CN103087131B

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

Info

Publication number: CN103087131B
Application number: CN201310015235.3A
Authority: CN
Inventors: 沈玉梅; 赵小东; 邵志峰; 龚兵; 汤道年; 李小卫; 彭丽娜; 邢宇洋; 庄园; 黎庆; 伍新燕
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2013-01-15
Filing date: 2013-01-15
Publication date: 2015-07-08
Anticipated expiration: 2033-01-15
Also published as: CN103087131A

Abstract

The invention discloses a reversible terminal and synthesis and use in DNA synthesis sequencing thereof. The structural formula of the reversible terminal is described according to formula (I), wherein R1 is fluorescein and R2 is a connection unit. The cracking reversible terminal of the invention is available for DNA synthesis sequencing. At the same time, as synthesis required material is easily available and synthesis processes are all conventional chemical reactions, the reversible terminal disclosed by the invention is applicable for large-scale promotion and has good practical prospect due to a biological evaluation result showing that the reversible terminal is capable of totally satisfying biochemical reaction requirements of high-energy sequencing.

Description

Reversible terminal and synthesis thereof and the purposes in DNA synthesis order-checking

Technical field

The present invention relates to chemosynthesis and biochemical field, be specifically related to a class Reversible terminal and synthesis thereof and synthesize the purposes in order-checking at DNA.

Background technology

DNA sequencing technology is one of means important in modern biology research.After the Human Genome Project completes, DNA sequencing technology obtains and develops rapidly.DNA sequencing (DNA sequencing) refers to the base sequence analyzing specific DNA fragments, namely the arrangement mode of VITAMIN B4 (A), thymus pyrimidine (T), cytosine(Cyt) (C) and guanine (G).Development is accurate, the DNA sequencing method of high-throughput, low cost has very important significance for biology, medical science etc.

Synthesis method order-checking (Sequencing By Synthesis, SBS) is one of DNA sequencing technology of new generation.Synthesis method sequence measurement is by being fixed template DNA fragment tested in a large number, and hybridization, in conjunction with general DNA primer, controls four kinds of Nucleotide extensions on DNA primer respectively in immobilized DNA sequencing template.By detecting extension process or extending Nucleotide, realize the detection of the DNA sequence dna information of high-flux parallel.

In synthesis method order-checking, first want four kinds of nucleotide material of synthetic DNA chain extension, be again " Reversible terminal " (reversible terminator).This kind of nucleotide derivative must have the connector element of a cleavable that Nucleotide and fluorescein are coupled together.Then, before next one instruction Nucleotide is incorporated to, make this connector element rupture under mild conditions, so that next Reversible terminal can be incorporated to smoothly, thus read DNA base sequence successively.To reading of checking order of synthesis method, long and efficiency has material impact to this connector element, and therefore, people are also devoted to develop new cleavable connector element always, improve the efficiency of DNA sequencing.The connector element reported at present has photodestruciton, Pd catalytic pyrolysis, fluorochemical cleavable etc., but these connector elements are only limitted to fundamental research at present, can not practical application (Accounts ofChemical Research 2010,43,551-563.).

Summary of the invention

The object of the present invention is to provide a kind of Reversible terminal and synthesis thereof and synthesize the purposes in order-checking at DNA.This Reversible terminal has reduction sensitivity or sensitivity to acid.

The object of the invention is to be achieved through the following technical solutions:

The present invention relates to a kind of Reversible terminal, its structural formula is such as formula shown in (I):

Wherein R ₁for fluorescein, R ₂for connector element.This connector element is the connector element that can rupture completely that two ends are active function groups.

Preferably, the structural formula of described Reversible terminal is such as formula shown in (II):

Preferably, described Reversible terminal is (as shown in Figure 2) of synthesizing as follows:

A, compound F 17-hydroxy-corticosterone ₂synthesis: under ice-water bath agitation condition, mol ratio is 1.0: the propargylamine of (1.2 ~ 2) and trifluoro-acetate react, and obtain compound F 17-hydroxy-corticosterone ₂

B, compound F 17-hydroxy-corticosterone ₃synthesis: at CuI, Pd (PPh ₃) ₄under (tetrakis triphenylphosphine palladium) and TEA (triethylamine) existent condition, compound F 17-hydroxy-corticosterone ₂and F ₁ reaction, obtains compound F 17-hydroxy-corticosterone ₃ described F _1,f _2,cuI, Pd (PPh ₃) ₄be 1 with the mol ratio of TEA: (2 ~ 3): 0.072: 0.025: (1.5 ~ 2);

C, compound dUTP-NH ₂synthesis: compound F 17-hydroxy-corticosterone ₃with tri-n-butylamine pyrophosphate salt (E-4), the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone (E-3) reaction under triethylamine and iodine exist, reaction product goes protection, obtains compound dUTP-NH ₂ described E-4, E-3 and F ₃mol ratio be 2: 2: 1;

The synthesis of D, compound dUTP-SPDP: under TEA existent condition, compound dUTP-NH ₂in sodium carbonate sodium bicarbonate buffer liquid, with the SPDP taking anhydrous acetonitrile as solvent reaction, obtains compound dUTP-SPDP described dUTP-NH ₂the mol ratio of (i.e. Nucleotide U) and SPDP is 1: (1.5 ~ 3);

The synthesis of E, compound R DM-SH: under DTT (dithiothreitol (DTT)) existent condition, cysteamine in sodium carbonate sodium bicarbonate buffer liquid with compound TAMRA (5/6) lucifuge is reacted, and obtains compound R DM-SH the mol ratio of described TAMRA (5/6), cysteamine and DTT is 1: (10 ~ 50): (40 ~ 70);

The synthesis of F, compound dUTP-T: with Na ₃pO ₄-edta buffer liquid (sodium phosphate-ethylenediamine tetraacetic acid (EDTA) damping fluid) and acetonitrile are solvent, and compound dUTP-SPDP and RDM-SH reacts, and obtains compound dUTP-T; The mol ratio of described RDM-SH, dUTP-SPDP is 1: (1 ~ 2); Namely described compound dUTP-T has the cleavable connector element of eliminant shown in formula (II).

Preferably, described Reversible terminal (II) is (as shown in Figure 5) of synthesizing as follows:

B, compound F 17-hydroxy-corticosterone ₃synthesis: at CuI, Pd (PPh ₃) ₄with under TEA existent condition, compound F 17-hydroxy-corticosterone ₁ and F ₂reaction, obtains compound F 17-hydroxy-corticosterone ₃ described F _1,f ₂, CuI, Pd (PPh ₃) ₄be 1 with the mol ratio of TEA: (2 ~ 3): 0.072: 0.025: (1.5 ~ 2);

C, compound G ₁synthesis: take methyl alcohol as solvent, compound F 17-hydroxy-corticosterone ₃react with strong aqua, obtain compound described F ₃be 1 with the mol ratio of ammoniacal liquor: (50 ~ 100);

D, compound G ₂synthesis: make solvent with methyl alcohol and anhydrous acetonitrile, compound G ₁with SPDP reaction, obtains compound described G ₁be 1 with the mol ratio of SPDP: (1 ~ 2);

The synthesis of E, compound R DM-SH: under DTT existent condition, cysteamine in sodium carbonate sodium bicarbonate buffer liquid with compound TAMRA (5/6) lucifuge is reacted, and obtains compound R DM-SH the mol ratio of described TAMRA (5/6), cysteamine and DTT is 1: (10 ~ 50): (40 ~ 70);

F, compound G ₃synthesis: using methyl alcohol and acetonitrile as solvent, compound R DM-SH under the nitrogen protection of aluminium foil parcel with G ₂reaction, obtains compound G ₃ described RDM-SH and G ₂mol ratio be 1: (1.2 ~ 2);

The synthesis of G, compound dUTP-T: compound G ₃with tri-n-butylamine pyrophosphate salt (E-4), the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone (E-3) reaction under triethylamine and iodine exist, reaction product goes protection, obtains compound dUTP-T; Described E-4, E-3 and G ₃mol ratio be 2: 2: 1; Namely described compound dUTP-T has the cleavable connector element of eliminant shown in formula (II).

Preferably, when the structural formula of described Reversible terminal is such as formula shown in (III):

Preferably, described Reversible terminal is (as shown in Figure 7) of synthesizing 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: with dry DMF (DMF) for 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 reacts with DSC (N, N-succinimidyl carbonate) under nitrogen protection condition, obtains compound N-3 the mol ratio of described N-2, DSC and TEA is 1: (4 ~ 6): (5 ~ 15);

The synthesis of D, compound N-4: with NaHCO ₃/ Na ₂cO ₃the buffered soln of (pH is 8.73) is 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).

Preferably, the structural formula of described Reversible terminal is such as formula shown in (IV):

Preferably, described Reversible terminal is (as shown in Figure 9) of synthesizing as follows:

The synthesis of A, compound T-1: under hydrochloric acid existent condition, Pidolidone reacts with Sodium Nitrite under ice bath agitation condition, obtains compound the mol ratio of described Pidolidone, hydrochloric acid and Sodium Nitrite is 1: (1.5 ~ 2): (1.5 ~ 2);

The synthesis of B, compound T-2: make solvent with anhydrous tetrahydro furan, compound T-1 reacts with the borine being dissolved in dimethyl sulphide under nitrogen protection and ice-water bath, obtains compound T-2 the mol ratio of described T-1 and borine is 1: (1.5 ~ 5);

The synthesis of C, compound T-3: make solvent with methylene dichloride, under imidazoles existent condition, compound T-2 reacts with TBSCl (dimethyl tertiary butyl chlorosilane) under nitrogen protection, obtains compound T-3 the mol ratio of described T-2 and TBSCl is 1: (1.5 ~ 2.5);

The synthesis of D, compound T-4: make solvent with methylene dichloride, compound T-3 reacts with DIBAL-H (diisobutyl aluminium hydride) under nitrogen protection and cryosel bath, obtains compound T-4 the mol ratio of described T-3 and DIBAL-H is 1: (1.5 ~ 2.5);

The synthesis of E, compound T-6: under A-15 (A-15 type storng-acid cation exchange resin) catalyzer existent condition, compound T-4 and bromoethanol amine react, and obtain compound T-6 the mol ratio of described T-4 and bromoethanol amine is 1: (1.5 ~ 3);

The synthesis of F, compound T-7: under tetrabutyl fluoride amine (TBAF) existent condition, compound T-6 at room temperature dehydroxylation protection, obtains compound T-7 the mol ratio of described T-6 and TBAF is 1: (1.5 ~ 2);

The synthesis of G, compound T-8: compound T-7 and excess of ammonia water react, and obtain compound T-8 the mol ratio of described T-7 and ammoniacal liquor is 1: (50 ~ 100);

The synthesis of H, compound T-9: take dry DMF as solvent, under TEA existent condition, compound T-8 and TAMRA (5/6) reaction, obtains compound T-9 the mol ratio of described TAMRA (5/6), T-8 and TEA is 1: (3 ~ 6): (5 ~ 20);

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

The synthesis of J, compound T-11: compound T-10 and dUTP-NH ₂(Nucleotide U) reaction, obtains compound T-11; Described T-10 and dUTP-NH ₂mol ratio be 1: (1.5 ~ 3); Described compound T-11 and the Reversible terminal shown in structural formula (IV).

Preferably, the structural formula of described Reversible terminal is as shown in formula V:

A, Na ₂se ₂the preparation of alkaline aqueous solution: under ice bath cooling, by NaBH ₄solid is dissolved in water and forms NaBH ₄solution; After water-soluble for NaOH solid, add selenium powder and cetyl trimethylammonium bromide, under N2 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 is reacted with DSC under nitrogen protection, obtains Compound D-5 the mol ratio of described D-4, DSC and TEA is 1: (4 ~ 6): (5 ~ 15);

The synthesis of G, Compound D-6: with NaHCO ₃/ Na ₂cO ₃the buffered soln of (pH is 8.73) is 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 structure formula V.

The invention still further relates to the purposes of a kind of aforesaid cleavable connector element in DNA synthesis order-checking.

The present invention has following beneficial effect: the present invention has synthesized the new Reversible terminal of a class; Such Reversible terminal can be used for DNA synthesis order-checking; Meanwhile, its synthesis desired raw material is simple and easy to get, and building-up process is conventional chemical reaction, can be used for large-scale promotion and uses.

Accompanying drawing explanation

By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:

Fig. 1 is total synthesis process schematic of Reversible terminal of the present invention, wherein R ₁for fluorescein, R ₂for connector element (linker);

Fig. 2 is the building-up process schematic diagram of the Reversible terminal of embodiment 1;

Fig. 3 is compound dUTP-NH in embodiment 1 ₂building-up process schematic diagram;

Fig. 4 is the process schematic that in embodiment 1, TAMRA (5/6) is Thiolation;

Fig. 5 is the building-up process schematic diagram of the Reversible terminal of embodiment 2;

Fig. 6 is the building-up process schematic diagram of compound dUTP-T in embodiment 2;

Fig. 7 is the building-up process schematic diagram of the Reversible terminal of embodiment 3;

Fig. 8 is the building-up process schematic diagram of the Reversible terminal of embodiment 5;

Fig. 9 is the building-up process schematic diagram of the Reversible terminal of embodiment 4;

Figure 10 is the test result of in embodiment 6 6.1, wherein (a) is DNA chain extension reaction PAGE electrophorogram, b () is cleavage reaction fluorescent scanning result schematic diagram, wherein, M is DNA marker, and 1 is contrast template, and 2 is DNA chain extension reaction positive control, 3 is the fracture of chain extension product pH 2.0 containing Reversible terminal, and 4 be the fracture of the chain extension product pH2.2 containing Reversible terminal;

Figure 11 is the test result of in embodiment 6 6.2, wherein (a) is DNA chain extension reaction PAGE electrophorogram, b () is cleavage reaction fluorescent scanning result schematic diagram, wherein, M is DNA marker, and 1 is contrast template, and 2 is DNA chain extension reaction positive control, 3 is the fracture of chain extension product pH1.7 containing Reversible terminal, and 4 be the fracture of the chain extension product pH1.5 containing Reversible terminal;

Figure 12 is the test result of in embodiment 6 6.3, wherein (a) is DNA chain extension reaction PAGE electrophorogram, b () is cleavage reaction fluorescent scanning result schematic diagram, wherein, M is DNA marker, and 1 is contrast template, and 2 is DNA chain extension reaction positive control, 3 is the fracture of chain extension product pH1.9 containing Reversible terminal, and 4 be the fracture of the chain extension product pH1.5 containing Reversible terminal;

Figure 13 is the test result of in embodiment 6 6.4, wherein (a) is DNA chain extension reaction PAGE electrophorogram, b () is cleavage reaction fluorescent scanning result schematic diagram, wherein, M is DNA marker, 1 is contrast template, 2 is DNA chain extension reaction positive control, 3 for containing the fracture of the chain extension product 10uM DTT room temperature effect 2h of Reversible terminal, 4 for containing the fracture of the chain extension product 8mM DTT room temperature effect 2h of Reversible terminal, and 5-9 is respectively the fracture of the effect of chain extension product 10mM DTT room temperature 10min, 20min, 30min, 1h and 2h containing Reversible terminal;

Figure 14 is the rupture test result of the DNA chain extension product containing disulfide linkage Reversible terminal under 10mM DTT different action time of in embodiment 6 6.5, wherein (a) is DNA chain extension reaction PAGE electrophorogram, b () is cleavage reaction fluorescent scanning result schematic diagram, wherein, M is DNA marker, 1 is contrast template, 2 is DNA chain extension reaction positive control, and 3-7 is respectively the fracture processing 3min, 5min, 8min, 10min and 15min containing disulfide linkage Reversible terminal chain extension product 10mM DTT respectively;

Figure 15 is the rupture test result of the DNA chain extension product containing disulfide linkage Reversible terminal respectively 20, under 30mMDTT different action time of in embodiment 6 6.5, wherein, M is DNA marker, 4 is contrast template, 6 is DNA chain extension reaction positive control, 1-3 is respectively the fracture processing 8min, 5min and 3min containing disulfide linkage Reversible terminal chain extension product 20mM DTT respectively, and 7-8 is respectively the fracture processing 3min and 5min containing disulfide linkage Reversible terminal chain extension product 30mM DTT respectively.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make certain adjustments and improvements.These all belong to protection scope of the present invention.The present invention's raw material used, reagent are commercially available AR, CP level.Gained intermediate product of the present invention and final product adopt NMR etc. to characterize; Total synthesis process schematic of Reversible terminal of the present invention as shown in Figure 1; It is the Reversible terminal that the different fluorescein of employing four kinds marks respectively containing four kinds of different IPs thuja acids (A, G, C, U).

embodiment 1

The structural formula of the Reversible terminal of the present embodiment is as shown in the formula shown in (II):

The synthetic route of its correspondence as shown in Figure 2; Specifically comprise the steps:

1.1 compound F 17-hydroxy-corticosterone ₂synthesis

Trifluoro-acetate and propargylamine are obtained by reacting compound F 17-hydroxy-corticosterone in organic solvent ₂be specially: in a single port bottle, add 60ml methyl alcohol, stir under ice-water bath, add propargylamine (60mmol, 3.3042g), stir after 15 minutes and slowly add trifluoro-acetate (86.7mmol, 11.0957g), the water-bath of 10 minutes recession deicings, reacts 24 hours under room temperature.Reaction TLC plate is monitored, and PE: EA=8: 1, baking sheet, it is product F2 that Rf=0.5 produces new point.Underpressure distillation (51 DEG C, 280Pa), obtains 3.53g, productive rate 39%.

¹H NMR(CDCl ₃，300MHz)：δ2.32(t，J＝4.0Hz，1H)，4.13-4.15(m，2H)，6.92(s，1H)。

In above-mentioned synthesis, the trifluoro-acetate added can be the arbitrary value in 72 ~ 120mmol.

1.2 compound F 17-hydroxy-corticosterone ₃synthesis

In single port bottle, add F1 (0.7mmol, 247mg), then take 9.7mgCuI and 20.3mg Pd (PPh ₃) ₄add in reaction flask, vacuumize, nitrogen protection, aluminium foil wraps up, and adds 2.3ml DMF, stirring and dissolving, adds 0.2mlTEA, and take after F2 (254mg, 1.7mmol) DMF dissolves and add in above-mentioned reaction flask, stirring at room temperature, reaction is spent the night.TLC plate is monitored, and EA is developping agent, and Rf=0.35 is raw material F1, Rf=0.32 is product F3, and 2 positions closely.After question response terminates, evaporated under reduced pressure solvent, direct column chromatography for separation, 20: 1 DCM: MeOH is eluent, obtains 214mg, productive rate 61%.

¹H 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 above-mentioned synthesis, the F added ₂can be the arbitrary value in 1.4 ~ 2.1mmol, TEA can be the value in 1.05 ~ 1.4mmol.

1.3 compound dUTP-NH ₂synthesis

The synthesis of compound dUTP-NH2 is concrete as shown in Figure 3, and the reaction conditions that in Fig. 3, each step is corresponding is: i) DMF, tributylamine ii) DMF, F ₃iii) I ₂, Py, H ₂o iV) NH ₃.

Difference Weigh Compound F in glove box ₃60mg (0.16mmol), tri-n-butylamine pyrophosphate salt 150mg (0.32mmol), the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone 66mg (0.32mmol) is placed in three reaction tubess.Tri-n-butylamine pyrophosphate salt is dissolved in 0.5mL dry DMF, then adds the tri-n-butylamine that 0.6mL newly steams, stir half an hour.Chloro-for 2-4H-1,3,2-benzo dioxy phosphorus-4-ketone is dissolved in 0.5mL dry DMF, under high degree of agitation, adds above-mentioned tri-n-butylamine pyrophosphate solution by syringe, stir half an hour.Then this mixed solution is injected into F ₃in, stir 1.5h.Add 5mL 3% iodine (9: 1 Py/H2O) solution.Add 4mL water after 15min, stir 2h.Add 0.5mL 3M NaCl solution, then add 30mL dehydrated alcohol ,-20 DEG C of freeze overnight, centrifugal (3200r/min, 25 DEG C) 20min.Incline supernatant liquor, obtains precipitation, drains solvent.Add TEAB solution and strong aqua successively again, stirred overnight at room temperature., there is white solid in evaporated under reduced pressure solvent, obtains dUTP-NH ₂.Analyze with analysis mode HPLC, condition: pillar: C18,10 μm, 4.6 × 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAAc and CH ₃cH ₂oH, gradient wash, 0%-20%CH3CH2OH (35min); UV-detector: 254nm.There is product peak to generate when t=13.5min.

¹H NMR(D ₂O，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 forC12H18N3O14P3 Na[M+Na]+ 543.9899，found 543.9883。

The synthesis of 1.4 compound dUTP-SPDP

DUTP (AP is added in the single port bottle of 10mL ₃) (i.e. dUTP-NH ₂) 24.4mg (0.026mmol), then add 600 μ l Na ₂cO ₃/ NaHCO ₃damping fluid, stirring at room temperature, is dissolved in the anhydrous CH of 400 μ l SPDP12.3mg (0.039mmol) ₃cN, adds above-mentioned solution, adds 3 μ l Et ₃n.Stirring at room temperature is reacted, and analysis mode HPLC tracks to raw material and disappears.Condition: pillar: C18,10 μm, 4.6 × 250m; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH ₃cN, 100%TEAA (5min), gradient wash 0% ~ 10% CH ₃cN (5min), 10% ~ 50%CH ₃cN (50min); UV-detector: 293nm; There is product peak to generate when 27.55min.After 8 hours, stopped reaction, preparation HPLC separation and purification product, obtains 5mg.Condition: pillar: C18,5 μm, 9.4 × 250mm; Flow velocity: 4mL/min; Moving phase: 100mM TEAA and CH ₃cN, 100%TEAA (5min), gradient wash 0% ~ 5% CH ₃cN (5min), 5%-30%CH ₃cN (50min); UV-detector: 293nm.

¹H 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 above-mentioned synthesis, the SPDP added can be the arbitrary value in 0.039 ~ 0.078mmol.

1.5 fluoresceins (rhodamine TAMRA (5/6)) are Thiolation

Fluorescein (rhodamine TAMRA (5/6)) is Thiolation obtains compound R DM-SH as shown in Figure 4, specific as follows:

Get cysteamine (73.5mg, 0.95mmol) in 10mL single port bottle, add 400 μ l Na ₂cO ₃/ NaHCO ₃damping fluid stirring and dissolving, aluminium foil wraps up; Get TAMRA (5/6) (10mg, 0.019mmol), add in above-mentioned reaction flask after dissolving by 0.95mL dry DMF, after lucifuge stirring at room temperature 1h, add 1.33mL 1M DTT, stirring at room temperature 2.5h.TLC plate is monitored: there is product dot generation at DCM: MeOH=5: 1, Rf=0.7 place.Be separated and adopt preparation HPLC to purify, obtain 8.8mg, productive rate 94.6%.Condition: pillar: C18,5 μm, 9.4 × 250mm; Flow velocity: 4mL/min; Moving phase: 0.1%TFA and CH ₃cN, 100%TFA (5min), gradient wash 0%-8% CH ₃cN (5min), 8%-50%CH ₃cN (50min); UV-detector: 293nm and 546nm, collects 41min and goes out product peak.

¹H 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 above-mentioned synthesis, the cysteamine added can be the arbitrary value in 0.19 ~ 0.95mmol, and DTT can be the arbitrary value in 0.76 ~ 1.33mmol.

The synthesis of 1.6 compound dUTP-T

Get 5mg RDM-SH (5mg, 0.01mmol) in 10ml single port bottle, vacuumize, nitrogen protection, aluminium foil wraps up; Get dUTP (AP3)-SPDP (22mg, 0.02mmol) in 10mL single port bottle, add 2ml Na ₃pO ₄-edta buffer liquid and acetonitrile 0.5ml, inject RDM-SH reaction flask after stirring and dissolving; Stirring at room temperature, analysis mode HPLC detection reaction.Condition: pillar: C18,10 μm, 4.6 × 250mm; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH ₃cN, 5% CH ₃cN (5min), gradient wash 5%-35% CH3CN (60min); UV-detector: 293nm and 546nm; There is product peak to generate when 49.74min.Stopped reaction after 10h, preparation HPLC separation and purification.Obtain 2.76mg. condition: pillar: C18,5 μm, 9.4 × 250mm; Flow velocity: 4mL/min; Moving phase: 100mM TEAA and CH ₃cN, 5% TEAA (5min), gradient wash 5%-25% CH ₃cN (45min); 80% CH ₃cN (10min), UV-detector: 293nm and 546nm.Collect product peak, 51min place.

¹H NMR(D ₂O，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 ₄₂H ₄₇N ₆O ₁₉P ₃S ₂[M+Na+2H] ³⁺1121.1604，found 1121.1655。

In above-mentioned synthesis, the dUTP-SPDP added can be arbitrary value in 0.01 ~ 0.02mmol.

embodiment 2

The synthetic route of its correspondence as shown in Figure 5; Specifically comprise the steps:

2.1 compound F 17-hydroxy-corticosterone ₂, F ₃synthesis with embodiment 1

The synthesis of 2.2 compound G1

Get 23mg F ₃(0.06mmol) in the single port bottle of 10mL, add 1mL dissolve with methanol, add 0.1mL strong aqua (6mmol), stirred overnight at room temperature.TLC plate is monitored: DCM: MeOH=3: 1, product G1 Rf=0.15.Be separated and adopt TLC plate layer chromatography, MeOH: EA: NH3=6: 6: 1, collect Rf=0.6 ultraviolet color development area.ESI-HRMS：cals for C ₁₂H ₁₅N ₃O ₅[M]281.1012，found 281.1015.

In above-mentioned synthesis, the ammoniacal liquor added can be arbitrary value in 3 ~ 6mmol.

The synthesis of 2.3 compound G2

Get 8.5mg G1 (0.03mmol), use 0.5mL dissolve with methanol; Get 9.4mg SPDP (0.03mmol), add the methanol solution of above-mentioned G1 after dissolving with 0.5mL anhydrous acetonitrile, stirring at room temperature 10h.TLC plate is monitored: MeOH: EA=1: 6, product Rf=0.55.Stopped reaction, screw out solvent, plate layer chromatography obtains 9.5mg product.ESI-HRMS：cals for C ₂₀H ₂₂N ₄O ₆S ₂[M]478.0981，found 478.0974.

In above-mentioned synthesis, the SPDP added can be arbitrary value in 0.03 ~ 0.06mmol.

The synthesis of 2.4 compound G3

Get 6.8mg RDM-SH (0.013mmol, its synthesis is with embodiment 1) and, in 10mL single port bottle, vacuumize nitrogen protection, aluminium foil wraps up; Separately get 9mg G ₂(0.019mmol) in 10ml single port bottle, 0.5ml CH is used ₃cN, does not dissolve completely, then adds 1ml methyl alcohol, dissolves completely, it is injected in above-mentioned reaction flask, stirring at room temperature 9h.Analysis mode HPLC detection reaction, condition: pillar: C18,10 μm, 4.6 × 250mm; Flow velocity: 1mL/min; Moving phase: H ₂o and CH ₃oH, gradient wash 0%-10% CH ₃oH (5min), 10%-70% CH ₃oH (55min); UV-detector: 293nm and 546nm; There is product peak to generate when 49min.Preparative HPLC is separated to obtain 5mg product.

¹H 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 above-mentioned synthesis, the G added ₂it can be arbitrary value in 0.016 ~ 0.026mmol.

The synthesis of 2.5 compound dUTP-T

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

Difference Weigh Compound G in glove box ₃6mg (0.007mmol), tri-n-butylamine pyrophosphate salt 7.7mg (0.014mmol), the chloro-4-H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone 2.8mg (0.014mmol) is placed in three reaction tubess.Tri-n-butylamine pyrophosphate salt is dissolved in 0.15mL dry DMF, then adds the tri-n-butylamine that 0.15mL newly steams, stir half an hour.Chloro-for 2-4H-1,3,2-benzo dioxy phosphorus-4-ketone is dissolved in 0.15mL dry DMF, under high degree of agitation, adds above-mentioned tri-n-butylamine pyrophosphate solution by syringe, stir half an hour.Then this mixed solution is injected in G3, stirs 1.5h.Add 1mL 3% iodine (9: 1Py/H2O) solution.Add 1mL water after 15min, stir 2h.Add 0.5mL 3M NaCl solution, then add 9mL dehydrated alcohol ,-20 DEG C of freeze overnight, centrifugal (3200r/min, 25 DEG C) 20min.Incline supernatant liquor, obtains precipitation, drains solvent, obtains dUTP-T.Analyze with analysis mode HPLC, condition: pillar: C18,10 μm, 4.6 × 250mm; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH ₃cN, 5% CH3CN (5min), gradient wash 5%-35% CH3CN (60min); UV-detector: 293nm and 546nm; There is product peak to generate when 49.7min.Stopped reaction after 10h, preparation HPLC separation and purification.Condition: pillar: C18,5 μm, 9.4 × 250mm; Flow velocity: 4mL/min; Moving phase: 100mM TEAA and CH ₃cN, 5%TEAA (5min), gradient wash 5%-25% CH ₃cN (45min); 80% CH ₃cN (10min), UV-detector: 293nm and 546nm.Collect product peak, 51min place.

From embodiment 1,2, embodiment 1,2 correspond to same Reversible terminal, is all to select a kind of fluorescein to mark Reversible terminal containing a kind of Nucleotide (U) respectively; Particularly, chemically structure and synthetic method aspect, first marks dUTP Reversible terminal with fluorescein TAMRA, has synthesized this Reversible terminal by two kinds of different synthetic routes.Wherein, the synthetic method of embodiment 1 just introduces triphosphoric acid in second step reaction, so just must use reverse preparative HPLC separation and purification product from this step, causes that building-up process is loaded down with trivial details, efficiency is low; And the synthetic method of embodiment 2 first connects fluorescein, in the end step reaction just connects triphosphoric acid, so only have the reaction product of an in the end step just must use HPLC separation and purification.

embodiment 3

The structural formula of the Reversible terminal of the present embodiment is such as formula shown in (III):

As shown in Figure 7, concrete steps are as follows for the synthetic route of its correspondence:

The synthesis of 3.1 compound Ns-1

Get a 100ml single port bottle, add Mercaptamine 0.75g (6.6mmol), use 4ml dissolve with methanol, under ice-water bath stirs, drip the mixed solution of 2-HEDS 2.04g (6.6mmol, 50% aqueous solution are dissolved in 3ml methyl alcohol) and 1.85mlTEA (13.2mmol), the water-bath of 30min recession deicing, stirring at room temperature.TLC follows the tracks of reaction process, stopped reaction after 24h, and screw out solvent, plate layer chromatography, MeOH: EA=1: 1, obtaining 44mg product, is yellow oily liquid.

¹H NMR(D ₂O，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＝6.0Hz，2H)。

In above-mentioned synthesis, the 2-HEDS added can be arbitrary value in 6.6 ~ 13.2mmol, and TEA can be arbitrary value in 13.2 ~ 19.8mmol.

The synthesis of 3.2 compound Ns-2

2mL dry DMF is added in the single port bottle of 10mL, add 22mg (84 μm of ol) compound N-1 again, lucifuge, stirred at ambient temperature, by 20mg (38 μm of ol) TAMRA (5/6)) be dissolved in 4mL dry DMF, inject, then add 80 μ L (570 μm of ol) triethylamine.Stirred at ambient temperature reacts, and TLC tracks to raw material and disappears.After question response terminates, remove DMF under decompression, with 3: 1 DCM/MeOH for developping agent, the separation and purification of TLC plate obtains product 20mg.ESI-HRMS：cals forC ₂₉H ₃₁N ₃O ₅S ₂[M]565.1705，found 565.1717.

In above-mentioned synthesis, the N-1 added can be arbitrary value in 38 ~ 152 μm of ol, and triethylamine can be arbitrary value in 380 ~ 570 μm of ol.

The synthesis of 3.3 compound Ns-3

In reaction flask, add compound N-2 9.6mg (0.017mmol), inject 0.5mL anhydrous acetonitrile and 20 μ L triethylamines under nitrogen protection, stirred at ambient temperature.In another reaction flask, add N under nitrogen protection, N-succinimidyl carbonate (DSC) 27mg (0.105mmol), then merged by above-mentioned mixed solution, stirred at ambient temperature reacts.TLC follows the tracks of reaction to raw material and disappears.After raw material disappears, stopped reaction, is not treatedly directly used in the next step.ESI-HRMS：calsfor C ₃₄H ₃₄N ₄O ₉S ₂[M]706.1767，found 706.1761.

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

The synthesis of 3.4 compound Ns-4

Compound dUTP-NH ₂synthesis with embodiment 1.

By compound dUTP-NH ₂(25.9mg, 0.028mmol) is dissolved in 0.5mL NaHCO ₃/ Na ₂cO ₃in the buffered soln of (pH is 8.73), then join in above-mentioned buffered soln by the reaction solution (starting material compound N-3 9.6mg (0.014mmol)) of N-3, stirred at ambient temperature reacts.Analysis mode HPLC detection reaction k.Condition: pillar: C18,10 μm, 4.6 × 250mm; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH ₃cN, 5% CH3CN (5min), gradient wash 5%-35% CH3CN (60min); UV-detector: 293nm and 546nm; There is product peak to generate when 49.1min.Condition: pillar: C18,5 μm, 9.4 × 250mm; Flow velocity: 4mL/min; Moving phase: 100mMTEAA and CH ₃cN, 5% TEAA (5min), gradient wash 5%-25% CH ₃cN (45min); 80% CH ₃cN (10min), UV-detector: 293nm and 546nm.Collect product peak, 53min place.ESI-HRMS：cals for C ₄₂H ₄₃N ₆O ₂₀P ₃S ₂ ^4-[M+2H]1110.1186，found 1110.1180.

In above-mentioned synthesis, the dUTP-NH added ₂it can be arbitrary value in 0.014 ~ 0.028mmol.

In the present embodiment, first synthesize connector element N-2, then N-2 directly react with the fluorescein active ester bought, and avoids and uses price higher and the SPDP that side reaction easily occurs, more then with DSC and the new dUTP-NH synthesized ₂reaction, can obtain expecting product; The product that the reaction product that this method obtains structurally obtains with embodiment 1,2 is structurally different, can be advantageously applied to DNA sequencing through biological assessment these two kinds containing in the evaluation at DNA sequencing of the Reversible terminal of different structure of disulfide linkage.

embodiment 4

Shown in the following formula V of structural formula of the Reversible terminal of the present embodiment:

As shown in Figure 9, concrete steps are as follows for its synthetic route:

4.1 Na ₂se ₂the synthesis of alkaline aqueous solution

2g (50mmol) NaOH solid is dissolved in 25ml water, after add 3.95g (50mmol) selenium powder and 100mg cetyl trimethylammonium bromide.Separately take 0.25g (6.6mmol) NaBH ₄with 0.2gNaOH solid, under ice bath cooling, add 5ml water dissolution, under N2 protection, this solution be under agitation added drop-wise in above-mentioned selenium solution, room temperature reaction 1h, after react at 90 DEG C and make half an hour reaction tend to completely, obtain that there is the henna Na of feature ₂se ₂alkaline aqueous solution, this solution does not need to process the synthesis that namely can be used for next step diselenide.

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

The synthesis of 4.2 dihydroxy ethyl diselenides (Compound D-1)

Get a 10ml flask, add 0.25g bromoethanol (2.0mmol), 2mlTHF, vacuumize, under nitrogen protection, add freshly prepd Na ₂se ₂alkaline aqueous solution 2.4ml (2.0mmol), oil bath 50 DEG C stirring.TLC follows the tracks of reaction process, and stirring is spent the night.After question response terminates, screw out solvent, column chromatography, PE: EA=1: 1, obtain 104mg sterling.

¹H NMR(CDCl ₃，400MHz)：δ2.34(s，2H)，3.10(t，J＝6.0Hz，4H)，3.92(t，J＝6.0Hz，4H)。

In above-mentioned synthesis, the Na added ₂se ₂it can be arbitrary value in 2.0 ~ 4.0mmol.

The synthesis of 4.3 Compound D-2, D-3

Make solvent with dimethylbenzene, compound 100mg D-1 (0.40mmol) and excessive HBr (162mg, 2.0mmol) at room temperature stir 6h, then excessive concentrated ammonia (2.3ml, 30mmol) is added, stirring at room temperature, silica-gel plate separation and purification, obtains yellow liquid.

In above-mentioned synthesis, the HBr added can be arbitrary value in 1.6 ~ 2.4mmol, and ammoniacal liquor can be arbitrary value in 20 ~ 40mmol.

The synthesis of 4.4 Compound D-4

2mL dry DMF is added in the single port bottle of 10mL, add 26mg Compound D-3 (104 μm of ol) again, lucifuge, stirred at ambient temperature, by 20mg (38 μm of ol) TAMRA (5/6)) be dissolved in 4mL dry DMF, inject, then add 80 μ L (570 μm of ol) triethylamine.Stirred at ambient temperature reacts, and TLC tracks to raw material and disappears.After question response terminates, remove DMF under decompression, with 3: 1 DCM/MeOH for developping agent, the separation and purification of TLC plate obtains product 21mg.ESI-HRMS：cals forC ₂₉H ₃₁N ₃O ₅Se ₂[M+H]662.0594，found 662.0582.

In above-mentioned synthesis, the D-3 added can be arbitrary value in 38 ~ 152 μm of ol, and triethylamine can be arbitrary value in 380 ~ 570 μm of ol.

The synthesis of 4.5 Compound D-5

In reaction flask, add Compound D-4 11mg (17 μm of ol), inject 0.5mL anhydrous acetonitrile and 20 μ L triethylamines (143 μm of ol) under nitrogen protection, stirred at ambient temperature.In another reaction flask, add N under nitrogen protection, N-succinimidyl carbonate (DSC) 22mg (85 μm of ol), then merged by above-mentioned mixed solution, stirred at ambient temperature reacts.TLC follows the tracks of reaction to raw material and disappears.After raw material disappears, stopped reaction, reaction product is directly used in next step reaction.

In above-mentioned synthesis, the DSC added can be arbitrary value in 68 ~ 102 μm of ol, and triethylamine can be arbitrary value in 85 ~ 255 μm of ol.

The synthesis of 4.6 Compound D-6

By compound 19mg dUTP-NH ₂(20 μm of ol) is dissolved in 0.5mL NaHCO ₃/ Na ₂cO ₃in the buffered soln of (pH is 8.73), then join in above-mentioned buffered soln by the reaction solution (starting material compound D-4 11mg (17 μm of ol)) of whole D-5 of above-mentioned synthesis, stirred at ambient temperature reacts.Analysis mode HPLC detection reaction.Condition: pillar: C18,10 μm, 4.6 × 250mm; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH ₃cN, 5% CH3CN (5min), gradient wash 5%-35% CH3CN (60min); UV-detector: 293nm and 546nm; There is product peak to generate when 49.1min.Condition: pillar: C18,5 μm, 9.4 × 250mm; Flow velocity: 4mL/min; Moving phase: 100mMTEAA and CH ₃cN, 5% TEAA (5min), gradient wash 5%-25% CH ₃cN (45min); 80% CH ₃cN (10min), UV-detector: 293nm and 546nm.Collect product peak.ESI-HRMS：cals for C ₄₂H ₄₃N ₆O ₂₀P ₃Se ₂ ^4-[M+H]1202.95，found 1202.87.

In above-mentioned synthesis, the dUTP-NH added ₂it can be arbitrary value in 17 ~ 34 μm of ol.

embodiment 5

The structural formula of the Reversible terminal of the present embodiment is as shown in the formula shown in (IV):

As shown in Figure 8, concrete steps are as follows for its synthetic route:

The synthesis of 5.1 compound T-1

Be raw material with Pidolidone, add hydrochloric acid, under ice bath stirs, drip sodium nitrite solution, after dropwising, continue to react 3 ~ 5h under ice bath, then move to overnight at room temperature; After reaction terminates, pressure reducing and steaming water, adds acetic acid ethyl dissolution, filters, filtrate anhydrous sodium sulfate drying, and filter, be spin-dried for solvent and obtain T-1, described step is specially:

In 500mL single port bottle, add 10.00g (68mmol) Pidolidone, then add hydrochloric acid soln (14mL concentrated hydrochloric acid is dissolved in 28mL water) by dissolution of solid.Reaction solution stirs 30min under ice-water bath, then keeps temperature to drip sodium nitrite in aqueous solution (7.00g, 100mmol are dissolved in 30mL water), has the gas of reddish-brown to produce in dropping process.After dropwising, continue to stir 3h under ice-water bath, then rise to stirred overnight at room temperature., there is white solid and pale yellow oily liquid body, add 150mL acetic acid ethyl dissolution in pressure reducing and steaming water, the white solid that elimination is insoluble, filtrate anhydrous sodium sulfate drying, filters, evaporated under reduced pressure solvent obtains 9.52g pale yellow oily liquid body, not purified be directly used in next step reaction.

In above-mentioned synthesis, the hydrochloric acid added can be arbitrary value in 102 ~ 136mmol, and Sodium Nitrite can be arbitrary value in 102 ~ 136mmol.

The synthesis of 5.2 compound T-2

Get T-1, make solvent with anhydrous tetrahydro furan, nitrogen protection, under ice-water bath, slowly drip borine/dimethyl sulphide solution, after dropwising, continue at room temperature to react 4 ~ 5h; After reaction terminates, add methyl alcohol cancellation reaction, revolve desolventizing, then add methyl alcohol, be spin-dried for obtain T-2, described step is specially:

Get 9.52g (68mmol) compound T1-1 (crude product) and be placed in 500mL two-mouth bottle, inject 150mL anhydrous tetrahydro furan under nitrogen protection, stirred at ambient temperature makes T1-1 dissolve completely.Under ice-water bath, in 4h, slowly in reaction system, drip 9mL 10M borine/dimethyl sulphide solution.After dropwising, continue at room temperature to stir 4h, then add 100mL methyl alcohol cancellation reaction.Pressure reducing and steaming solvent, then add 100mL methyl alcohol, revolve desolventizing and obtain 8.30g yellow oily liquid.Take a morsel, silica gel column chromatography be separated, 20: 1 DCM/MeOH are eluent, obtain sterling for ¹h NMR analyzes.Remaining is not purified is directly used in next step reaction.

¹H 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 above-mentioned synthesis, the borine added can be arbitrary value in 102 ~ 340mmol.

The synthesis of 5.3 compound T-3

Get T-2, make solvent with methylene dichloride, add imidazoles, under nitrogen protection, at room temperature react 17 ~ 20h with TBSCl; After reaction terminates, add dchloromethane, use 2mol/L hydrochloric acid, water and saturated sodium bicarbonate washing reaction liquid respectively, anhydrous sodium sulfate drying, is spin-dried for, and column chromatography obtains T-3 sterling, and described step is specially:

Get compound T-2 crude product 8.30g (68mmol) and be placed in 250mL two-mouth bottle, add 5.90g (86mmol) imidazoles.100mL methylene dichloride is injected under nitrogen protection.Dimethyl tertiary butyl chlorosilane (TBSCl) 16.51g (110mmol) is dissolved in 50mL methylene dichloride, is expelled in above-mentioned system, stirred at ambient temperature reaction 17h.Add dchloromethane after completion of the reaction, use 2M HCl, water and saturated NaHCO successively ₃solution washing, anhydrous sodium sulfate drying, filter, evaporated under reduced pressure solvent, obtains 12.49g yellow oily liquid.Silica gel column chromatography is separated, and 15: 1 petrol ether/ethyl acetate drip washing, obtain faint yellow compound T-3 4.32g.

¹H 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 above-mentioned synthesis, the TBSCl added can be arbitrary value in 102 ~ 170mmol.

The synthesis of 5.4 compound T-4

Make solvent with methylene dichloride, T-3 reacts with DIBAL-H under nitrogen protection and cryosel bath, and TLC tracks to and reacts completely. after reaction terminates; add 15% sodium hydroxide solution cancellation reaction; anhydrous sodium sulfate drying, filtration rotary evaporation removes desolventizing and obtains T-4, and described step is specially:

Get compound T-3 4.32g (19.0mmol) in two mouthfuls of flasks, N ₂protection is lower injects 45mL methylene dichloride, stirring and dissolving, under cryosel bath (-15 DEG C), slowly injects 30.0mL diisobutyl aluminium hydride (DIBAL-H) (1M/L at toluene, 30.0mmol).After stirring 30min, TLC monitoring display raw material disappears.Stop stirring, add 120mL 0.2M HCl cancellation reaction, dichloromethane extraction three times, merge the saturated NaHCO of organic phase ₃solution is washed once, anhydrous sodium sulfate drying, and filter, evaporated under reduced pressure solvent obtains colourless liquid 3.44g, productive rate 78%.

¹H 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＝10.8Hz，1H)，2.16(s，2H)，1.92-1.98(m，2H)，0.92(s，9H)，0.11(s，6H)。

In above-mentioned synthesis, the DIBAL-H added can be arbitrary value in 28.5 ~ 47.5mmol.

The synthesis of 5.5 compound T-6

T-4 obtains T-6 with bromoethanol amine under A-15 catalyzer, and described step is specially:

Get compound T-4 1.07g (4.6mmol) in single port flask, add bromoethanol 1.15g (9.2mmol), A-15 catalyzer 230mg, reflux stirs, and after 2h, TLC detection display disappeared originally.Stop stirring, cross and filter A-15, screw out solvent, column chromatography obtains T-6-1 100mg, T-6-2 70mg.

T-6-1： ¹H 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＝2.0Hz，6H)。

¹³C NMR(CDCl ₃，100MHz)：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 C ₁₃H ₂₇O ₃SiBrNa[M+Na] ⁺361.0811，found 361.0835。

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

T-6-2： ¹H 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)。

¹³C NMR(CDCl ₃，100MHz)：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 C ₁₃H ₂₇O ₃SiBrNa[M+Na] ⁺361.0811，found 361.0836。

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

In above-mentioned synthesis, the bromoethanol added can be arbitrary value in 6.9 ~ 13.8mmol.

5.6.1 the synthesis of compound T-7-1

Under tetrabutyl fluoride amine TBAF effect, T-6-1 at room temperature dehydroxylation protection, obtain T-7-1, described step is specially:

Get compound T-6-1 110mg (0.32mmol) obtained in the previous step, add 5mL THF, stir 10min, then add 0.64mL (0.64mmol, 1M in THF) tetrabutyl ammonium fluoride (TBAF) solution.Stirred at ambient temperature 60min, TLC follow the tracks of reaction process, and after question response terminates, evaporated under reduced pressure solvent, direct silica gel column chromatography is separated, and 5: 1PE/EA is eluent, obtains oil product compound T-7-1 60mg, productive rate 83.3%.

¹H 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)。

¹³C NMR(CDCl ₃，100MHz)：δ104.59，78.62，67.34，64.78，32.41，30.94，24.87。HRMS：calc for C ₇H ₁₃BrO ₃Na[M+Na] ⁺246.9946，found 246.9929。

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

In above-mentioned synthesis, the tetrabutyl fluoride amine added can be arbitrary value in 0.48 ~ 0.64mmol.

5.6.2 the synthesis of compound T-7-2

Under tetrabutyl fluoride amine TBAF effect, T-6-2 at room temperature dehydroxylation protection, obtain T-7-2, described step is specially:

Get compound T-6-2 70mg (0.21mmol) obtained in the previous step, add 5mL THF, stir 10min, then add 0.41mL (0.41mmol, 1M in THF) tetrabutyl ammonium fluoride (TBAF) solution.Stirred at ambient temperature 40min, TLC follow the tracks of reaction process, and after question response terminates, evaporated under reduced pressure solvent, direct silica gel column chromatography is separated, and 5: 1PE/EA is eluent, obtains compound T-7-2 42mg, productive rate 87.9%.

¹H 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＝12.0Hz，1H)，3.49(t，J＝6.0Hz，2H)，1.92-2.07(m，4H)。

¹³C NMR(CDCl ₃，100MHz)：δ104.74，81.55，67.93，65.61，33.25，30.79，24.33。

HRMS：calc for C ₇H ₁₃BrO ₃Na[M+Na] ⁺246.9946，found 246.9938。

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

In above-mentioned synthesis, the tetrabutyl fluoride amine added can be arbitrary value in 0.32 ~ 0.42mmol.

5.7.1 the synthesis of compound T-8-1

T-7-1 is dissolved in excess of ammonia water, reacts, obtain T-8-1 under room temperature, described step is specially:

Get compound T-7-1 40mg (0.18mmol) obtained in the previous step, be dissolved in 2mL ammoniacal liquor, stirred at ambient temperature 40h, TLC follows the tracks of reaction process, after question response terminates, adds ethanol in proper amount, evaporated under reduced pressure solvent, obtains compound T-8-1 crude product 28mg, productive rate 97%.

¹H NMR(CD ₃OD，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)。

¹³C NMR(CD ₃OD，100MHz)：δ104.72，79.11，63.87，62.95，39.53，31.54，24.86。

HRMS：calc for C ₇H ₁₆NO ₃[M+H] ⁺162.1130，found 162.1135。

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

In above-mentioned synthesis, the ammoniacal liquor added can be arbitrary value in 9 ~ 18mmol.

5.7.2 the synthesis of compound T-8-2

T-7-2 is dissolved in excess of ammonia water, reacts, obtain T-8-1 under room temperature, described step is specially:

Get compound T-7-2 136mg (0.60mmol) obtained in the previous step, be dissolved in 5mL ammoniacal liquor, under room temperature, react 60h, TLC follows the tracks of reaction process, after question response terminates, adds ethanol in proper amount, evaporated under reduced pressure solvent, obtains compound T-8-2 crude product 90mg, productive rate 93%.

¹H 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)。

¹³C NMR(CD ₃OD，100MHz)：δ105.02，81.38，64.01，63.49，39.78，32.68，24.20。

HRMS：calc for C ₇H ₁₆NO ₃[M+H] ⁺162.1130，found 162.1128。

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

In above-mentioned synthesis, the ammoniacal liquor added can be arbitrary value in 30 ~ 60mmol.

In the present embodiment, for these two kinds of non-corresponding isomer, select a kind of non-corresponding isomer T-8-2 wherein used as following synthesis.

The synthesis of 5.8 compound T-9-2

T-8-2 and fluorescein generation substitution reaction, obtain compound T-9-2, described concrete steps are:

2mL dry DMF is added in the single port bottle of 10mL, add 30mg (84 μm of ol) compound T-8-2 again, lucifuge, stirred at ambient temperature, 20mg (38 μm of ol) TAMRA (5/6) is dissolved in 4mL dry DMF, inject, then add 80 μ L (570 μm of ol) triethylamine (TEA).Stirred at ambient temperature reacts, and TLC tracks to raw material and disappears.After question response terminates, remove DMF under decompression, with 3: 1 DCM/MeOH for developping agent, the separation and purification of TLC plate obtains product 20mg, productive rate 96%.

¹H-NMR(CD ₃OD，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，J ₁＝1.6，J ₂＝9.6Hz)，6.99(dd，2H，J ₁＝2.0，J ₂＝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 ₃₂H ₃₅N ₃O ₇+H]574.2553，found 574.2531；calc for[C ₃₂H ₃₅N ₃O ₇+Na]596.2373，found 596.2340。

In above-mentioned synthesis, the T-8 added can be arbitrary value in 114 ~ 228 μm of ol, and TEA can be arbitrary value in 190 ~ 760 μm of ol.

The synthesis of 5.9 compound F 17-hydroxy-corticosterones-2

Trifluoro-acetate and propargylamine are obtained by reacting compound F 17-hydroxy-corticosterone-2 in organic solvent, and described step is specially:

In a single port bottle, add 15ml methyl alcohol, stir under ice-water bath, add propargylamine E-2 (30mmol, 1.65g), stir after 20 minutes and slowly add trifluoro-acetate E-1 (39mmol, 4.99g), the water-bath of 10 minutes recession deicings, reacts 24 hours under room temperature.Reaction TLC plate is monitored.After reaction terminates, evaporated under reduced pressure solvent, adds 30ml chloroform, saturated NaHCO ₃solution washing twice (2 × 30ml), saturated NaCl solution washing (30ml), anhydrous sodium sulfate drying, filter, evaporated under reduced pressure solvent, underpressure distillation obtains product F-2 3.02g, productive rate 66.6%.

¹H NMR(300MHz，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)。

The synthesis of 5.10 compound F 17-hydroxy-corticosterones-3

There is linked reaction and obtain compound F 17-hydroxy-corticosterone-3 in compound F 17-hydroxy-corticosterone-2 and the iodo-2 '-deoxyuridine F1 of 5-, described step is specially under cuprous iodide effect:

The iodo-2 '-deoxyuridine F-1 (0.7mmol, 247.9mg) of 5-is added, 8mL DMF, stirring and dissolving, lucifuge in a single port bottle.Add cuprous iodide (0.14mmol, 26.7mg), nitrogen protection, stir and cuprous iodide was fully dissolved in 20 minutes.Add 0.25mL TEA, trifluoroacetyl propargylamine F-2 (2.8mmol, 423.0mg), Pd (PPh successively ₃) ₄(0.07mmol, 80.9mg), under room temperature, reaction is spent the night.Reaction TLC plate monitoring.Evaporated under reduced pressure solvent, column chromatography, be eluent at DCM: MeOH=20: 1, obtains product F-3 158mg, productive rate 60%.

¹H 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＝5.3Hz)，11.63(1H，s)。

5.11 compound dUTP-NH ₂synthesis

Compound F 17-hydroxy-corticosterone-3 and the chloro-4H-1 of tri-n-butylamine pyrophosphate salt E-4,2-, 3,2-benzo dioxy phosphorus-4-ketone E-3, in DMF solvent, are obtained by reacting compound F 17-hydroxy-corticosterone-4 under triethylamine and iodine exist, then go protection, obtain compound dUTP-NH ₂, described step is specially:

Weigh Compound F-3 (one) 60mg (0.16mmol) in glove box, tri-n-butylamine pyrophosphate salt E-4 (two) 150mg (0.32mmol), the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone E-3 (three) 66mg (0.32mmol), is placed in three reaction tubess respectively.(1) is dissolved in 0.5ml dry DMF, then adds the tri-n-butylamine that 0.6ml newly steams, stir half an hour.(2) are dissolved in 0.5ml dry DMF, under vigorous stirring, (one) are injected in (two), stir half an hour.Above-mentioned mixed solution is injected (three), stirs 1.5h.Add 5ml 3% iodine (py/H ₂o, 9/1), add 4ml water after in 15min, stir 2h.Add 0.9ml 3M NaCl solution, then add 30ml dehydrated alcohol, subzero 20 DEG C of freeze overnight, centrifugal (3200r/min, 25 DEG C) 20min.Incline supernatant liquor, drains solvent.Add the TEAB solution of 1ml 1M again, add 4ml strong aqua, stirred overnight at room temperature., there is white solid in evaporated under reduced pressure solvent.First analyze with analysis mode HPLC, condition: pillar: C18,5 μm, 4.6 × 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAAc and ethanol, 0min 20mM TEAAc, 35min 20% ethanol; UV-detector: 260nm.Preparative HPLC is separated, and obtains 26mg white solid, productive rate 18%.Pillar: C18,5 μm, 9.4 × 250mm; Flow velocity: 6mL/min; Moving phase: 20mM TEAAc and ethanol, 0min 20mM TEAAc, 40min 5% ethanol; UV-detector: 260nm.

¹H NMR(400MHz，D ₂O)：1.27(Et ₃N-CH ₃，t，J＝8.0Hz)，2.33～2.48(2H，m)，3.18(Et ₃N-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)。

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

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

The synthesis of 5.12 compound T-10-2

Compound T-9-2 and DSC reacts to obtain compound T-10-2 in the basic conditions, and described step is specially:

In a reaction flask, add compound T-9-2 10mg (0.017mmol), vacuumize, nitrogen protection, inject 0.5ml anhydrous acetonitrile with syringe, add 20 μ l triethylamines, stirred at ambient temperature.In another reaction flask, add N, N-succinimidyl carbonate (DSC) 27mg (0.105mmol), vacuumizes, nitrogen protection, is injected by above-mentioned mixed solution, and stirred at ambient temperature reacts.TLC follows the tracks of reaction to raw material and disappears.After raw material disappears, stopped reaction, is directly used in next step.

In above-mentioned synthesis, the DSC added can be arbitrary value in 0.085 ~ 0.136mmol.

The synthesis of 5.13 compound T-11-2

Compound T-10-2 and compound dUTP-NH ₂substitution reaction occurs in the basic conditions and obtains compound T-11-2, described step is specially:

Compound dUTP-NH ₂(22mg, 0.024mmol) is dissolved in 0.5ml NaHCO ₃/ Na ₂cO ₃in the buffered soln of (pH is 8.73), the reaction solution of compound T-10-2 (starting material compound T-9-2 (7mg, 0.012mmol)) is added compound dUTP-NH ₂buffered soln in, stirred at ambient temperature react.First be separated unreacted compound T-10-2 with the large plate of TLC after reaction terminates, be then separated with preparative HPLC, obtain compound T-11-2 sterling 4.5mg, productive rate 33.3%.TR is 28.5min. condition: pillar: C18,5 μm, 9.4 × 250mm; Flow velocity: 4mL/min; Moving phase: 20mM TEAAc and methyl alcohol, 0min 0% methyl alcohol, 10min 0% methyl alcohol, 30min 50% methyl alcohol, 50min 50% methyl alcohol; UV-detector: 546nm.Analysis tR is 32.8min.Condition: pillar: C18,10 μm, 4.6 × 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAAc and methyl alcohol, 0min 0% methyl alcohol, 10min 0% methyl alcohol, 30min 50% methyl alcohol, 50min 50% methyl alcohol; UV-detector: 260nm, 546nm.

¹H NMR(400MHz，D ₂O)：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 ₃N-CH ₂，q)，2.26～2.90(m，1H)，2.09～2.11(m，3H)，1.67～1.72(m，2H)，1.31(Et ₃N-CH ₃，t)。

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

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

In above-mentioned synthesis, the dUTP-NH added ₂it can be arbitrary value in 0.018 ~ 0.036mmol.

embodiment 6, the biological assessment of Reversible terminal to synthesis

In order to whether the Reversible terminal detected synthesized by the present invention can be applied to DNA sequencing, the present embodiment have detected the characteristic of Reversible terminal two aspects of embodiment 1 ~ 5:

1) whether can identify by archaeal dna polymerase, the substrate as archaeal dna polymerase participates in the extension of DNA;

2) fluorophor entrained by this Reversible terminal can be removed after participating in DNA chain extension, so that the extension of next round.

These two aspects is the core of high-throughput synthesis order-checking (sequencing by synthesis).Therefore DNA extension system is prepared: Reversible terminal fully mixed with DNA profiling, Klenow (exo-) archaeal dna polymerase, Klenow damping fluid, 30 DEG C leave standstill 15 minutes, 75 DEG C of process 10 minutes are with deactivation klenow DNA polymerase activity, and under then have detected different acidic conditions respectively for acid-sensitive Reversible terminal and disulfide linkage Reversible terminal, under (pH2.0, pH1.7, pH1.5, Dowex 50Wx acidic resins) and different concns reductive agent condition, whether these two kinds dissimilar fluorophors entrained by Reversible terminal can rupture.Specific as follows:

Fracture test (Reversible terminal of embodiment 5) of 6.1 acid-sensitive Reversible terminal under DNA chain extension reaction and pH2.0 condition thereof

1) in eppendorf pipe, the DNA chain extension reaction of Reversible terminal is set up according to 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, cumulative volume 100uL.

Reaction system is placed in 30 DEG C of water baths process 15 minutes, then is placed in 75 DEG C of water-baths process 10 minutes with deactivation archaeal dna polymerase.Reaction product is used for the cleavage reaction of follow-up Reversible terminal fluorophor.

2) cleavage reaction of acid-sensitive Reversible terminal fluorophor

13.5uL 0.24M HCl is added in DNA chain extension reaction system, regulate pH to 2.0, room temperature treatment 30 minutes, pH to 8.0 is regulated again with 1M Tris, get cleavage reaction product and carry out 12%PAGE electrophoretic analysis, as shown in Figure 10, as shown in Figure 10, acid-sensitive Reversible terminal can by archaeal dna polymerase identification, the extension of DNA chain is participated in as its substrate, but under the acidic conditions of pH2.0 and pH2.2, the fluorophor breaking effect entrained by Reversible terminal is not good, also needs to adjust failure condition further.

(Reversible terminal of embodiment 5) is tested in 6.2 fractures of DNA chain extension product under pH1.7 acidic conditions containing acid-sensitive Reversible terminal

DNA chain extension reaction is set up according to the method in 6.1,9uL 0.48M HCl is added in DNA chain extension reaction system, regulate pH to 1.7, room temperature treatment 30 minutes, pH to 8.0 is regulated again with 1M Tris, get cleavage reaction product and carry out 12%PAGE electrophoretic analysis, as shown in figure 11, as shown in Figure 11, DNA chain extension product containing Reversible terminal, under pH1.7 condition, breaking effect is better than pH2.0, and the extension products fluorophor of about about 50% is ruptured, but still have considerable part not rupture, thus await further optimization.

(Reversible terminal of embodiment 5) is tested in 6.3 fractures of DNA chain extension product under Dowex 50Wx8 environment containing acid-sensitive Reversible terminal

DNA chain extension reaction is set up according to the method in 6.1, regulate through Dowex 50Wx8 in DNA chain extension reaction system, the pH of reaction system is made to be respectively pH 1.9 and pH 1.5, room temperature treatment 30 minutes, pH to 8.0 is regulated again with 1M Tris, get cleavage reaction product and carry out 12%PAGE electrophoretic analysis, as shown in figure 12, as shown in Figure 12, DNA chain extension product containing Reversible terminal, fluorophor breaking effect under pH1.5 acidic conditions entrained by Reversible terminal is all better than pH2.0 and pH1.9, major part fluorophor is ruptured, although not exclusively, but may be used for order-checking.

6.4 disulfide linkage Reversible terminal are in DNA chain extension reaction and the test of the fracture under different DTT concentration (Reversible terminal of embodiment 1,2,3) thereof

1) in eppendorf pipe, the DNA chain extension reaction containing disulfide linkage Reversible terminal is set up according to following system: 10 × Klenow buffer10uL, 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, cumulative volume 100uL.

2) cleavage reaction of disulfide linkage Reversible terminal fluorophor

At room temperature respectively with the DNA chain extension reaction product of DTT process containing disulfide linkage Reversible terminal of 10uM, 8mM and 10mM, action time was from 10 minutes to 2 hours.Get cleavage reaction product and carry out 12%PAGE electrophoretic analysis, as shown in figure 13, as shown in Figure 13, acid-sensitive Reversible terminal by archaeal dna polymerase identification, can participate in the extension of DNA chain as its substrate.10uM DTT process DNA chain extension product, can not effectively rupture disulfide linkage Reversible terminal; And under 8mM and 10mM DTT room temperature, act on 10 minutes to 2 hours respectively, all can effectively rupture the reversible group of disulfide linkage, illustrates that it can be applied to high-flux sequence reaction completely.

6.5 fractures of DNA chain extension product respectively under 10mM, 20mM action time different from 30mM DTT containing disulfide linkage Reversible terminal are tested

Test be the Reversible terminal of embodiment 1,2,3, evaluation method and the effect of the Reversible terminal of these two kinds of structures are just the same; Specific as follows:

In order to optimize the failure condition of the DNA chain extension product containing disulfide linkage Reversible terminal further, shortening rupture time, testing the breaking effect of different concns DTT under the different treatment time respectively:

1) 3 minutes to 15 minutes are acted under 10mM DTT room temperature respectively, and detection of broken effect: set up DNA chain extension reaction according to the method in 6.4, in DNA chain extension reaction system, add final concentration is that the DTT of 10mM processes different time respectively, get cleavage reaction product and carry out 12%PAGE electrophoretic analysis, as shown in figure 14, as shown in Figure 14, DNA chain extension product containing disulfide linkage Reversible terminal is at the DTT room temperature effect 3min of 10mM, 5min, after 8min, the display of fluorescent scanning result still has fluorescent signal, under this concentration is described, DTT can not completely by disulfide bonds, have faint fluorescent signal after effect 10min, after 15min, fluorescent signal can't detect substantially, and during the DTT process 15 minutes of display 10mM, fracture disulfide linkage effect is better.

2) 3 to 8 minutes are acted on respectively under 20mM and 30mM DTT room temperature, and detection of broken effect: set up DNA chain extension reaction according to the method described above, in DNA chain extension reaction system, add final concentration is respectively that the DTT of 20mM and 30mM processes different time respectively, get cleavage reaction product and carry out 12%PAGE electrophoretic analysis, as shown in figure 15, as shown in Figure 15, DNA chain extension product containing disulfide linkage Reversible terminal is at the DTT room temperature effect 3min of 20mM, 5min, after 8min, fluorescent scanning result can't detect fluorescent signal, the DTT effect 3min that 20mM is described just can completely by containing Reversible terminal disulfide bonds.Similar, the effect of 30mM DTT room temperature 3min, 5min also can completely by the disulfide bonds of Reversible terminal.Can rupture under reductive agent DTT effect equally for two selenium key Reversible terminal, just the DTT concentration of fracture needs is larger, and the time also can be longer.

The biological assessment of embodiment 4 Reversible terminal and embodiment 1,2,3 identical, just the used in amounts of reductive agent DTT will be increased to 10 times; Its result illustrates that its Reversible terminal of synthesizing can be applied to DNA sequencing equally.

In sum, the present invention adopts the fluorescein-labelled Reversible terminal containing Nucleotide U.The test result of embodiment 6 demonstrates Reversible terminal of the present invention further and has completed the biochemical reaction requirement meeting high-flux sequence, possesses good practical prospect.

Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.

Claims

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.