sequence of a target single-stranded polynucleotide comprising the steps of:

[0083] (a) providing a duplex, a nucleotide, a polymerase, and an excision reagent; the duplex comprising a growing nucleic acid strand and a nucleic acid molecule to be sequenced;

[0084] (b) performing a reaction cycle comprising steps (i), (ii), and (iii):

[0085] Step (i): using the polymerase to incorporate the nucleotide into the growing nucleic acid strand to form a nucleic acid intermediate comprising a blocking group and a detectable label;

[0086] Step (ii): detecting the detectable label on the nucleic acid intermediate;

[0087] Step (iii): using the excision reagent to remove the blocking group on the nucleic acid intermediate.

[0088] In certain embodiments, the reaction cycle further comprises step (iv): using the excision reagent to remove the detectable label on the nucleic acid intermediate.

[0089] The sequencing method of the present application can optionally employ a four-color fluorescent sequencing technique, a three-color fluorescent sequencing technique, or a two-color fluorescent sequencing technique. The four-color fluorescent sequencing technique includes using four fluorescent substances that can excite to produce different colors to label dATP, dGTP, dTTP, and dCTP, respectively. The three-color fluorescent sequencing technique includes using three fluorescent substances that can excite to produce different colors to label three of dATP, dGTP, dTTP, dCTP, respectively, and the remaining one nucleotide is not labeled. The two-color fluorescent sequencing technique includes using two fluorescent substances that can excite to produce different colors to label dATP, dGTP, dTTP, dCTP, wherein the two fluorescent substances are used to label the first nucleotide in a mixed manner (i.e., dual labeling, such as 50% of dATP labeled dye AF532 and the other 50% of dATP labeled dye Cy5), the second and third nucleotides are labeled with the two fluorescent substances, respectively, and the remaining one nucleotide is not labeled.

[0090] In the present invention, a nucleic acid can include a nucleotide or a nucleotide analog. A nucleotide typically contains a sugar, a nucleobase, and at least one phosphate group. Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include, for example, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), 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), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP). Nucleotide analogs comprising modified nucleobases can also be used in the methods described herein. Exemplary modified nucleobases that can be included in a polynucleotide, whether having a natural backbone or an analog 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-propynylcytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8-halo-adenine or guanine, 8-amino-adenine or guanine, 8-thioladenine or guanine, 8-thiolalkyladenine or guanine, 8-hydroxyladenine or guanine, 5-halogen substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, and the like. Certain nucleotide analogs, such as adenosine 5'-phosphorothioate, cannot be incorporated into a polynucleotide, as known in the art.

[0091] In the methods of the application, the nucleic acid molecules to be sequenced are not limited by their length. In certain preferred embodiments, the nucleic acid molecules to be sequenced can 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 length. In certain preferred embodiments, the nucleic acid molecules to be sequenced can 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 more than 2000 bp in length. In certain preferred embodiments, the nucleic acid molecules to be sequenced can be 10-1000 bp in length, to facilitate high-throughput sequencing.

[0092] In certain preferred embodiments, the nucleic acid molecules can be pre-processed prior to immobilization on the support. Such pre-processing includes, but is not limited to, fragmentation of the nucleic acid molecules, end-filling, addition of adapters, addition of tags, amplification of the nucleic acid molecules, isolation and purification of the nucleic acid molecules, and any combination thereof.

[0093] In certain embodiments, the solid support surface can bear reactive functional groups that react with complementary functional groups on the polynucleotide molecules to form covalent bonds, for example, in the same manner as used for attaching cDNA to microarrays, for example, see Smirnov et al. (2004), Genes, Chromosomes & Cancer, 40:72-77 and Beaucage (2001), Current Medicinal Chemistry, 8: 1213_1244, both of which are incorporated herein by reference. DNBs can also be effectively attached to hydrophobic surfaces, for example, clean glass surfaces bearing low concentrations of various reactive functional groups, for example, -OH groups. Attachment via covalent bonds formed between the reactive functional groups on the polynucleotide molecules and the surface is also referred to herein as "chemical attachment." In other embodiments, the polynucleotide molecules can be adsorbed to the surface. In this implementation, the polynucleotides are immobilized by non-specific interactions with the surface, or by non-covalent interactions such as hydrogen bonds, van der Waals forces, and the like.

[0094] In other embodiments, the nucleic acid library can be double-stranded nucleic acid fragments, immobilized on the surface of the solid support by ligation to oligonucleotides immobilized on the surface of the solid support, followed by rolling circle amplification to prepare the sequencing library.

[0095] Kit

[0096] In another aspect, the present application provides a kit comprising nucleosides and / or nucleotides labeled with a dye compound of the present application. In certain embodiments, the kit comprises one or more nucleotides, wherein at least one nucleotide is a nucleotide labeled with a dye compound of the present application. In certain embodiments, the kit can comprise two or more labeled nucleotides. The fluorescent dye compounds, labeled nucleotides, or kits of the present application can be used for sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, or protein binding assays. The use can be performed on an automated sequencing instrument. The sequencing instrument can include two lasers operating at different wavelengths.

[0097] In the case where the kit comprises a plurality of nucleotides labeled with a dye compound, particularly two nucleotides and four nucleotides, the different nucleotides can be labeled with the same or different dye compounds, or one nucleotide can be unlabeled with a dye compound. In the case where the different nucleotides are labeled with the same or different dye compounds, the kit is characterized in that the dye compound-labeled nucleotides can be distinguished by fluorescence spectra and algorithms. When two nucleotides labeled with fluorescent dye compounds are supplied in a kit, in certain embodiments, the spectrally distinguishable fluorescent dyes can be excited at the same wavelength (such as, for example, by the same laser). When four nucleotides labeled with fluorescent dye compounds are supplied in a kit, in certain embodiments, two of the spectrally distinguishable fluorescent dyes can both be excited at one wavelength, and the other two spectrally distinguishable dyes can both be excited at another wavelength.

[0098] The dye compounds of the present application can be excited at the maximum excitation wavelength of blue light (e.g., about 470 nm) and emit at a wavelength that falls in the cyan region (e.g., about 490 nm). Thus, in certain embodiments, the kits of the present application can comprise at least one fluorescent dye that can be excited at the maximum excitation wavelength of blue light (e.g., about 470 nm) and emits at a wavelength that falls outside the cyan region, in addition to two spectrally distinguishable dyes that are excited at another wavelength.

[0099] In certain embodiments, the kits of the present application can further comprise: a reagent for immobilizing (e.g., by covalent or non-covalent linkage) a nucleic acid molecule to be sequenced to a support; a primer for initiating nucleotide polymerization reactions; a polymerase for conducting nucleotide polymerization reactions; one or more buffer solutions; one or more wash solutions; or any combination thereof.

[0100] In certain embodiments, the kit of the present application can further comprise reagents and / or devices for extracting nucleic acid molecules from a sample. Methods for extracting nucleic acid molecules from a sample are well known in the art. Thus, various reagents and / or devices for extracting nucleic acid molecules can be provided in the kit of the present application as necessary, such as reagents for disrupting cells, reagents for precipitating DNA, reagents for washing DNA, reagents for dissolving DNA, reagents for precipitating RNA, reagents for washing RNA, reagents for dissolving RNA, reagents for removing proteins, reagents for removing DNA (e.g., when the nucleic acid molecule of interest is RNA), reagents for removing RNA (e.g., when the nucleic acid molecule of interest is DNA), and any combination thereof.

[0101] In certain embodiments, the kit of the present application further comprises reagents for pretreating nucleic acid molecules. The reagents for pretreating nucleic acid molecules in the kit of the present application are not additionally limited, and can be selected as necessary. The reagents for pretreating nucleic acid molecules include, for example, reagents for fragmenting nucleic acid molecules (e.g., DNase I), reagents for filling in the ends of nucleic acid molecules (e.g., DNA polymerases such as T4 DNA polymerase, Pfu DNA polymerase, Klenow DNA polymerase), linker molecules, tag molecules, reagents for ligating linker molecules to nucleic acid molecules of interest (e.g., ligases such as T4 DNA ligase), reagents for repairing the ends of nucleic acids (e.g., DNA polymerases that lack 3'-5' exonuclease activity but exhibit 5'-3' exonuclease activity), reagents for amplifying nucleic acid molecules (e.g., DNA polymerases, primers, dNTPs), reagents for isolating and purifying nucleic acid molecules (e.g., chromatography columns), and any combination thereof.

[0102] In certain embodiments, the kit of the present application further comprises a support for immobilizing nucleic acid molecules to be sequenced. In general, the support for immobilizing nucleic acid molecules to be sequenced is in a solid phase, so as to facilitate handling. Thus, in the present disclosure, the "support" is sometimes also referred to as a "solid support" or a "solid-phase support". However, it should be understood that the "support" referred to herein is not limited to a solid, but can also be a semi-solid (e.g., a gel).

[0103] As used herein, the terms "loading," "immobilizing," and "attaching" when used in reference to nucleic acids means attachment, either directly or indirectly, to a solid support, via covalent or non-covalent bonds. In certain embodiments of the present disclosure, the methods of the present invention comprise immobilizing nucleic acids on a solid support via covalent attachment. However, generally, all that is required is that the nucleic acid remains immobilized or attached to the solid support under conditions in which it is desired to use the solid support (e.g., in applications in which nucleic acid amplification and / or sequencing is desired). In certain embodiments, immobilizing a nucleic acid on a solid support can comprise immobilizing an oligonucleotide to be used as a capture primer or amplification primer on a solid support such that the 3' end is available for enzymatic extension and at least a portion of the primer sequence is capable of hybridizing to a complementary nucleic acid sequence; then hybridizing the nucleic acid to be immobilized to the oligonucleotide, in which case the immobilized oligonucleotide or polynucleotide can be in the 3'-5' orientation. In certain embodiments, immobilizing a nucleic acid on a solid support can comprise binding a nucleic acid binding protein to a solid support by way of an aminated modification, and capturing a nucleic acid molecule by the nucleic acid binding protein. Alternatively, loading can occur by other means than base-pairing hybridization, such as the covalent attachment described above. Non-limiting examples of ways in which nucleic acids can be attached to a solid support include nucleic acid hybridization, biotin streptavidin binding, thiol binding, photoactivated binding, covalent binding, antibody-antigen, physical confinement via hydrogels or other porous polymers, and the like. Various exemplary methods for immobilizing nucleic acids on a solid support 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 Applications US 5639603, US 5641658, US2010248991; International Patent Applications WO 2001062982, WO 2001012862, WO 2007111937, WO0006770, all of which are incorporated herein by reference in their entireties, specifically for all teachings related to the preparation of solid supports on which nucleic acids are immobilized.

[0104] In the present application, the support can be made of various suitable materials. Such materials include, for example: inorganics, 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, polyphenylacetylene cross-linked with divinylbenzene (see, e.g., Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamide, latex, dextran, rubber, silicon, plastic, natural sponge, metal plastic, cross-linked dextran (e.g., Sephadex TM ), agarose gel (Sepharose TM ), and other supports known to those skilled in the art.

[0105] In certain preferred embodiments, the support for immobilization of nucleic acid molecules to be sequenced can be a solid support comprising an inert substrate or matrix (e.g., a glass slide, a polymeric bead, etc.) that has been functionalized, e.g., by applying an intermediate material containing reactive groups that allow covalent attachment of biological molecules such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels loaded on an inert substrate such as glass, in particular the polyacrylamide hydrogels described in WO 2005 / 065814 and US 2008 / 0280773, the contents of which are incorporated herein by reference in their entirety. In such embodiments, the biological molecules (e.g., polynucleotides) can be covalently attached directly to the intermediate material (e.g., hydrogel), while the intermediate material itself can be non-covalently attached to the substrate or matrix (e.g., a glass substrate). In certain preferred embodiments, the support is a glass or silicon slide that is surface-modified with a layer of avidin, amino, acrylamide silane, or aldehyde groups.

[0106] In the present application, the support or solid support is not limited in size, shape, and configuration. In some embodiments, the support or solid support is a planar structure, e.g., a slide, a chip, a microchip, and / or an array. The surface of such a support can be in the form of a planar layer.

[0107] In certain preferred embodiments, the support for immobilization of nucleic acid molecules to be sequenced is an array of beads or wells (which is also referred to as a chip). 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 immobilization of nucleic acid molecules. The number of beads or wells on the array is not limited. For example, each array can comprise 10- 10 2 , 10 2-10 3 、10 3 -10 4 、10 4 -10 5 、10 5 -10 6 、10 6 -10 7 、10 7 -10 8 、10 8 -10 9 、10 10 -10 11 、10 11 -10 12 or more beads or wells. In certain exemplary embodiments, the surface of each bead or well can immobilize one or more nucleic acid molecules. Accordingly, each array can immobilize 10-10 2 、10 2 -10 3 、10 3 -10 4 、10 4 -10 5 、10 5 -10 6 、10 6 -10 7 、10 7 -10 8 、10 8 -10 9 、10 10 -10 11 、10 11 -10 12 or more nucleic acid molecules. Such arrays can thus be particularly advantageous for high-throughput sequencing of nucleic acid molecules.

[0108] In certain preferred embodiments, the kits of the application further comprise reagents for immobilizing (e.g., by covalent or non-covalent linkage) the nucleic acid molecule to be sequenced to a support. Such reagents include, for example, reagents that activate or modify the nucleic acid molecule (e.g., at its 5' end), such as a phosphate, a thiol, an amine, a carboxylic acid, or an aldehyde; reagents that activate or modify the surface of the support, such as an amino-alkoxysilane (e.g., aminopropyltrimethoxysilane, aminopropyltriethoxysilane, 4-aminobutyltriethoxysilane, etc.); a crosslinking agent, such as succinic anhydride, phenyl diisothiocyanate (Guo et al., 1994), maleic anhydride (Yang et al., 1998), l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), m-maleimidobenzoic acid-N-hydroxysuccinimide ester (MBS), N-succinimidyl [4-iodoacetyl]aminobenzoate (SIAB), 4-(N-maleimidomethyl)cyclohexane-l-carboxylate succinimidyl ester (SMCC), N-γ-maleimidobutyryloxy-succinimide ester (GMBS), 4-(p-maleimidophenyl)butyric acid succinimidyl ester (SMPB); and any combination thereof.

[0109] In certain preferred embodiments, the kits of the application further comprise a primer for initiating nucleotide polymerization. In the present application, the primer is not additionally limited as long as it is capable of annealing specifically to a region of the target nucleic acid molecule. In some exemplary embodiments, the primer can be 5-50 bp in length, such as 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50 bp. In some exemplary embodiments, the primer can comprise naturally-occurring or non-naturally-occurring nucleotides. In some exemplary embodiments, the primer comprises or consists of naturally-occurring nucleotides. In some exemplary embodiments, the primer comprises modified nucleotides, such as locked nucleic acids (LNAs). In certain preferred embodiments, the primer comprises a universal primer sequence.

[0110] In certain preferred embodiments, the kits of the application further comprise a polymerase for performing a nucleotide polymerization reaction. In the present application, various suitable polymerases can be used to perform a polymerization reaction. In some exemplary embodiments, the polymerase is capable of synthesizing a new DNA strand using DNA as a template (e.g., a DNA polymerase). In some exemplary embodiments, the polymerase is capable of synthesizing a new DNA strand using RNA as a template (e.g., a reverse transcriptase). In some exemplary embodiments, the polymerase is capable of synthesizing a new RNA strand using DNA or RNA as a template (e.g., an RNA polymerase). Thus, in certain preferred embodiments, the polymerase is selected from the group consisting of a DNA polymerase, an RNA polymerase, and a reverse transcriptase.

[0111] In certain preferred embodiments, the kits of the application further comprise one or more excision reagents. In certain embodiments, the excision reagent is selected from the group consisting of Endo IV and alkaline phosphatase.

[0112] In certain preferred embodiments, the kits of the application further comprise one or more buffer solutions. Such buffer solutions include, but are not limited to, a buffer solution for DNAse I, a buffer solution for DNA polymerase, a buffer solution for ligase, a buffer solution for eluting nucleic acid molecules, a buffer solution for dissolving nucleic acid molecules, a buffer solution for performing a nucleotide polymerization reaction (e.g., PCR), and a buffer solution for performing a ligation reaction. The kits of the application can comprise any one or more of the above-mentioned buffer solutions.

[0113] In certain embodiments, the buffer solution for DNA polymerase comprises monovalent salt ions (e.g., sodium ions, chloride ions) and / or divalent salt ions (e.g., magnesium ions, sulfate ions, manganese ions). In certain embodiments, the concentration of the monovalent salt ions or divalent salt ions in the buffer solution is 10 μΜ-200 mM, e.g., 10 μΜ, 50 μΜ, 100 μΜ, 200 μΜ, 500 μΜ, 1 mM, 3 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, or 200 mM.

[0114] In certain embodiments, the buffer solution for DNA polymerase comprises tris(hydroxymethyl)aminomethane (Tris). In certain embodiments, the concentration of Tris in the buffer solution is 10 mM-200 mM, e.g., 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, or 200 mM.

[0115] In certain embodiments, the buffer solution for DNA polymerase comprises an organic solvent, such as DMSO or glycerol (glycerin). In certain embodiments, the mass content of the organic solvent in the buffer solution is 0.01%-10%, such as 0.01%, 0.02%, 0.05%, 1%, 2%, 5%, or 10%.

[0116] In certain embodiments, the buffer solution for DNA polymerase has a pH of 7.0-9.0, such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0.

[0117] In certain embodiments, the buffer solution for DNA polymerase comprises monovalent salt ions (such as sodium ions, chloride ions), divalent salt ions (such as magnesium ions, sulfate ions, manganese ions), Tris, and an organic solvent (such as DMSO or glycerol). In certain embodiments, the pH of the buffer solution phase is 8.8.

[0118] In certain preferred embodiments, the kit of the present application further comprises one or more wash solutions. Examples of such wash solutions include, but are not limited to, phosphate buffer, citrate buffer, Tris-HCl buffer, acetate buffer, carbonate buffer, and the like. The kit of the present application can comprise any one or more of the above-mentioned wash solutions.

[0119] In certain preferred embodiments, the kit of the present application comprises sequencing reagents. In certain preferred embodiments, the sequencing reagents comprise at least one of: a dNTPs mix, a nucleic acid polymerase mix, an elution solution.

[0120] In certain preferred embodiments, the dNTPs mix contains at least one modified or labeled nucleotide of the present application.

[0121] In certain preferred embodiments, the elution solution contains a cleavage reagent; optionally, the cleavage reagent is selected from one or more of the following: Endo IV, alkaline phosphatase, an organic phosphine (such as tris(3-hydroxypropyl)phosphine (THPP), tris(2-carboxyethyl)phosphine hydrochloride (TCEP)), or a PdCl2complex with sulfonated triphenylphosphine.

[0122] In another aspect, the present application provides use of the fluorescent dye, labeled nucleotide or oligonucleotide, or kit of the present application for determining the sequence of a target polynucleotide.

[0123] The present application also provides the use of the fluorescent dye in sequencing, expression analysis, hybridization analysis, gene analysis, RNA analysis, protein binding assay, in vitro diagnosis, immunoassay, molecular marker field. In some embodiments, the molecular marker is used for cell imaging, tissue imaging or biological imaging.

[0124] The present application also provides the use of the fluorescent dye in the fluorescent labeling, quantification or detection of proteins, enzymes or nucleic acids.

[0125] Method for preparing dye compounds

[0126] The present application also provides a method for preparing the dye compounds of the present application, which comprises: subjecting compound A and compound B to a nucleophilic substitution reaction in the presence of acetic acid, acetic anhydride and a base (such as potassium carbonate). The reaction scheme of the method is shown as follows:

[0127] R 1 -R 18 As defined above. The reaction can be carried out at room temperature to 70°C.

[0128] Method for preparing labeled nucleotides

[0129] The present application provides a method for preparing the labeled nucleotides of the present application, which comprises: subjecting the dye compounds of the present application to a condensation reaction with nucleotides with a linking group to form an amide bond.

[0130] In some embodiments, the reaction scheme of the method is shown as follows:

[0131] Each n is independently selected from an integer between 0-5 (such as 0, 1, 2, 3, 4, 5).

[0132] The present application also provides another method for preparing the labeled nucleotides of the present application, which comprises: subjecting nucleotides to a condensation reaction with the dye compounds of the present application with a linking group to form an amide bond.

[0133] In some embodiments, the reaction scheme of the method is shown as follows:

[0134] Each n is independently selected from an integer between 0-5 (such as 0, 1, 2, 3, 4, 5). Advantages

[0135] The present application develops new dyes in the red light region and uses them to label bases, which helps to improve the sequencing accuracy in the field of gene sequencing.

[0136] The method for preparing the dye of the present application has the advantages of simple steps and mild conditions.

[0137] Embodiments of the present application will now be described in detail in connection with the accompanying drawings and examples, but it will be understood that the following examples and drawings are merely illustrative of the present application and should not be taken as limiting the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the following detailed description, taken in connection with the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0138] Figure 1 is a synthetic route for compound 32 and 32-dCTP.

[0139] Figure 2 is a synthetic route for compound 33 and 33-dCTP.

[0140] Figure 3 is the1H NMR spectrum of compound 33. 1

[0141] Figure 4 is the1H NMR spectrum of compound 33-dCTP. 1

[0142] Figure 5 is the19F NMR spectrum of compound 33-dCTP. 19

[0143] Figure 6 is the31P NMR spectrum of compound 33-dCTP. 31

[0144] Figure 7 shows the effect of compound 32-dCTP and 33-dCTP on sequencing Q30.

[0145] Figure 8 shows the effect of compound 33-dCTP on fluorescence crosstalk.

[0146] Figure 9 shows the fluorescence spectra of compound 32 and 32-dCTP. DETAILED DESCRIPTION

[0147] Embodiments of the present application will now be described in detail in connection with the accompanying drawings and examples, but it will be understood that the following examples and drawings are merely illustrative of the present application and should not be taken as limiting the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the following detailed description, taken in connection with the accompanying drawings and preferred embodiments.

[0148] As used herein, the following common organic abbreviations have the following meanings:

[0149] HBF4 tetrafluoroboric acid

[0150] °C degrees Celsius

[0151] dATP deoxyadenosine triphosphate

[0152] ​​​​dTTP deoxythymidine triphosphate

[0153] dCTP deoxycytidine triphosphate

[0154] dGTP deoxyguanosine triphosphate

[0155] DIPEA N’N-diisopropylethylamine acetonitrile

[0156] DMAP 4-dimethylaminopyridine

[0157] DMF N’N-dimethylformamide

[0158] DMSO dimethyl sulfoxide

[0159] DMSO-d6 deuterated dimethyl sulfoxide

[0160] DSC N,N’-disuccinimidyl carbonate

[0161] g gram

[0162] LCMS liquid chromatography mass spectrometry

[0163] mg milligram

[0164] mL milliliter

[0165] m / z mass to charge ratio

[0166] PBS phosphate buffered saline solution

[0167] TEAB triethylamine-bicarbonate buffer solution

[0168] THF tetrahydrofuran

[0169] DCM dichloromethane

[0170] LiHMDS lithium bis(trimethylsilyl)amide

[0171] Preparation of Compound 1 of Example 1

[0172] Take 100 mL three-necked flask, add anhydrous tetrahydrofuran (20 mL), ice water bath 0-5 ℃, sodium hydride (2.77 g, 60%, 69.36 mmol) is added in batches, then 2-methyl acetoacetate (10 g, 69.36 mmol) is added dropwise in tetrahydrofuran solution (20 mL), 0-5 ℃, electromagnetic stirring for 1 hour, a large amount of white solid is precipitated. Then add propyl sulfone lactone (8.47 g, 69.36 mmol) in tetrahydrofuran solution (20 mL), after adding, warm to 40 ℃, electromagnetic stirring for 4 hours. The reaction solution is concentrated under reduced pressure to obtain a crude product, water (50 mL) is added, extracted with ethyl acetate (50 mL*2), the aqueous phase is separated and directly used in the next step reaction. The aqueous solution of compound 1 is obtained. LCMS: calcd for C 10 H 18 O6S[M+H] + :267.08.Found,m / z,[M+H] + :267.23。

[0173] Preparation of compound 2 in example 2

[0174] Take 250 mL flask, add the aqueous solution of compound 1 (50 mL), then add concentrated hydrochloric acid (50 mL), warm to 120 ℃, electromagnetic stirring for 4 hours. The reaction solution is concentrated under reduced pressure to obtain a crude product, which is purified by column chromatography (dichloromethane / methanol=0:1-2:1) to obtain compound 2. LCMS: calcd for C 14 O4S[M-H] - :193.06.Found,m / z,[M-H] - :193.10。

[0175] Preparation of compound 3 in example 3

[0176] Take 100 mL flask, weigh compound 2 (5 g, 25.74 mmol), add acetic acid (25 mL), then add 4-sulfonic acid phenylhydrazine (4.48 g, 25.74 mmol), warm to 140 ℃, electromagnetic stirring for 4 hours. The reaction solution is concentrated under reduced pressure to obtain a crude product, the pH is adjusted to 9-10 with aqueous potassium hydroxide solution, filtered, and the filtrate is purified by fast preparation liquid chromatography (water / acetonitrile). The preparation solution is concentrated under reduced pressure to obtain compound 3. LCMS: calcd for C 13 H 17 NO6S2[M+H] + :348.05.Found,m / z,[M+H] + :348.06。

[0177] Preparation of compound 4 of Example 4

[0178] Into a 100 mL flask, compound 3 (3 g, 7.08 mmol) was weighed, and then cyclobutanesulfone (30 mL) was added, followed by 6-bromohexanoic acid (2.76 g, 14.16 mmol), potassium iodide (235 mg, 1.42 mmol), and the temperature was raised to 135 °C, and electromagnetic stirring was performed for 15 hours. The reaction solution was added to water (30 mL), filtered, and the filtrate was purified by flash preparative liquid chromatography (0.1% hydrochloric acid / acetonitrile), and the preparation solution was concentrated under reduced pressure to remove water and acetonitrile, and then ethyl acetate (50 mL) was added to make a slurry, filtered, and dried to obtain compound 4. LCMS: calcd for C 19 H 27 NO8S2[M+H] + : 462.12. Found, m / z, [M+H] + : 462.10.

[0179] Preparation of compound 5 of Example 5

[0180] Into a 40 mL sample bottle, compound 4 (600 mg, 1.30 mmol) was weighed, and then acetic anhydride (1.32 g, 12.97 mmol), acetic acid (5 mL), and acetonitrile (10 mL) were dissolved, followed by the addition of N,N-diisopropylethylamine (671 mg, 5.19 mmol), and malondialdehyde bisphenylimine monohydrochloride (336 g, 1.30 mmol), and electromagnetic stirring was performed at 20 °C for 4 hours. The reaction solution was filtered, purified by flash preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile), and the preparation solution was concentrated under reduced pressure, and then freeze-dried to obtain compound 5. LCMS: calcd for C 30 H 36 N2O9S2[M+H] + : 633.19. Found, m / z, [M+H] + : 633.18.

[0181] Preparation of compound 6 of Example 6

[0182] Into a 250 mL flask, weighed 4-sulfonic acid phenylhydrazine (12 g, 63.76 mmol), added acetic acid (30 mL), then added 3-methyl-2-butanone (16.48 g, 191.28 mmol), warmed to 130 °C, electromagnetic stirring for 4 hours. The reaction liquid was cooled to room temperature, slowly poured into ethyl acetate (500 mL), a large amount of solid was precipitated. Filtered, washed with ethyl acetate, dried to give compound 7. LCMS: calcd for C 15 H 13 BrN2[M]: 300.03, 302.02. Found, m / z, [M+H] + : 301.07, 303.07.

[0183] Preparation of compound 7 in example 7

[0184] Into a 250 mL flask, weighed 4-sulfonic acid phenylhydrazine (12 g, 63.76 mmol), added acetic acid (30 mL), then added 3-methyl-2-butanone (16.48 g, 191.28 mmol), warmed to 130 °C, electromagnetic stirring for 4 hours. The reaction liquid was cooled to room temperature, slowly poured into ethyl acetate (500 mL), a large amount of solid was precipitated. Filtered, washed with ethyl acetate, dried to give compound 7. LCMS: calcd for C 11 H 13 NO3S[M+H] + : 240.06. Found, m / z, [M+H] + : 240.09.

[0185] Preparation of compound 8 in example 8

[0186] Into a 250 mL flask, weighed compound 7 (14 g, 58.51 mmol), added methanol (20 mL) and ethanol (20 mL), then added potassium hydroxide (3.94 g, 70.21 mmol), warmed to 70 °C, electromagnetic stirring for half an hour. The reaction liquid was cooled to room temperature, filtered, washed with a small amount of ethanol, dried to give compound 8. LCMS: calcd for C 11 H 13 NO3S[M+H] + : 240.06. Found, m / z, [M+H] + : 240.09.

[0187] Preparation of compound 9 in example 9

[0188] Into a 100 mL flask, weigh compound 8 (6 g, 21.60 mmol), add cyclobutan sulfone (20 mL), then add 1,3-propane sultone (7.9 g, 64.70 mmol), heat to 110 °C, magnetically stir for 5 hours. Cool the reaction to room temperature, pour into ethyl acetate (100 mL), suction filter, dissolve the filter cake in water, purify by preparative liquid chromatography (0.1% trifluoroacetic acid in acetonitrile), concentrate the preparative solution under reduced pressure to remove water and acetonitrile, slurry with ethyl acetate (500 mL) and methanol (50 mL), filter, dry to give compound 9. LCMS: Calculated for C 14 H 20 NO6S2[M] + :362.07.Found, m / z, [M] + :362.11.

[0189] Preparation of compound 10 of example 10

[0190] Into a 100 mL flask, weigh compound 6 (1.59 g, 4.12 mmol), add acetonitrile (22.5 mL), then add acetic anhydride (2.55 g, 25.00 mmol) DIPEA (564 mg, 4.38 mmol), magnetically stir at room temperature for half an hour, then cool to 0 °C, dissolve compound 9 (0.9 g, 2.50 mmol) in a mixture of acetonitrile (4 mL) and acetic acid (2 mL), add dropwise to the solution of compound 6, magnetically stir at room temperature for 15 hours. Concentrate the reaction under reduced pressure to remove acetonitrile, add ethyl acetate (100 mL), suction filter the resulting precipitate, dissolve the filter cake in a mixture of water and acetonitrile, filter through a fritted funnel, purify the filtrate by reverse phase biotage autopurifier (welflash C18-I, regular C18 20-40 μm, 330 g, 0.1 M TEAB / acetonitrile, acetonitrile content 5-95%), concentrate the preparative solution under reduced pressure, freeze dry to give compound 10. LCMS: Calculated for C 25 H 27 BrN2O7S2[M]:610.04.Found, m / z, [M-Ac] + :569.16, 571.17.

[0191] Preparation of compound 11 of example 11

[0192] Into a 100 mL flask, compound 8 (5 g, 18.03 mmol) was weighed, and then cyclobutanesulfone (40 mL) and 6-bromohexanoic acid (7.03 g, 36.05 mmol) were added, followed by potassium iodide (300 mg, 1.81 mmol). The mixture was heated to 130 °C and magnetically stirred for 15 h. The reaction solution was poured into water (100 mL) and extracted with ethyl acetate (50 mL*2). The aqueous phase was separated and purified by flash preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile). The prepared solution was concentrated under reduced pressure to remove water and acetonitrile. Ethyl acetate (100 mL) was added to the slurry, filtered, and dried to obtain compound 11. LCMS: calcd for C 17 H 23 NO5S[M-H] - : 352.13. Found, m / z, [M-H] - : 352.20.

[0193] Preparation of compound 12 in Example 12

[0194] Into a 250 mL flask, compound 11 (2 g, 5.64 mmol) was weighed, and then acetic anhydride (5.76 g, 56.43 mmol), acetic acid (20 mL), and acetonitrile (40 mL) were added to dissolve the compound. Then N,N-diisopropylethylamine (2.92 g, 22.57 mmol) and propanedial bisphenylimine monohydrochloride (1.46 g, 5.64 mmol) were added. The mixture was magnetically stirred at 25 °C for 3 h. The reaction solution was poured into water (400 mL) to quench the reaction. The mixture was purified by flash preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile). The prepared solution was concentrated under reduced pressure to obtain compound 12. LCMS: calcd for C 28 H 32 N2O6S[M+H] + : 525.20. Found, m / z, [M+H] + : 525.33.

[0195] Preparation of compound 13 in Example 13

[0196] Into a 250 mL flask, weighed 2,4-dihydroxyacetophenone (10 g, 65.80 mmol), dissolved with pyridine (70 mL), added pivaloyl chloride (19.80 g, 164.50 mmol) under ice bath, after the addition, 25 °C, electromagnetic stirring for 15 hours. The reaction solution was poured into a mixture of ice 100 g and concentrated hydrochloric acid (60 mL), extracted with ethyl acetate (200 mL*3), the combined organic phase was washed with saturated brine (200 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 1:0~10:1) to give compound 13.

[0197] Example 14 Preparation of compound 14

[0198] Into a 250 mL flask, weighed compound 13 (6 g, 18.73 mmol), dissolved with anhydrous N’N-dimethylformamide (60 mL), ice water bath 0~5 °C, added sodium hydride (1.5 g, 60%, 37.45 mmol) in batches, after the addition, 0~5 °C, electromagnetic stirring for 10 minutes, then warmed to 25 °C, electromagnetic stirring for 2 hours. The reaction solution was poured into water (400 mL) to quench the reaction, added dilute hydrochloric acid to adjust pH=5-6, extracted with ethyl acetate (100 mL*3), the combined organic phase was washed with water (100 mL*3), concentrated under reduced pressure to remove ethyl acetate, then added methanol (60 mL), dissolved in 1,4-dioxane (60 mL), added concentrated hydrochloric acid (60 mL), warmed to 60 °C, electromagnetic stirring for 5 hours. The reaction solution was concentrated under reduced pressure to remove the organic solvent, and a significant amount of solid was precipitated. Filtration, washed with a small amount of water, dried to give compound 14.

[0199] Example 15 Preparation of compound 15

[0200] Into a 250 mL flask, weighed compound 14 (2 g, 9.16 mmol), dissolved with anhydrous tetrahydrofuran (100 mL), ice water bath 0~5 °C, added methyl magnesium bromide (12 mL, 3 mol / L in 2-methyltetrahydrofuran, 36.00 mmol), after the addition, warmed to 50 °C, electromagnetic stirring for 2 hours. The reaction solution was poured into water (100 mL) to quench the reaction, added tetrafluoroboric acid (4 mL, 50% aqueous solution), the reaction solution was concentrated under reduced pressure to remove the organic solvent, and a large amount of solid was precipitated. Filtration, washed with a small amount of water, dried to give compound 15.

[0201] Example 16 Preparation of compound 16

[0202] Into a 100 mL round-bottom flask, 4-fluoro-resorcinol (10 g, 78.0 mmol) was dissolved in boron trifluoride etherate (60 mL), and glacial acetic acid (8.9 mL, 156.13 mmol) was added. The reaction was stirred at 90 °C for 16 h under nitrogen protection. 10% sodium acetate aqueous solution (100 mL) was added, and the mixture was extracted with ethyl acetate (100 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 16.

[0203] Example 17 Preparation of compound 17

[0204] Into a 500 mL round-bottom flask, compound 16 (13 g, 76.41 mmol) was dissolved in pyridine (60 mL), and trimethylacetyl chloride (46 g, 382.04 mmol) was added dropwise by syringe. The reaction was stirred at 25 °C for 16 h. Saturated ammonium chloride aqueous solution was added to quench the reaction, and the mixture was extracted with ethyl acetate (100 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 1:0 to 20:1) to give compound 17.

[0205] Example 18 Preparation of compound 18

[0206] Into a 500 mL round-bottom flask, compound 17 (10 g, 0.13 mmol) was dissolved in DMF (100 mL), and the reaction was cooled to 0 °C in an ice bath. NaH (60%, 2.4 g, 103.44 mmol) was added portionwise, and the reaction was stirred at 25 °C for 16 h. Saturated ammonium chloride aqueous solution was added to quench the reaction, and the mixture was extracted with ethyl acetate (100 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. 10 mL of methanol and 5 mL of hydrochloric acid were added, and the reaction was stirred at 45 °C for 1.5 h. The crude product was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA = 1:0 to 2:1) to give compound 18. LCMS: calcd for C 13 H 13 FO3 Exat Mass: 236.1; Found, m / z, [M+H] + : 237.16.

[0207] Example 19 Preparation of compound 19

[0208] Take 100 mL brown reaction bottle, weighed compound 18 (1 g, 4.23 mmol), dissolved in anhydrous THF (15 mL), added CH3MgBr (3M, 7.05 mL, 21.16 mmol) by syringe dropwise, stirred at 25°C for 3h, then added HFB4 solution (50%, 4 mL), extracted with ethyl acetate (50 mL x 3), dried with anhydrous sodium sulfate, concentrated under reduced pressure to obtain compound 19. LCMS: calcd for C 14 H 16 BF5O2Excat Mass:322.1;Found,m / z,[M-BF4 - ] + :235.13.

[0209] Preparation of compound 20 of example 20

[0210] Take 250 mL flask, weighed 2,3-dihydroxyacetophenone (1.5 g, 9.86 mmol), dissolved in anhydrous pyridine (40 mL), cooled to 0°C by ice bath, slowly added pivaloyl chloride (4.85 ml, 39.44 mmol), stirred at room temperature for 15h. Added 50 ml saturated NH4Cl to quench the reaction, then removed the solvent in the reaction solution by reduced pressure concentration. Added 70 ml ethyl acetate to dissolve the product, washed with saturated brine (3 x 60 ml), separated the organic phase, added anhydrous magnesium sulfate for drying, filtered the solution, then concentrated under reduced pressure to obtain compound 20. LCMS: calcd for C 18 H 24 O5[M+NH4] + :338.17.Found,m / z,[M+NH4] + :338.21.

[0211] Preparation of compound 21 of example 21

[0212] Into a 100 mL flask, weighed compound 20 (1.3 g, 4.06 mmol) and dissolved in DMF (10 mL), cooled to 0 °C by ice bath, added sodium hydride (568 mg, 14.20 mmol, 60%) portion wise, stirred magnetically at room temperature for 15 h. Added 50 ml saturated NH4Cl to quench the reaction, extracted with ethyl acetate (3 x 60 ml), combined the organic phase, added anhydrous magnesium sulfate to dry, concentrated the solution under reduced pressure. Added methanol (2 ml) and hydrochloric acid solution (3 ml, 1 M), heated slowly from room temperature to 45 °C, stirred magnetically for 1.5 h, extracted with ethyl acetate (30 ml), washed with saturated brine (3 x 30 ml), separated the organic phase, added anhydrous magnesium sulfate to dry, filtered the solution, concentrated again under reduced pressure, obtained compound 21. LCMS: calcd for C 13 H 14 O3[M+1] + :219.09. Found, m / z, [M] + :219.14.

[0213] Preparation of compound 22 of example 22

[0214] Into a 100 mL flask, weighed compound 21 (600 mg, 2.75 mmol) and dissolved in THF (10 mL), cooled to 0 °C by ice bath, added methyl magnesium bromide (2.29 ml, 6.87 mmol, 3 M) drop wise slowly, stirred magnetically at room temperature for 15 h. Added HFB4 (10 ml, 50 wt. % in H2O) to quench the reaction, extracted with ethyl acetate (3 x 50 ml), combined the organic phase, added anhydrous magnesium sulfate to dry, concentrated the solution under reduced pressure. Purified by reverse phase biotage autopurifier (welflash C18-I, regular C18 20-40 μm, 330 g, 0.1 M TFA / acetonitrile, acetonitrile content 5-95 %), concentrated the solution under reduced pressure, freeze dried to obtain compound 22. LCMS: calcd for C 14 H 17 O2 + [M] + :217.12. Found, m / z, [M] + :217.29.

[0215] Preparation of compound 23 of example 23

[0216] Weigh 11.34 g (60.0 mmol) of 4-bromoresorcinol into a 100 mL round-bottom flask and dissolve it in 37 mL of boron trifluoride diethyl ether. Then add 7.2 g (120.0 mmol) of glacial acetic acid and stir at 90 °C for 16 h under nitrogen protection. After cooling the reaction solution, pour in 150 mL of 10% sodium acetate aqueous solution and stir at room temperature for 2 hours. Extract with ethyl acetate (200 mL x 2). Wash the organic phase with 150 mL of water, collect the organic phase, dry it with anhydrous sodium sulfate, and concentrate it under reduced pressure to obtain the crude product. Analyze the crude product by silica gel column chromatography (PE:EA = 5:1 to 3:1) to give 9.6 g of compound 23 as a pale brown solid.

[0217] Example 24 Preparation of Compound 24

[0218] Take a 250 mL round-bottom flask, weigh compound 23 (9.6 g, 41.74 mmol), dissolve it in pyridine (45 mL), and add trimethylacetyl chloride (12.5 g, 104.35 mmol) dropwise with a syringe. Stir the reaction mixture at 25 °C for 16 h. The next day, pour the reaction mixture into a mixture of 60 g ice and 40 mL concentrated hydrochloric acid, extract with ethyl acetate (150 mL x 2), collect the organic phase, dry it with anhydrous sodium sulfate, and concentrate it under reduced pressure to obtain the crude product. Spectrophotometry of the crude product on silica gel column (PE:EA = 30:1–15:1) yields 12 g of compound 24 as a yellow solid.

[0219] Example 25 Preparation of Compound 25

[0220] Take a 500 mL round-bottom flask, add NaH (60%, 2.12 g, 53 mmol), dissolve in THF (45 mL), and then cool to 0 °C. Weigh compound 24 (12 g, 30.15 mmol) and dissolve in THF (45 mL). Under nitrogen protection, slowly add the solution dropwise to the NaH solution and stir for 2 h. Disperse the reaction solution with 450 mL of aqueous acetic acid solution (containing 3.15 g acetic acid), extract with ethyl acetate (300 mL x 2), combine the organic phases, wash with 300 mL of saturated brine, dry with anhydrous sodium sulfate, and concentrate under reduced pressure. Add 210 mL of methanol and 105 mL of hydrochloric acid, heat to 60 °C, and stir for 4 h. Concentrate under reduced pressure to obtain the crude product. Slurry the crude product with 200 mL of petroleum ether and 10 mL of ethyl acetate to obtain 2.89 g of compound 25 as a powdery white solid. LCMS: Calculated for C 13 H 13 BrO3Exact Mass:296.0;Found,m / z,[M+H] + :297.04,299.06.

[0221] Example 26 Preparation of Compound 26

[0222] Into a 100 mL three-necked flask, compound 25 (1.8 g, 6 mmol) was dissolved in anhydrous THF (60 mL) under nitrogen atmosphere. CH3MgBr (3 M, 8 mL, 24 mmol) was added dropwise by syringe at 0 °C under ice-bath. The mixture was stirred at 50 °C for 2.5 h. After cooling, HFB4 solution (50%, 10 mL) was added. The mixture was extracted with dichloromethane (300 mL x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 26 as a green solid, 1.96 g. LCMS: Calcd for C 14 H 16 Exact Mass: 382.04; Found, m / z, [M-BF4 - ] + : 295.03, 297.06.

[0223] Preparation of compound 27

[0224] Into a 500 mL three-necked flask, 1,3-dimethoxy-2,4-difluorobenzene (10 g, 57.42 mmol) was dissolved in DCM (10 mL), and then BBr3 (1.0 M, 114.85 mL, 114.85 mmol) was added dropwise. The reaction was stirred at 20 °C for 16 h. H2O (100 mL) was added, and the mixture was extracted with ethyl acetate (100 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 27.

[0225] Preparation of compound 28

[0226] Into a 100 mL three-necked flask, compound 27 (10 g, 68.45 mmol) was dissolved in boron trifluoride etherate (60 mL), and then glacial acetic acid (12.0 mL, 209.82 mmol) was added. The mixture was stirred at 120 °C for 16 h under nitrogen protection. 10% aqueous sodium acetate solution (100 mL) was added, and the mixture was extracted with ethyl acetate (100 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was slurried with petroleum ether: ethyl acetate = 5: 1 to give compound 28. LCMS: calcd for C8H6F2O3Excat Mass: 188.1; Found, m / z, [M-H] - : 187.04.

[0227] Preparation of compound 29

[0228] Into a 500 mL round-bottom flask, compound 28 (6 g, 31.89 mmol) was dissolved in pyridine (30 mL), and pivaloyl chloride (19 g, 159.46 mmol) was added dropwise by syringe. The reaction was stirred at 25 °C for 16 h. New spots were generated on the plate (PE:EA = 5:1), and the reaction was completed. The reaction was quenched by the addition of saturated aqueous ammonium chloride solution, extracted with ethyl acetate (100 mL x 3), washed with 1 M HCl solution (100 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 29.

[0229] Example 30 Preparation of compound 30

[0230] Into a 500 mL round-bottom flask, compound 29 (10 g, 0.13 mmol) was dissolved in THF (100 mL), and the reaction was cooled to 0 °C in an ice bath. LiHMDS (1.0 M, 78.57 mL, 78.57 mmol) was added dropwise, and the reaction was stirred at 25 °C for 16 h. The reaction was quenched by the addition of saturated aqueous ammonium chloride solution, extracted with ethyl acetate (100 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Then 10 mL of methanol and 10 mL of hydrochloric acid were added, and the reaction was stirred at 75 °C for 48 h. The reaction was concentrated under reduced pressure to give the crude product. The crude product was purified by reverse-phase biotage automated column machine (welflash C18-I, regular C18 20-40 μm, 330 g, 0.1% TFA in water / acetonitrile, acetonitrile content 5-95%), and the product solution was concentrated under reduced pressure and lyophilized to give compound 30. LCMS: calcd for C 13 H 12 F2O3Excat Mass:254.2;Found,m / z,[M+H] + :255.15.

[0231] Example 31 Preparation of compound 31

[0232] Into a 100 mL round-bottom flask, compound 30 (470 mg, 2.0 mmol) was dissolved in THF (5 mL), and the reaction was cooled to 0 °C in an ice bath. CH3MgBr (3 M, 3.32 mL, 9.95 mmol) was added dropwise, and the reaction was stirred at 25 °C for 16 h. Then HFB4 solution (50%, 4 mL) was added, and the reaction was concentrated under reduced pressure. The reaction was filtered, and the filter cake was washed with a small amount of ethyl acetate and dried to give compound 31. LCMS: calcd for C 14 H 15 BF6O3Excat Mass:253.3;Found,m / z,[M+H] + :253.12.

[0233] Preparation of compound 32

[0234] Take 40 mL sample bottle, weigh compound 15 (50 mg, 164.42 umol), add acetic anhydride (2 mL) and acetic acid (2 mL), then add potassium carbonate (91 mg, 658.44 umol), compound 12 (86 mg, 163.92 umol), 25℃, electromagnetic stirring for 15 hours. The reaction solution is added with water (5 mL), heated to 60℃, and electromagnetically stirred for 12 hours. The reaction solution is filtered, first purified by fast preparation liquid chromatography (water / acetonitrile), then purified by preparation liquid chromatography (0.1M TEAB / acetonitrile), the preparation solution is concentrated under reduced pressure, freeze-dried to obtain compound 32. The synthetic route is shown in Figure 1.

[0235] LCMS: calcd for C 34 H 39 NO7S[M+H] + :606.24.Found,m / z,[M+H] + :606.35.

[0236] 1 H NMR (400 MHz, dmso) δ 8.16-8.07 (m, 1H), 8.00-7.89 (m, 2H), 7.75 (s, 1H), 7.60 (dd, J = 8.3, 1.3 Hz, 1H), 7.25 (d, J = 8.2 Hz, 1H), 6.94 (d, J = 13.6 Hz, 1H), 6.83 (s, 1H), 6.69 (dd, J = 9.1, 1.9 Hz, 1H), 6.62 (t, J = 12.5 Hz, 1H), 6.44 (s, 1H), 6.30 (d, J = 13.9 Hz, 1H), 4.10-3.98 (m, 2H), 2.86 (d, J = 6.8 Hz, 4H), 2.20 (t, J = 7.2 Hz, 2H), 1.66 (s, 6H), 1.55 (dt, J = 14.7, 7.2 Hz, 2H), 1.36 (s, 9H).

[0237] Preparation of compound 32-dCTP

[0238] Take 15 mL sample bottle, weigh compound 32 (20 mg, 33.02 umol), add anhydrous N'N-dimethylformamide (3 mL) to dissolve, then add N,N'-disuccinimidyl carbonate (17 mg, 66.36 umol), 4-dimethylaminopyridine (8 mg, 65.48 umol), 21 ℃, electromagnetic stirring for 2 hours. Then add dCTP (61 mg, 65.90 umol), triethylamine (17 mg, 168.00 umol), 21 ℃, continue electromagnetic stirring for 15 hours. The reaction solution is filtered, first purified by preparative liquid chromatography (0.1M TEAB / acetonitrile), the preparation solution is concentrated under reduced pressure, freeze-dried to get blue solid, then purified by preparative liquid chromatography (0.1M TEAB / acetonitrile), the preparation solution is concentrated under reduced pressure, freeze-dried to get compound 32-dCTP.

[0239] LCMS: calcd for C 62 H 75 N 12 O 25 P3S[(M-2) / 2] - :755.19.Found,m / z,[(M-2) / 2] - :754.89.

[0240] 1H NMR (400 MHz, dmso) δ 8.84 - 8.63 (m, 2H), 8.22 - 7.85 (m, 4H), 7.76 (d, J = 12.9 Hz, 1H), 7.62 (d, J = 7.1 Hz, 1H), 7.49 - 7.42 (m, 1H), 7.39 - 7.22 (m, 2H), 7.08 (dd, J = 8.2, 2.0 Hz, 1H), 6.91 (d, J = 12.8 Hz, 1H), 6.83 (s, 1H), 6.77 (dd, J = 9.4, 1.0 Hz, 1H), 6.73 - 6.61 (m, 1H), 6.52 (s, 1H), 6.38 (d, J = 14.7 Hz, 1H), 6.17 - 6.08 (m, 1H), 5.36 - 5.29 (m, 1H), 5.18 - 5.11 (m, 1H), 4.90 (d, J = 8.8 Hz, 1H), 4.80 (d, J = 8.9 Hz, 1H), 4.48 - 4.39 (m, 1H), 4.26 - 3.96 (m, 6H), 3.94 - 3.80 (m, 2H), 3.65 (d, J = 4.1 Hz, 2H), 3.20 (dd, J = 12.2, 6.4 Hz, 3H), 2.81 (s, 8H), 2.29 (d, J = 4.7 Hz, 1H), 2.19 - 2.11 (m, 1H), 2.10 - 2.04 (m, 1H), 2.03 - 1.95 (m, 1H), 1.67 (s, 3H), 1.55 (dd, J = 14.7, 8.5 Hz, 2H), 1.35 (s, 3H), 1.23 (s, 2H), 1.07 (t, J = 7.0 Hz, 9H). 31 P NMR (162 MHz, dmso) δ -10.48 (d, J = 19.8 Hz), -12.86 (d, J = 24.0 Hz), -22.96 (dd, J = 24.0, 19.8 Hz).

[0241] Preparation of compound 33 of example 34

[0242] Take 40 mL sample bottle, weigh compound 12 (105 mg, 0.34 mmol), compound 19 (170 mg, 0.32 mmol), dissolve in AcOH / Ac2O (v:v = 1:1, 2 mL), add K2CO3 (223 mg, 1.62 mmol), stir at 25 °C for 16 h. Add H2O (2 mL), warm to 70 °C and stir for 24 h. Filter the reaction solution, and purify the filtrate by reverse phase biotage automatic column machine (welflash C18-I, regular C18 20-40 μm, 330 g, 0.1 M TEAB / acetonitrile, acetonitrile content 5-95%), prepare a solution and concentrate under reduced pressure, freeze-dried to obtain compound 33. The synthetic route is shown in Figure 2.

[0243] LCMS: calcd for C 34 H 38 FNO7S Excat Mass:623.20.Found,m / z,[M+H] + :624.56.

[0244] 1 H NMR (400 MHz, DMSO-d6) δ = 8.05 (t, J = 12.9, 1H), 7.81 (dd, J = 30.7, 13.0, 2H), 7.66 (s, 1H), 7.55 (d, J = 8.3, 1H), 7.09 (d, J = 8.5, 1H), 6.95 (d, J = 14.0, 1H), 6.90 (s, 1H), 6.53 (t, J = 12.4, 1H), 6.45 (d, J = 7.9, 1H), 6.05 (d, J = 13.0, 1H), 2.20 (t, J = 7.2, 2H), 1.63 (s, 8H), 1.57-1.51 (m, 2H), 1.36 (s, 11H), 1.28-1.20 (m, 2H).

[0245] Preparation of compound 33-dCTP of Example 35

[0246] According to the synthesis of compound 32-dCTP, compound 33-dCTP was prepared from compound 33.

[0247] LCMS: calcd for C 62 H 75 FN 13 O 24 P3S Excat Mass:1529.40.Found,m / z,[M-H] - :1528.45.

[0248] 1 H NMR (600 MHz, DMSO-d6) δ = 8.70 (d, J = 23.9, 1H), 7.96 (d, J = 12.9, 1H), 7.79 (d, J = 13.1, 2H), 7.64 (t, J = 13.1, 1H), 7.57 (s, 1H), 7.35 (t, J = 8.0, 1H), 7.10 (d, J = 6.9, 1H), 6.98 (d, J = 14.3, 1H), 6.91 (m, 3H), 6.45 (t, J = 12.4, 1H), 6.30 (d, J = 7.9, 1H), 6.17-6.07 (m, 1H), 5.80 (d, J = 12.8, 1H), 5.14 (m, 1H), 4.90 (d, J = 8.9, 1H), 4.82 (d, J = 8.8, 1H), 4.43 (m, 1H), 4.23 (dd, J = 10.2, 3.9, 2H), 4.15 (m 4H), 3.98 (s, 2H), 3.91 (m, 2H), 3.86-3.82 (m, 2H), 3.77 (m, 2H), 3.68 (m, 2H), 3.29 (m, 2H), 3.20 (m, 2H), 2.32 (m, 1H), 2.24-2.12 (m, 1H), 2.07 (t, J = 7.2, 2H), 1.60 (s, 6H), 1.56-1.51 (m, 2H), 1.36 (s, 9H), 1.30 (s, 2H), 1.25-1.15 (m, 3H).

[0249] 19 F NMR (376 MHz, DMSO-d6) δ = -128.22. (s, 1F)

[0250] 31 P NMR (162 MHz, DMSO-d6) δ = -11.22 (d, J = 22.4), -12.09 (d, J = 22.7), -23.68 (t, J = 22.6).

[0251] Preparation of compound 34 of example 36

[0252] Into a 100 mL flask, compound 19 (102 mg, 316.69 umol) was weighed, acetic anhydride (6 mL) and acetic acid (6 mL) were added, then potassium carbonate (175 mg, 1.27 mmol) and compound 5 (200 mg, 316.08 umol) were added, the temperature was raised to 50 °C, and electromagnetic stirring was performed for 2 hours. The reaction solution was added to water (12 mL), the temperature was raised to 70 °C, and electromagnetic stirring was performed for 15 hours. The reaction solution was filtered, purified by flash preparative liquid chromatography (0.1% trifluoroacetic acid / acetonitrile) first, and then the prepared solution was concentrated under reduced pressure to obtain a crude product, which was further purified by preparative liquid chromatography (0.1 M TEAB / acetonitrile), the prepared solution was concentrated under reduced pressure, and freeze-drying was performed to obtain compound 34.

[0253] LCMS: calcd for C 36 H 42 FNO 10 S2[M+H] + : 732.22. Found, m / z, [M+H] + : 732.32.

[0254] 1 H NMR (400 MHz, dmso) δ = 8.01 - 7.87 (m, 1H), 7.77 (d, J = 12.9, 1H), 7.58 (t, J = 13.1, 1H), 7.50 - 7.44 (m, 2H), 6.99 - 6.87 (m, 3H), 6.43 (t, J = 12.4, 1H), 6.27 (d, J = 7.8, 1H), 5.84 (d, J = 12.6, 1H), 3.81 (s, 2H), 2.30 - 2.24 (m, 2H), 2.19 (dd, J = 7.5, 1.9, 2H), 2.05 - 1.96 (m, 1H), 1.62 - 1.50 (m, 7H), 1.35 - 1.32 (m, 11H), 1.25 - 1.18 (m, 1H), 1.14 - 1.10 (m, 1H), 0.90 - 0.83 (m, 1H).

[0255] 19 F NMR (376 MHz, dmso) δ = -128.29.

[0256] Preparation of compound 34-dCTP of example 37

[0257] According to the synthesis of compound 32-dCTP, compound 34 was used as raw material to prepare compound 34-dCTP.

[0258] LCMS: calcd for C 64 H 79 FN 13 O27 P3S2[(M-2) / 2] - :817.69Found, m / z, [(M-2) / 2] - :817.53.

[0259] 1 H NMR (400 MHz, dmso) δ = 8.58 (d, J = 28.4, 2H), 8.04 (s, 1H), 7.99 - 7.85 (m, 2H), 7.75 - 7.70 (m, 2H), 7.62 (t, J = 12.9, 1H), 7.54 - 7.46 (m, 2H), 7.44 - 7.35 (m, 2H), 7.25 (t, J = 7.8, 1H), 7.04 (d, J = 8.6, 1H), 6.94 - 6.85 (m, 4H), 6.44 (t, J = 12.4, 1H), 6.31 (d, J = 8.1, 1H), 6.14 - 6.04 (m, 1H), 5.90 (d, J = 12.5, 1H), 5.09 (s, 1H), 4.89 (d, J = 9.7, 1H), 4.79 (d, J = 8.8, 1H), 4.39 (s, 1H), 4.16 (dd, J = 10.4, 4.4, 1H), 4.12 - 4.06 (m, 4H), 3.92 - 3.86 (m, 2H), 3.84 - 3.80 (m, 3H), 3.24 - 3.20 (m, 2H), 3.16 (t, J = 5.8, 1H), 3.10 - 3.06 (m, 1H), 2.36 - 2.20 (m, 4H), 2.19 - 2.10 (m, 1H), 2.07 - 1.95 (m, 3H), 1.64 - 1.56 (m, 2H), 1.54 (s, 3H), 1.50 - 1.39 (m, 2H), 1.33 (s, 9H), 1.30 - 1.19 (m, 4H), 1.18 - 1.15 (m, 1H), 0.92 - 0.83 (m, 2H).

[0260] 19 F NMR (376 MHz, dmso) δ = -128.77.

[0261] 31 P NMR (162 MHz, dmso) δ = -10.70, -12.71, -23.32.

[0262] Preparation of compound 35 of example 38

[0263] Take 40 mL sample bottle, weigh compound 19 (113 mg, 350.84 umol), add acetic anhydride (6 mL) and acetic acid (6 mL), then add potassium carbonate (194 mg, 1.40 mmol), compound 10 (200 mg, 350.57 umol), 25°C, electromagnetic stirring for 15 hours. The reaction liquid is filtered, first purified by fast preparation liquid chromatography (0.1% trifluoroacetic acid / acetonitrile), then purified by preparation liquid chromatography (0.1M TEAB / acetonitrile), the preparation solution is concentrated under reduced pressure, freeze-dried to obtain compound 35.

[0264] LCMS: calcd for C 33 H 35 BrFNO9S2[M+H] + : 754.10. Found, m / z, [M+H] + : 754.24.

[0265] Preparation of compound 36 of example 39

[0266] Take 15 mL sample bottle, weigh compound 35 (50 mg, 66.34 umol), dissolve in ethanol (4 mL) and water (4 mL), then add 4-(2-carboxyethyl)phenylboronic acid (26 mg, 134.03 umol), tetrakis triphenyl phosphine palladium (8 mg, 6.92 umol), cesium carbonate (43 mg, 131.98 umol), nitrogen protection, heating to 90°C, electromagnetic stirring for 1.5 hours. The reaction liquid is filtered, first purified by fast preparation liquid chromatography (0.1% trifluoroacetic acid / acetonitrile), then purified by preparation liquid chromatography (0.1M TEAB / acetonitrile), the preparation solution is concentrated under reduced pressure, freeze-dried to obtain compound 36.

[0267] LCMS: calcd for C 40 H 42 FNO 10 S2[M+H] + : 780.23. Found, m / z, [M+H] + : 780.40.

[0268] 1H NMR (400 MHz, dmso) δ = 8.22 (d, J = 14.1, 1H), 7.80 (s, 1H), 7.58 (s, 1H), 7.47 (d, J = 8.3, 1H), 7.40 (d, J = 8.1, 2H), 7.17 (d, J = 8.0, 2H), 7.06 (s, 1H), 7.02 - 6.93 (m, 2H), 6.31 (d, J = 7.9, 2H), 5.37 (s, 1H), 3.82 - 3.57 (m, 2H), 2.91 (t, J = 7.6, 2H), 2.62 (t, J = 7.6, 2H), 2.42 - 2.34 (m, 2H), 1.84 - 1.71 (m, 2H), 1.59 (s, 6H), 1.37 (s, 9H).

[0269] 19 F NMR (376 MHz, dmso) δ = -128.35.

[0270] Example 40 Preparation of compound 36-dCTP

[0271] Compound 36-dCTP was prepared according to the synthesis of compound 32-dCTP, using compound 36 as the starting material.

[0272] LCMS: calcd for C 68 H 79 FN 13 O 27 P3S2[(M-2) / 2] - : 841.69 Found, m / z, [(M-2) / 2] - : 841.53.

[0273] 1H NMR (400 MHz, dmso) δ = 8.58 (d, J = 4.4, 2H), 8.13 (d, J = 5.5, 2H), 7.96 (s, 1H), 7.74 (s, 1H), 7.57 (s, 1H), 7.48 - 7.41 (m, 3H), 7.39 - 7.29 (m, 3H), 7.17 - 7.02 (m, 4H), 6.99 - 6.91 (m, 2H), 6.88 (s, 1H), 6.72 (d, J = 6.9, 2H), 6.30 (d, J = 7.9, 1H), 6.09 (t, J = 6.6, 1H), 5.31 (s, 1H), 5.11 (t, J = 4.6, 1H), 4.88 (d, J = 8.9, 1H), 4.78 (d, J = 8.9, 1H), 4.38 (s, 1H), 4.21 (dd, J = 10.5, 4.1, 1H), 4.15 - 4.04 (m, 6H), 4.00 - 3.91 (m, 2H), 3.90 - 3.86 (m, 2H), 3.84 - 3.78 (m, 4H), 3.33 - 3.29 (m, 2H), 3.25 - 3.20 (m, 2H), 2.91 - 2.87 (m, 2H), 2.53 - 2.48 (m, 2H), 2.42 - 2.35 (m, 2H), 2.31 - 2.26 (m, 1H), 2.20 - 2.09 (m, 1H), 1.84 - 1.70 (m, 2H), 1.59 (s, 6H), 1.37 (s, 9H).

[0274] 19 F NMR (376 MHz, dmso) δ = -128.09.

[0275] 31 P NMR (162 MHz, dmso) δ = -10.62 (d, J = 19.1), -12.92 (d, J = 24.0), -23.22 (dd, J = 24.0, 19.7).

[0276] Preparation of compound 37 of example 41

[0277] Compound 37 was prepared according to the synthesis of compound 34, using compound 5 and compound 26 as starting materials.

[0278] LCMS: Calculated for C 36 H 42 BrNO 10 S2Exact Mass: 791.14; Found, m / z, [M+H] + : 792.14, 794.09.

[0279] Preparation of compound 37-dCTP according to the synthesis of compound 32-dCTP, using compound 37 as the starting material.

[0280] Preparation of compound 37-dCTP according to the synthesis of compound 32-dCTP, using compound 37 as the starting material.

[0281] LCMS: Calcd for C 64 H 79 BrN 13 O 27 P3S2Exact Mass: 1697.30; Found, m / z, [M-2H] 2- : 848.96.

[0282] Preparation of compound 38 according to the synthesis of compound 34, using compound 5 and compound 22 as the starting materials.

[0283] Preparation of compound 38 according to the synthesis of compound 34, using compound 5 and compound 22 as the starting materials.

[0284] LCMS: calcd for C 36 H 44 NO 10 S2 + [M] + : 714.24. Found, m / z, [M] + : 714.41.

[0285] Preparation of compound 38-dCTP according to the synthesis of compound 32-dCTP, using compound 38 as the starting material.

[0286] Preparation of compound 38-dCTP according to the synthesis of compound 32-dCTP, using compound 38 as the starting material.

[0287] LCMS: calcd for C 64 H 81 N 13 O 27 P3S2 + [M / 2] + : 810.20. Found, m / z, [M / 2] + : 810.37.

[0288] Preparation of compound 39 according to the synthesis of compound 36, using compound 10 and compound 22 as the starting materials.

[0289] Preparation of compound 39 according to the synthesis of compound 36, using compound 10 and compound 22 as the starting materials.

[0290] LCMS: calcd for C 40 H44 NO 10 S2 + [M] + :762.24.Found,m / z,[M] + :762.20.

[0291] Preparation of compound 39-dCTP

[0292] Compound 39-dCTP was prepared according to the synthesis of compound 32-dCTP using compound 39 as the starting material.

[0293] LCMS: calcd for C 68 H 81 N 13 O 27 P3S2 + [M / 2] + :834.20.Found,m / z,[M / 2] + :834.58.

[0294] Preparation of compound 40

[0295] Compound 40 was prepared according to the synthesis of compound 34 using compound 5 and compound 31 as the starting materials.

[0296] LCMS: calcd for C 36 H 41 F2NO 10 S2 - Excat Mass:749.80.Found,m / z,[M+H] + :749.7.

[0297] 1 H NMR (400 MHz, DMSO-d6) δ = 8.30 (t, J = 12.9, 1H), 8.19 (t, J = 13.1, 1H), 8.00 (d, J = 9.3, 1H), 7.86 (d, J = 1.3, 1H), 7.67 (dd, J = 8.3, 1.4, 1H), 7.62 (t, J = 6.7, 1H), 7.44 (d, J = 8.4, 1H), 6.96 (dd, J = 9.0, 2.2, 1H), 6.94 - 6.84 (m, 3H), 6.72 (t, J = 12.4, 1H), 6.57 (d, J = 14.1, 1H), 4.19 (q, J = 8.7, 4H), 2.20 (t, J = 7.2, 2H), 1.70 (s, 8H), 1.55 (p, J = 7.1, 3H), 1.38 (s, 11H).

[0298] 19 F NMR (376 MHz, DMSO-d6) d = -127.66 (dd, J = 20.4, 12.7), -163.79 (d, J = 20.5).

[0299] Preparation of compound 40-dCTP

[0300] Compound 40-dCTP was prepared according to the synthesis of compound 32-dCTP, using compound 40 as the starting material.

[0301] LCMS: calcd for C 64 H 78 F2N 13 O 27 P3S2 - Excat Mass: 1656.4. Found, m / z, [M-H] - : 1655.4.

[0302] 1 H NMR (400 MHz, DMSO-d6) d = 8.55 (s, 1H), 8.10 - 7.96 (m, 2H), 7.73 (dt, J = 24.6, 12.9, 3H), 7.59 - 7.48 (m, 2H), 7.42 (d, J = 9.5, 2H), 7.26 (d, J = 8.6, 1H), 6.94 (d, J = 12.3, 3H), 6.46 (d, J = 12.2, 1H), 6.10 (s, 1H), 5.95 (s, 1H), 5.11 (s, 1H), 4.91 (s, 1H), 4.81 (d, J = 9.6, 1H), 4.38 (s, 2H), 4.11 (s, 5H), 3.97 (d, J = 9.8, 4H), 3.85 (s, 5H), 3.67 (s, 4H), 3.24 (s, 4H), 2.49 (d, J = 9.2, 5H), 2.33 (s, 4H), 2.17 (s, 1H), 2.04 (s, 3H), 1.37 (dd, J = 9.6, 3.5, 10H).

[0303] 19 F NMR (376 MHz, DMSO-d6) d = -127.69 (dd, J = 20.4, 12.7), -163.80 (d, J = 20.6).

[0304] Preparation of compound 41

[0305] Compound 41 was prepared according to the synthesis of compound 36, using compound 10 and compound 31 as starting materials.

[0306] LCMS: calcd for C40H40F2NO10S 2- Excat Mass: 796.90. Found, m / z, [M-H] - : 796.17.

[0307] 1 H NMR (400 MHz, DMSO-d6) d = 8.24 (s, 1H), 7.83 (d, J = 33.6, 1H), 7.61 (s, 1H), 7.50 (dd, J = 8.2, 1.6, 1H), 7.43 (d, J = 7.7, 2H), 7.23 - 7.09 (m, 3H), 7.05 (d, J = 8.2, 1H), 6.94 (d, J = 12.2, 1H), 6.35 (d, J = 14.5, 1H), 5.37 (d, J = 13.2, 1H), 2.50 (p, J = 1.8, 7H), 2.35 (s, 2H), 1.58 (s, 1H), 1.42 (s, 11H), 1.25 (d, J = 16.7, 1H), 1.18 (d, J = 7.5, 2H), 0.88 (dd, J = 8.5, 6.4, 1H).

[0308] 19 F NMR (376 MHz, DMSO-d6) d = -127.08, -163.61 (d, J = 20.8).

[0309] Example 50 Preparation of compound 41-dCTP

[0310] Compound 41-dCTP was prepared according to the synthesis of compound 32-dCTP, using compound 41 as starting material.

[0311] LCMS: calcd for C 68 H 77 F2N 13 O 27 P3S 2- Excat Mass: 1703.50. Found, m / z, [M+H] + : 1704.49.

[0312] 1H NMR (400 MHz, DMSO-d6) δ = 8.63 (s, 1H), 8.16 (s, 1H), 7.96 (s, 1H), 7.74 (s, 1H), 7.58 (s, 1H), 7.50-7.41 (m, 3H), 7.40-7.26 (m, 3H), 7.08 (dd, J = 17.1, 7.9, 3H), 7.02-6.85 (m, 3H), 6.33 (d, J = 14.4, 1H), 6.08 (t, J = 7.1, 1H), 5.32 (d, J = 13.2, 1H), 5.09 (s, 1H), 4.87 (d, J = 8.9, 1H), 4.78 (d, J = 8.9, 1H), 4.38 (d, J = 5.6, 1H), 4.18 (s, 2H), 4.14-4.05 (m, 5H), 3.94 (s, 6H), 3.84 (d, J = 23.4, 5H), 3.64 (s, 3H), 3.30 (s, 2H), 3.22 (d, J = 6.6, 2H), 2.57-2.41 (m, 6H), 2.40-2.23 (m, 3H), 2.21-2.09 (m, 1H), 1.38 (s, 9H), 0.83 (t, J = 7.6, 1H).

[0313] 19 F NMR (376 MHz, DMSO-d6) δ = -127.11, -163.59 (d, J = 20.8).

[0314] 31 P NMR (162 MHz, DMSO-d6) δ = -11.23 (d, J = 22.1), -12.09 (d, J = 22.6), -23.70 (t, J = 22.4).

[0315] Example 51, Application of the dye in gene sequencing

[0316] 1. Experimental apparatus

[0317] MGISEQ-2000RS sequencer, MGIDL-200H loading instrument, MGISEQ-2000RS sequencing slide, MGISEQ-2000RS high-throughput sequencing reagent kit FCLPE150, excitation wavelength of the instrument: 532 nm, 650 nm, respectively.

[0318] 2. Reagents and raw materials used in the experiment

[0319] The MGISEQ-2000RS high-throughput sequencing kit has a product number of 1000012536 and a brand of Huada Zhi Zao. The E. coli single-stranded loop DNA is used as a template, namely the standard library reagent V3.0. The primer sequence is CAACTCCTTGGCTCACAGAACATGGCTACGATCCGACTT. The DNA polymerase (Cpas DNA Polymerase) is from BGI. The DNB nanoballs are derived from BGI, dATP-1, which refers to an adenine nucleotide with both reversible blocking group modification and AF532 fluorescent modification; dTTP-1, which refers to a thymine nucleotide with both reversible blocking group modification and ROX fluorescent modification; dGTP-1, which refers to a guanine nucleotide with both reversible blocking group modification and Cy5 fluorescent modification; and dCTP-1, which refers to a cytosine nucleotide with both reversible blocking group modification and IF700 fluorescent modification, all from BGI.

[0320] 3. Experimental method:

[0321] (a) Preparation of reagents

[0322] The MGISEQ-2000RS high-throughput sequencing reagent kit FCLPE150 is used as a sequencing control experiment. In the dNTP mixture in the MGISEQ-2000RS high-throughput sequencing reagent kit FCLPE150, the C base is replaced with 32-dCTP or 33-dCTP provided by the present application, which is used as test experiment 1 and test experiment 2. The above kit is prepared for sequencing.

[0323] (b) DNA sequencing method:

[0324] Sequencing process: First, load the DNB nanoballs onto the prepared sequencing chip.

[0325] Second, pump the prepared dNTP molecular mixture solution into the chip with DNA polymerase, and add dNTP to the complementary strand of the DNA parent strand.

[0326] Third, take a photo scan. Since the dNTP is a modified molecule with a fluorescent group, a laser is used as the excitation wavelength for photographing. Since the laser has a photo-damage effect on DNA, a scanning reagent is used as a protective agent for photographing in this step. After photographing, the base type is confirmed.

[0327] Fourth, use the excision reagent to excise and wash away the base end fluorescent group and the 3' block, so that the 3'-OH is exposed, thereby performing the next round of reaction.

[0328] 4. Experimental results and discussion

[0329] (1) Effect of 32-dCTP and 33-dCTP on sequencing error rate

[0330] The experimental results are shown in Figure 7, in which the higher the value of Q30, the lower the error rate. As can be seen from Figure 7, all the labeled dNTPs of 33, 32 performed better in sequencing than the control group, and the Q30 of both single-strand sequencing and double-strand sequencing was higher than that of the control experiment, significantly reducing the overall error rate of sequencing.

[0331] (2) Effect of 33-dCTP on fluorescence crosstalk

[0332] The experimental results are shown in Figure 8, in which the greater the angle between the two optical images, the lower the degree of fluorescence crosstalk. As can be seen from Figure 8, the angle between the optical images of C and G bases of the 33-dCTP sequencing image is greater than that of the control group.

[0333] Example 52, Emission spectrum of dye

[0334] Preparation of PBS buffer: 9 mL of 10X PBS buffer was measured with a graduated cylinder and dissolved in 36 mL of ultrapure water to prepare a 20 mmol / L PBS buffer.

[0335] Preparation of stock solution: The dried compounds 32 and 33 were accurately weighed with a one-hundredth balance and prepared into 2.5 mmol / L DMSO stock solutions in brown sample bottles, which were stored in a -20°C refrigerator for standby use.

[0336] Preparation of test solution: 4.8 μL of dye stock solution was measured with a pipette and dissolved in PBS buffer to dilute into 10 mL of test solution containing 1.2 μmol / L of concentration.

[0337] Fluorescence spectrum determination of compounds 32 and 33: 3.5 mL of test solution was transferred to a quartz cuvette, and the fluorescence emission spectra of both were measured at excitation wavelengths of 700 nm and 705 nm, respectively. As shown in Figure 9, the maximum emission wavelengths of compounds 32 and 33 are 732 nm and 734 nm, respectively.

[0338] Although the present application has been described with reference to the preferred embodiments, various modifications can be made to it without departing from the scope of the application, and equivalent components can be substituted therefor. In particular, the technical features mentioned in each embodiment can be combined in any manner as long as there is no structural conflict. The present application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A compound represented by the formula (I), an ester thereof, or a salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 are each the same or different and are each independently selected from the group consisting of hydrogen, C1-C6alkyl, halogenated C1-C6alkyl, hydroxyl, halogen, nitro, sulfonic acid group, phosphoric acid group; R 7 , R 8 , R 9 , R 10 , R 11 each is the same or different and each is independently selected from the group consisting of hydrogen, C1-C6alkyl, phenyl, substituted phenyl, nitrogen containing heterocycle; R 12 , R 13 each is the same or different and each is independently selected from the group consisting of alkyl having 1-18 carbons, carboxyalkyl having 1-18 carbons, aminealkyl having 1-18 carbons, alkyl sulfonate having 1-18 carbons, polyethylene glycol substituted alkyl; R 14 , R 15 , R 16 , R 17 each is the same or different, and each is independently selected from the group consisting of hydrogen, C1-C6alkyl, haloC1-C6alkyl, hydroxyl, halogen, nitro, sulfonic acid group, phosphoric acid group; R 18 selected from hydrogen, alkyl of 1-18 carbons, carboxyalkyl of 1-18 carbons, aminealkyl of 1-18 carbons, alkylsulfonate of 1-18 carbons, polyethylene glycol substituted alkyl; each m is independently selected from an integer between 1 and 5 (e.g. 1, 2, 3, 4, 5).

2. The compound, ester or salt thereof of claim 1, wherein, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 are each the same or different and are each independently selected from the group consisting of hydrogen, C1-C6alkyl, hydroxyl, halogen, sulfonic acid group, phosphoric acid group; Preferably, R 2 is Ci-C6alkyl; Preferably, R 3 is hydrogen, hydroxy or halogen; Preferably, R 4 is hydrogen or hydroxy; Preferably, R 5 is hydrogen or halogen; Preferably, R 6 is hydrogen.

3. The compound, ester thereof, or salt thereof of claim 1 or 2, wherein, R 7 , R 8 , R 9 , R 10 , R 11 are each independently selected from the group consisting of hydrogen, substituted phenyl.

4. The compound, ester or salt thereof of any one of claims 1-3, wherein, R 12 , R 13 each independently is selected from the group consisting of C1-C6alkyl, sulfonic acid substituted C1-C6alkyl.

5. The compound, ester or salt thereof of any one of claims 1-4, wherein, R 14 , R 15 , R 16 , R 17 each is the same or different and each is independently selected from the group consisting of hydrogen, sulfo; Preferably, R 15 is a sulfonic acid group.

6. The compound of any one of claims 1-5, its ester or salt thereof, wherein the compound has a structure as shown in formula (II): wherein, R 2 , R 3 , R 4 , R 5 , R 9 , R 10 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 as defined in any one of claims 1 to 5.

7. The compound of any one of claims 1-6, an ester thereof, or a salt thereof, having a structure according to Formula (III): ###0003### (III) ​ wherein, R 2 , R 3 , R 4 , R 5 , R 9 , R 10 , R 12 , R 13 , R 15 , R 18 as defined in any one of claims 1 to 6.

8. The compound of any one of claims 1-7, an ester thereof, or a salt thereof, having a structure selected from the group consisting of:

9. The compound, ester or salt thereof of any one of claims 1 to 8, which is covalently attached to a nucleotide or oligonucleotide via the carboxyl group.

10. A labelled nucleoside, nucleotide or oligonucleotide, which is labelled with the compound, ester or salt thereof of any one of claims 1 to 9.

11. The labelled nucleoside, nucleotide or oligonucleotide of claim 10, wherein the compound is covalently attached to the nucleoside, nucleotide or oligonucleotide via the carboxyl group; Preferably, the compound is covalently attached to the nucleoside, nucleotide or oligonucleotide via a cleavable linker; Preferably, the cleavable linker is selected from the group consisting of linkers of the following structures:

12. The labelled nucleoside, nucleotide or oligonucleotide of claim 10 or 11, which nucleotide is a dNTP, i.e. a deoxyribonucleoside triphosphate, for example selected from dATP, dGTP, dTTP, dCTP; or which nucleotide is a rNTP, i.e. a ribonucleoside triphosphate, for example selected from ATP, GTP, CTP, UTP.

13. The labelled nucleoside, nucleotide or oligonucleotide of any one of claims 10 to 12, which nucleotide is modified with a reversible blocking group, for example with an azidomethylene (-CH2-N3) or an allyl group at the 3'-0 of the deoxyribose.

14. The labeled nucleotide or oligonucleotide of any one of claims 10-13, said labeled nucleotide selected from the group consisting of dye-labeled dNTPs, for example: wherein each L is independently absent or a linker, for example a cleavable linker as described above, and each dNTP is independently selected from dATP, dGTP, dTTP or dCTP; Preferably, the dye-labeled dCTP has a structure selected from the group consisting of: Preferably, the dye-labeled dATP has a structure selected from the group consisting of: Preferably, the dye-labeled dTTP has a structure selected from the group consisting of: Preferably, the dye-labeled dGTP has a structure selected from the group consisting of:

15. A method of sequencing, comprising incorporating a labelled nucleotide of any one of claims 10 to 14 into a sequencing assay; Preferably, the method further comprises detecting the labelled nucleotide. Preferably, the sequencing assay is performed on an automated sequencing instrument, and wherein the automated sequencing instrument comprises two light sources operating at different wavelengths.

16. The method of sequencing of claim 15, which method comprises: (a) incorporating at least one labelled nucleotide of any one of claims 10 to 14 into a polynucleotide; and (b) detecting the labelled nucleotide incorporated into the polynucleotide by detecting a fluorescent signal from the new fluorescent dye attached to the modified nucleotide.

17. The method of sequencing of claim 15 or 16, which method comprises the steps of: (a) providing a duplex, nucleotides, a polymerase and an excision reagent; the duplex comprising a growing nucleic acid chain and a nucleic acid molecule to be sequenced; (b) performing a reaction cycle comprising the steps (i), (ii) and (iii): step (i): using the polymerase to incorporate a nucleotide into the growing nucleic acid chain to form a nucleic acid intermediate comprising a blocking group and a detectable label; step (ii): detecting the detectable label on the nucleic acid intermediate; step (iii): using the excision reagent to remove the blocking group on the nucleic acid intermediate; Preferably, the reaction cycle further comprises the step (iv): using the excision reagent to remove the detectable label on the nucleic acid intermediate.

18. The method of sequencing according to any one of claims 15-17, which method employs a four-color fluorescent sequencing technique, a three-color fluorescent sequencing technique or a two-color fluorescent sequencing technique; The four-color fluorescent sequencing technology includes: the four nucleotides dATP, dGTP, dTTP and dCTP are labeled separately using four fluorescent substances that can be excited to produce different colors; the three-color fluorescent sequencing technique comprises labeling three of the four nucleotides dATP, dGTP, dTTP, dCTP separately using three fluorescent substances that can be excited to produce different colors, the remaining one nucleotide being unlabeled; the two-color fluorescent sequencing technique comprises labeling the four nucleotides dATP, dGTP, dTTP, dCTP using two fluorescent substances that can be excited to produce different colors, wherein the first nucleotide is labeled mixed (i.e. dual-labeled, e.g. 50% of the dATP is labeled with dye AF532 and the other 50% of the dATP is labeled with dye Cy5) using the two fluorescent substances, the second and third nucleotides are labeled separately using the two fluorescent substances, and the remaining one nucleotide is unlabeled.

19. A kit comprising one or more nucleotides, wherein at least one nucleotide is a labeled nucleotide according to any one of claims 10-14; Preferably, the kit comprises two or more labeled nucleotides. Preferably, the kit comprises sequencing reagents; Preferably, the sequencing reagents comprise at least one of a dNTPs mix, a nucleic acid polymerase mix, an elution solution; Preferably, the dNTPs mix contains at least one labeled nucleotide according to any one of claims 13-17; Preferably, the elution solution contains a cleavage reagent; Optionally, the cleavage reagent is selected from one or more of the following reagents: Endo IV, alkaline phosphatase, an organic phosphine (e.g. tris(3-hydroxypropyl)phosphine (THPP), tris(2-carboxyethyl)phosphine hydrochloride (TCEP)) or a PdCl2 complex with sulfonated triphenylphosphine.

20. Use of a compound, an ester thereof or a salt thereof according to any one of claims 1-9 in sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, protein binding assays, in vitro diagnostics, immunoassays, molecular labeling; Preferably, the molecular labeling is used for cell imaging, tissue imaging or imaging of a living organism.

21. Use of a compound, an ester thereof or a salt thereof according to any one of claims 1-9 for fluorescent labeling, quantification or detection of proteins, enzymes or nucleic acids.

22. Use of a labeled nucleoside, nucleotide or oligonucleotide according to any one of claims 10-14 or a kit according to claim 19 in sequencing. ​

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