CN103087131B - Reversible terminal, synthesis and use in DNA synthesis sequencing thereof - Google Patents

Abstract

The invention discloses a reversible terminal, its synthesis and its application in DNA synthesis sequencing. The structural formula of the reversible terminal is shown in formula (I): Wherein _R1 is fluorescein, and _R2 is a linking unit. The cleavable reversible terminal of the present invention can be used for DNA synthesis and sequencing; at the same time, the raw materials required for the synthesis of the reversible terminal of the present invention are simple and easy to obtain, and the synthesis process is a conventional chemical reaction, which can be used for large-scale promotion and use, and the biological evaluation results It shows that this type of reversible terminal can fully meet the biochemical reaction requirements of high-throughput sequencing, and has a good practical prospect.

Description

Reversible terminal and its synthesis and application in DNA synthesis sequencing

technical field

The invention relates to the fields of chemical synthesis and biochemistry, in particular to a type of reversible terminal and its synthesis and application in DNA synthesis sequencing.

Background technique

DNA sequencing technology is one of the important means in modern biological research. After the completion of the Human Genome Project, DNA sequencing technology has developed rapidly. DNA sequencing refers to the analysis of the base sequence of a specific DNA fragment, that is, the arrangement of adenine (A), thymine (T), cytosine (C) and guanine (G). The development of accurate, high-throughput, and low-cost DNA sequencing methods is of great significance to biological and medical sciences.

Sequencing By Synthesis (SBS) is one of the next-generation DNA sequencing technologies. The sequencing by synthesis method immobilizes a large number of template DNA fragments to be tested, hybridizes and combines universal DNA primers on the immobilized DNA sequencing template, and controls the extension of four nucleotides on the DNA primers respectively. By detecting the extension reaction process or extended nucleotides, high-throughput parallel detection of DNA sequence information is realized.

In the sequencing by synthesis method, the four nucleotide raw materials for DNA chain extension must first be synthesized, also called "reversible terminator". Such nucleotide derivatives must have a cleavable linker linking the nucleotide and fluorescein. Then, before the next indicator nucleotide is incorporated, the linking unit is broken under mild conditions, so that the next reversible terminal can be smoothly incorporated, thereby reading out the DNA base sequence sequentially. The junction unit has an important impact on the read length and efficiency of sequencing by synthesis. Therefore, people have been working on the development of new cleavable junction units to improve the efficiency of DNA sequencing. The linking units that have been reported so far include photocleavage, Pd catalytic cracking, fluoride cleavage, etc., but these linking units are currently limited to basic research and cannot be practically applied (Accounts of Chemical Research 2010, 43, 551-563.).

Contents of the invention

The object of the present invention is to provide a reversible terminal and its synthesis and application in DNA synthesis sequencing. The reversible terminal is either reduction sensitive or acid sensitive.

The purpose of the present invention is achieved through the following technical solutions:

The present invention relates to a reversible terminal, the structural formula of which is shown in formula (I):

Wherein _R1 is fluorescein, and _R2 is a linking unit. The linking unit is a fully cleavable linking unit with active functional groups at both ends.

Preferably, the structural formula of the reversible terminal is shown in formula (II):

Preferably, the reversible terminal is synthesized through the following steps (as shown in Figure 2):

A, the synthesis of compound F ₂ : under ice-water bath stirring condition, the molar ratio is 1.0: (1.2～2) propargyl amine reacts with methyl trifluoroacetate, obtains compound F ₂

B. Synthesis of compound F ₃ : in the presence of CuI, Pd(PPh ₃ ) ₄ (tetrakis(triphenylphosphine) palladium) and TEA (triethylamine), compounds F ₂ and F ₁ reaction to obtain compound F ₃ The molar ratio of F _{1 ,} F _{2 ,} CuI, Pd(PPh ₃ ) ₄ and TEA is 1:(2-3):0.072:0.025:(1.5-2);

C, the synthesis of compound dUTP-NH ₂ : compound F ₃ and tri-n-butylamine pyrophosphate (E-4), 2-chloro-4H-1,3,2-benzodioxophosphor-4-ketone (E-4) -3) react in the presence of triethylamine and iodine, the reaction product is deprotected, and the compound dUTP-NH ₂ is obtained The molar ratio of said E-4, E-3 and _F3 is 2:2:1;

D, the synthesis of compound dUTP-SPDP: under the condition that TEA exists, compound dUTP-NH ₂ in sodium carbonate sodium bicarbonate buffer solution, with anhydrous acetonitrile as solvent SPDP Reaction, get compound dUTP-SPDP The molar ratio of the dUTP-NH ₂ (i.e. nucleotide U) to SPDP is 1: (1.5-3);

E, the synthesis of compound RDM-SH: under the condition that DTT (dithiothreitol) exists, cysteamine is mixed with compound TAMRA (5/6) in sodium carbonate sodium bicarbonate buffer solution Avoid light reaction to get compound RDM-SH The molar ratio of TAMRA (5/6), cysteamine and DTT is 1: (10-50): (40-70);

F. Synthesis of compound dUTP-T: Using Na ₃ PO ₄ -EDTA buffer (sodium phosphate-ethylenediaminetetraacetic acid buffer) and acetonitrile as solvents, the compound dUTP-SPDP was reacted with RDM-SH to obtain the compound dUTP-T ; The molar ratio of RDM-SH and dUTP-SPDP is 1: (1-2); the compound dUTP-T is a cleavable linking unit having the knot shown in formula (II).

Preferably, the reversible terminal (II) is synthesized through the following steps (as shown in Figure 5):

A, the synthesis of compound F ₂ : under ice-water bath stirring condition, the molar ratio is 1.0: (1.2～2) propargyl amine reacts with methyl trifluoroacetate, obtains compound F ₂

B. Synthesis of compound F ₃ : in the presence of CuI, Pd(PPh ₃ ) ₄ and TEA, compound F ₁ React with F ₂ to get compound F ₃ The molar ratio of F _{1 ,} F ₂ , CuI, Pd(PPh ₃ ) ₄ and TEA is 1:(2-3):0.072:0.025:(1.5-2);

C, the synthesis of compound G ₁ : using methanol as a solvent, compound F ₃ reacts with concentrated ammonia water to obtain compound The mol ratio of described _F3 and ammoniacal liquor is 1: (50～100);

D, the synthesis of compound G ₂ : with methanol and anhydrous acetonitrile as solvent, compound G ₁ and SPDP reaction, compound The molar ratio of _G1 and SPDP is 1: (1～2);

E, the synthesis of compound RDM-SH: under the condition that DTT exists, cysteamine is mixed with compound TAMRA (5/6) in sodium carbonate sodium bicarbonate buffer solution Avoid light reaction to get compound RDM-SH The molar ratio of TAMRA (5/6), cysteamine and DTT is 1: (10-50): (40-70);

F. Synthesis of compound G ₃ : using methanol and acetonitrile as solvents, compound RDM-SH reacted with G ₂ under nitrogen protection wrapped in aluminum foil to obtain compound G ₃ The molar ratio of RDM-SH and _G2 is 1: (1.2～2);

G, the synthesis of compound dUTP-T: Compound G ₃ and tri-n-butylamine pyrophosphate (E-4), 2-chloro-4H-1,3,2-benzodioxophosphor-4-ketone (E- 3) react in the presence of triethylamine and iodine, and the reaction product is deprotected to obtain compound dUTP-T; the molar ratio of E-4, E-3 and _G3 is 2:2:1; the compound dUTP- T is the cleavable linking unit having the knot formula shown in formula (II).

Preferably, when the structural formula of the reversible terminal is shown in formula (III):

Preferably, the reversible terminal is synthesized through the following steps (as shown in Figure 7):

A, the synthesis of compound N-1: using methanol as solvent, in the presence of TEA, cysteamine hydrochloride and 2-hydroxyethyl disulfide Reaction, get compound N-1 The molar ratio of cysteamine hydrochloride, 2-hydroxyethyl disulfide and TEA is 1: (1～2): (2～3);

B, the synthesis of compound N-2: with anhydrous DMF (N, N-dimethylformamide) as solvent, under the condition that TEA exists, compound N-1 and TAMRA (5/6) Protected from light reaction to obtain compound N-2 The molar ratio of TAMRA (5/6), N-1 and TEA is 1: (1～4): (10～15);

C, the synthesis of compound N-3: using anhydrous acetonitrile as a solvent, under the condition that TEA exists, compound N-2 reacts with DSC (N, N-succinimidyl carbonate) under nitrogen protection conditions to obtain Compound N-3 The molar ratio of said N-2, DSC and TEA is 1: (4～6): (5～15);

D. Synthesis of compound N-4: using a buffer solution of NaHCO ₃ /Na ₂ CO ₃ (pH 8.73) as solvent, compound dUTP-NH ₂ React with N-3, obtain compound N-4; Described N-3 and dUTP- _NH The mol ratio is 1: (1～2); Described compound N-4 promptly has the knot shown in formula (III) reversible terminal.

Preferably, the structural formula of the reversible terminal is shown in formula (IV):

Preferably, the reversible terminal is synthesized through the following steps (as shown in Figure 9):

A. Synthesis of compound T-1: In the presence of hydrochloric acid, L-glutamic acid reacts with sodium nitrite under ice-bath stirring conditions to obtain compound The mol ratio of described L-glutamic acid, hydrochloric acid and sodium nitrite is 1: (1.5～2): (1.5～2);

B. Synthesis of compound T-2: using anhydrous tetrahydrofuran as a solvent, compound T-1 reacts with borane dissolved in dimethyl sulfide under nitrogen protection and an ice-water bath to obtain compound T-2 The molar ratio of described T-1 and borane is 1: (1.5～5);

C, the synthesis of compound T-3: using dichloromethane as a solvent, in the presence of imidazole, compound T-2 reacts with TBSCl (dimethyl tert-butylchlorosilane) under nitrogen protection to obtain compound T-3 The molar ratio of T-2 and TBSCl is 1: (1.5～2.5);

D. Synthesis of compound T-4: using dichloromethane as solvent, compound T-3 reacts with DIBAL-H (diisobutylaluminum hydride) under nitrogen protection and ice-salt bath to obtain compound T-4 The molar ratio of T-3 and DIBAL-H is 1: (1.5～2.5);

E, the synthesis of compound T-6: under the condition that A-15 (A-15 type strongly acidic cation exchange resin) catalyst exists, compound T-4 reacts with bromoethanolamine to obtain compound T-6 The mol ratio of described T-4 and bromoethanolamine is 1: (1.5～3);

F, Synthesis of Compound T-7: In the presence of tetrabutylammonium fluoride (TBAF), Compound T-6 was dehydroxylated and protected at room temperature to obtain Compound T-7 The mol ratio of described T-6 and TBAF is 1: (1.5～2);

G. Synthesis of Compound T-8: Compound T-7 reacts with excess ammonia water to obtain Compound T-8 The mol ratio of described T-7 and ammoniacal liquor is 1: (50～100);

H, the synthesis of compound T-9: with anhydrous DMF as solvent, under the condition that TEA exists, compound T-8 and TAMRA (5/6) Reaction, get compound T-9 The molar ratio of TAMRA (5/6), T-8 and TEA is 1: (3～6): (5～20);

I, the synthesis of compound T-10: compound T-9 obtains compound T-10 under the condition that DSC exists The mol ratio of described T-9 and DSC is 1: (5～8);

J, the synthesis of compound T-11: compound T-10 and dUTP-NH ₂ (nucleotide U) reaction to obtain compound T-11; the molar ratio of T-10 and dUTP-NH ₂ is 1: (1.5-3); the compound T-11 is the reversible terminal shown in structural formula (IV).

Preferably, the structural formula of the reversible terminal is shown in formula (V):

Preferably, the reversible terminal is synthesized through the following steps (as shown in Figure 7):

A. Preparation of Na ₂ Se ₂ alkaline aqueous solution: under ice bath cooling, dissolve NaBH ₄ solid with water to form NaBH ₄ solution; dissolve NaOH solid in water, add selenium powder and cetyltrimethylammonium bromide, Under the protection of N2, add the NaBH ₄ solution, react at room temperature for 0.5-1.5 h, then react at 85-95°C for 0.5-1 h to obtain Na ₂ Se ₂ alkaline aqueous solution; the NaBH ₄ , selenium powder and NaOH The molar ratio is 1: (7～8): (8～9);

B. Synthesis of compound D-1: using THF as solvent, under the protection of nitrogen, bromoethanol reacted with Na ₂ Se ₂ alkaline aqueous solution in an oil bath at 45-55°C to obtain compound D-1 The molar _ratio of the bromoethanol to _Na2Se2 is 1: (1-2);

C, the synthesis of compound D-2: with xylene as solvent, compound D-1 reacts with HBr to obtain compound D-2 The molar ratio of D-1 and HBr is 1: (4～6);

D. Synthesis of Compound D-3: Compound D-2 reacts with concentrated ammonia water to obtain Compound D-3 The mol ratio of described D-2 and ammoniacal liquor is 1: (50～100);

E, the synthesis of compound D-4: with anhydrous DMF as solvent, under the condition that TEA exists, compound D-3 and TAMRA (5/6) Light-shielding reaction yields compound D-4 The molar ratio of TAMRA (5/6), D-3 and TEA is 1: (1～4): (10～15);

F. Synthesis of compound D-5: using anhydrous acetonitrile as solvent, in the presence of TEA, compound D-4 was reacted with DSC under nitrogen protection to obtain compound D-5 The molar ratio of D-4, DSC and TEA is 1: (4～6): (5～15);

G. Synthesis of compound D-6: Using a buffer solution of NaHCO ₃ /Na ₂ CO ₃ (pH 8.73) as solvent, compound dUTP-NH ₂ Reaction with D-5, obtains compound D-6; Described D-5 and dUTP- _NH The mol ratio is 1: (1～2); Described compound D-6 is the reversible terminal shown in structural formula (V) .

The present invention also relates to the use of the aforementioned cleavable linking unit in DNA synthesis sequencing.

The invention has the following beneficial effects: the invention synthesizes a new type of reversible terminal; this type of reversible terminal can be used for DNA synthesis and sequencing; at the same time, the raw materials required for its synthesis are simple and easy to obtain, and the synthesis process is all conventional chemical reactions, which can be used in large Use on a large scale.

Description of drawings

Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

1 is a schematic diagram of the overall synthesis process of the reversible terminal of the present invention, wherein R ₁ is fluorescein, and R ₂ is a linker;

Figure 2 is a schematic diagram of the synthesis process of the reversible terminal of Example 1;

Fig. 3 is compound dUTP-NH in embodiment ₁ Synthesis process schematic diagram;

Fig. 4 is the schematic diagram of the process of TAMRA (5/6) thiolation in embodiment 1;

Figure 5 is a schematic diagram of the synthesis process of the reversible terminal of Example 2;

6 is a schematic diagram of the synthesis process of compound dUTP-T in Example 2;

Fig. 7 is a schematic diagram of the synthesis process of the reversible terminal of embodiment 3;

Figure 8 is a schematic diagram of the synthesis process of the reversible terminal of Example 5;

Figure 9 is a schematic diagram of the synthesis process of the reversible terminal of Example 4;

Figure 10 is the test result of 6.1 in Example 6, wherein (a) is the PAGE electrophoresis image of the DNA chain extension reaction, and (b) is a schematic diagram of the fluorescence scanning result of the fragmentation reaction, wherein, M is a DNA marker, 1 is a control template, and 2 is a DNA chain extension reaction positive control, 3 is the fragmentation of the chain extension product with reversible terminal at pH 2.0, 4 is the fragmentation of the chain extension product with reversible terminal at pH 2.2;

Figure 11 is the test result of 6.2 in Example 6, wherein (a) is the PAGE electrophoresis image of the DNA chain extension reaction, and (b) is a schematic diagram of the fluorescence scanning result of the fragmentation reaction, wherein, M is a DNA marker, 1 is a control template, and 2 is a DNA chain extension reaction positive control, 3 is the fragmentation of the chain extension product with reversible terminal at pH 1.7, and 4 is the fragmentation of the chain extension product with reversible terminal at pH 1.5;

Figure 12 is the test result of 6.3 in Example 6, wherein (a) is the PAGE electrophoresis image of the DNA chain extension reaction, and (b) is a schematic diagram of the fluorescence scanning result of the fragmentation reaction, wherein, M is a DNA marker, 1 is a control template, and 2 is a DNA chain extension reaction positive control, 3 is the fragmentation of the chain extension product with reversible terminal at pH 1.9, and 4 is the fragmentation of the chain extension product with reversible terminal at pH 1.5;

Figure 13 is the test result of 6.4 in Example 6, wherein (a) is the PAGE electrophoresis image of the DNA chain extension reaction, and (b) is a schematic diagram of the fluorescence scanning result of the fragmentation reaction, wherein, M is a DNA marker, 1 is a control template, and 2 is a DNA chain elongation reaction positive control, 3 is the fragmentation of the chain extension product containing reversible terminal 10uM DTT at room temperature for 2h, 4 is the fragmentation of the chain extension product containing reversible terminal 8mM DTT at room temperature for 2h, 5-9 are respectively containing reversible terminal Fragmentation of chain extension products by 10mM DTT at room temperature for 10min, 20min, 30min, 1h and 2h;

Figure 14 is the fragmentation test result of the DNA chain extension product containing the reversible terminal of the disulfide bond of 6.5 in Example 6 under different action times of 10mM DTT, wherein (a) is the PAGE electrophoresis pattern of the DNA chain extension reaction, and (b) is the fragmentation Schematic diagram of reaction fluorescence scanning results, in which, M is DNA marker, 1 is the control template, 2 is the positive control of DNA chain extension reaction, 3-7 are the reversible terminal chain extension products containing disulfide bonds, respectively, treated with 10mM DTT for 3min, 5min, and 8min , 10min and 15min breaks;

Figure 15 is the breakage test results of 6.5 DNA chain extension products containing reversible disulfide bond terminals in Example 6 under different action times of 20 and 30 mMDTT, wherein M is a DNA marker, 4 is a control template, and 6 is a DNA chain Positive controls for extension reaction, 1-3 are the breakages containing disulfide bond reversible terminal chain extension products treated with 20mM DTT for 8min, 5min and 3min respectively, and 7-8 are the disulfide bond reversible terminal chain extension products respectively treated with 30mM DTT for 3min and a 5min break.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make some adjustments and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available AR and CP grades. The obtained intermediate product and the final product of the present invention are characterized by NMR, etc.; the schematic diagram of the total synthesis process of the reversible terminal of the present invention is shown in Figure 1; four different fluoresceins are used to mark the four different nucleotides (A, G, C, U) reversible terminals.

Example 1

The structural formula of the reversible terminal in this embodiment is shown in the following formula (II):

Its corresponding synthetic route is as shown in Figure 2; Specifically comprises the following steps:

1.1 Synthesis of compound _F2

Methyl trifluoroacetate reacts with propargyl amine in an organic solvent to obtain compound F ₂ , specifically: add 60 ml of methanol to a single-necked bottle, stir in an ice-water bath, add propargyl amine (60 mmol, 3.3042 g), and stir for 15 minutes Then slowly add methyl trifluoroacetate (86.7mmol, 11.0957g), remove the ice-water bath after 10 minutes, and react at room temperature for 24 hours. The reaction was monitored with a TLC plate, PE:EA=8:1, baking plate, Rf=0.5 to produce a new spot as product F2. Distillation under reduced pressure (51°C, 280Pa) yielded 3.53g with a yield of 39%.

¹ H NMR (CDCl ₃ , 300 MHz): δ 2.32 (t, J=4.0 Hz, 1H), 4.13-4.15 (m, 2H), 6.92 (s, 1H).

In the above synthesis, the added methyl trifluoroacetate can be any value in 72-120 mmol.

1.2 Synthesis of compound _F3

Add F1 (0.7mmol, 247mg) to the single-necked flask, weigh 9.7mgCuI and 20.3mg Pd(PPh ₃ ) ₄ into the reaction flask, vacuumize, protect with nitrogen, wrap with aluminum foil, add 2.3ml DMF, stir to dissolve, add Weigh 0.2ml of TEA, weigh F2 (254mg, 1.7mmol) and dissolve it in DMF, add it to the reaction bottle, stir at room temperature, and react overnight. TLC plate monitoring, EA is the developer, Rf=0.35 is the raw material F1, Rf=0.32 is the product F3, and the two points are very close. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure, and directly separated by column chromatography, 20:1 DCM:MeOH was used as eluent to obtain 214 mg, with a yield of 61%.

¹ H NMR (DMSO-D ₆ , 300MHz): δ2.11(t, J=5.1Hz, 2H), 3.56-3.58(m, 2H), 3.78(m, 1H), 4.21(d, J=5.1Hz , 3H), 5.08(t, J=5.1Hz, 1H), 5.23(d, J=4.2Hz, 1H), 6.09(t, J=6.6Hz, 1H), 8.18(s, 1H), 10.05(t , J=4.8Hz, 1H), 11.63(s, 1H).

In the above synthesis, the added _F2 can be any value in 1.4-2.1 mmol, and the TEA can be any value in 1.05-1.4 mmol.

1.3 Synthesis of compound dUTP-NH ₂

The synthesis of compound dUTP-NH2 is shown in Figure 3. The reaction conditions corresponding to each step in Figure 3 are: i) DMF, tributylamine ii) DMF, F ₃ iii) I ₂ , Py, H ₂ O iV) NH ₃ .

Weigh 60 mg (0.16 mmol) of compound F ₃ , 150 mg (0.32 mmol) of tri-n-butylamine pyrophosphate, 2-chloro-4H-1,3,2-benzodioxophosphor-4-one in the glove box 66 mg (0.32 mmol) were placed in three reaction tubes. Dissolve tri-n-butylamine pyrophosphate in 0.5 mL of anhydrous DMF, then add 0.6 mL of freshly distilled tri-n-butylamine, and stir for half an hour. Dissolve 2-chloro-4H-1,3,2-benzodioxophosphor-4-one in 0.5 mL of anhydrous DMF, add the above-mentioned tri-n-butylamine pyrophosphate solution through a syringe under vigorous stirring, and stir for half an hour . Then the mixture was injected into _F3 and stirred for 1.5h. 5 mL of 3% iodine (9:1 Py/H2O) solution was added. After 15 min, 4 mL of water was added and stirred for 2 h. Add 0.5mL 3M NaCl solution, then add 30mL absolute ethanol, freeze overnight at -20°C, and centrifuge (3200r/min, 25°C) for 20min. The supernatant was poured off to obtain a precipitate, and the solvent was drained. Then add TEAB solution and concentrated ammonia water in turn, and stir overnight at room temperature. The solvent was evaporated to dryness under reduced pressure, and a white solid appeared to obtain dUTP-NH ₂ . Analytical HPLC was used for analysis, conditions: column: C18, 10μm, 4.6×250mm; flow rate: 1mL/min; mobile phase: 20mM TEAAc and CH ₃ CH ₂ OH, gradient washing, 0%-20% CH3CH2OH (35min); UV detector: 254nm. A product peak was formed at t=13.5 min.

¹ H NMR (D ₂ O, 400MHz): δ2.34-2.48(m, 2H), 4.03(s, 2H), 4.20-4.29(m, 3H), 4.61-4.64(m, 1H), 6.27(t , J=6.4Hz, 1H), 8.38(s, 1H).

31P NMR (D2O, 161MHz): δ-22.22, -11.45, -9.90.

HRMS: calc for C12H19N3O14P3 [M+H]+ 522.0080, found 522.0070; calc for C12H18N3O14P3 Na[M+Na]+ 543.9899, found 543.9883.

1.4 Synthesis of compound dUTP-SPDP

Add 24.4mg (0.026mmol) of dUTP(AP ₃ ) (i.e. dUTP-NH ₂ ) to a 10mL single-mouth bottle, then add 600μl Na ₂ CO ₃ /NaHCO ₃ buffer solution, stir at room temperature, and mix SPDP12.3mg (0.039mmol) Dissolve in 400 μl anhydrous CH ₃ CN, add the above solution, add 3 μl Et ₃ N. The reaction was stirred at room temperature, followed by analytical HPLC until the disappearance of the starting material. Conditions: column: C18, 10μm, 4.6×250m; flow rate: 1mL/min; mobile phase: 100mM TEAA and CH ₃ CN, 100% TEAA (5min), gradient washing 0% ~ 10% CH ₃ CN (5min), 10 %～50% CH ₃ CN (50min); UV detector: 293nm; a product peak was formed at 27.55min. After 8 hours, the reaction was stopped, and the product was separated and purified by preparative HPLC to obtain 5 mg. Conditions: column: C18, 5μm, 9.4×250mm; flow rate: 4mL/min; mobile phase: 100mM TEAA and CH ₃ CN, 100% TEAA (5min), gradient washing 0% ~ 5% CH _3CN (5min), 5 %-30% _CH3CN (50min); UV detector: 293nm.

¹ H NMR (MeOH, 400MHz): δ1.61(q, J=7.6Hz, J=15.2Hz, 1H), 2.89-2.34(m, 3H), 2.64(dd, J=2.8Hz, J=9.6Hz , 2H), 4.05(s, 2H), 4.13(s, 3H), 4.53(d, J=0.8Hz, 1H), 6.18(t, J=6.4Hz, 1H), 7.23(s, 1H), 7.79 (d, J=6.8Hz, 2H), 8.07(d, J=4.0Hz, 1H), 8.32-8.37(m, 1H).

In the above synthesis, the added SPDP can be any value in 0.039-0.078 mmol.

1.5 Thiolation of fluorescein (rhodamine TAMRA(5/6))

Fluorescein (rhodamine TAMRA (5/6)) is thiolated to obtain the compound RDM-SH as shown in Figure 4, specifically as follows:

Take cysteamine (73.5mg, 0.95mmol) in a 10mL one-mouth bottle, add 400μl Na ₂ CO ₃ /NaHCO ₃ buffer solution, stir to dissolve, and wrap it in aluminum foil; take TAMRA (5/6) (10mg, 0.019mmol) Anhydrous DMF was dissolved and added to the above reaction flask, and after stirring at room temperature for 1 h in the dark, 1.33 mL of 1M DTT was added and stirred at room temperature for 2.5 h. TLC plate monitoring: DCM:MeOH=5:1, a product point was formed at Rf=0.7. It was isolated and purified by preparative HPLC to obtain 8.8 mg with a yield of 94.6%. Conditions: column: C18, 5μm, 9.4×250mm; flow rate: 4mL/min; mobile phase: 0.1% TFA and CH ₃ CN, 100% TFA (5min), gradient washing 0%-8% CH ₃ CN (5min), 8%-50% CH ₃ CN (50min); UV detector: 293nm and 546nm, the product peak was collected for 41min.

¹ H NMR (MeOH, 400MHz): δ1.26-1.31(m, 12H), 2.70(t, J=7.2Hz, 2H), 3.53(t, J=6.8Hz, 2H), 6.97(d, J= 2.4Hz, 2H), 7.04(dd, J=2.4Hz, J=9.6Hz, 2H), 7.13(d, J=9.2Hz, 2H), 7.81(d, J=1.2Hz, 1H), 8.19(dd , J=1.6Hz, J=8.4Hz, 1H), 8.39(d, J=8.4Hz, 1H).

In the above synthesis, the added cysteamine can be any value in 0.19-0.95 mmol, and the DTT can be any value in 0.76-1.33 mmol.

1.6 Synthesis of compound dUTP-T

Take 5mg RDM-SH (5mg, 0.01mmol) in a 10ml one-mouth bottle, vacuumize, nitrogen protection, and wrap in aluminum foil; take dUTP(AP3)-SPDP (22mg, 0.02mmol) in a 10mL one-mouth bottle, add 2ml Na ₃ PO ₄ - EDTA buffer solution and acetonitrile 0.5ml, stir to dissolve and pour into the RDM-SH reaction bottle; stir at room temperature, and analyze the reaction by HPLC. Conditions: Column: C18, 10μm, 4.6×250mm; flow rate: 1mL/min; mobile phase: 100mM TEAA and CH ₃ CN, 5% CH ₃ CN (5min), gradient washing 5%-35% CH3CN (60min); UV Detectors: 293nm and 546nm; product peaks formed at 49.74min. After 10 h, the reaction was stopped, and the product was separated and purified by preparative HPLC. 2.76mg was obtained. Conditions: column: C18, 5μm, 9.4×250mm; flow rate: 4mL/min; mobile phase: 100mM TEAA and CH ₃ CN, 5% TEAA (5min), gradient washing 5%-25% CH ₃ CN ( 45 min); 80% _CH3CN (10 min), UV detector: 293 nm and 546 nm. The product peak at 51 min was collected.

¹ H NMR (D ₂ O, 400MHz): δ1.31(s, 12H), 2.15-2.26(m, 1H), 2.27-2.41(m, 1H), 2.61-2.66(m, 2H), 3.73-3.80 (m, 2H), 3.89(s, 2H), 4.14-4.18(m, 3H), 6.05-6.09(m, 1H), 6.71-6.74(m, 2H), 6.88-6.95(m, 2H), 7.23 (dd, J=9.6Hz, J=13.6Hz, 2H), 7.85(s, 1H), 7.91(s, 1H), 8.01(d, J=6.8Hz, 1H), 8.09-8.12(m, 1H) .ESI-HRMS: calc for C ₄₂ H ₄₇ N ₆ O ₁₉ P ₃ S ₂ [M+Na+2H] ³⁺ 1121.1604, found 1121.1655.

In the above synthesis, the added dUTP-SPDP can be any value in the range of 0.01-0.02 mmol.

Example 2

The structural formula of the reversible terminal in this embodiment is shown in the following formula (II):

Its corresponding synthetic route is as shown in Figure 5; Specifically comprises the following steps:

2.1 The synthesis of compound F ₂ , F ₃ is the same as in Example 1

2.2 Synthesis of compound G1

Take 23mg of F ₃ (0.06mmol) in a 10mL single-necked bottle, add 1mL of methanol to dissolve, add 0.1mL of concentrated ammonia water (6mmol), and stir overnight at room temperature. TLC plate monitoring: DCM:MeOH=3:1, product G1 Rf=0.15. Separation adopts TLC plate chromatography, MeOH:EA:NH3=6:6:1, and collects Rf=0.6 ultraviolet color region. ESI-HRMS: cals for C ₁₂ H ₁₅ N ₃ O ₅ [M] 281.1012, found 281.1015.

In the above synthesis, the ammonia water added can be any value in 3-6 mmol.

2.3 Synthesis of Compound G2

Take 8.5mg G1 (0.03mmol) and dissolve it with 0.5mL methanol; take 9.4mg SPDP (0.03mmol) and dissolve it with 0.5mL anhydrous acetonitrile, then add the methanol solution of G1 above, and stir at room temperature for 10h. TLC plate monitoring: MeOH:EA=1:6, product Rf=0.55. The reaction was stopped, the solvent was spun out, and 9.5 mg of product was obtained by plate chromatography. ESI-HRMS: cals for C ₂₀ H ₂₂ N ₄ O ₆ S ₂ [M] 478.0981, found 478.0974.

In the above synthesis, the added SPDP can be any value in the range of 0.03-0.06 mmol.

2.4 Synthesis of Compound G3

Take 6.8mg RDM-SH (0.013mmol, its synthesis is the same as in Example 1) in a 10mL single-mouth bottle, vacuum nitrogen protection, and wrap it in aluminum foil; take another 9mg _G2 (0.019mmol) in a 10ml single-mouth bottle, and use 0.5ml _CH3 CN, if it is not completely dissolved, add 1ml of methanol to dissolve completely, pour it into the above reaction flask, and stir at room temperature for 9h. Analytical HPLC detection reaction, conditions: column: C18, 10μm, 4.6×250mm; flow rate: 1mL/min; mobile phase: H ₂ O and CH ₃ OH, gradient washing 0%-10% CH ₃ OH (5min), 10 %-70% _CH3OH (55min); UV detector: 293nm and 546nm; product peaks formed at 49min. Preparative HPLC isolated 5 mg of product.

¹ H NMR (MeOD, 400MHz): δ1.33(s, 12H), 2.11-2.42(m, 3H), 2.25-2.33(m, 1H), 2.61(t, J=6.4Hz, 2H), 3.68( s, 3H), 3.71-3.76(m, 3H), 3.92(d, J=3.2Hz, 1H), 4.01(d, J=2.4Hz, 2H), 4.36-4.40(m, 1H), 6.17-6.23 (m, 1H), 6.93(d, J=2.4Hz, 2H), 7.00-7.09(m, 2H), 7.29(dd, J=6.4Hz, J=9.6Hz, 2H), 7.84(d, J= 1.6Hz, 1H), 8.13(dd, J=1.6Hz, J=8.0Hz, 1H), 8.18(d, J=8.0Hz, 1H), 8.21(s, 1H).

In the above synthesis, the added _G2 can be any value in the range of 0.016 to 0.026 mmol.

2.5 Synthesis of compound dUTP-T

The synthesis of the compound dUTP-T is shown in Figure 6. The reaction conditions corresponding to each step in Figure 6 are: i) DMF, tributylamine ii) DMF, G ₃ iii) I ₂ , Py, H2O iV) NH3.

In the glove box, weigh 6 mg (0.007 mmol) of compound G ₃ , 7.7 mg (0.014 mmol) of tri-n-butylamine pyrophosphate, 2-chloro-4-H-1,3,2-benzodioxophosphate- 2.8 mg (0.014 mmol) of 4-ketone was placed in three reaction tubes. Dissolve tri-n-butylamine pyrophosphate in 0.15 mL of anhydrous DMF, then add 0.15 mL of freshly distilled tri-n-butylamine, and stir for half an hour. Dissolve 2-chloro-4H-1,3,2-benzodioxophosphor-4-one in 0.15 mL of anhydrous DMF, add the above-mentioned tri-n-butylamine pyrophosphate solution through a syringe under vigorous stirring, and stir for half an hour . Then the mixture was injected into G3 and stirred for 1.5h. 1 mL of 3% iodine (9:1 Py/H2O) solution was added. After 15 min, 1 mL of water was added and stirred for 2 h. Add 0.5mL 3M NaCl solution, then add 9mL absolute ethanol, freeze overnight at -20°C, and centrifuge (3200r/min, 25°C) for 20min. Pour off the supernatant to obtain a precipitate, and drain the solvent to obtain dUTP-T. Analytical HPLC was used for analysis, conditions: column: C18, 10μm, 4.6×250mm; flow rate: 1mL/min; mobile phase: 100mM TEAA and CH ₃ CN, 5% CH3CN (5min), gradient washing 5%-35% CH3CN (60 min); UV detector: 293nm and 546nm; product peaks formed at 49.7min. After 10 h, the reaction was stopped, and the product was separated and purified by preparative HPLC. Conditions: column: C18, 5μm, 9.4×250mm; flow rate: 4mL/min; mobile phase: 100mM TEAA and CH ₃ CN, 5% TEAA (5min), gradient washing 5%-25% CH ₃ CN (45min); 80 % _CH3CN (10 min), UV detector: 293nm and 546nm. The product peak at 51 min was collected.

¹ H NMR (D ₂ O, 400MHz): δ1.31(s, 12H), 2.15-2.26(m, 1H), 2.27-2.41(m, 1H), 2.61-2.66(m, 2H), 3.73-3.80 (m, 2H), 3.89(s, 2H), 4.14-4.18(m, 3H), 6.05-6.09(m, 1H), 6.71-6.74(m, 2H), 6.88-6.95(m, 2H), 7.23 (dd, J=9.6Hz, J=13.6Hz, 2H), 7.85(s, 1H), 7.91(s, 1H), 8.01(d, J=6.8Hz, 1H), 8.09-8.12(m, 1H) .ESI-HRMS: calc for C ₄₂ H ₄₇ N ₆ O ₁₉ P ₃ S ₂ [M+Na+2H] ³⁺ 1121.1604, found 1121.1655.

It can be seen from Examples 1 and 2 that Examples 1 and 2 correspond to the same reversible terminal, and a kind of fluorescein is used to mark the reversible terminal containing a nucleotide (U) respectively; specifically, from the chemical structure and synthesis In terms of methods, firstly, the reversible terminal of dUTP was labeled with fluorescein TAMRA, and the reversible terminal was synthesized by two different synthetic routes. Wherein, the synthetic method of embodiment 1 just introduces triphosphoric acid in the second step reaction, so just must use reverse preparation HPLC to separate and purify the product from this step, causes synthetic process loaded down with trivial details, inefficiency; And the synthetic method of embodiment 2 first Fluorescein is connected, and triphosphate is connected in the last step of the reaction, so only the reaction product in the last step must be separated and purified by HPLC.

Example 3

The structural formula of the reversible terminal of this embodiment is shown in formula (III):

The corresponding synthetic route is shown in Figure 7, and the specific steps are as follows:

3.1 Synthesis of Compound N-1

Take a 100ml one-mouth bottle, add 0.75g (6.6mmol) of cysteamine hydrochloride, dissolve it with 4ml of methanol, and add 2.04g (6.6mmol, 50% aqueous solution, Dissolve in 3ml of methanol) and 1.85ml of TEA (13.2mmol) mixture, remove the ice-water bath after 30min, and stir at room temperature. The reaction progress was tracked by TLC, and the reaction was stopped after 24 h. The solvent was spun out, and plate chromatography, MeOH:EA=1:1, gave 44 mg of the product as a yellow oily liquid.

¹ H NMR (D ₂ O, 400MHz): δ2.92(t, J=6.0Hz, 2H), 3.00(t, J=6.4Hz, 2H), 3.40(t, J=6.4Hz, 2H), 3.87 (t, J = 6.0 Hz, 2H).

In the above synthesis, the added 2-hydroxyethyl disulfide can be any value in 6.6-13.2 mmol, and the TEA can be any value in 13.2-19.8 mmol.

3.2 Synthesis of Compound N-2

Add 2mL of anhydrous DMF to a 10mL single-mouth bottle, then add 22mg (84μmol) of compound N-1, keep away from light, stir at room temperature, dissolve 20mg (38μmol) TAMRA (5/6)) in 4mL of anhydrous DMF, inject , and then added 80 μL (570 μmol) of triethylamine. The reaction was stirred at room temperature, followed by TLC until the starting material disappeared. After the reaction was completed, DMF was removed under reduced pressure, and 3:1 DCM/MeOH was used as a developing solvent, and 20 mg of the product was obtained by separation and purification on a TLC plate. ESI-HRMS: cals for C ₂₉ H ₃₁ N ₃ O ₅ S ₂ [M] 565.1705, found 565.1717.

In the above synthesis, the N-1 added can be any value in 38-152 μmol, and the triethylamine can be added in any value in 380-570 μmol.

3.3 Synthesis of Compound N-3

Add 9.6 mg (0.017 mmol) of compound N-2 into the reaction flask, inject 0.5 mL of anhydrous acetonitrile and 20 μL of triethylamine under nitrogen protection, and stir at room temperature. Under nitrogen protection, 27 mg (0.105 mmol) of N,N-succinimidyl carbonate (DSC) was added to another reaction flask, and then the above mixtures were combined and stirred at room temperature for reaction. TLC followed the reaction until the starting material disappeared. After the raw materials disappeared, the reaction was stopped, and it was directly used in the next step reaction without treatment. ESI-HRMS: cals for C ₃₄ H ₃₄ N ₄ O ₉ S ₂ [M] 706.1767, found 706.1761.

In the above synthesis, the added DSC can be any value in 0.068-0.105 mmol, and the triethylamine can be added in any value in 0.085-0.255 mmol.

3.4 Synthesis of compound N-4

The synthesis of compound dUTP-NH ₂ is the same as in Example 1.

Compound dUTP-NH ₂ (25.9mg, 0.028mmol) was dissolved in a buffer solution of 0.5mL NaHCO ₃ /Na ₂ CO ₃ (pH 8.73), and then the reaction solution of N-3 (starting material compound N-3 9.6 mg (0.014 mmol)) was added to the above buffer solution, and the reaction was stirred at room temperature. Analytical HPLC detection reaction k. Conditions: Column: C18, 10μm, 4.6×250mm; Flow rate: 1mL/min; Mobile phase: 100mM TEAA and CH ₃ CN, 5% CH3CN (5min), gradient washing 5%-35% CH3CN (60min); UV detector : 293nm and 546nm; a product peak was generated at 49.1min. Conditions: Column: C18, 5μm, 9.4×250mm; flow rate: 4mL/min; mobile phase: 100mM TEAA and CH ₃ CN, 5% TEAA (5min), gradient washing 5%-25% CH ₃ CN (45min); 80% _CH3CN (10 min), UV detector: 293nm and 546nm. The product peak at 53 min was collected. ESI-HRMS: cals for C ₄₂ H ₄₃ N ₆ O ₂₀ P ₃ S ₂ ^4- [M+2H] 1110.1186, found 1110.1180.

In the above synthesis, the added dUTP-NH ₂ can be any value in the range of 0.014-0.028 mmol.

In this example, the linking unit N-2 was first synthesized, and then N-2 directly reacted with the purchased fluorescein active ester, avoiding the use of SPDP, which is expensive and prone to side reactions, and then reacted with DSC and newly synthesized dUTP-NH ₂ reaction, the expected product can be obtained; the reaction product obtained by this method is structurally different from the product obtained in Examples 1 and 2, and these two disulfide bond-containing The reversible terminals with different structures can be well applied to DNA sequencing in the evaluation of DNA sequencing.

Example 4

The structural formula of the reversible terminal of this embodiment is shown in the following formula (V):

Its synthetic route is shown in Figure 9, and the specific steps are as follows:

4.1 Synthesis of Na ₂ Se ₂ Alkaline Aqueous Solution

2g (50mmol) of NaOH solid was dissolved in 25ml of water, and then 3.95g (50mmol) of selenium powder and 100mg of cetyltrimethylammonium bromide were added. Weigh 0.25g (6.6mmol) NaBH ₄ and 0.2g NaOH solid, add 5ml water to dissolve under ice-bath cooling, under the protection of N2, add this solution dropwise to the above-mentioned selenium solution under stirring, react at room temperature for 1h, and then React at 90°C for half an hour to complete the reaction, and obtain a characteristic brown-red Na ₂ Se ₂ alkaline aqueous solution, which can be used for the next step of diselenide synthesis without treatment.

In the above synthesis, the added selenium powder can be any value in 46.2-52.8 mmol, and the NaOH can be any value in 52.8-59.4 mmol.

4.2 Synthesis of dihydroxyethyl diselenide (compound D-1)

Take a 10ml flask, add 0.25g bromoethanol (2.0mmol), 2mlTHF, vacuumize, under the protection of nitrogen, add 2.4ml (2.0mmol) of freshly prepared Na ₂ Se ₂ alkaline aqueous solution, stir in an oil bath at 50°C. TLC followed the progress of the reaction and stirred overnight. After the reaction was completed, the solvent was spun out, and column chromatography, PE: EA = 1: 1, yielded 104 mg of pure product.

¹ H NMR (CDCl ₃ , 400 MHz): δ 2.34 (s, 2H), 3.10 (t, J=6.0 Hz, 4H), 3.92 (t, J=6.0 Hz, 4H).

In the above synthesis, the added Na ₂ Se ₂ can be any value in the range of 2.0-4.0 mmol.

4.3 Synthesis of compounds D-2 and D-3

Using xylene as a solvent, compound 100mg D-1 (0.40mmol) and excess HBr (162mg, 2.0mmol) were stirred at room temperature for 6h, then added excess concentrated ammonia water (2.3ml, 30mmol), stirred at room temperature, separated and purified on a silica gel plate , a yellow liquid was obtained.

In the above synthesis, the added HBr can be any value in 1.6-2.4 mmol, and the ammonia water can be any value in 20-40 mmol.

4.4 Synthesis of compound D-4

Add 2mL of anhydrous DMF to a 10mL single-mouth bottle, then add 26mg of compound D-3 (104μmol), keep away from light, stir at room temperature, dissolve 20mg (38μmol) TAMRA (5/6)) in 4mL of anhydrous DMF, inject , and then added 80 μL (570 μmol) of triethylamine. The reaction was stirred at room temperature, followed by TLC until the starting material disappeared. After the reaction was completed, DMF was removed under reduced pressure, and 3:1 DCM/MeOH was used as the developing solvent, and 21 mg of the product was obtained by separation and purification on a TLC plate. ESI-HRMS: cals for C ₂₉ H ₃₁ N ₃ O ₅ Se ₂ [M+H] 662.0594, found 662.0582.

In the above synthesis, the D-3 added can be any value in 38-152 μmol, and the triethylamine can be added in any value in 380-570 μmol.

4.5 Synthesis of Compound D-5

Add 11 mg (17 μmol) of compound D-4 into the reaction flask, inject 0.5 mL of anhydrous acetonitrile and 20 μL of triethylamine (143 μmol) under nitrogen protection, and stir at room temperature. Under the protection of nitrogen, 22 mg (85 μmol) of N,N-succinimidyl carbonate (DSC) was added into another reaction flask, and the above mixtures were combined, and stirred at room temperature for reaction. TLC followed the reaction until the starting material disappeared. After the raw materials disappeared, the reaction was stopped, and the reaction product was directly used for the next reaction.

In the above synthesis, the added DSC can be any value in 68-102 μmol, and the triethylamine can be added in any value in 85-255 μmol.

4.6 Synthesis of Compound D-6

Compound 19 mg dUTP-NH ₂ (20 μmol) was dissolved in a buffer solution of 0.5 mL NaHCO ₃ /Na ₂ CO ₃ (pH 8.73), and then all the reaction solutions of D-5 synthesized above (starting material compound D- 4 11 mg (17 μmol)) was added to the above buffer solution, and the reaction was stirred at room temperature. Analytical HPLC detection reaction. Conditions: Column: C18, 10μm, 4.6×250mm; Flow rate: 1mL/min; Mobile phase: 100mM TEAA and CH ₃ CN, 5% CH3CN (5min), gradient washing 5%-35% CH3CN (60min); UV detector : 293nm and 546nm; a product peak was generated at 49.1min. Conditions: Column: C18, 5μm, 9.4×250mm; flow rate: 4mL/min; mobile phase: 100mM TEAA and CH ₃ CN, 5% TEAA (5min), gradient washing 5%-25% CH ₃ CN (45min); 80% _CH3CN (10 min), UV detector: 293nm and 546nm. Collect the product peaks. ESI-HRMS: cals for C ₄₂ H ₄₃ N ₆ O ₂₀ P ₃ Se ₂ ^4- [M+H] 1202.95, found 1202.87.

In the above synthesis, the added dUTP-NH ₂ can be any value in the range of 17-34 μmol.

Example 5

The structural formula of the reversible terminal of this embodiment is shown in the following formula (IV):

Its synthetic route is shown in Figure 8, and the specific steps are as follows:

5.1 Synthesis of Compound T-1

Use L-glutamic acid as raw material, add hydrochloric acid, add sodium nitrite solution dropwise under ice bath stirring, after the dropwise addition, continue to react under ice bath for 3~5h, and then move to room temperature overnight; after the reaction, Evaporate the water under reduced pressure, add ethyl acetate to dissolve, filter, dry the filtrate with anhydrous sodium sulfate, filter, and spin the solvent to obtain T-1. The steps are as follows:

Add 10.00 g (68 mmol) of L-glutamic acid into a 500 mL single-necked bottle, and then add hydrochloric acid solution (14 mL of concentrated hydrochloric acid dissolved in 28 mL of water) to dissolve the solid. The reaction solution was stirred in an ice-water bath for 30 min, and then sodium nitrite aqueous solution (7.00 g, 100 mmol, dissolved in 30 mL of water) was added dropwise while maintaining the temperature, during which reddish-brown gas was generated. After the dropwise addition was completed, the mixture was stirred for 3 h in an ice-water bath, then raised to room temperature and stirred overnight. Evaporate the water under reduced pressure, white solid and light yellow oily liquid appear, add 150mL ethyl acetate to dissolve, filter off the insoluble white solid, dry the filtrate with anhydrous sodium sulfate, filter, evaporate the solvent under reduced pressure to obtain 9.52g light yellow oil The liquid was directly used in the next reaction without purification.

In the above synthesis, the added hydrochloric acid can be any value in 102-136 mmol, and the sodium nitrite can be any value in 102-136 mmol.

5.2 Synthesis of Compound T-2

Take T-1, use anhydrous tetrahydrofuran as solvent, nitrogen protection, slowly add borane/dimethyl sulfide solution dropwise under ice-water bath, after the dropwise addition, continue to react at room temperature for 4~5h; after the reaction, add Methanol was used to quench the reaction, the solvent was spun off, methanol was added, and T-2 was spin-dried to obtain T-2. The steps were as follows:

Take 9.52g (68mmol) of compound T1-1 (crude product) into a 500mL two-neck flask, inject 150mL of anhydrous tetrahydrofuran under nitrogen protection, and stir at room temperature to completely dissolve T1-1. Under an ice-water bath, 9 mL of 10M borane/dimethyl sulfide solution was slowly added dropwise to the reaction system within 4 h. After the dropwise addition, continue to stir at room temperature for 4 h, and then add 100 mL of methanol to quench the reaction. The solvent was distilled off under reduced pressure, and then 100 mL of methanol was added, and the solvent was spun off to obtain 8.30 g of a yellow oily liquid. A small amount was taken and separated by silica gel column chromatography with 20:1 DCM/MeOH as the eluent to obtain a pure product for ¹ H NMR analysis. The rest were directly used in the next reaction without purification.

¹ H NMR (CDCl ₃ , 300MHz): δ4.58-4.66 (m, 1H), 3.89 (dd, J ₁ =3.0, J ₂ =12.6Hz, 1H), 3.63 (dd, J ₁ =4.5, J ₂ =12.6Hz, 1H), 2.46-2.68(m, 2H), 2.11-2.29(m, 2H).

In the above synthesis, the borane added can be any value in 102-340 mmol.

5.3 Synthesis of Compound T-3

Take T-2, use dichloromethane as solvent, add imidazole, and react with TBSCl at room temperature for 17-20h under the protection of nitrogen; The reaction solution was washed with sodium, dried over anhydrous sodium sulfate, spin-dried, and the pure product T-3 was obtained by column chromatography. The steps are as follows:

Take 8.30 g (68 mmol) of the crude compound T-2 and place it in a 250 mL two-necked bottle, and add 5.90 g (86 mmol) of imidazole. Inject 100 mL of dichloromethane under nitrogen protection. 16.51 g (110 mmol) of dimethyl tert-butylchlorosilane (TBSCl) was dissolved in 50 mL of dichloromethane, injected into the above system, and stirred at room temperature for 17 h. After the reaction was completed, dichloromethane was added for dilution, washed successively with 2M HCl, water and saturated NaHCO ₃ solution, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure to obtain 12.49 g of a yellow oily liquid. Separation by silica gel column chromatography, eluting with 15:1 petroleum ether/ethyl acetate gave 4.32 g of light yellow compound T-3.

¹ H NMR (CDCl ₃ , 400MHz): δ4.54-4.59 (m, 1H), 3.84 (dd, J ₁ =3.2, J ₂ =11.2Hz, 1H), 3.67 (dd, J ₁ =3.2, J ₂ =11.2Hz, 1H), 2.40-2.61(m, 2H), 2.14-2.26(m, 2H), 0.87(s, 9H), 0.05(d, J=4.0Hz, 6H).

In the above synthesis, the added TBSCl can be any value in 102-170 mmol.

5.4 Synthesis of Compound T-4

Using dichloromethane as solvent, T-3 reacted with DIBAL-H under nitrogen protection and ice-salt bath, followed by TLC until the reaction was complete. After the reaction was completed, 15% sodium hydroxide solution was added to quench the reaction, and dried over anhydrous sodium sulfate , filter rotary evaporation to remove solvent to get T-4, the steps are specifically:

Take 4.32g (19.0mmol) of compound T-3 in a two-necked flask, inject 45mL of dichloromethane under the protection of _N2 , stir to dissolve, and slowly inject 30.0mL of diisobutylaluminum hydride ( DIBAL-H) (1 M/L in toluene, 30.0 mmol). After stirring for 30 min, TLC monitoring showed that the starting material had disappeared. Stirring was stopped, 120mL 0.2M HCl was added to quench the reaction, dichloromethane was extracted three times, the combined organic phase was washed once with saturated _NaHCO solution, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness under reduced pressure to obtain 3.44 g of a colorless liquid. The rate is 78%.

¹ H NMR (CDCl ₃ , 400MHz): δ5.38-5.56 (m, 1H), 4.25-4.29 (m, 1H), 3.80 (dd, J ₁ =2.8, J ₂ =10.4Hz, 1H), 3.57 ( dd, J ₁ =2.8, J ₂ =10.8 Hz, 1H), 2.16 (s, 2H), 1.92-1.98 (m, 2H), 0.92 (s, 9H), 0.11 (s, 6H).

In the above synthesis, the added DIBAL-H can be any value in the range of 28.5-47.5 mmol.

5.5 Synthesis of Compound T-6

T-4 obtains T-6 with bromoethanolamine under the A-15 catalyst, and the steps are specifically:

Take 1.07g (4.6mmol) of compound T-4 in a single-necked flask, add 1.15g (9.2mmol) of bromoethanol, 230mg of A-15 catalyst, heat and reflux and stir, after 2h, TLC detection shows that the original has disappeared. Stop stirring, remove A-15 by filtration, spin out the solvent, and obtain T-6-1 100mg and T-6-2 70mg by column chromatography.

T-6-1: ¹ H NMR (CDCl ₃ , 400MHz): δ5.18 (d, J=4.8Hz, 1H), 4.16-4.19 (m, 1H), 3.92-3.96 (m, 1H), 3.73- 3.76(m, 1H), 3.61(d, J=4.4Hz, 2H), 3.46-3.50(m, 2H), 1.89-2.08(m, 3H), 1.69-1.73(m, 1H), 0.89(s, 9H), 0.06 (d, J = 2.0 Hz, 6H).

¹³ C NMR (CDCl ₃ , 100 MHz): δ104.67, 79.02, 67.29, 65.41, 32.08, 31.10, 25.93, 25.31, 18.36, -5.26, -5.31.

HRMS: _calc for _{C13H27O3SiBrNa} [M+Na] ⁺ 361.0811, _found 361.0835.

IR (KBr, cm ^-1 ): 2954, 2929, 2858, 1465, 1254, 1102, 839, 777.

T-6-2: ¹ H NMR (CDCl ₃ , 400MHz): δ5.13 (d, J=4.0Hz, 1H), 4.11-4.14 (m, 1H), 3.92-3.97 (m, 1H), 3.68- 3.75(m, 2H), 3.57-3.61(m, 1H), 3.44-3.50(m, 2H), 1.93-2.01(m, 3H), 1.78-1.80(m, 1H), 0.90(s, 9H), 0.07(s, 6H).

¹³ C NMR (CDCl ₃ , 100 MHz): δ104.36, 81.26, 67.21, 67.19, 32.82, 30.97, 26.24, 25.93, 18.36, -5.25, -5.28.

HRMS: _calc for _{C13H27O3SiBrNa} [M+Na] ⁺ 361.0811, found _361.0836 .

IR (KBr, cm ^-1 ): 2928, 2858, 1465, 1254, 1098, 840, 777.

In the above synthesis, the added bromoethanol can be any value in the range of 6.9-13.8 mmol.

5.6.1 Synthesis of Compound T-7-1

Under the action of tetrabutylammonium fluoride TBAF, T-6-1 is dehydroxylated and protected at room temperature to obtain T-7-1. The steps are as follows:

Take 110mg (0.32mmol) of compound T-6-1 obtained in the previous step, add 5mL THF, stir for 10min, then add 0.64mL (0.64mmol, 1M in THF) tetrabutylammonium fluoride (TBAF) solution. Stir at room temperature for 60 min, follow the reaction process by TLC, after the reaction is over, evaporate the solvent to dryness under reduced pressure, and directly separate by silica gel column chromatography, using 5:1PE/EA as the eluent, to obtain 60 mg of the oily product compound T-7-1. The rate is 83.3%.

¹ H NMR (CDCl ₃ , 400MHz): δ5.19(d, J=4.8Hz, 1H), 4.20-4.26(m, 1H), 3.93-3.96(m, 1H), 3.69-3.76(m, 2H) , 3.44-3.52 (m, 3H), 1.95-2.06 (m, 3H), 1.64-1.68 (m, 1H).

¹³ C NMR (CDCl ₃ , 100 MHz): δ 104.59, 78.62, 67.34, 64.78, 32.41, 30.94, 24.87. HRMS: calc for _C7H13BrO3Na [M+Na] ⁺ 246.9946 _, found _246.9929 .

IR (KBr, cm ^-1 ): 3449, 2924, 1189, 1104, 1028.

In the above synthesis, the added tetrabutylammonium fluoride can be any value in the range of 0.48-0.64 mmol.

5.6.2 Synthesis of Compound T-7-2

Under the action of tetrabutylammonium fluoride TBAF, T-6-2 is dehydroxylated and protected at room temperature to obtain T-7-2. The steps are as follows:

Take 70mg (0.21mmol) of compound T-6-2 obtained in the previous step, add 5mL THF, stir for 10min, then add 0.41mL (0.41mmol, 1M in THF) tetrabutylammonium fluoride (TBAF) solution. Stir at room temperature for 40 min, follow the reaction progress by TLC, after the reaction is over, evaporate the solvent under reduced pressure, and directly separate by silica gel column chromatography, 5:1PE/EA is used as eluent to obtain 42 mg of compound T-7-2, with a yield of 87.9% %.

¹ H NMR (CDCl ₃ , 400MHz): δ5.15(d, J=4.4Hz, 1H), 4.26-4.32(m, 1H), 3.99-4.02(m, 1H), 3.74-3.81(m, 2H) , 3.55 (dd, J ₁ =5.2, J ₂ =12.0 Hz, 1H), 3.49 (t, J = 6.0 Hz, 2H), 1.92-2.07 (m, 4H).

¹³ C NMR (CDCl ₃ , 100 MHz): δ 104.74, 81.55, 67.93, 65.61, 33.25, 30.79, 24.33.

HRMS: calc for _C7H13BrO3Na [M+Na] ⁺ _{246.9946 ,} found 246.9938.

IR (KBr, cm ^-1 ): 3448, 2930, 1197, 1058, 1028.

In the above synthesis, the added tetrabutylammonium fluoride can be any value in the range of 0.32-0.42 mmol.

5.7.1 Synthesis of Compound T-8-1

Dissolve T-7-1 in excess ammonia water and react at room temperature to obtain T-8-1. The steps are as follows:

Take 40mg (0.18mmol) of the compound T-7-1 obtained in the previous step, dissolve it in 2mL ammonia water, stir at room temperature for 40h, follow the reaction progress by TLC, after the reaction is finished, add an appropriate amount of ethanol, and evaporate the solvent under reduced pressure to obtain compound T -8-1 crude product 28mg, yield 97%.

¹ H NMR (CD ₃ OD, 400MHz): δ5.20-5.22(m, 1H), 4.17-4.23(m, 1H), 3.87-3.95(m, 1H), 3.64-3.78(m, 1H), 3.56 -3.61 (m, 1H), 3.47-3.53 (m, 1H), 3.15 (t, J=4.8Hz, 1H), 1.91-2.10 (m, 3H), 1.64-1.70 (m, 1H).

¹³ C NMR (CD ₃ OD, 100 MHz): δ 104.72, 79.11, 63.87, 62.95, 39.53, 31.54, 24.86.

HRMS: calc for _C7H16NO3 [M+H] ⁺ _162.1130 , found 162.1135 _.

IR (KBr, cm ^-1 ): 3382, 2951, 1607, 1497, 1459, 1194, 1097, 1056, 1021, 829.

In the above synthesis, the ammonia water added can be any value in 9-18 mmol.

5.7.2 Synthesis of Compound T-8-2

Dissolve T-7-2 in excess ammonia water and react at room temperature to obtain T-8-1. The steps are as follows:

Take 136mg (0.60mmol) of the compound T-7-2 obtained in the previous step, dissolve it in 5mL of ammonia water, react at room temperature for 60h, follow the reaction progress by TLC, after the reaction is completed, add an appropriate amount of ethanol, and evaporate the solvent under reduced pressure to obtain compound T -8-2 crude product 90mg, yield 93%.

¹ H NMR (CDCl ₃ , 400MHz): δ5.15 (d, J = 4.0Hz, 1H), 4.22-4.23 (m, 1H), 3.97-4.02 (m, 2H), 3.89 (dd, J ₁ = 2.4 , J ₂ =12.0Hz, 1H), 3.67(dd, J ₁ =4.8, J ₂ =12.0Hz, 1H), 3.38-3.44(m, 1H), 3.19-3.25(m, 1H), 1.97-2.05( m, 3H), 1.81-1.87 (m, 1H).

¹³ C NMR (CD ₃ OD, 100 MHz): δ 105.02, 81.38, 64.01, 63.49, 39.78, 32.68, 24.20.

HRMS: calc for _C7H16NO3 [M+H] ⁺ _162.1130 , found 162.1128 _.

IR (KBr, cm ^-1 ): 3416, 2925, 1619, 1499, 1458, 1195, 1094, 1057, 1021, 815.

In the above synthesis, the ammonia water added can be any value in 30-60 mmol.

In this example, for the two diastereoisomers, one of the diastereoisomers T-8-2 was selected for the following synthesis.

5.8 Synthesis of Compound T-9-2

T-8-2 undergoes a substitution reaction with fluorescein to obtain compound T-9-2, and the specific steps are:

Add 2 mL of anhydrous DMF to a 10 mL single-necked bottle, then add 30 mg (84 μmol) of compound T-8-2, keep away from light, stir at room temperature, dissolve 20 mg (38 μmol) of TAMRA (5/6) in 4 mL of anhydrous DMF, Inject, and then add 80 μL (570 μmol) of triethylamine (TEA). The reaction was stirred at room temperature, followed by TLC until the starting material disappeared. After the reaction was finished, DMF was removed under reduced pressure, and 3:1 DCM/MeOH was used as the developing solvent, and the product was separated and purified by TLC plate to obtain 20 mg of the product, with a yield of 96%.

¹ H-NMR (CD ₃ OD, 400M): δ8.13 (d, 1H, J=8.0Hz), 8.08 (dd, 1H, J ₁ =1.6, J ₂ =8.0Hz), 7.73 (d, 1H, J=1.2Hz), 7.25(dd, 2H, _J1 =1.6, J2=9.6Hz), 6.99(dd, _2H , _J1 =2.0, _J2 =9.2Hz), 6.89(d, 2H, J= 2.4Hz), 5.10(d, 1H, J=1.6Hz), 4.07～4.11(m, 1H), 3.78～3.85(m, 1H), 3.46～3.61(m, 5H), 3.26(s, 12H), 1.87-1.95 (m, 3H), 1.68-1.76 (m, 1H). ESI-HRMS: calc for [C ₃₂ H ₃₅ N ₃ O ₇ +H] 574.2553, found 574.2531; calc for [C ₃₂ H ₃₅ N ₃ O ₇ +Na] 596.2373, found 596.2340.

In the above synthesis, the added T-8 can be any value in 114-228 μmol, and the TEA can be any value in 190-760 μmol.

5.9 Synthesis of Compound F-2

Methyl trifluoroacetate reacts with propargylamine in an organic solvent to obtain compound F-2, and the steps are specifically:

Add 15ml of methanol to a single-necked bottle, stir under an ice-water bath, add propargylamine E-2 (30mmol, 1.65g), and slowly add methyl trifluoroacetate E-1 (39mmol, 4.99g) after stirring for 20 minutes, 10 Minutes later, the ice-water bath was removed, and the reaction was carried out at room temperature for 24 hours. Reactions were monitored with TLC plates. After the reaction, evaporate the solvent to dryness under reduced pressure, add 30ml chloroform, wash with saturated _NaHCO solution twice (2×30ml), wash with saturated NaCl solution (30ml), dry over anhydrous sodium sulfate, filter, evaporate the solvent to dryness under reduced pressure, reduce Pressure distillation gave 3.02 g of product F-2 with a yield of 66.6%.

¹ H NMR (300 MHz, CDCl ₃ ): 2.32 (t, 1H, J=2.7Hz), 4.14 (2H, dd, J ₁ =2.7, J ₂ =5.4Hz), 6.92 (s, 1H).

5.10 Synthesis of Compound F-3

Compound F-2 and 5-iodo-2'-deoxyuridine F1 undergo a coupling reaction under the action of cuprous iodide to obtain compound F-3, and the steps are as follows:

Add 5-iodo-2'-deoxyuridine F-1 (0.7mmol, 247.9mg) and 8mL DMF into a single-necked bottle, stir to dissolve, and protect from light. Cuprous iodide (0.14 mmol, 26.7 mg) was added, under nitrogen protection, and stirred for 20 minutes to fully dissolve the cuprous iodide. Add 0.25mL TEA, trifluoroacetylpropargylamine F-2 (2.8mmol, 423.0mg), Pd(PPh ₃ ) ₄ (0.07mmol, 80.9mg) in sequence, and react overnight at room temperature. Reactions were monitored with TLC plates. The solvent was evaporated to dryness under reduced pressure, followed by column chromatography with DCM:MeOH=20:1 as the eluent to obtain 158 mg of product F-3 with a yield of 60%.

¹ H NMR (300MHz; DMSO-d6): 2.11 (2H, t, J = 5.4Hz), 3.56～3.58 (2H, m), 3.79 (1H, m), 4.21 (3H, d, J = 5.4Hz) , 5.08(1H, t, J=4.3Hz), 5.23(1H, d, J=3.7Hz), 6.09(1H, t, J=6.4Hz), 8.18(1H, s), 10.05(1H, t, J = 5.3 Hz), 11.63 (1H, s).

5.11 Synthesis of Compound dUTP-NH ₂

Compound F-3 and tri-n-butylamine pyrophosphate E-4, 2-chloro-4H-1,3,2-benzodioxophosphor-4-one E-3 in DMF solvent, triethylamine and iodine React in the presence of compound F-4 to obtain compound F-4, and then deprotect to obtain compound dUTP-NH ₂ , the steps are as follows:

Weigh compound F-3 (one) 60mg (0.16mmol), tri-n-butylamine pyrophosphate E-4 (two) 150mg (0.32mmol), 2-chloro-4H-1,3,2- 66 mg (0.32 mmol) of benzodioxophosphor-4-one E-3 (tri) was placed in three reaction tubes respectively. Dissolve (1) in 0.5ml of anhydrous DMF, then add 0.6ml of freshly distilled tri-n-butylamine, and stir for half an hour. Dissolve (2) in 0.5ml of anhydrous DMF, inject (1) into (2) under vigorous stirring, and stir for half an hour. Inject the above mixed solution into (3) and stir for 1.5h. Add 5ml of 3% iodine (py/H ₂ O, 9/1), add 4ml of water after 15min, and stir for 2h. Add 0.9ml of 3M NaCl solution, then add 30ml of absolute ethanol, freeze overnight at minus 20°C, and centrifuge (3200r/min, 25°C) for 20min. Pour off the supernatant and drain the solvent. Then add 1ml of 1M TEAB solution, add 4ml of concentrated ammonia water, and stir overnight at room temperature. The solvent was evaporated to dryness under reduced pressure, and a white solid appeared. Analytical HPLC was first used for analysis, conditions: column: C18, 5 μm, 4.6 × 250mm; flow rate: 1mL/min; mobile phase: 20mM TEAAc and ethanol, 0min 20mM TEAAc, 35min 20% ethanol; UV detector: 260nm. After preparative HPLC separation, 26 mg of white solid was obtained with a yield of 18%. Column: C18, 5 μm, 9.4×250 mm; flow rate: 6 mL/min; mobile phase: 20 mM TEAAc and ethanol, 0 min 20 mM TEAAc, 40 min 5% ethanol; UV detector: 260 nm.

¹ H NMR (400MHz, D ₂ O): 1.27 (Et ₃ N-CH ₃ , t, J=8.0Hz), 2.33～2.48 (2H, m), 3.18 (Et ₃ N-CH ₂ , q, J= 8.0Hz), 4.03(2H, s), 4.20~4.26(3H, m), 4.58~4.64(1H, m), 6.27(1H, t, J=8.0Hz), 8.38(s, 1H).

³¹ P NMR (162 MHz, D ₂ O): -22.22 (1P), -11.45 (1P), -9.90 (1P).

ESI-HRMS: calc for [C ₁₂ H ₁₈ N ₃ O ₁₄ P ₃ +H] 522.0080, found 522.0070; calc for [C ₁₂ H ₁₈ N ₃ O ₁₄ P ₃ +Na] 543.9899, found 543.9883.

5.12 Synthesis of compound T-10-2

Compound T-9-2 is reacted with DSC under alkaline conditions to obtain compound T-10-2, and the steps are as follows:

Add 10mg (0.017mmol) of compound T-9-2 into a reaction flask, vacuumize, nitrogen protection, inject 0.5ml of anhydrous acetonitrile with a syringe, add 20μl of triethylamine, and stir at room temperature. Add 27 mg (0.105 mmol) of N,N-succinimidyl carbonate (DSC) into another reaction bottle, vacuumize, and inject the above mixed solution under nitrogen protection, and stir the reaction at room temperature. TLC followed the reaction until the starting material disappeared. After disappearance of the starting material, the reaction was stopped and used directly in the next step.

In the above synthesis, the added DSC can be any value in the range of 0.085-0.136 mmol.

5.13 Synthesis of Compound T-11-2

Compound T-10-2 and compound dUTP-NH ₂ undergo a substitution reaction under alkaline conditions to obtain compound T-11-2, and the steps are as follows:

The compound dUTP-NH ₂ (22mg, 0.024mmol) was dissolved in the buffer solution of 0.5ml NaHCO ₃ /Na ₂ CO ₃ (pH is 8.73), the reaction solution of compound T-10-2 (starting material compound T- 9-2 (7 mg, 0.012 mmol)) was added into the buffer solution of compound dUTP-NH ₂ , and the reaction was stirred at room temperature. After the reaction was completed, the unreacted compound T-10-2 was firstly separated by TLC plate, and then separated by preparative HPLC to obtain 4.5 mg of pure compound T-11-2 with a yield of 33.3%. tR is 28.5min. Conditions: column: C18, 5μm, 9.4×250mm; flow rate: 4mL/min; mobile phase: 20mM TEAAc and methanol, 0min 0% methanol, 10min 0% methanol, 30min 50% methanol, 50min 50% methanol ; UV detector: 546 nm. The analytical tR was 32.8 min. Conditions: Column: C18, 10μm, 4.6×250mm; Flow rate: 1mL/min; Mobile phase: 20mM TEAAc and methanol, 0% methanol for 0min, 0% methanol for 10min, 50% methanol for 30min, 50% methanol for 50min; UV detector: 260nm, 546nm.

¹ H NMR (400MHz, D ₂ O): δ8.13(d, 1H, J=4.0Hz), 8.00(d, 1H, J=8.0Hz), 7.95(s, 1H), 7.87(s, 1H) , 7.18～7.26(m, 2H), 6.93～7.01(m, 2H), 6.81(s, 1H), 6.76(s, 1H), 6.14(t, 1H, J=4.0Hz), 5.25(s, 1H ), 4.45～4.56(m, 2H), 4.29～4.38(m, 2H), 4.13～4.27(m, 4H), 4.64～4.80(m, 5H), 3.30(s, 12H), 3.22(Et ₃ N -CH ₂ , q), 2.26-2.90 (m, 1H), 2.09-2.11 (m, 3H), 1.67-1.72 (m, 2H), 1.31 (Et ₃ N-CH ₃ , t).

³¹ P NMR (162 MHz, D ₂ O): -20.63 (1P), -10.92 (1P), -8.32 (1P).

ESI-HRMS: calc for [C ₄₅ H ₅₁ N ₆ O ₂₂ P ₃ -H] 1119.2191, found 1119.2216; calc for [C ₄₅ H ₅₁ N ₆ O ₂₂ P ₃ -PO ₃ H ₂ ] ^- 1039.2528, found 1039.2551; calc for [C ₄₅ H ₅₁ N ₆ O ₂₂ P ₃ +Na-2H] ^- 1141.2010, found 1141.1981.

In the above synthesis, the added dUTP-NH ₂ can be any value in the range of 0.018-0.036 mmol.

Example 6, Biological Evaluation of Synthetic Reversible Terminals

In order to detect whether the reversible terminal synthesized by the present invention can be applied to DNA sequencing, this embodiment detects the characteristics of the reversible terminal in Examples 1-5 in two aspects:

1) Whether it can be recognized by DNA polymerase and participate in DNA extension reaction as a substrate of DNA polymerase;

2) Whether the fluorescent group carried by the reversible terminal can be removed after participating in DNA chain extension, so as to facilitate the next round of extension reaction.

These two aspects are the core of high-throughput sequencing by synthesis. Therefore, the DNA extension reaction system was prepared: fully mix the reversible terminal with the DNA template, Klenow (exo-) DNA polymerase, and Klenow buffer, stand at 30°C for 15 minutes, and treat at 75°C for 10 minutes to inactivate the activity of klenow DNA polymerase. Then, the acid-sensitive reversible terminal and disulfide bond reversible terminal were detected under different acidic conditions (pH2.0, pH1.7, pH1.5, Dowex 50Wx acidic resin) and the conditions of different concentrations of reducing agent. Whether the fluorophore carried by the reversible terminal can be broken. details as follows:

6.1 Fragmentation test of acid-sensitive reversible terminal in DNA chain extension reaction and pH 2.0 conditions (reversible terminal in Example 5)

1) Set up a reversible terminal DNA chain extension reaction in an eppendorf tube according to the following system: 10×Klenowbuffer 10uL, BSA (10mg/mL) 1uL, DMSO 20uL, NaCl(1M) 25uL, Klenow(exo-)pol(5U/uL ) 1.32uL, dUTP (10uM) 6uL, template DNA (853ng/uL) 1.25uL, ddH2O 35.43uL, total volume 100uL.

The reaction system was treated in a 30°C water bath for 15 minutes, and then placed in a 75°C water bath for 10 minutes to inactivate the DNA polymerase. The reaction product is used for the subsequent cleavage reaction of the reversible terminal fluorophore.

2) Fragmentation reaction of acid-sensitive reversible terminal fluorophore

Add 13.5uL 0.24M HCl to the DNA chain extension reaction system, adjust the pH to 2.0, treat at room temperature for 30 minutes, then adjust the pH to 8.0 with 1M Tris, and take the fragmentation reaction product for 12% PAGE electrophoresis analysis, as shown in Figure 10. It can be seen from Figure 10 that the acid-sensitive reversible terminal can be recognized by DNA polymerase as its substrate to participate in the extension of the DNA chain, but under the acidic conditions of pH2.0 and pH2.2, the cleavage effect of the fluorescent group carried by the reversible terminal Not good, need to further adjust the fracture conditions.

6.2 Fragmentation Test of DNA Chain Extension Products Containing Acid-Sensitive Reversible Terminals Under Acidic Conditions at pH 1.7 (Reversible Terminals in Example 5)

Set up the DNA chain extension reaction according to the method in 6.1, add 9uL 0.48M HCl to the DNA chain extension reaction system, adjust the pH to 1.7, treat at room temperature for 30 minutes, then adjust the pH to 8.0 with 1M Tris, and take the fragmentation reaction product for 12% PAGE electrophoresis analysis, as shown in Figure 11, can be seen from Figure 11, DNA chain extension products containing reversible terminals, the fragmentation effect is better at pH 1.7 than at pH 2.0, and about 50% of the fluorescent groups of the extension products are broken , but there are still quite a few parts that are not broken, so it needs to be further optimized.

6.3 Fragmentation Test of DNA Chain Extension Products Containing Acid-sensitive Reversible Terminals in Dowex 50Wx8 Environment (Reversible Terminals of Example 5)

Set up the DNA chain extension reaction according to the method in 6.1, adjust the pH of the reaction system to pH 1.9 and pH 1.5 in the DNA chain extension reaction system by Dowex 50Wx8, treat at room temperature for 30 minutes, and then adjust the pH to 8.0 with 1M Tris, Take the fragmentation reaction product and carry out 12% PAGE electrophoresis analysis, as shown in Figure 12. From Figure 12, it can be known that the DNA chain extension product containing the reversible terminal has a higher fragmentation effect of the fluorescent group carried by the reversible terminal under acidic conditions at pH 1.5 than that at pH 2 .0 and pH 1.9 are both good, most of the fluorophore is broken, although not completely, it can be used for sequencing.

6.4 Disulfide bond reversible terminal in DNA chain extension reaction and its fragmentation test at different DTT concentrations (reversible terminal of Examples 1, 2, and 3)

1) Set up a DNA chain extension reaction containing a disulfide bond reversible terminal in an eppendorf tube according to the following system: 10×Klenow buffer 10uL, BSA (10mg/mL) 1uL, DMSO 20uL, NaCl(1M) 25uL, Klenow(exo-)pol (5U/uL) 1.32uL, dUTP (10uM) 6uL, template DNA (853ng/uL) 1.25uL, ddH2O 35.43uL, total volume 100uL.

The reaction system was treated in a 30°C water bath for 15 minutes, and then placed in a 75°C water bath for 10 minutes to inactivate the DNA polymerase. The reaction product is used for the subsequent cleavage reaction of the reversible terminal fluorophore.

2) The cleavage reaction of the disulfide bond reversible terminal fluorescent group

The DNA chain extension reaction products containing the reversible end of the disulfide bond were treated with 10uM, 8mM and 10mM DTT respectively at room temperature for 10 minutes to 2 hours. The cleavage reaction product was subjected to 12% PAGE electrophoresis analysis, as shown in Figure 13. It can be seen from Figure 13 that the acid-sensitive reversible terminal can be recognized by DNA polymerase and participate in the extension of the DNA chain as its substrate. 10uM DTT treatment of DNA chain extension products cannot effectively break the reversible terminal of the disulfide bond; while 8mM and 10mM DTT acted at room temperature for 10 minutes to 2 hours, respectively, can effectively break the reversible group of the disulfide bond, indicating that it can be applied to high Throughput sequencing reactions.

6.5 Breakage test of DNA chain extension products containing reversible terminal disulfide bonds under different action times of 10mM, 20mM and 30mM DTT

The reversible terminals of Examples 1, 2, and 3 were tested. The evaluation methods and effects of the reversible terminals of the two structures are exactly the same; the details are as follows:

In order to further optimize the fragmentation conditions of DNA chain extension products containing reversible disulfide bonds and shorten the fragmentation time, the fragmentation effects of different concentrations of DTT under different treatment times were tested:

1) 10mM DTT was applied at room temperature for 3 minutes to 15 minutes, and the fragmentation effect was detected: set up the DNA chain extension reaction according to the method in 6.4, and add DTT with a final concentration of 10mM to the DNA chain extension reaction system for different time respectively, and take The cleavage reaction product was analyzed by 12% PAGE electrophoresis, as shown in Figure 14. It can be seen from Figure 14 that the DNA chain extension product containing the reversible terminal of the disulfide bond was exposed to 10 mM DTT at room temperature for 3 min, 5 min, and 8 min. Fluorescent signal, indicating that DTT at this concentration cannot completely break the disulfide bond; there is a weak fluorescent signal after 10 minutes of action, and the fluorescent signal is basically undetectable after 15 minutes, indicating that 10mM DTT has a better effect of breaking the disulfide bond when treated for 15 minutes.

2) 20mM and 30mM DTT were applied at room temperature for 3 to 8 minutes respectively, and the fragmentation effect was detected: set up a DNA chain extension reaction according to the above method, and add DTT with a final concentration of 20mM and 30mM to the DNA chain extension reaction system for different time respectively 12% PAGE electrophoresis analysis was performed on the fragmentation reaction product, as shown in Figure 15. From Figure 15, it can be seen that the DNA chain extension product containing the reversible terminal of the disulfide bond was exposed to 20mM DTT at room temperature for 3min, 5min, and 8min after the fluorescence scanning results were detected. If there is no fluorescent signal, it means that 20mM DTT can completely break the disulfide bond containing the reversible terminal after acting for 3 minutes. Similarly, 30mM DTT at room temperature for 3min and 5min can also completely break the disulfide bond at the reversible terminal. The reversible terminal of the diselenide bond can also be broken under the action of the reducing agent DTT, but the concentration of DTT required for the break is greater and the time will be longer.

The biological evaluation of the reversible terminal in Example 4 is the same as in Examples 1, 2, and 3, except that the amount of the reducing agent DTT needs to be increased by 10 times; the results also indicate that the synthesized reversible terminal can be applied to DNA sequencing.

In summary, the present invention uses fluorescein to label the reversible terminal containing nucleotide U. The test results of Example 6 further verified that the reversible terminal of the present invention has met the biochemical reaction requirements of high-throughput sequencing, and has a good practical prospect.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims (4)

1. a preparation method for Reversible terminal, is characterized in that, the structural formula of described Reversible terminal is as shown in formula III:

Described Reversible terminal is synthesized as follows:

The synthesis of A, compound N-1: take methyl alcohol as solvent, under TEA existent condition, Mercaptamine under ice bath agitation condition with 2-HEDS reaction, obtains compound N-1 the mol ratio of described Mercaptamine, 2-HEDS and TEA is 1:(1 ~ 2): (2 ~ 3);

The synthesis of B, compound N-2: take dry DMF as solvent, under TEA existent condition, compound N-1 and TAMRA (5/6) lucifuge is reacted, and obtains compound N-2 the mol ratio of described TAMRA (5/6), N-1 and TEA is 1:(1 ~ 4): (10 ~ 15);

The synthesis of C, compound N-3: take anhydrous acetonitrile as solvent, under TEA existent condition, compound N-2 under nitrogen protection condition with N, N'-bis-succinimidyl carbonate react, obtain compound

N-3 described N-2, N, the mol ratio of N'-bis-succinimidyl carbonate and TEA is 1:(4 ~ 6): (5 ~ 15);

The synthesis of D, compound N-4: with NaHCO ₃/ Na ₂cO ₃buffered soln be solvent, compound dUTP-NH ₂ react with N-3, obtain compound N-4; Described N-3 and dUTP-NH ₂mol ratio be 1:(1 ~ 2); Namely described compound N-4 has the Reversible terminal of eliminant shown in formula III.

2. a Reversible terminal, is characterized in that, the structural formula of described Reversible terminal is such as formula shown in (V):

3. the preparation method of Reversible terminal as claimed in claim 2, it is characterized in that, described Reversible terminal is synthesized as follows:

A, Na ₂se ₂the preparation of alkaline aqueous solution: under ice bath cooling, by NaBH ₄solid is dissolved in water and forms NaBH ₄solution; Selenium powder and cetyl trimethylammonium bromide is added, at N after water-soluble for NaOH solid ₂under protection, then add described NaBH ₄solution, reacts 0.5 ~ 1h after room temperature reaction 0.5 ~ 1.5h, obtains Na at 85 ~ 95 DEG C ₂se ₂alkaline aqueous solution; Described NaBH ₄, selenium powder and NaOH mol ratio be 1:(7 ~ 8): (8 ~ 9);

The synthesis of B, Compound D-1: be solvent with THF, bromoethanol and Na under nitrogen protection ₂se ₂alkaline aqueous solution oil bath 45 ~ 55 DEG C of stirring reactions, obtain Compound D-1 described bromoethanol and Na ₂se ₂mol ratio be 1:(1 ~ 2);

The synthesis of C, Compound D-2: take dimethylbenzene as solvent, Compound D-1 and HBr react, and obtain Compound D-2 the mol ratio of described D-1 and HBr is 1:(4 ~ 6);

The synthesis of D, Compound D-3: Compound D-2 and strong aqua react, and obtain Compound D-3 the mol ratio of described D-2 and ammoniacal liquor is 1:(50 ~ 100);

The synthesis of E, Compound D-4: take dry DMF as solvent, under TEA existent condition, Compound D-3 and TAMRA (5/6) lucifuge is reacted, and obtains Compound D-4 the mol ratio of described TAMRA (5/6), D-3 and TEA is 1:(1 ~ 4): (10 ~ 15);

The synthesis of F, Compound D-5: take anhydrous acetonitrile as solvent, under TEA existent condition, Compound D-4 under nitrogen protection with N, N'-bis-succinimidyl carbonate react, obtain compound

D-5 described D-4, N, the mol ratio of N'-bis-succinimidyl carbonate and TEA is 1:(4 ~ 6): (5 ~ 15);

The synthesis of G, Compound D-6: with NaHCO ₃/ Na ₂cO ₃buffered soln be solvent, compound dUTP-NH ₂ with D-5 reaction, obtain Compound D-6; Described D-5 and dUTP-NH ₂mol ratio be 1:(1 ~ 2); Described Compound D-6 is the Reversible terminal shown in structural formula (V).

4. the purposes of a Reversible terminal as claimed in claim 2 in DNA synthesis order-checking.