3'-o-reversibly blocked nucleotides and their use in template-free enzymatic nucleic acid synthesis

Abstract

The present application relates to the field of enzymatic nucleic acid synthesis, and in particular, provides a 3'-O-reversible blocked nucleotide and its application in template-free enzymatic nucleic acid synthesis. The 3'-O-blocked nucleotide provided by the present application has a cyanovinyl as blocking group. The cyanovinyl can reversibly block the 3'-OH of the nucleotide. The cyanovinyl has a small volume, a relatively stable structure, is easy to synthesize, and has a low cost. Meanwhile, the cyanovinyl can interact with the catalytic site of TdT enzyme through hydrogen bond, stably combine the conformation, and is very conducive to the catalytic reaction of TdT enzyme, thereby improving the reaction rate of enzymatic nucleic acid synthesis.

Description

Technical Field

[0001] This invention relates to the field of enzymatic nucleic acid synthesis, and more specifically, to a 3'-O-reversibly blocked nucleotide and its application in template-free enzymatic nucleic acid synthesis. Background Technology

[0002] Currently, the widely used oligonucleotide synthesis method is a chemical synthesis technique based on a four-step process of solid-phase phosphorous amide: deprotection, coupling, capping, and oxidation. However, due to the limitations of synthetic chemistry, its maximum synthetic length is approximately 200 nt, which is insufficient to fully meet the demands of synthetic biology and DNA storage technologies for high-throughput, high-fidelity, long-fragment, and low-cost DNA synthesis. Therefore, further development of new enzymatic DNA synthesis techniques is needed.

[0003] In enzymatic DNA synthesis, TdT enzyme (Terminal deoxynucleotidyltransferase) is a DNA polymerase that catalyzes the binding of deoxyribonucleic acid (dNTPs) to the 3'-OH end of a single-stranded DNA molecule under template-free conditions. Compared with chemical methods, enzymatic DNA synthesis based on TdT enzymes has great potential: (1) TdT enzymes have high catalytic efficiency, and their template-free DNA synthesis can reach a single-strand length of 8000 nt, which can increase the DNA single-strand synthesis length by several orders of magnitude, breaking the limitation of chemical synthesis length; (2) The enzymatic synthesis process has mild reaction conditions, which reduces damage to DNA and helps to improve the accuracy of DNA synthesis products; (3) The reaction process is carried out in an aqueous phase, without the need for toxic compounds; (4) There are fewer synthesis steps, faster cycle rate, and higher synthesis efficiency, which can significantly reduce the cost of DNA synthesis. In addition, DNA enzymatic synthesis technology is fully compatible with natural DNA (DNA with unprotected 3'-OH), thereby enabling artificial modification of natural DNA. The development of DNA enzymatic synthesis technology will greatly enhance synthetic biology's ability to design and assemble gene networks, and will also bring about significant changes in fields such as DNA data storage, the design and manufacture of DNA nanomaterials.

[0004] However, natural TdT enzymes can only randomly add dNTPs to the 3' end of the DNA strand, making it impossible to precisely control the enzymatic elongation process and thus failing to meet the requirements of artificial DNA synthesis. Therefore, the key to enzymatic DNA synthesis technology is to construct an appropriate reaction system to achieve precise control over the synthesis of DNA strands by TdT enzymes.

[0005] Based on the catalytic specificity of TdT enzymes, an important control strategy is to utilize reversible termination groups to block the 3'-OH bonding site of dNTPs, thereby controlling enzymatic DNA elongation. Specifically, when the 3'-OH group required for DNA elongation is blocked by a reversible termination group, the TdT enzyme-catalyzed DNA chain elongation will terminate, thus achieving precise control over enzymatic DNA synthesis. Subsequently, the reversible termination group is deprotected, releasing the 3'-OH group of the DNA chain, and the next round of enzymatic elongation reaction continues. This "stop-restart" cycle is repeated to achieve template-free synthesis of the target DNA chain catalyzed by TdT enzymes.

[0006] Introducing a protecting group onto the 3'-OH of a nucleotide affects substrate-enzyme binding and reduces the TdT enzyme's catalytic elongation rate. Therefore, selecting a suitable reversible termination group to control TdT enzymatic elongation is crucial for template-free DNA synthesis. Methyl, 2-nitrobenzyl, allyl, azidomethyl, amino, and tert-butoxyethoxy groups can all serve as reversible termination protecting groups. However, these protecting groups suffer from problems such as low catalytic elongation rate, insufficient precision, low deprotection efficiency, complex dNTP synthesis processes, and high costs. Currently, they cannot meet the requirements of industrial DNA synthesis and have not been widely applied to genome synthesis. Therefore, enzymatic DNA synthesis technology requires further development and optimization, and there is an urgent need to develop template-free TdT enzymatic DNA synthesis technology based on novel reversible termination groups.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The primary objective of this invention is to provide a 3'-O-blocked nucleotide.

[0009] A second objective of this invention is to provide the application of the above-mentioned 3'-O-blocked nucleotides in enzymatic nucleic acid synthesis.

[0010] A third objective of this invention is to provide a method for preparing the above-mentioned 3'-O-blocked nucleotide.

[0011] A fourth objective of this invention is to provide a method for deprotecting the 3'-O-blocked nucleotides described above.

[0012] The fifth objective of this invention is to provide a method for enzyme-catalyzed nucleic acid synthesis.

[0013] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0014] A 3'-O-blocked nucleotide, wherein the blocking group is cyanovinyl.

[0015] Furthermore, the nucleotide is selected from any one of deoxyadenosine monophosphate and its derivatives, deoxyguanosine monophosphate and its derivatives, deoxycytidine monophosphate and its derivatives, deoxythymidine monophosphate and its derivatives, adenosine monophosphate and its derivatives, guanosine monophosphate and its derivatives, cytidine monophosphate and its derivatives, and uridine monophosphate and its derivatives.

[0016] Applications of the above-mentioned 3'-O-blocked nucleotides in enzymatic nucleic acid synthesis;

[0017] Preferably, the enzyme is a TdT enzyme;

[0018] Preferably, the enzymatic nucleic acid synthesis is a template-free enzymatic nucleic acid synthesis that is reversibly terminated.

[0019] The above method for preparing 3'-O-blocked nucleotides involves replacing the 3'-OH group of the nucleotide with 3'-O-cyanovinyl to obtain 3'-O-blocked nucleotides.

[0020] Furthermore, the 3'-OH of the nucleotide first undergoes an esterification reaction with formic acid to generate a methyl ester, and then reacts with triphenylphosphine acetonitrile to generate a 3'-O-cyanovinyl ester, thus obtaining a 3'-O-blocked nucleotide.

[0021] Furthermore, using 5'-OTBDMS-protected nucleosides as raw materials, the 3'-OH of the 5'-OTBDMS-protected nucleosides first undergoes an esterification reaction with formic acid to generate a methyl ester, and then reacts with triphenylphosphine acetonitrile to generate 3'-O-cyanovinyl. After obtaining 3'-O-cyanovinyl, it is then subjected to the removal of 5'-OTBDMS and phosphorylation to obtain a 3'-O-blocked nucleotide.

[0022] The above-mentioned method for deprotecting 3'-O-blocked nucleotides involves using a cleavage agent to cut the blocking group in the 3'-O-blocked nucleotides to obtain 3'-OH nucleotides.

[0023] Furthermore, the pyrolysis agent is a palladium complex, preferably palladium dichloride diacetonitrile;

[0024] Preferably, the concentration of palladium dichloride diacetonitrile is 0.04-0.06M, and the cutting conditions are a reaction at 55-65℃ for 0.5-10 min.

[0025] A method for enzyme-catalyzed nucleic acid synthesis, wherein the raw materials of the method include the above-mentioned 3'-O-blocked nucleotides.

[0026] Furthermore, the enzymatic nucleic acid synthesis is a template-free enzymatic nucleic acid synthesis that is reversibly terminated;

[0027] Preferably, the enzyme is a TdT enzyme;

[0028] Preferably, the above-mentioned deprotection method is used to restart the termination of nucleic acid extension.

[0029] A synthesis instrument for generating nucleic acids based on the above method.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] This invention provides a 3'-O-blocked nucleotide with a cyanovinyl blocking group. The cyanovinyl group serves to reversibly block the 3'-OH group of the nucleotide. During enzymatic nucleic acid synthesis, using this 3'-O-blocked nucleotide allows for the cessation of enzymatic nucleic acid synthesis because its 3'-OH group is blocked. Simultaneously, the cyanovinyl group can be cleaved, converting the 3'-O-cyanovinyl group to 3'-OH, thus restarting the enzymatic nucleic acid synthesis. By repeating this "stop-restart" process, efficient and precise control over the synthesis of specific nucleic acid sequences can be achieved.

[0032] Cyanovinyl groups are small in size, relatively stable in structure, easy to synthesize, and low in cost. Furthermore, the 3'-O-cyanovinyl group (3'-O-Cyanovinyl), as a vinyl ether, can be conveniently and efficiently cleaved by [Pd] catalysis, achieving orthogonal deprotection. In addition, for example, against TdT enzymes, the 3'-O-blocked nucleotide exhibits a unique binding effect: the small size and high electronegativity of the hydrophilic cyano group on the 3'-O-cyanovinyl group are highly favorable for TdT enzyme catalysis, increasing the reaction rate of enzymatic nucleic acid synthesis. For instance, under the same conditions, using 3'-O-cyanovinyl-dTTP (3'-O-Cyanovinyl-dTTP, CTTP) as a substrate, primer extension can be completed in 1 hour, while the control substrate 3'-O-azidomethyl-dTTP (3'-O-Azidomethyl-dTTP, ATTP) requires 4.5 hours. Attached Figure Description

[0033] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 This is a schematic diagram illustrating the principle of 3'-O-blocked nucleotide deprotection in an embodiment of the present invention.

[0035] Figure 2 This is a schematic diagram illustrating the principle of template-free enzymatic DNA synthesis in an embodiment of the present invention.

[0036] Figure 3The results of gel electrophoresis detection of the TdT enzymatic DNA extension reaction in Example 1 of this invention;

[0037] Figure 4 The results of gel electrophoresis detection of the deprotection reaction in Example 2 of this invention;

[0038] Figure 5 This is a schematic diagram of the synthesis process of 3'-O-cyanovinyl-dNTP in Example 3 of the present invention;

[0039] Figure 6 This is a molecular docking diagram of the 3'-O-cyanovinyl-dNTP substrate of the present invention with the TdT enzyme (PDB: 4I2J). Detailed Implementation

[0040] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply.

[0041] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be used in this invention.

[0042] This invention achieves reversible blocking of the 3'-OH of nucleotides by utilizing cyanovinyl (also known as cyanovinyl), that is, the 3'-O-blocked nucleotide of this invention is the nucleotide whose 3'-OH is replaced by 3'-O-cyanovinyl.

[0043] Cyanovinyl groups are small in size, relatively stable in structure, easy to synthesize, and low in cost. Furthermore, the 3'-O-cyanovinyl group (3'-O-Cyanovinyl), as a vinyl ether, can be conveniently and efficiently deprotected by [Pd]-catalyzed cleavage. In addition, docking analysis for TdT enzymes shows (e.g.) Figure 6The 3'-O-blocked nucleotide has a unique binding effect with TdT enzymes: the small, highly electronegative hydrophilic cyano group on the 3'-O-cyanovinyl group can extend into the positively charged region and solvation cavity formed by residues Lys338, Arg336, Ser453, Gly452, and Arg454 on the TdT enzyme (PDB: 4I2J), and interact with phosphate groups, water molecules, and Arg454 through hydrogen bonding, stabilizing the 3'-O-cyanovinyl-d The NTP conformation and its binding to TdT enzymes are highly favorable for TdT enzyme-catalyzed reactions, increasing the reaction rate of enzymatic nucleic acid synthesis. For example, under the same conditions, using 3'-O-cyanovinyl-dTTP (CTTP) as a substrate, primer extension can be completed in 1 hour, while the control substrate 3'-O-azidomethyl-dTTP (ATTP) requires 4.5 hours.

[0044] It should be noted that "3'-O-blocked nucleotides" refer to nucleotides used in nucleic acid synthesis whose 3'-OH terminus is protected by an additional group (blocking group, also known as a protecting group) to prevent further addition of nucleotides and thus stop nucleic acid synthesis. In this invention, the blocking group of the 3'-O-blocked nucleotide is cyanovinyl, and the nucleotide can be, for example, any one of deoxyadenosine monophosphate and its derivatives, deoxyguanosine monophosphate and its derivatives, deoxycytidine monophosphate and its derivatives, deoxythymidine monophosphate and its derivatives, adenosine monophosphate and its derivatives, guanosine monophosphate and its derivatives, cytidine monophosphate and its derivatives, and uridine monophosphate and its derivatives, and is further preferably dATP, dGTP, dCTP, dTTP, ATP, GTP, CTP, or TTP.

[0045] The 3'-O-blocked nucleotide provided by this invention can be used as a raw material for enzymatic nucleic acid synthesis. Its blocking group is cyanovinyl, which reversibly blocks the 3'-OH group of the nucleotide. During enzymatic nucleic acid synthesis, using this 3'-O-blocked nucleotide, the 3'-OH group is blocked, thus stopping the synthesis. Simultaneously, the cyanovinyl group can be cleaved, converting the 3'-O-cyanovinyl group to 3'-OH, restarting the synthesis. Repeating this "stop-restart" process allows for efficient and precise control of the synthesis of specific nucleic acid sequences. The enzyme used for enzymatic nucleic acid synthesis is preferably a TdT enzyme, more preferably a template-free enzymatic nucleic acid synthesis method with reversible termination, and even more preferably template-independent enzymatic oligonucleotide synthesis (TiEOS). Enzymatic nucleic acid synthesis refers to the method of synthesizing DNA length using enzymes, or the method of synthesizing RNA length using enzymes, by introducing nucleotides (n+1) to extend the nucleic acid (n) chain.

[0046] The method for preparing 3'-O-blocked nucleotides provided by this invention involves replacing the 3'-OH group of a nucleotide with a 3'-O-cyanovinyl group to obtain the 3'-O-blocked nucleotide. Specifically, the 3'-OH group of the nucleotide first undergoes an esterification reaction with formic acid to generate a methyl ester, and then reacts with triphenylphosphine acetonitrile to generate a 3'-O-cyanovinyl group, thus obtaining the 3'-O-blocked nucleotide. To ensure that only the 3'-terminal hydroxyl group of the nucleotide participates in the reaction, 5'-OTBDMS-protected nucleosides are used as raw materials. The 3'-OH group of the 5'-OTBDMS-protected nucleosides first undergoes an esterification reaction with formic acid to generate a methyl ester, and then reacts with triphenylphosphine acetonitrile to generate a 3'-O-cyanovinyl group. After obtaining the 3'-O-cyanovinyl group, it is subsequently subjected to removal of 5'-OTBDMS and phosphorylation to obtain the 3'-O-blocked nucleotide.

[0047] The 3'-O-blocked nucleotide provided by this invention has a cyanovinyl blocking group at its 3' end that can be cleaved by a cleaving agent to become 3'-OH, thus deprotecting it and allowing it to be added to nucleotides to complete nucleic acid elongation. The cleaving agent is preferably a palladium complex, more preferably palladium diacetonitrile dichloride, with a concentration of 0.04-0.06 M, and the cleavage conditions are 55-65°C for 0.5-10 min. The concentration of palladium diacetonitrile dichloride can be, but is not limited to, 0.04 M, 0.05 M, or 0.06 M; the cleavage conditions can be, but are not limited to, 55°C, 57°C, 59°C, 61°C, 63°C, or 65°C; and the cleavage time can be, but is not limited to, 0.5 min, 1 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min. The deprotection method provided by this invention is illustrated in the schematic diagram below. Figure 1 As shown.

[0048] This invention provides a method for enzymatic nucleic acid synthesis, wherein the raw materials for nucleic acid synthesis include the 3'-O-blocked nucleotides provided by this invention. The preferred enzyme is TdT enzyme.

[0049] For example, this method is a template-free nucleic acid synthesis method based on a strategy of reversibly blocking 3'-OH bonding sites, as illustrated in the schematic diagram below. Figure 2 As shown, the method is as follows:

[0050] (1) Provide a starting sequence, (2) add the 3'-O-blocked nucleotide of the present invention, and in the presence of TdT enzyme, the starting sequence is extended. Then, remove all reagents, use a cleavage agent to cleave the cyanovinyl group of the 3'-O-blocked nucleotide, and then remove the cleavage agent. Repeating step (2) can achieve the addition of more than one nucleotide in the starting sequence.

[0051] Finally, this invention provides a nucleic acid synthesis instrument based on the above-described enzymatic nucleic acid synthesis method.

[0052] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0053] The amino acid sequence of the TdT enzyme used in the embodiments of the present invention is shown in SEQ ID NO.1.

[0054] MGHHHHHHHHHHSSGHIDDDDKHMSPSPVPGSQNVPAPAVKKISQYACQRRTTLNNYNQLFTDALDILAENDELRENEGSCLAFMRASSVLKSLPFPITSM KDTEGIPCLGDKVKSIIEGIIEDGESSEAKAVLNDERYKSFKLFTSVFGVGLKTAEKWFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCVNRPEAEA VSMLVKEAVVTFLPDALVTMTGGFRRGKMTGHDVDFLITSPEATEDEEQQLLHKVTDFWKQQGLLLYCDILESTFEKFKQPSRKVDALDHFQKCFLILKLDH GRVHSEKSGQQEGKGWKAIRVDLVMCPYDRRAFALLGWTGSRQFERDLRRYATHERKMMLDNHALYDRTKRVFLEAESEEEIFAHLGLDYIEPWERNA(SEQ ID NO.1).

[0055] Docking analysis shows that the 3'-O-blocked nucleotides (e.g., 3'-O-cyanovinyl-dNTP substrates) provided by this invention have a unique binding effect with TdT enzymes: the hydrophilic cyano group on the 3'-O-cyanovinyl group is small in size and highly electronegative, and can extend into the positively charged region and solvation cavity formed by residues Lys338, Arg336, Ser453, Gly452, Arg454 on the TdT enzyme (PDB: 4I2J), and interact with phosphate groups, water molecules, and Arg454 through hydrogen bonding, stabilizing the conformation of 3'-O-cyanovinyl-dNTP and its binding to TdT enzymes, which is very beneficial to TdT enzyme catalysis and improves the rate of enzymatic DNA reaction based on 3'-O-cyanovinyl-dNTP substrates.

[0056] To illustrate the template-free TdT enzymatic DNA elongation and orthogonal deprotection performance based on 3'-O-cyanovinyl-dNTP substrate, this invention uses 3'-O-cyanovinyl-dTTP as the substrate for performance testing, with 3'-O-azidomethyl-dTTP (US10059929B2), native dNTPs, and a blank as controls. For rapid and convenient detection of the OligoDNA reaction, this invention uses the addition of native dNTPs to the reaction system. The principle is that the TdT enzyme catalyzes the addition of native dNTP substrates to the 3'-OH end of the DNA strand at a much higher rate than with non-native 3'-O-substituted dNTP substrates. When dNTPmix is ​​added to the reaction system and incubated for a certain period, unreacted oligoDNA undergoes rapid elongation, randomly adding nucleotides to generate DNA strands of different lengths. However, the 3'-OH of already reacted oligoDNA is blocked by a reversible termination group, preventing further nucleotide addition. Specific implementation schemes are as follows:

[0057] Example 1: TdT Enzymatic DNA Extension Experiment

[0058] (I) Experimental Materials

[0059] 1. OligoDNA sequence: 5'-GCAGA TAATA CGACT CACTA TAGGG ATTTA GACTA CCCCAAAAAC GAAGG GGACT AAAAC-3' (60nt, SEQ ID NO.2);

[0060] 2.TdT (0.12mg / mL) (SEQ ID NO.1);

[0061] 3. TdT reaction buffer: 10×TdT reaction buffer, 10×CoCl2 (2.5mM) (NEB, BO315S), of which 1×TdT buffer: 20mM Tris-Acetate, 50mM KAc, 10mM MgAc2, pH 7.9@25℃;

[0062] 4. Substrate: 3'-O-cyanovinyl-dTTP (CTTP, 10mM), 3'-O-Azidomethyl-dTTP (ATTP, 10mM), dNTP mix (dNTP, 10mM);

[0063] 5.ddH2O;

[0064] (II) Experimental Procedure

[0065] (1) Prepare four TdT-catalyzed DNA extension reaction systems:

[0066] OligoDNA (5 μL, 2.5 mM, 60 nt), 10×TdT reaction buffer (5 μL), 2.5 mM CoCl2 solution (5 μL), 8.3 μL TdT (0.12 mg / mL), and the corresponding nucleotide substrate were added to a microcentrifuge tube (1.5 mL), and the mixture was diluted to 50 μL with ddH2O.

[0067]

[0068]

[0069] (2) Dilute the mixture to 50 μL with ddH2O. Make 3 copies of each of the CTTP system and ATTP system, and group them according to the subsequent incubation time.

[0070] (3) The mixture was incubated at 37°C for 1 h, 4.5 h and 5 h, depending on the different substrates.

[0071] (4) Place 50 μL of the mixture on a 3% agar gel (prepare the gel by adding 80 mL of 1×TBE to 2.4 g of solute) and perform gel electrophoresis detection;

[0072] (5) For the CTTP and ATTP systems, after incubation, no inactivation operation is performed. 2 μL of dNTP is added to each system and incubated for 3 h before gel electrophoresis is performed to detect whether the oligoDNA in the system has reacted completely.

[0073] (III) Experimental Results

[0074] The results of the TdT enzyme-catalyzed DNA extension reaction are as follows: Figure 3 As shown (Lan 1: CTTP system, incubated for 5 h; Lan 2: ATTP system, incubated for 5 h; Lan 3: CTTP system, dNTP added after 5 h incubation; Lan 4: ATTP system, dNTP added after 5 h incubation; Lan 5: CTTP system, dNTP added after 4.5 h incubation; Lan 6: ATTP system, dNTP added after 4.5 h incubation; Lan 7: CTTP system, dNTP added after 1 h incubation; Lan 8: ATTP system, dNTP added after 1 h incubation; Lan 9: blank system; Lan 10: dNTP system, incubated for 3 h; Lan 11: 760 bp marker), the 3'-O-cyanovinyl-dTTP (CTTP) substrate provided by this invention can complete oligo DNA extension in 1 h, while the control 3'-O-azidomethyl-dTTP (ATTP) requires 4.5 h.

[0075] Example 2, Deprotection Experiment

[0076] (I) Experimental Materials

[0077] Deprotection reagents: 0.5M TCEP (pH=10) solution was used for ATTP substrates, and 0.05M palladium dichloride diacetonitrile (PdCl2(CH3CN)2) solution was used for CTTP substrates;

[0078] (II) Experimental Procedure

[0079] (1) Prepare four TdT-catalyzed DNA extension reaction systems:

[0080] OligoDNA (15 μL, 2.5 mM, 60 nt), 10×TdT reaction buffer (15 μL), 2.5 mM CoCl2 solution (15 μL), 16.6 μL TdT (0.12 mg / mL), and the corresponding nucleotide substrate were added to a microcentrifuge tube (1.5 mL), and the mixture was diluted to 150 μL with ddH2O.

[0081] 1 CTTP 1.5 2 ATTP 1.5 3 dNTP 12 4 blank --(No addition)

[0082] (2) The mixture was incubated at 37°C for 1 h, 4.5 h and 5 h, depending on the different substrates;

[0083] (3) Take out the reaction solution, add 2.5 μL of 0.5 M TCEP (pH = 10) solution to the ATTP system, and add 0.05 M PdCl2 (CH3CN)2 solution to the CTTP system;

[0084] (4) Heat to 60°C and react for 5 minutes to achieve deprotection;

[0085] (5) The product was purified using a DNA purification column (EZ-10, Sangon Biotech), the eluent was collected and concentrated;

[0086] (6) Add 10×TdT reaction buffer (15μL), CoCl2 solution (2.5mM, 15μL), dNTP (10mM, 12μL) and 16.6μL TdT (0.12mg / ml) to the concentrated ATTP system and CTTP system respectively, and incubate at 37℃ for 1.5h. Dilute the mixture to 150μL with ddH2O.

[0087] (7) Place 50 μL of the mixture on a 3% agar gel (prepared by adding 2.4 g of solute to 80 ml of 1×TBE) and perform the test.

[0088] (III) Experimental Results

[0089] Deprotection reaction test results as follows Figure 4 As shown in the figures (Lan 1: blank system; Lan 2: blank system, deprotected with Pd reagent; Lan 3: ATTP system, deprotected with TCEP reagent after 5 h incubation; Lan 4: CTTP system, deprotected with Pd reagent after 5 h incubation; Lan 5: ATTP system, deprotected with TCEP reagent after 5 h incubation, then dNTP added; Lan 6: CTTP system, deprotected with Pd reagent after 5 h incubation, then dNTP added; Lan 7: CTTP system, deprotected with Pd reagent after 1 h incubation, then dNTP added; Lan 8: 760 bp marker), the 3'-O-cyanovinyl-dTTP (CTTP) substrate provided by this invention can be orthogonally deprotected at 60 °C for 5 min.

[0090] Example 3: Synthesis of 3'-O-cyanovinyl-dNTP

[0091] Using 5'-OTBDMS-protected nucleosides as starting materials, the synthesis first involves a selective esterification reaction at the 3'-OH position with formic acid via a condensation reaction to generate the corresponding methyl ester. Utilizing the wittig reactivity of the methyl ester, it reacts with a phosphorus ylide reagent (triphenylphosphine)acetonitrile to generate 3'-O-cyanovinyl. After removing 5'-OTBDMS, the 3'-O-cyanovinyl-dNTP precursor is obtained, followed by triphosphorylation and deprotection to synthesize the desired substrate. The specific synthetic flow chart is shown below. Figure 5 Shown ((i) HCOOH, EDCI, DMAP, DCM, 0℃-room temperature, 6h; (ii) (1) 2-(triphenylphosphaneylidene)acetonitrile (Wittigreagents), Toluene, 120℃, 9h; (2) TBAF·3H2O, THF, room temperature, 1h; (iv) (1) POCl3, 1,8-Bis(dimethyl amino)naphthalene, (MeO)3PO, 0℃, 2h; (2) Tributylammoniumpyrophosphate, Bu3N, CH3CN, 0℃, 10min; (3) NH4OH, 25℃, 16h).

[0092] a:B=T

[0093] 2a 3'-O-formyl-5'-O-tert-butyldimethylsilyl-dT

[0094] Under ice bath conditions, formic acid (2 g / 1.6 mL, 27.6 mmol) was slowly added dropwise to 30 mL of dichloromethane (DCM) solution containing 1a (5 g, 14 mmol), DMAP (180 mg, 10% mol), and EDCI (10.7 g, 56 mmol). After the addition was complete, the reaction was allowed to proceed at room temperature for 6 h. The reaction was confirmed to be complete by TLC [DCM / MeOH = 30:1]. The product was extracted with DCM, dried over anhydrous MgSO4, and purified by column chromatography [DCM / CH3OH = 0-10%] to obtain 4.7 g of red solid product 2a, with a yield of 87%.

[0095] 1 H NMR (400MHz, CDCl3) δ8.69 (br s,1H),8.06(s,1H),7.54(d,J=1.2Hz,1H),6.39(dd,J=9.2,5.2Hz,1H),5.39(d,J=5.6Hz,1H),4.15(d,J=1.2Hz,1H),3 .93(d,J=1.6Hz,2H),2.48(dd,J=14.0,5.2Hz,1H),2.19-2.10(m,1H),1.93(d,J=1.2Hz,3H),0.93(s,9H),0.14(s,6H).

[0096] 3a 3'-O-cyanovinyl-dT

[0097] Take a pressure-resistant bottle, add 2a (4g, 10.4mmol) and 2-cyanomethylenetriphenylphosphine (6.28g, 20.8mmol), add to 40mL of toluene solution, heat to 120℃, and react for 9h. TLC detection showed complete reaction [DCM / MeOH = 30:1]. Directly evaporate to dryness, and purify by column chromatography [CH3OH / DCM = 0-10%] to obtain 8g of intermediate product containing impurities. Dissolve it in THF (40mL), add TBAF·3H2O (6.08g, 16mmol), react for 1h, TLC detection showed complete reaction [DCM / MeOH = 10:1], extract with EA, dry to anhydrous Na2SO4, and purify by column chromatography [DCM / CH3OH = 0-10%] to obtain 2.12g of product 3a, yield 75%.

[0098] 1H NMR(400MHz,CH3OH-d3)δ7.78(d,J=1.2Hz,1H),7.46(d,J=12.8Hz,1H),6.24(dd,J=8.0,2.0Hz,1H),5 .05(d,J=12.8Hz,1H),4.16-4.13(m,1H),3.90-3.78(m,2H),2.43-2.38(m,2H),1.88(d,J=1.2Hz,3H).

[0099] 4a 3'-O-cyanovinyl-dTTP

[0100] Compound 3a (2.0 g, 7.16 mmol, 1.0 eq) and 1,8-bis(methylaminonaphthalene) (3.07 g, 14.3 mmol, 2.0 eq) were dissolved in 29 mL of trimethyl phosphate, purged three times with argon, and the system was cooled to 0 °C in an ice-water bath. POCl3 (1.61 g, 10.7 mmol, 1.5 eq) was dissolved in 2.4 mL of trimethyl phosphate and slowly added dropwise to the reaction system. After the addition was complete, the system was kept at 0 °C for 2 hours. In a fresh reaction flask, tributylpyrophosphoramide (7.8 g, 14.3 mmol, 2 eq) and Bu3N (6.62 g, 35.8 mmol, 5 eq) were dissolved in 50 mL of ACN, purged three times with argon, and cooled to 0 °C in an ice-water bath. The monophosphate generated earlier was slowly added dropwise to the fresh system, and the reaction was kept in an ice-water bath for 10 minutes. The reaction was quenched with TEAB (24 mL, 1 mol / L). The reaction solution was separated and eluted using DEAE (22g, H2O:TEAB = 1:0–4:1). The fraction containing the product was collected and concentrated under reduced pressure to 50 mL at 30 °C. Prep-HPLC was performed (mobile phase A: 50 mM triethylamine acetate; mobile phase B: chromatographically pure acetonitrile; elution program: 0–8 min from A to 20% B, 8–12 min holding at 20% B, 12–15 min from B to 40% and holding until product was obtained). The prepared product was concentrated, salt replaced, and lyophilized to obtain 440 mg of a white solid, yield: 10.5%.

[0101] 1 H NMR(400MHz,D2O)δ7.81(d,J=0.8Hz,1H),7.53(d,J=13.2Hz,1H),6.41(dd,J=9.0,5.8Hz,1H),5.16(d, J=4.8Hz,1H),5.08(d,J=13.0Hz,1H),4.49(s,1H),4.30-4.24(m,2H),2.56-2.50(m,2H),1.96(s,3H); 31PNMR(162MHz,D2O)δ-5.88(d),-11.37(d),-21.65(t).

[0102] b:B=A Bz

[0103] 2b 3'-O-formyl-5'-O-tert-butyldimethylsilyl-N6-benzoyl-dA

[0104] Under ice bath conditions, DMAP (0.124 g, 1.01 mmol) and EDCI (8.16 g, 42.59 mmol) were added to a 35 mL solution of DCM containing 5 g (10.65 mmol) of 1b. Formic acid (0.98 g / 0.803 mL, 21.29 mmol) in 10 mL of DCM was then slowly added dropwise. After the addition was complete, the reaction was allowed to proceed at room temperature for 6 h. TLC was used to confirm the completeness of the reaction [DCM / MeOH = 10:1]. The product was washed with water, extracted with DCM, dried over anhydrous MgSO4, and purified by column chromatography [CH3OH / DCM = 0-10%] to obtain 4.85 g of product 2b, yield: 91.5%.

[0105] 1 H NMR(400MHz,CDCl3)δ9.17(br s,1H),8.82(s,1H),8.40(s,1H),8.13(s,1H),8.06(d,J=7.6Hz,2H),7.64(t,J=7.2Hz,1H),7.56(t,J=7.6Hz,2H),6.63(dd ,J=8.2,6.0Hz,1H),5.60(d,J=5.2Hz,1H),4.29(s,1H),3.96(d,J=2.2Hz,2H),2.80-2.69(m,2H),0.93(s,9H),0.13(s,6H).

[0106] 3b 3'-O-cyanovinyl-N6-benzoyl-dA

[0107] Take a pressure-resistant bottle, add 2b (5.78 g, 12.2 mmol), dissolve in 40 mL of toluene, add triphenylphosphine acetonitrile (7.3 g, 24.4 mmol), and heat in an oil bath to 120-130 °C for 9 h. TLC detection showed the reaction was complete [DCM / EA = 1 mL: 0.4 mL]. Directly evaporate to dryness, and purify by column chromatography [CH3OH / DCM = 0-10%] to obtain 8.27 g of impurity intermediate. Dissolve the intermediate in THF (40 mL), add TBAF·3H2O (11.5 g, 36.6 mmol), and react at room temperature for 1 h. TLC detection showed the reaction was complete [DCM / MeOH = 10:1]. Wash several times with water, extract with EA, dry with anhydrous MgSO4, and purify by column chromatography [CH3OH / DCM = 0-10%] to obtain 3.16 g of product 3b. Overall yield of the two steps: 79.8%.

[0108] 1 H NMR (400MHz, CDCl3) δ9.28 (br s,1H),8.76(s,1H),8.13(s,1H),8.04(d,J=7.2Hz,2H),7.65(t,J=7.4Hz,1H),7.55(t,J=7 .6Hz,2H),7.21(d,J=13.0Hz,0.75H),6.86(d,J=6.4Hz,0.25H),6.46-6.34(m,1H),6.06(br s,1H),5.04-5.00(m,1H),4.81(d,J=13.0Hz,0.75H),4.62(d,J=6.4Hz,0.25H),4.44-4.39( m,1H),4.05(d,J=12.8Hz,1H),3.84(d,J=12.8Hz,1H),3.25-3.18(m,1H),2.61-2.58(m,1H).

[0109] 4b 3'-O-cyanovinyl-dATP

[0110] Compound 3b (2.2 g, 5.42 mmol, 1.0 eq) and 1,8-bis(methylaminonaphthalene) (2.31 g, 10.8 mmol, 2.0 eq) were dissolved in 22 mL of trimethyl phosphate, purged three times with argon, and the system was cooled to 0 °C in an ice-water bath. POCl3 (1.22 g, 8.13 mmol, 1.5 eq) was dissolved in 2.4 mL of trimethyl phosphate and slowly added dropwise to the reaction system. After the addition was complete, the system was kept at 0 °C for 2 hours. In a fresh reaction flask, tributylpyrophosphoramide (5.9 g, 10.8 mmol, 2 eq) and Bu3N (5.01 g, 27.1 mmol, 5 eq) were dissolved in 50 mL of ACN, purged three times with argon, and cooled to 0 °C in an ice-water bath. The monophosphate generated earlier was slowly added dropwise to the fresh system, and the reaction was kept in an ice-water bath for 10 minutes. The reaction was quenched with TEAB (18 mL, 1 mol / L). The reaction solution was separated and eluted using DEAE (22g, H2O:TEAB = 1:0–4:1). The fraction containing the product was collected and concentrated to 200mL under reduced pressure at 30°C. 22mL of ammonia was added, and the reaction was carried out at room temperature for 16 hours. The reaction was stopped, and the reaction system was concentrated to 50mL under reduced pressure at 30°C. Prep-HPLC was performed (mobile phase A: 50mM triethylamine acetate; mobile phase B: chromatographically pure acetonitrile; elution program: 0–8 min from A to 20% B, 8–12 min holding at 20% B, 12–15 min from B to 40% and holding until product was obtained). The prepared product was concentrated and lyophilized to obtain 453mg of a pale yellow solid with a yield of 9.8%. The product was confirmed by LC-MS, and the HPLC purity was 98.14%. MS: calcd for C 13 H 17 N6O 12 P3[MH] - 541.0, found 541.2.

[0111] c:B=C Bz

[0112] 2c 3'-O-formyl-5'-O-tert-butyldimethylsilyl-N4-benzoyl-dC

[0113] Under ice bath conditions, DMAP (0.143 g, 1.17 mmol) and EDCI (9.46 g, 49.4 mmol) were added to a 35 mL solution of DCM containing 5.5 g (12.3 mmol) of product 1c. Formic acid (1.14 g / 0.931 mL, 24.68 mmol) in DCM was then slowly added dropwise. After the addition was complete, the reaction was allowed to proceed at room temperature for 6 h. TLC was used to confirm the completeness of the reaction [DCM / MeOH = 10:1]. The product was washed with water, extracted with DCM, dried over anhydrous MgSO4, and purified by column chromatography [DCM / MeOH = 0-10%] to give 5.78 g of product 2c, yield: 99%.

[0114] 1 H NMR (400MHz, CDCl3) δ8.72 (br s,1H),8.37(d,J=7.4Hz,1H),8.08(s,1H),7.92(d,J=7.6Hz,2H),7.66(t,J=7.4Hz,1H),7.56(t,J=7.6Hz,2H),6.41(dd,J=7.6,5.9Hz ,1H),5.43(d,J=6.0Hz,1H),4.29(d,J=1.3Hz,1H),4.02-3.92(m,2H),2.84-2.79(m,1H),2.26-2.18(m,1H),0.95(s,9H),0.15(s,6H).

[0115] 3c 3'-O-cyanovinyl-N4-benzoyl-dC

[0116] Take a pressure-resistant bottle, add 2c (5.78 g, 12.2 mmol), dissolve in 40 mL of toluene, add triphenylphosphine acetonitrile (7.3 g, 24.4 mmol), and heat in an oil bath to 120 °C for 9 h. TLC detection showed the reaction was complete [DCM / EA = 1 mL: 0.4 mL]. Directly evaporate to dryness, and purify by column chromatography [DCM / MeOH = 0-10%] to obtain 12.27 g of impurity-containing intermediate. Dissolve the intermediate in THF (40 mL), add TBAF·3H2O (11.5 g, 36.6 mmol), and react at room temperature for 3 h. TLC detection showed the reaction was complete [DCM / MeOH = 10:1]. Wash with water, extract with EA, dry to anhydrous MgSO4, and purify by column chromatography [MeOH / DCM = 0-10%] to obtain 1.6 g of product. Overall yield of the two steps: 34.3%.

[0117] 1H NMR (400MHz, DMSO-d6) δ11.29(s,1H),8.37(d,J=7.2Hz,1H),8.02(d,J=7.6Hz ,2H),7.65-7.50(m,4H),7.39(d,J=7.2Hz,1H),6.15(t,J=7.2Hz,1H),5.36(br s, 1H), 5.24 (d, J = 13.0Hz, 1H), 4.88 (t, J = 2.8Hz, 1H), 4.19 (d, J = 2.4Hz, 1H), 3.68 (d, J = 3.0Hz, 2H), 2.60-2.31 (m, 2H).

[0118] 4c 3'-O-cyanovinyl-dCTP

[0119] Compound 3c (1.9 g, 4.96 mmol, 1.0 eq) and 1,8-bis(methylaminonaphthalene) (2.12 g, 9.9 mmol, 2.0 eq) were dissolved in 19 mL of trimethyl phosphate, purged three times with argon, and the system was cooled to 0 °C in an ice-water bath. POCl3 (1.14 g, 7.4 mmol, 1.5 eq) was dissolved in 2.5 mL of trimethyl phosphate and slowly added dropwise to the reaction system. After the addition was complete, the system was kept at 0 °C for 2 hours. In a fresh reaction flask, tributylpyrophosphoramide (5.45 g, 9.9 mmol, 2 eq) and Bu3N (4.59 g, 24.8 mmol, 5 eq) were dissolved in 50 mL of ACN, purged three times with argon, and cooled to 0 °C in an ice-water bath. The monophosphate generated earlier was slowly added dropwise to the fresh system, and the reaction was kept in an ice-water bath for 10 minutes. The reaction was quenched with TEAB (19 mL, 1 mol / L). The reaction solution was separated and eluted using DEAE (22g, H2O:TEAB = 1:0–4:1). The fraction containing the product was collected and concentrated to 190mL under reduced pressure at 30°C. 57mL of ammonia was added, and the reaction was carried out at room temperature for 16 hours. The reaction was stopped, and the reaction system was concentrated to 25mL under reduced pressure at 30°C. Prep-HPLC was performed (mobile phase A: 50mM triethylamine acetate; mobile phase B: chromatographically pure acetonitrile; elution program: 0–8 min from A to 20% B, 8–12 min holding at 20% B, 12–15 min from B to 40% and holding until product was obtained). The prepared product was concentrated and lyophilized to obtain 436mg of a pale yellow solid with a yield of 10.7%. The product was confirmed by LC-MS, and the HPLC purity was 97.23%. MS: calcd for C 12 H 17 N4O 13 P3[MH] - 517.2, found 517.2.

[0120] d:B=GiBu

[0121] 2d 3'-O-formyl-5'-O-tert-butyldimethylsilyl-N2-isobutyl-dG

[0122] Under ice bath conditions, DMAP (0.129 g, 1.05 mmol) and EDCI (8.49 g, 44.29 mmol) were added to a 35 mL solution of DCM containing 5 g (11.07 mmol) of 1 d, followed by the slow addition of a 10 mL solution of DCM containing 1.01 g (0.835 mL, 22.14 mmol) of formic acid. After the addition was complete, the reaction was allowed to proceed at room temperature for 6 h. The reaction was confirmed to be complete by TLC [DCM / MeOH = 10:1]. The product was washed with water, extracted with DCM, dried over anhydrous MgSO4, and purified by column chromatography [MeOH / DCM = 0-10%] to give 4.91 g of product 2 d, yield: 92.7%.

[0123] 1 H NMR (400MHz, CDCl3) δ12.21(s,1H),9.69(s,1H),8.09(s,1H),8.01(s,1H),6.22(dd,J=8.2,5.8Hz,1H),5.53(d,J =5.4Hz,1H),4.21(s,1H),3.91-3.83(m,2H),2.83-2.51(m,3H),1.27(t,J=6.6Hz,6H),0.89(s,9H),0.09(s,6H).

[0124] 3d 3'-O-cyanovinyl-N2-isobutyl-dG

[0125] Take a pressure-resistant bottle, add 2d (4.91g, 10.23mmol), dissolve in 40mL toluene, add triphenylphosphine acetonitrile (6.17g, 20.5mmol), heat in an oil bath to 120℃, and react for 9h. TLC detection showed the reaction was complete [DCM / EA = 1mL:0.4mL]. Directly evaporate to dryness, and purify by column chromatography [MeOH / DCM = 0-10%] to obtain 4.6g of impurity intermediate. Dissolve the intermediate in THF (40mL), add TBAF·3H2O (9.68g, 30.69mmol), and react at room temperature for 1h. TLC detection showed the reaction was complete [DCM / MeOH = 15:2]. Wash with water several times, extract with EA, dry with anhydrous MgSO4, and purify by column chromatography [MeOH / DCM = 0-10%] to obtain 2.2g of product. Overall yield of the two steps: 55.4%.

[0126] 1H NMR (400MHz, DMSO-d6) δ12.08(s,1H),11.69(s,1H),8.27(s,1H),7.63(d,J=13.0Hz,1H),6.21(dd,J=8.2,5.9Hz,1H),5.25- 5.21(m,2H),4.99(d,J=4.2Hz,1H),4.09(s,1H),3.59(s,2H),2.92-2.72(m,2H),2.61-2.2.51(m,1H),1.13(d,J=6.8Hz,6H).

[0127] 4d 3'-O-cyanovinyl-dGTP

[0128] Compound 3d (1.8 g, 4.6 mmol, 1.0 eq) and 1,8-bis(methylaminonaphthalene) (1.98 g, 9.2 mmol, 2.0 eq) were dissolved in 18 mL of trimethyl phosphate, purged three times with argon, and the system was cooled to 0 °C in an ice-water bath. POCl3 (1.06 g, 6.9 mmol, 1.5 eq) was dissolved in 2 mL of trimethyl phosphate and slowly added dropwise to the reaction system. After the addition was complete, the system was kept at 0 °C for 2 hours. In a fresh reaction flask, tributylpyrophosphoramide (5.08 g, 9.2 mmol, 2 eq) and Bu3N (4.28 g, 23.2 mmol, 5 eq) were dissolved in ACN, purged three times with argon, and cooled to 0 °C in an ice-water bath. The monophosphate generated earlier was slowly added dropwise to the fresh system, and the reaction was kept in an ice-water bath for 10 minutes. The reaction was quenched with TEAB (18 mL, 1 mol / L). The reaction solution was separated and eluted using DEAE (22g, H2O:TEAB = 1:0–4:1). The fraction containing the product was collected and concentrated to 180mL under reduced pressure at 30°C. 54mL of ammonia was added, and the reaction was carried out at room temperature for 16 hours. The reaction was stopped, and the reaction system was concentrated to 25mL under reduced pressure at 30°C. Prep-HPLC was performed (mobile phase A: 50mM triethylamine acetate; mobile phase B: chromatographically pure acetonitrile; elution program: 0–8 min from A to 20% B, 8–12 min holding at 20% B, 12–15 min from B to 40% and holding until product was obtained). The prepared product was concentrated and lyophilized to obtain 311mg of a pale yellow solid with a yield of 7.8%. The product was confirmed by LC-MS, and the HPLC purity was 99.32%. MS: calcd for C 13 H 17 N6O 13 P3[MH] - 557.2, found 557.4.

[0129] Although the invention has been illustrated and described with reference to specific embodiments, it should be understood that many other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications falling within the scope of the invention be included in the appended claims. SEQUENCE LISTING <110> Military Medical Research Institute of the Academy of Military Sciences of the Chinese People's Liberation Army <120> 3'-O-reversibly blocked nucleotides and their application in template-free enzymatic nucleic acid synthesis <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 403 <212> PRT <213> TdT enzyme <400> 1 Met Gly His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Asp Asp Asp Asp Lys His Met Ser Pro Ser Pro Val Pro Gly Ser 20 25 30 Gln Asn Val Pro Ala Pro Ala Val Lys Lys Ile Ser Gln Tyr Ala Cys 35 40 45 Gln Arg Arg Thr Thr Leu Asn Asn Tyr Asn Gln Leu Phe Thr Asp Ala 50 55 60 Leu Asp Ile Leu Ala Glu Asn Asp Glu Leu Arg Glu Asn Glu Gly Ser 65 70 75 80 Cys Leu Ala Phe Met Arg Ala Ser Ser Val Leu Lys Ser Leu Pro Phe 85 90 95 Pro Ile Thr Ser Met Lys Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp 100 105 110 Lys Val Lys Ser Ile Ile Glu Gly Ile Ile Glu Asp Gly Glu Ser Ser 115 120 125 Glu Ala Lys Ala Val Leu Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu 130 135 140 Phe Thr Ser Val Phe Gly Val Gly Leu Lys Thr Ala Glu Lys Trp Phe 145 150 155 160 Arg Met Gly Phe Arg Thr Leu Ser Lys Ile Gln Ser Asp Lys Ser Leu 165 170 175 Arg Phe Thr Gln Met Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu 180 185 190 Val Ser Cys Val Asn Arg Pro Glu Ala Glu Ala Val Ser Met Leu Val 195 200 205 Lys Glu Ala Val Val Thr Phe Leu Pro Asp Ala Leu Val Thr Met Thr 210 215 220 Gly Gly Phe Arg Arg Gly Lys Met Thr Gly His Asp Val Asp Phe Leu 225 230 235 240 Ile Thr Ser Pro Glu Ala Thr Glu Asp Glu Glu Gln Gln Leu Leu His 245 250 255 Lys Val Thr Asp Phe Trp Lys Gln Gln Gly Leu Leu Leu Tyr Cys Asp 260 265 270 Ile Leu Glu Ser Thr Phe Glu Lys Phe Lys Gln Pro Ser Arg Lys Val 275 280 285 Asp Ala Leu Asp His Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu Asp 290 295 300 His Gly Arg Val His Ser Glu Lys Ser Gly Gln Gln Glu Gly Lys Gly 305 310 315 320 Trp Lys Ala Ile Arg Val Asp Leu Val Met Cys Pro Tyr Asp Arg Arg 325 330 335 Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp 340 345 350 Leu Arg Arg Tyr Ala Thr His Glu Arg Lys Met Met Leu Asp Asn His 355 360 365 Ala Leu Tyr Asp Arg Thr Lys Arg Val Phe Leu Glu Ala Glu Ser Glu 370 375 380 Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu 385 390 395 400 Arg Asn Ala <210> 2 <211> 60 <212> DNA <213> Artificial sequence <400> 2 gcagataata cgactcacta tagggattta gactacccca aaaacgaagg ggactaaaac 60

Claims

1. A method for enzyme-catalyzed nucleic acid synthesis, characterized in that, The raw materials for the method include 3'-O-blocked nucleotides; The blocking group of the 3'-O-blocked nucleotide is cyanovinyl; the nucleotide includes deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxycytidine monophosphate, and deoxythymidine monophosphate. The enzyme is a TdT enzyme; the enzymatic nucleic acid synthesis is a template-free enzymatic DNA synthesis that is reversibly terminated. The following deprotection method was used to restart the termination of nucleic acid extension: the blocking group in the 3'-O-blocked nucleotide was cleaved with a cleavage agent, which was a palladium complex, to obtain 3'-OH nucleotide.

2. The method for enzyme-catalyzed nucleic acid synthesis according to claim 1, characterized in that, The pyrolysis agent is palladium diacetonitrile.

3. The method for enzymatic nucleic acid synthesis according to claim 2, characterized in that, The concentration of palladium dichloride in diacetonitrile is 0.04-0.06 M, and the cutting conditions are 55-65℃ reaction for 0.5-10 min.

4. The method for enzyme-catalyzed nucleic acid synthesis according to any one of claims 1-3, characterized in that, The 3'-O-blocked nucleotide was prepared by replacing the 3'-OH of the nucleotide with 3'-O-cyanovinyl to obtain the 3'-O-blocked nucleotide.

5. The method for enzyme-catalyzed nucleic acid synthesis according to claim 4, characterized in that, The 3'-OH of the nucleotide first undergoes an esterification reaction with formic acid to form a methyl ester, and then reacts with triphenylphosphine acetonitrile to form a 3'-O-cyanovinyl ester, yielding a 3'-O-blocked nucleotide.

6. The method for enzymatic nucleic acid synthesis according to claim 5, characterized in that, Using 5'-OTBDMS-protected nucleosides as raw materials, the 3'-OH of the 5'-OTBDMS-protected nucleosides first undergoes an esterification reaction with formic acid to generate a methyl ester, and then reacts with triphenylphosphine acetonitrile to generate 3'-O-cyanovinyl. After obtaining 3'-O-cyanovinyl, it is successively subjected to removal of 5'-OTBDMS and phosphorylation to obtain a 3'-O-blocked nucleotide.

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