Nucleotide analogs, methods of making and using the same

Abstract

This application provides a nucleotide analogue, its preparation method, and its application. The nucleotide analogue provided in this application has the structure shown in formula (I). The nucleotide analogue provided in this application has good stability; as a sequencing blocking group, it can rapidly expose the 3' hydroxyl group during sequencing and hardly interfere with other reactions in the system. The excision reagent and reaction byproducts can be rapidly removed by the washing buffer after the reaction, showing broad application prospects.

Description

Technical Field

[0001] This application relates to the field of nucleic acid sequencing, and more specifically, to a nucleotide analogue, its preparation method, and its application. Background Technology

[0002] Since the 1950s, numerous researchers have attempted to synthesize DNA using chemical and enzymatic methods. The chemical method employs a four-step phosphorus amide synthesis, involving deprotection, coupling, capping, and oxidation cycles. Phosphorus amide-modified nucleotide monomers are coupled one by one to the 5' end of the target oligonucleotide chain, extending the chain from the 3' to the 5' end. The trivalent phosphine monomers used in chemical synthesis require an anhydrous and oxygen-free environment to prevent monomer decomposition. This stringent environmental requirements increase the complexity and difficulty of the operation, as well as the risk of synthesis failure. Furthermore, the coupling of trivalent phosphine monomers necessitates oxidation after each subsequent coupling step to obtain the desired phosphodiester bond. Therefore, overcoming these challenges and breaking through bottlenecks in oligonucleotide synthesis may require finding methods other than chemical synthesis. In the past decade, enzymatic synthesis of oligonucleotides has gained increasing popularity and public attention.

[0003] 3'-reversibly blocked nucleotide monomers are one of the key raw materials for the enzymatic synthesis of oligonucleotides. Depending on the requirements of enzymatic synthesis, the 3' end of the nucleotide monomer may be protected or deprotected. The mechanism of action of these compounds is that, under the action of a template-free terminal transferase, the phosphate group at the 5' end of the free reversible terminator forms a phosphodiester bond with the 3' hydroxyl group of the previous nucleotide. The blocked 3' end of the reversible terminator terminates the nucleic acid chain elongation, ensuring that only one nucleotide is extended per round of reaction. After this round of elongation, a cleavage reagent is added to release the hydroxyl group at the 3' end of the nucleotide, thus initiating the next round of synthesis.

[0004] To meet the requirements of enzymatic synthesis, these compounds need to have good stability for transportation and storage; they need to be recognized by terminal transferases for initiation chain extension; the 3' protecting group needs to be rapidly removed under relatively mild conditions to ensure that the initiating nucleic acid chain is not damaged and to shorten the single-base synthesis coupling time; and the compound formed after the 3' protecting group leaves should not negatively affect the activity of enzymes or other components in the synthesis system.

[0005] Currently reported reversible blocking monomers for enzymatic synthesis of oligonucleotides include 3'-OH azidomethyl derivatives and 3'-OH hydroxylamine-blocked nucleoside triphosphates. Both offer advantages such as small size, mild excision conditions, and rapid excision speed. The excision mechanism of the 3'-OH azidomethyl derivative involves the Staudinger reaction of the azido group with a phosphine reagent to generate an intermediate containing a 3'-OH methyleneamine group, followed by rapid hydrolysis to release the 3'-OH group. The excision mechanism of the 3'-OH hydroxylamine-blocking monomer involves the release of nitrous oxide from hydroxylamine in a sodium nitrite buffer solution, followed by hydrolysis to release the 3'-OH group. However, in the chemical structure of the azido group, the three nitrogen atoms form a conjugated structure in a single plane, resulting in a zigzag structure with a certain angle. The conjugated structure formed by the three nitrogen atoms in the azido group has multiple resonance patterns, which helps to improve its structural stability. However, due to the superposition of three nitrogen atoms, the azide group is unstable under specific reaction conditions, readily reacting and rapidly releasing nitrogen gas. Therefore, the azide group exhibits highly reactive and explosive properties. Hydroxylamine blocking substrates, on the other hand, have poor stability and require storage and use under anhydrous and oxygen-free conditions. Otherwise, they are prone to hydroxylamine hydrolysis or side reactions with aldehydes and ketones in the environment to form oximes, leading to reduced purity and decreased synthesis efficiency.

[0006] In view of the above, this application is hereby submitted. Summary of the Invention

[0007] The main objective of this application is to provide a nucleotide analogue, its preparation method, and its application, in order to improve the problems of the unstable azido group in 3'-OH azidomethyl derivatives, which easily reacts to release nitrogen gas, and the relatively high stability of 3'-OH hydroxylamine-blocked nucleoside triphosphates, which require harsh storage conditions and are prone to side reactions.

[0008] To achieve the above objectives, according to one aspect of this application, a nucleotide analog is provided having the structure shown in formula (I):

[0009]

[0010] Wherein, R1, R2, R3, and R4 are each independently selected from H, halogen, nitro, amino, sulfonyl, carboxyl, C1-C20 alkyl, C3-C20 cycloalkyl, C6-C20 aryl, C6-C20 arylC1-C20 alkyl, and C1-C20 alkoxy; R5 and R6 are each independently substituted or unsubstituted C6-C20 aryl; when R5 and R6 are each independently substituted C6-C10 aryl, the substituent is selected from halogen, nitro, amino, sulfonyl, carboxyl, C1-C20 alkyl, C3-C20 cycloalkyl, C6-C20 aryl, C6-C20 arylC1-C20 alkyl, and C1-C20 alkoxy; R7 is selected from H, Base is selected from bases, denitrogenated bases, or their tautomers.

[0011] Furthermore, R1, R2, R3, and R4 are each independently selected from H, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, C6-C10 arylC1-C6 alkyl, and C1-C6 alkoxy.

[0012] Furthermore, R1, R2, R3, and R4 are each independently selected from H, C1-C4 alkyl, C3-C6 epoxy, C6-C10 aryl, phenyl C1-C3 alkyl, and C1-C4 alkoxy.

[0013] Furthermore, R1, R2, R3, and R4 are each independently selected from H, methyl, ethyl, phenyl, methoxy, ethoxy, benzyl, and phenethyl.

[0014] Furthermore, R5 and R6 are each independently substituted or unsubstituted phenyl groups; when R5 and R6 are each independently substituted phenyl groups, the substituents are selected from halogen, nitro, amino, sulfonyl, carboxyl, C1-C10 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, C6-C10 arylC1-C6 alkyl, and C1-C6 alkoxy.

[0015] Furthermore, R5 and R6 are each independently phenyl or substituted phenyl. When R5 and R6 are each independently substituted phenyl, the substituent is selected from halogen, nitro, amino, sulfonyl, carboxyl, C1-C4 alkyl, C3-C6 epoxy, C6-C10 aryl, phenyl C1-C3 alkyl, and C1-C4 alkoxy.

[0016] Furthermore, R5 and R6 are each independently phenyl or substituted phenyl. When R5 and R6 are each independently substituted phenyl, the substituent is selected from methyl, ethyl, phenyl, methoxy, ethoxy, benzyl, and phenethyl.

[0017] Furthermore, R7 is selected from

[0018] Furthermore, R7 is selected from

[0019] Furthermore, Base is selected from bases, adenine, 7-deadenine, thymine, uracil, cytosine, guanine, 7-deadenine, or their tautomers.

[0020] Furthermore, Base is selected from bases.

[0021] According to a second aspect of the present invention, a method for preparing the nucleotide analog provided in the first aspect is also provided, the method comprising: step S1, providing a compound of formula (A), and subjecting the compound of formula (A) to an esterification reaction with a compound of formula (B) to obtain a compound of formula (C); wherein, in formula (A), P represents a protecting group; step S2, removing the protecting group P from the compound of formula (C) and optionally subjecting it to a phosphorylation reaction to obtain a nucleotide analog.

[0022]

[0023]

[0024] Further, in step S1, the molar ratio of compound (B) to compound (A) is 1:1.1-1.5, preferably 1:1.1-1.3.

[0025] Furthermore, the esterification reaction is carried out under the activation of an activator, which includes at least one of dicyclohexylcarbodiimide, 4-dimethylaminopyridine, and 2,4,6-trichlorobenzoyl chloride.

[0026] Further, step S2 includes: removing the protecting group P from the compound of formula (C) to obtain the compound of formula (D), and subjecting the compound of formula (D) to a phosphorylation reaction to obtain a nucleotide analog.

[0027]

[0028] According to a third aspect of this application, the nucleotide analogues provided in the first aspect are provided for use in the enzymatic synthesis of oligonucleotides.

[0029] According to the fourth aspect of this application, the application of the nucleotide analogues provided in the first aspect above in sequencing is provided.

[0030] By applying the technical solution of this application, the nucleotide analog provided by this application has good stability. As a sequencing blocking group, it can quickly expose the 3' hydroxyl group during the sequencing process and hardly interfere with other reactions in the system. The excision reagent and reaction byproducts can be quickly removed by the washing buffer after the reaction, which has broad application prospects. Attached Figure Description

[0031] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0032] Figure 1 The mass spectrum of compound 8 provided in Example 1 of this application is shown;

[0033] Figure 2(a) shows the high performance liquid chromatogram of compound 8 provided in Example 1 of this application before the excision experiment;

[0034] Figure 2(b) shows the high performance liquid chromatogram of compound 8 provided in Example 1 of this application after an excision experiment;

[0035] Figure 3 The image shown is a gel electrophoresis diagram of compound 8 provided in Example 1 of this application after base extension.

[0036] Figure 4(a) shows the high performance liquid chromatogram of the pure solution of compound 8 provided in Example 1 of this application before stability testing;

[0037] Figure 4(b) shows the high-performance liquid chromatogram of compound 8 provided in Example 1 of this application after stability testing;

[0038] Figure 4(c) shows the high performance liquid chromatogram of the pure nucleotide analog solution provided in Comparative Example 1 of this application before stability testing;

[0039] Figure 4(d) shows a high-performance liquid chromatogram of the nucleotide analog provided in Comparative Example 1 of this application after stability testing. Detailed Implementation

[0040] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present application will now be described in detail with reference to the embodiments.

[0041] As analyzed in the background section of this application, 3'-OH azidomethyl derivatives and 3'-OH hydroxylamine-blocked nucleoside triphosphates both possess advantages such as small size, mild excision conditions, and rapid excision speed when used as reversible blocking monomers for the enzymatic synthesis of oligonucleotides. However, the azido group in 3'-OH azidomethyl derivatives is unstable and readily reacts to release nitrogen gas, while 3'-OH hydroxylamine-blocked nucleoside triphosphates have poor stability, require harsh storage conditions, and are prone to side reactions. To address these issues, this application provides a nucleotide analog, its preparation method, and its applications.

[0042] In one typical embodiment of this application, a nucleotide analog is provided having the structure shown in formula (I):

[0043]

[0044] Wherein, R1, R2, R3, and R4 are each independently selected from H, halogen, nitro, amino, sulfonyl, carboxyl, C1-C20 alkyl, C3-C20 cycloalkyl, C6-C20 aryl, C6-C20 arylC1-C20 alkyl, and C1-C20 alkoxy; R5 and R6 are each independently substituted or unsubstituted C6-C20 aryl; when R and R6 are each independently substituted C6-C10 aryl, the substituent is selected from halogen, nitro, amino, sulfonyl, carboxyl, C1-C20 alkyl, C3-C20 cycloalkyl, C6-C20 aryl, C6-C20 arylC1-C20 alkyl, and C1-C20 alkoxy; R7 is selected from H, (monophosphate group) (bisphosphate group) (triphosphate group) (Tetraphosphate group); Base is selected from bases, denitrogenated bases or their tautomers.

[0045] In this application, the term "C1-C20 alkyl" means having 1 to 20 alkyl groups, said groups being straight chains or branched chains having one or more branches, such as butyl, such as n-butyl, sec-butyl, isobutyl, tert-butyl; propyl, such as n-propyl or isopropyl; ethyl or methyl; more particularly, methyl, isopropyl or tert-butyl.

[0046] The term “C1-C20 alkoxy” refers to “C1-C20 alkyl-O-”, particularly C1-C6 alkoxy, such as methoxy, ethoxy, isopropoxy, or tert-butoxy.

[0047] The term "C3-C20 cycloalkyl" refers to a cycloalkyl group having 3 to 20 carbon atoms, particularly a C3-C6 epoxy group, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. C3-C6 cycloalkyl groups may optionally be substituted with C1-6 alkyl groups and / or halogens. Furthermore, "C3-C20 cycloalkyl" also includes epoxyalkyl groups with a total carbon number of 4 to 20, as well as alkylepoxy groups.

[0048] The term "C6-C20 aryl" refers to monocyclic, bicyclic, or tricyclic aromatic ring systems having 6 to 20 carbon atoms. Examples include phenyl, biphenyl, bitriphenyl, naphthyl, binatyl, phenylnaphthyl, naphthylphenyl, geniyl, phenylgeniyl, benzo[a]geniyl, dibenzo[a]geniyl, phenanthryl, phenylphenanthryl, anthracene, indole, bitriphenylene, pyrene, tetraphenyl, peryl, kinel, naphthanoyl, and propadienylgeniyl.

[0049] The term "halogen" or "halogen" refers to one or more of fluorine, chlorine, bromine, and iodine, especially fluorine or chlorine.

[0050] The term "C6-C20 aryl-C1-C20 alkyl" refers to C6-C20 arylene-C1-C20 alkyl and C1-C20 alkylene-C6-C20 aryl.

[0051] The nucleotide analog provided in this application has good stability. As a sequencing blocking group, it can rapidly expose the 3' hydroxyl group during sequencing and hardly interfere with other reactions in the system. The excision reagent and reaction byproducts can be quickly removed by the washing buffer after the reaction, which has broad application prospects.

[0052] It should be noted that the nucleotide analogs provided in this application use the Staudinger reaction as a reversible terminator with a 3' end blocking group cleavage mechanism. The Staudinger reaction is the reaction of an organic azide compound with a tertiary phosphine (e.g., triarylphosphine or trialkylphosphine) to yield the corresponding azaphosphine ylide (phosphineimine). The main characteristics of this reaction are: the reaction is usually very rapid, yielding almost equivalent products with almost no byproducts; generally, any trivalent phosphine compound can undergo this reaction; organic azides of various structures can undergo this reaction; and the phosphineimine compounds formed by alkyl or aryl azides and trialkylphosphine or triarylphosphine are very stable and easily separated.

[0053] In some embodiments, R1, R2, R3, and R4 are each independently selected from H, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, C6-C10 arylC1-C6 alkyl, and C1-C6 alkoxy. In particular, when R1, R2, R3, and R4 are each independently selected from H, C1-C4 alkyl, C3-C6 epoxy, C6-C10 aryl, phenylC1-C3 alkyl, and C1-C4 alkoxy, it is more conducive to improving structural stability. Specifically, when R1, R2, R3, and R4 are each independently selected from H, methyl, ethyl, phenyl, methoxy, ethoxy, benzyl, and phenethyl, the nucleotide analog exhibits even higher structural stability.

[0054] In some embodiments, R5 and R6 are each independently substituted or unsubstituted phenyl groups; and when R5 and R6 are each independently substituted phenyl groups, the substituents are selected from halogen, nitro, amino, sulfonyl, carboxyl, C1-C10 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, C6-C10 arylC1-C6 alkyl, and C1-C6 alkoxy groups; especially when R5 and R6 are each independently phenyl or substituted phenyl groups, and the substituents are selected from halogen, nitro, amino, sulfonyl, carboxyl, C1-C4 alkyl, C3-C6 epoxy, C6-C10 aryl, phenylC1-C3 alkyl, and C1-C4 alkoxy groups, it is more conducive to improving structural stability, especially when R5 and R6 are each independently phenyl or substituted phenyl groups of methyl, ethyl, phenyl, methoxy, ethoxy, benzyl, or phenethyl, their structural stability is even higher.

[0055] In some embodiments, R7 is selected from (monophosphate group) (bisphosphate group) (triphosphate group) (Tetraphosphate group) is more conducive to improving sequencing efficiency, especially R7, which is a triphosphate group, which has higher sequencing efficiency.

[0056] In some embodiments, Base is selected from bases, adenine, 7-deadenine, thymine, uracil, cytosine, guanine, 7-deadenine, or their tautomers. In particular, when Base is a base, it is more conducive to improving sequencing efficiency and structural stability.

[0057] In a second typical embodiment of this application, a method for preparing the above-mentioned nucleotide analog is provided, the method comprising: step S1, providing a compound of formula (A), and subjecting the compound of formula (A) to an esterification reaction with a compound of formula (B) to obtain a compound of formula (C); wherein, in formula (A), M represents a hydroxyl protecting group; step S2, removing the protecting group P from the compound of formula (C) and optionally subjecting it to a phosphorylation reaction to obtain a nucleotide analog.

[0058]

[0059]

[0060] It should be noted that M refers to a hydroxyl protecting group, including but not limited to formyl, acetyl, propionyl, formyloxymethyl, carbonate, trimethoxysilyl, dimethoxysilyl, silyl, propionyl, propionylsilyl, propionyl alcohol, tert-butyldimethylsilyl, etc., preferably tert-butyldimethylsilyl.

[0061] This application provides a method for preparing nucleotide analogs that is not only simple in process and convenient and efficient in synthesis, but also produces nucleotide analogs with high stability and fast excision speed, which have great application potential and broad market prospects.

[0062] In some embodiments, in step S1, to further improve the yield of compound (C), the molar ratio of compound (B) to compound (A) is preferably 1:1.1-1.5, with compound (B) in slight excess, to further improve the yield of compound (C). In particular, a molar ratio of compound (B) to compound (A) of 1:1.1-1.3 is more conducive to reducing energy waste.

[0063] Typical, but not limiting, the molar ratio of compound (B) to compound (A) is, for example, 1:1.1, 1:1.15, 1:1.2, 1:1.25, 1:1.3, 1:1.35, 1:1.4, 1:1.5 or any range of two values.

[0064] In some embodiments, the compound of formula (A) is obtained by a substitution reaction of a nucleoside with a halide containing a hydroxyl protecting group.

[0065] In some specific embodiments, the nucleoside of formula (a) undergoes a substitution reaction with tert-butyldimethylchlorosilane (TBS-Cl) and imidazole in N,N-dimethylformamide (DMF) to obtain the compound of formula (A). The reaction process is shown below.

[0066]

[0067] For example, compound (A) was prepared by the following steps: 0.8 mol of the nucleoside of formula (a) was dissolved in 20 mL of anhydrous N,N-dimethylformamide (DMF). At room temperature, 0.8 mol of tert-butyldimethylchlorosilane (TBS-Cl) was added to the above solution, and the mixture was stirred overnight. After the reaction was complete, water was slowly added to the reaction system to quench the reaction, DMF was washed away with water, extraction was performed, the solvent was evaporated, and separation was carried out by silica gel column chromatography using a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether:ethyl acetate = 1:20, to obtain compound (A).

[0068] In some embodiments, to further improve the efficiency of the esterification reaction, the esterification reaction is preferably carried out under the activation of an activator, which includes, but is not limited to, any one or more of dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), and 2,4,6-trichlorobenzoyl chloride.

[0069] In some specific embodiments, the activator includes DCC and DMAP. Preferably, the molar ratio of compound (B) to DCC is 1:1.0-1.5, and the molar ratio of compound (B) to DMAP is 1:0.15-0.20, to facilitate the activation of compound (B) to generate the activated intermediate of carboxylic acid (E). The activated intermediate of carboxylic acid (E) then reacts with compound (A) to generate compound (C). The reaction process is shown below:

[0070]

[0071] Typical, but not limiting, the molar ratio of compound (B) to DCC is 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 or any range of two such values; the molar ratio of compound (B) to DMAP is 1:0.15, 1:0.16, 1:0.17, 1:0.18, 1:0.19, 1:0.20 or any range of two such values.

[0072] For example, step S1 above is performed as follows: 1.0 mol of compound (B) is reacted with 1.5 mol of dicyclohexylcarbodiimide (DCC) and 0.15 mol of 4-dimethylaminopyridine (DMAP) in dry dichloromethane at room temperature for 2 hours. Then, 1.2 mol of compound (A) is added to the reaction solution, and the reaction is carried out overnight. The solvent is removed by evaporation, and the mixture is separated by silica gel column chromatography using a mixed solvent of petroleum ether and ethyl acetate as the eluent, with a volume ratio of petroleum ether:ethyl acetate of 3:1, to obtain compound (C).

[0073] In some embodiments, step S2 above involves removing the hydroxyl protecting group M from the compound of formula (C) to generate 5'-OH, thereby preparing the compound of formula (D) with R7 being H.

[0074]

[0075] In some embodiments, the compound of formula (C) reacts with a tetrahydrofuran (THF) solution of tetrabutylammonium fluoride (TBAF) in tetrahydrofuran to remove the 5'-OH hydroxyl protecting group M, generating the compound of formula (D).

[0076] For example, the removal of the hydroxyl protecting group in compound (C) is carried out as follows: 1.0 mol of compound (C) is dissolved in tetrahydrofuran. At room temperature, 2.0 mol of tetrabutylammonium fluoride in tetrahydrofuran is added to the above tetrahydrofuran solution, and the reaction is stirred for 2 hours at the same temperature. After the reaction is complete, the solvent is evaporated, and the mixture is separated by silica gel column chromatography using a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether:ethyl acetate of 1:1, to obtain compound (D).

[0077] In some embodiments, when R7 is a phosphate ester group, step S2 includes: phosphorylating the compound of formula (D) to replace the H in 5'-OH with a phosphate ester group, which includes, but is not limited to, monophosphate groups, diphosphate groups, triphosphate groups, tetraphosphate groups, etc.

[0078] In some embodiments, the 5'-OH in compound (D) undergoes a triphosphorylation reaction comprising the following steps: the compound (D) reacts with 1,8-bis(dimethylaminonaphthalene) and phosphorus oxychloride in trimethyl phosphate to generate an intermediate, which is then added to an N,N-dimethylformamide solution of tributylamine pyrophosphate in the presence of N,N-diisopropylethylamine (DIPEA) to generate a nucleotide analog of R7 being a triphosphate. A schematic diagram of the reaction is shown below:

[0079]

[0080] For example, R7 is a nucleotide analog of a triphosphate prepared according to the following steps: 1.0 mol of compound (D) and 2.0 mol of 1,8-bis(dimethylaminonaphthalene) are dissolved in dry trimethyl phosphate. 2.0 mol of phosphorus oxychloride is added to the above system at -10°C, and the mixture is stirred for 2 hours at the same temperature to generate an intermediate. The reaction solution containing the intermediate is transferred to a solution of N,N-dimethylformamide containing 3.0 mol of N,N-diisopropylethylamine and 2.0 mol of tributylammonium pyrophosphate, and the reaction is continued at -10°C for 3 hours. After the reaction is complete, the reaction is quenched with 0.1 mol / L triethylamine-carbonate buffer (TEAB), and the mixture is stirred for 1 hour at this temperature. A liquid chromatography separation solution is prepared with a gradient of (CH3CN:TEAB (0.1 mol / L) = 2 / 98 to 98 / 2).

[0081] In some specific embodiments, the reaction process of nucleotide analogs is represented as follows:

[0082]

[0083] The beneficial effects of this application will be further illustrated below with reference to embodiments and comparative examples.

[0084] Example 1

[0085] This embodiment provides a nucleotide analogue, which is prepared according to the following steps:

[0086] (1) In a 500 mL round-bottom flask equipped with a magnetic stirrer, thymidine (compound 1, Aldrich, 2 g, 8.2 mmol), imidazole (Aldrich, 1.72 g, 16.4 mmol), and tert-butyldimethylchlorosilane (Aldrich, 1.24 g, 8.2 mmol) were added. At room temperature, anhydrous N,N-dimethylformamide (Aldrich, 20 mL) was added, and the mixture was magnetically stirred overnight. After the reaction was complete, 100 mL of water and 100 mL of ethyl acetate were slowly added to the reaction system. The N,N-dimethylformamide was washed away with water in a separatory funnel. The organic layer was collected, the solvent was evaporated, and the mixture was separated by silica gel column chromatography. The eluent was a mixture of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether:ethyl acetate = 1:20. A white solid, compound 2, was obtained with a yield of 65%. The reaction diagram is shown below:

[0087]

[0088] The NMR spectrum of compound 2 is shown below: 1 H NMR (400MHz, CDCl3) δ9.36(s,1H),7.53(d,J=1.4Hz,1H),6.39(dd,J=8.3,5.6Hz,1H),4.44(dt,J=5.9,2.2Hz,1H),4.06(q,J=2.5Hz,1H),3.93–

[0089] 3.76(m,2H),2.49-1.99(m,2H),1.89(s,3H),0.91(s,9H),0.10(s,6H).

[0090] (2) At room temperature, 2-diphenylphosphine benzoic acid (compound 3, Aldrich, 0.2 g, 0.6 mmol), dicyclohexylcarbodiimide (DCC, Aldrich, 0.2 g, 0.9 mmol), 4-dimethylaminopyridine (Aldrich, 0.012 g, 0.09 mmol), and anhydrous dichloromethane (Aldrich, 13 mL) were added to a 100 mL round-bottom flask equipped with a magnetic stirrer. The mixture was stirred for 2 hours. Compound 2 (0.28 g, 0.72 mmol) was then added to the reaction mixture, and the reaction was continued for another 4 hours at the same temperature. The solvent was removed by evaporation, and the mixture was separated by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate as the eluent (petroleum ether:ethyl acetate = 3:1 by volume) to give a white solid (compound 5) in 85% yield. The reaction diagram is shown below:

[0091]

[0092] The NMR spectrum of compound 5 is shown below: 1H NMR(400MHz, CDCl3)δ8.05(s,1H),8.04-7.99(ddd,J=6.9,3.6,2.0Hz,1H),7.51(d,J=1.4Hz,1H),7.45–7.27(m,12H),6.92(ddd,J=7.5,4.1,1 .9Hz,1H),6.29(dd,J=9.1,5.3Hz,1H),5.29(d,J=6.0Hz,1H),2.04–2.29(m,2H),1.91(s,3H),1.26(t,J=7.2Hz,1H),0.91(s,9H),0.10(s,6H).

[0093] Example 3:

[0094]

[0095] (3) Compound 5 (0.357 g, 0.56 mmol), a tetrahydrofuran solution of tetrabutylammonium fluoride (1.0 mol / L, 1.1 mL), and tetrahydrofuran (Aldrich, 10 mL) were added to a 100 mL round-bottom flask equipped with a magnetic stirrer. The mixture was stirred at room temperature for 2 hours. The solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate as the eluent (by volume) to give a white solid (compound 6) in 98% yield. W (Molecular weight) = 530.52. The reaction diagram is shown below:

[0096]

[0097] The NMR spectrum of compound 6 is shown below: 1H NMR (400MHz, CDCl3) δ 8.10 (s, 1H), 8.04 (ddd, J = 6.9, 3.6, 2.0Hz, 1H), 7.42–7.27 (m, 12H), 6.93 (ddd, J = 7.7, 4.2, 1.8Hz, 1H), 6.03 (dd, J = 8.4, 5.9Hz, 1H), 5.44 (dt, J = 6.6, 2.4Hz, 1H), 3.89 (q, J = 2.5Hz, 1H), 3.85–3.76 (m, 2H), 2.51–2.16 (m, 3H), 1.92 (s, 3H).

[0098] (4) Prepare a dry 100mL two-necked flask (No. 1) equipped with a magnetic inlet and a three-way valve with an argon balloon. Weigh compound 6 (0.29g, 0.55mmol) and 1,8-bis(dimethylaminonaphthalene) (0.23g, 1.1mmol) and add them to flask No. 1. Prepare a dry 100mL two-necked flask (No. 2) equipped with a magnetic inlet and a three-way valve with an argon balloon. Weigh tributylammonium pyrophosphate (0.60g, 1.1mmol) and add it to flask No. 2. Inject argon gas into flask No. 1, add 10mL of trimethyl phosphate, stir until fully dissolved, and transfer the reaction mixture to 0℃. Slowly add phosphorus oxychloride (0.10mL, 1.1mmol) to the reaction system using a syringe. Stir at 0℃ for 1.5 hours. Inject argon gas into flask No. 2, and use a syringe to add 5mL of anhydrous DMF and DIPEA (0.45mL, 2.75mmol), stirring at 0℃ to dissolve. Maintaining the temperature at 0℃, the reaction solution in two-necked flask No. 1 was transferred to two-necked flask No. 2 using a syringe. After stirring for 3.5 hours, 5 mL of triethylamine-carbonate buffer (TEAB) was added to quench the reaction, and stirring was continued at this temperature for 1 hour. The reaction solution was prepared for liquid chromatography separation with a gradient of (CH3CN:TEAB (0.1 mol / L) = 2 / 98 to 98 / 2). The separated components were identified by mass spectrometry, and the corresponding mass spectrometry results are shown below. Figure 1 ( Figure 1 Molecular weight refers to molecular weight. The high-purity product solution was concentrated under vacuum using a rotary evaporator, transferred to centrifuge tubes, and freeze-dried until the product was a powdery solid (compound 8). Yield: 66%. W (Molecular weight) = 770.45. The reaction diagram is shown below:

[0099]

[0100] Example 2

[0101] This embodiment provides a nucleotide analog (compound 9), the structure of which is shown in the figure below.

[0102]

[0103] The difference between compound 9 and the preparation method of compound 8 provided in Example 1 is that, in step (4),

[0104] Prepare a dry 100 mL two-necked flask equipped with a magnetic inlet and a three-way valve with an argon balloon. Weigh compound 6 (0.29 g, 0.55 mmol) and 1,8-bis(dimethylaminonaphthalene) (0.23 g, 1.1 mmol) and add them to flask No. 1. Inject argon gas into flask No. 1, add 10 mL of trimethyl phosphate, and stir until fully dissolved. Transfer the reaction mixture to 0°C. Slowly add phosphorus oxychloride (0.10 mL, 1.1 mmol) to the reaction system using a syringe. Maintain stirring at 0°C for 1.5 hours. Add 5 mL of triethylamine-carbonate buffer (TEAB) to quench the reaction, and continue stirring at this temperature for 1 hour. Prepare a liquid chromatography separation solution with a gradient of (CH3CN:TEAB (0.1 mol / L) = 2 / 98 to 98 / 2). Concentrate the high-purity product solution under vacuum using a rotary evaporator, transfer it to centrifuge tubes, and freeze-dry until the product is a powdery solid (compound 9). Yield: 95%. W (Molecular weight) = 610.50. The reaction diagram is shown below:

[0105]

[0106] Example 3

[0107] This embodiment provides a nucleotide analog (compound 10), the structure of which is shown in the figure below.

[0108]

[0109] The difference between the preparation method of compound 10 and the compound of formula 8 provided in Example 1 is that, in step (4), when preparing the liquid chromatography separation reaction solution, LC-MS identification is used to separate the components corresponding to the molecular weight of compound 10, with a gradient of (CH3CN:TEAB (0.1mol / L) = 2 / 98 to 98 / 2). The separated components are identified by mass spectrometry. The high-purity product solution is concentrated under vacuum using a rotary evaporator, transferred to a centrifuge tube, and freeze-dried until the product is a powdery solid (compound 8). Yield: 18%. W (Weight-average molecular weight) = 690.47. The reaction diagram is shown below:

[0110]

[0111] Example 4

[0112] This embodiment provides a nucleotide analog (compound 11), the structure of which is shown in the figure below.

[0113]

[0114] The difference between the preparation method of compound 11 and the compound of formula 8 provided in Example 1 is that, in step (4), when preparing the liquid chromatography separation reaction solution, LC-MS identification is used to separate the components corresponding to the molecular weight of compound 11, with a gradient of (CH3CN:TEAB (0.1mol / L) = 2 / 98 to 98 / 2). The separated components are identified by mass spectrometry. The high-purity product solution is concentrated under vacuum using a rotary evaporator, transferred to a centrifuge tube, and freeze-dried until the product is a powdery solid (compound 8). Yield: 9%. W (Molecular weight) = 850.43. The reaction diagram is shown below:

[0115]

[0116]

[0117] Comparative Example 1

[0118] This comparative example provides a nucleotide analog with the following structure:

[0119]

[0120] Experimental Example 1

[0121] The reaction process of compound 8 provided in Example 1 was subjected to a cleavage experiment, and the schematic diagram of the reaction process is shown below:

[0122]

[0123] The specific operating method is as follows: The lyophilized compound 8 powder is prepared into a 1 mg / mL solution in a 1:1 mixture of pure water and acetonitrile, and mixed thoroughly. 0.5 mL of this solution is added to a 1 mg / mL aqueous solution of azidobutyric acid, and the mixture is inverted ten times. The mixture is allowed to stand at room temperature for 30 seconds, and the reaction is monitored by high-performance liquid chromatography (HPLC). The excision mechanism is shown in the figure below. The chromatograms of compound 8 before and after the excision experiment are shown in Figures 2(a) and 2(b). Figure 2(a) shows the chromatographic method (CH3CN:TEAB buffer (0.1 mol / L) = 2 / 98~98 / 2), injection concentration 5 mg / mL, and sample solvent pure water:acetonitrile = 1:1. Figure 2(b) shows the chromatographic method (CH3CN:TEAB buffer (0.1 mol / L) = 2 / 98~98 / 2), injection concentration 0.5 mg / mL, and sample solvent pure water:acetonitrile = 3:1.

[0124]

[0125] A schematic diagram illustrating the mechanism by which the trivalent phosphine blocking group is cleaved by azide compounds (where PTO represents a triphosphate group, and Base represents a base, a denitrogenated base, or its tautomer).

[0126] As can be seen from Figures 2(a) and 2(b), after reacting at room temperature for 30 seconds with the selected reagent containing an azide group, 58.05% of compound 8 had its protecting group on the 3'-OH cleaved.

[0127] Experimental Example 2

[0128] Compound 8 provided in Example 1 was reacted with a template-free terminal transferase to verify its ability to achieve single-base extension in one round of reaction. A schematic diagram of the reaction process is shown below.

[0129]

[0130] The specific steps include:

[0131] (1) Reaction system:

[0132] 50 mM NaCl, 0.25 mM CoCl2, 100 mM KAc, pH 7.0. Substrate: 0.1 OD oligo (14 nt continuous T bases), 1 mM of the nucleotide (T bases) with a modified group at the 3' end of this invention. Enzyme: 3 mg / mL wild-type template-free terminal transferase (TdT).

[0133] (2) Reaction conditions:

[0134] After reacting at 37°C for 30 minutes, 0.5M EDTA was immediately added to terminate the reaction, and the protein was denatured by heating at 70°C for 10 minutes. Then, urea denaturation PAGE gel was performed.

[0135] (3) Coupling result detection:

[0136] Analysis was performed using a 20% denaturing polyacrylamide gel electrophoresis tank. The prepared denaturing gel was placed on an appropriately sized electrophoresis tank filled with TBE buffer. Samples were loaded onto the gel. The gel was then subjected to 500V for 60 minutes. After the bands migrated to the appropriate height, the gel was released and transferred to a staining container. Staining with Syber Gold for 10 minutes allowed the gel running results to be observed under an electrophoresis apparatus. The results are shown below. Figure 3 As shown.

[0137] from Figure 3It can be seen that both reaction groups 1 and 2 have a 15nt band (14nt+1), indicating that the oligonucleotide chain (14T) can be extended by the terminal transferase. Since no bands longer than 15nt appear, this substrate can effectively block the extension of the next base before the 3' protecting group is removed. In summary, using the deoxyribonucleotide with a reversible blocking group at the 3' end provided in this application as a substrate, and utilizing wild-type template-free terminal transferases 1 (mouse) and 2 (white-throated sparrow) from different species, it is possible to extend the oligonucleotide chain (n+1) without a template chain, exhibiting both polymerization activity and good blocking effect. This demonstrates that this substrate can be used as a terminal transferase for extending oligonucleotide chains.

[0138] Experimental Example 3

[0139] The stability of the nucleotide analogs provided in Example 1 and Comparative Example 1 was tested. The specific steps were as follows: 1 mg of each lyophilized nucleotide analog powder was treated under the same conditions: dissolved in 1 mL of ultrapure water, kept at 80 °C for 12 h, and accelerated testing was carried out to compare the stability. The results are shown in the liquid chromatogram in Figure 4.

[0140] Figure 4(a) shows the high performance liquid chromatogram of compound 8 provided in Example 1 of this application before stability testing; Figure 4(b) shows the high performance liquid chromatogram of compound 8 provided in Example 1 of this application before stability testing.

[0141] Figure 4(c) shows the high-performance liquid chromatography (HPLC) chromatogram of the nucleotide analog provided by the compound provided in Comparative Example 1 before stability testing; Figure 4(d) shows the HPLC chromatogram of the nucleotide analog provided by the compound provided in Comparative Example 1 after stability testing.

[0142] Figure 4(a) uses the chromatographic method (CH3CN:TEAB buffer (0.1 mol / L) = 2 / 98~98 / 2), with an injection concentration of 0.3 mg / mL and a sample solvent of water:acetonitrile = 1:2; Figure 4(b) uses the chromatographic method (CH3CN:TEAB buffer (0.1 mol / L) = 2 / 98~98 / 2), with an injection concentration of 0.1 mg / mL and a sample solvent of pure water; Figure 4(c) uses the chromatographic method (CH3CN:TEAB buffer (0.1 mol / L) = 2 / 98~98 / 2), with an injection concentration of 0.3 mg / mL and a sample solvent of water:acetonitrile = 1:1; Figure 4(d) uses the chromatographic method (CH3CN:TEAB buffer (0.1 mol / L) = 2 / 98~98 / 2), with an injection concentration of 0.1 mg / mL and a sample solvent of pure water.

[0143] from Figures 4(a)-4(d)It can be seen that after treatment under the same conditions, the nucleotide analog I provided in Comparative Example 1 significantly decomposed into various impurities, while compound 8 did not significantly decompose. That is, the stability of compound 8 is higher than that of the nucleotide analog provided in Comparative Example 1.

[0144] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A nucleotide analogue, characterized in that, The nucleotide analog has the structure shown in formula (I): Among them, R1, R2, R3, and R4 are each independently selected from H, halogen, nitro, amino, sulfonyl, carboxyl, C1-C20 alkyl, C3-C20 cycloalkyl, C6-C20 aryl, C6-C20 arylC1-C20 alkyl, and C1-C20 alkoxy. R5 and R6 are each independently substituted or unsubstituted C6-C20 aryl groups; when R5 and R6 are each independently substituted C6-C10 aryl groups, the substituents are selected from halogens, nitro groups, amino groups, sulfonyl groups, carboxyl groups, C1-C20 alkyl groups, C3-C20 cycloalkyl groups, C6-C20 aryl groups, C6-C20 aryl-C1-C20 alkyl groups, and C1-C20 alkoxy groups. R7is selected from H, Base is selected from bases, denitrogenated bases, or their tautomers.

2. The nucleotide analogue of claim 1, wherein, R1, R2, R3, and R4 are each independently selected from H, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, C6-C10 arylC1-C6 alkyl, and C1-C6 alkoxy. Preferably, R1, R2, R3, and R4 are each independently selected from H, C1-C4 alkyl, C3-C6 epoxy, C6-C10 aryl, phenyl C1-C3 alkyl, and C1-C4 alkoxy. More preferably, R1, R2, R3, and R4 are each independently selected from H, methyl, ethyl, phenyl, methoxy, ethoxy, benzyl, and phenethyl.

3. The nucleotide analogue of claim 1, wherein R5 and R6 are each independently substituted or unsubstituted phenyl groups; when R5 and R6 are each independently substituted phenyl groups, the substituents are selected from halogen, nitro, amino, sulfonyl, carboxyl, C1-C10 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, C6-C10 arylC1-C6 alkyl, and C1-C6 alkoxy. Preferably, R5 and R6 are each independently phenyl or substituted phenyl. When R5 and R6 are each independently substituted phenyl, the substituent is selected from halogen, nitro, amino, sulfonyl, carboxyl, C1-C4 alkyl, C3-C6 epoxy, C6-C10 aryl, phenyl C1-C3 alkyl, and C1-C4 alkoxy. More preferably, R5 and R6 are each independently phenyl or substituted phenyl. When R5 and R6 are each independently substituted phenyl, the substituent is selected from methyl, ethyl, phenyl, methoxy, ethoxy, benzyl, and phenethyl.

4. The nucleotide analogue of claim 1, wherein R7is selected from Preferably, R7is selected from 5. The nucleotide analogue of claim 1, wherein Base is selected from bases, adenine, 7-deadenine, thymine, uracil, cytosine, guanine, 7-deadenine, or their tautomers; Preferably, Base is selected from bases.

6. The method of producing a nucleotide analogue according to any one of claims 1 to 5, wherein The preparation method includes: Step S1: Provide compound (A), and esterify compound (A) with compound (B) to obtain compound (C); wherein, in formula (A), P represents a protecting group; Step S2 involves removing the protecting group P from the compound of formula (C) and optionally subjecting it to phosphorylation to obtain the nucleotide analog.

7. The production method according to claim 6, characterized by, In step S1, the molar ratio of compound (B) to compound (A) is 1:1.1-1.5, preferably 1:1.1-1.3; Preferably, the esterification reaction is carried out under the activation of an activator, and the activator preferably includes at least one of dicyclohexylcarbodiimide, 4-dimethylaminopyridine, and 2,4,6-trichlorobenzoyl chloride.

8. The preparation method according to claim 6, characterized in that, Step S2 includes: removing the protecting group P from the compound of formula (C) to obtain the compound of formula (D), and subjecting the compound of formula (D) to a phosphorylation reaction to obtain the nucleotide analog; 9. The use of the nucleotide analogue according to any one of claims 1 to 5 in the enzymatic synthesis of oligonucleotides.

10. The use of the nucleotide analogue according to any one of claims 1 to 5 in sequencing.

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