CN103087131A

CN103087131A - Reversible terminal and synthesis and use in DNA synthesis sequencing thereof

Info

Publication number: CN103087131A
Application number: CN2013100152353A
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: 2013-05-08
Anticipated expiration: 2033-01-15
Also published as: CN103087131B

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 purposes synthetic and in the synthetic order-checking of DNA thereof

Technical field

The present invention relates to chemosynthesis and biochemical field, be specifically related to a reversible terminal of class and purposes synthetic and in the synthetic order-checking of DNA thereof.

Background technology

The DNA sequencing technology is one of means important in modern biology research.After the Human Genome Project was completed, the DNA sequencing technology had obtained developing rapidly.DNA sequencing (DNA sequencing) refers to analyze the base sequence of specific DNA fragments, namely the arrangement mode of VITAMIN B4 (A), thymus pyrimidine (T), cytosine(Cyt) (C) and guanine (G).Development accurately, high-throughput, the DNA sequencing method has very important significance for biology, medical science etc. cheaply.

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

In the synthesis method order-checking, at first want four kinds of Nucleotide raw materials of synthetic DNA chain extension, be again " reversible terminal " (reversible terminator).This class nucleotide derivative must have the connector element of a cleavable that Nucleotide and fluorescein are coupled together.Then, before next one indication Nucleotide is incorporated into, make this connector element fracture under the condition of gentleness, so that next reversible terminal can incorporate into smoothly, thereby read successively the DNA base sequence.This connector element to synthesis method order-checking read long and efficient has material impact, therefore, people also are devoted to develop new cleavable connector element always, improve the efficient of DNA sequencing.The connector element of having reported at present has photodestruciton, Pd catalytic pyrolysis, fluorochemical cleavable etc., but these connector elements only limit 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 purposes synthetic and in the synthetic order-checking of DNA thereof.This reversible terminal has reduction sensitivity or sensitivity to acid.

The objective 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 suc as formula shown in (I):

R wherein ₁Be fluorescein, R ₂Be connector element.This connector element is the connector element that can rupture fully that two ends are active function groups.

Preferably, the structural formula of described reversible terminal is suc 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 ₂Synthetic: under the ice-water bath agitation condition, mol ratio is 1.0: the propargylamine of (1.2～2) reacts with trifluoro-acetate, gets compound F 17-hydroxy-corticosterone ₂

B, compound F 17-hydroxy-corticosterone ₃Synthetic: at CuI, Pd (PPh ₃) ₄Under the condition that (tetrakis triphenylphosphine palladium) and TEA (triethylamine) exist, compound F 17-hydroxy-corticosterone ₂And F ₁ Reaction gets compound F 17-hydroxy-corticosterone ₃

Described F _1,F _2,CuI, Pd (PPh ₃) ₄With the mol ratio of TEA be 1: (2～3): 0.072: 0.025: (1.5～2);

C, compound dUTP-NH ₂Synthetic: compound F 17-hydroxy-corticosterone ₃With tri-n-butylamine pyrophosphate salt (E-4), 2-chloro-4H-1, the reaction under triethylamine and iodine existence of 3,2-benzo dioxy phosphorus-4-ketone (E-3), reaction product is gone protection, gets compound dUTP-NH ₂ Described E-4, E-3 and F ₃Mol ratio be 2: 2: 1;

D, compound dUTP-SPDP's is synthetic: under the condition that TEA exists, and compound dUTP-NH ₂In sodium carbonate sodium bicarbonate buffer liquid, with the SPDP take anhydrous acetonitrile as solvent Reaction gets compound dUTP-SPDP

Described dUTP-NH ₂The mol ratio of (being Nucleotide U) and SPDP is 1: (1.5～3);

E, compound R DM-SH's is synthetic: under the condition that DTT (dithiothreitol (DTT)) exists, cysteamine in sodium carbonate sodium bicarbonate buffer liquid with compound TAMRA (5/6)

The lucifuge reaction gets compound R DM-SH

The mol ratio of described TAMRA (5/6), cysteamine and DTT is 1: (10～50): (40～70);

F, compound dUTP-T's is synthetic: 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 reaction get compound dUTP-T; The mol ratio of described RDM-SH, dUTP-SPDP is 1: (1～2); Described compound dUTP-T namely 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 ₃Synthetic: at CuI, Pd (PPh ₃) ₄Under the condition that exists with TEA, compound F 17-hydroxy-corticosterone ₁

And F ₂Reaction gets compound F 17-hydroxy-corticosterone ₃ Described F _1,F ₂, CuI, Pd (PPh ₃) ₄With the mol ratio of TEA be 1: (2～3): 0.072: 0.025: (1.5～2);

C, compound G ₁Synthetic: take methyl alcohol as solvent, compound F 17-hydroxy-corticosterone ₃With the strong aqua reaction, get compound

Described F ₃With the mol ratio of ammoniacal liquor be 1: (50～100);

D, compound G ₂Synthetic: make solvent with methyl alcohol and anhydrous acetonitrile, compound G ₁With SPDP

Reaction gets compound

Described G ₁With the mol ratio of SPDP be 1: (1～2);

E, compound R DM-SH's is synthetic: under the condition that DTT exists, cysteamine in sodium carbonate sodium bicarbonate buffer liquid with compound TAMRA (5/6)

The lucifuge reaction gets compound R DM-SH

F, compound G ₃Synthetic: with methyl alcohol and acetonitrile as solvent, compound R DM-SH under the nitrogen protection of aluminium foil parcel with G ₂Reaction gets compound G ₃

Described RDM-SH and G ₂Mol ratio be 1: (1.2～2);

G, compound dUTP-T's is synthetic: compound G ₃With tri-n-butylamine pyrophosphate salt (E-4), 2-chloro-4H-1, the reaction under triethylamine and iodine existence of 3,2-benzo dioxy phosphorus-4-ketone (E-3), reaction product is gone protection, gets compound dUTP-T; Described E-4, E-3 and G ₃Mol ratio be 2: 2: 1; Described compound dUTP-T namely has the cleavable connector element of eliminant shown in formula (II).

Preferably, when the structural formula of described reversible terminal suc as formula shown in (III):

Preferably, described reversible terminal is (as shown in Figure 7) of synthesizing as follows:

A, compound N-1 synthetic: take methyl alcohol as solvent, under the condition that TEA exists, Mercaptamine under the ice bath agitation condition with the 2-HEDS

Reaction gets compound N-1

The mol ratio of described Mercaptamine, 2-HEDS and TEA is 1: (1～2): (2～3);

Synthesizing of B, compound N-2: take dry DMF (DMF) as solvent, under the condition that TEA exists, compound N-1 and TAMRA (5/6)

The lucifuge reaction gets compound N-2 The mol ratio of described TAMRA (5/6), N-1 and TEA is 1: (1～4): (10～15);

Synthesizing of C, compound N-3: take anhydrous acetonitrile as solvent, under the condition that TEA exists, react with DSC (N, N-succinimidyl carbonate) under the nitrogen protection condition compound N-2, gets compound N-3

The mol ratio of described N-2, DSC and TEA is 1: (4～6): (5～15);

Synthesizing of D, compound N-4: with NaHCO ₃/ Na ₂CO ₃The buffered soln of (pH is 8.73) is solvent, compound dUTP-NH ₂

With the N-3 reaction, get compound N-4; Described N-3 and dUTP-NH ₂Mol ratio be 1: (1～2); Described compound N-4 namely have the reversible terminal of eliminant shown in formula (III).

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

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

A, compound T-1's is synthetic: under the condition that hydrochloric acid exists, Pidolidone reacts with Sodium Nitrite under the ice bath agitation condition, gets compound

The mol ratio of described Pidolidone, hydrochloric acid and Sodium Nitrite is 1: (1.5～2): (1.5～2);

B, compound T-2's is synthetic: make solvent with anhydrous tetrahydro furan, compound T-1 reacts with the borine that is dissolved in dimethyl sulphide under nitrogen protection and ice-water bath, gets compound T-2

The mol ratio of described T-1 and borine is 1: (1.5～5);

C, compound T-3's is synthetic: make solvent with methylene dichloride, under the condition that imidazoles exists, compound T-2 under nitrogen protection and TBSCl (dimethyl tertiary butyl chloride silane) react, get compound T-3

The mol ratio of described T-2 and TBSCl is 1: (1.5～2.5);

D, compound T-4's is synthetic: make solvent with methylene dichloride, compound T-3 under nitrogen protection and cryosel are bathed and DIBAL-H (diisobutyl aluminium hydride) react, get compound T-4

The mol ratio of described T-3 and DIBAL-H is 1: (1.5～2.5);

E, compound T-6's is synthetic: under the condition that A-15 (A-15 type storng-acid cation exchange resin) catalyzer exists, compound T-4 and the reaction of bromoethanol amine get compound T-6

The mol ratio of described T-4 and bromoethanol amine is 1: (1.5～3);

F, compound T-7's is synthetic: under the condition that tetrabutyl fluoride amine (TBAF) exists, at room temperature dehydroxylation protection of compound T-6 gets compound T-7

The mol ratio of described T-6 and TBAF is 1: (1.5～2);

G, compound T-8's is synthetic: compound T-7 and the reaction of excess of ammonia water get compound T-8

The mol ratio of described T-7 and ammoniacal liquor is 1: (50～100);

H, compound T-9's is synthetic: take dry DMF as solvent, and under the condition that TEA exists, compound T-8 and TAMRA (5/6)

Reaction gets compound T-9

The mol ratio of described TAMRA (5/6), T-8 and TEA is 1: (3～6): (5～20);

I, compound T-10's is synthetic: compound T-9 gets compound T-10 under the condition that DSC exists The mol ratio of described T-9 and DSC is 1: (5～8);

J, compound T-11's is synthetic: compound T-10 and dUTP-NH ₂(Nucleotide U)

Reaction gets compound T-11; Described T-10 and dUTP-NH ₂Mol ratio be 1: (1.5～3); Described compound T-11 is 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 is cooling, with NaBH ₄Solid is dissolved in water and forms NaBH ₄Solution; Add selenium powder and cetyl trimethylammonium bromide after the NaOH solid is water-soluble, under the N2 protection, then add described NaBH ₄Solution reacts 0.5～1h after room temperature reaction 0.5～1.5h under 85～95 ℃, get Na ₂Se ₂Alkaline aqueous solution; Described NaBH ₄, selenium powder and NaOH mol ratio be 1: (7～8): (8～9);

Synthesizing of B, Compound D-1: take THF as solvent, bromoethanol and Na under nitrogen protection ₂Se ₂45～55 ℃ of stirring reactions of alkaline aqueous solution oil bath get Compound D-1 Described bromoethanol and Na ₂Se ₂Mol ratio be 1: (1～2);

Synthesizing of C, Compound D-2: take dimethylbenzene as solvent, Compound D-1 and HBr reaction get Compound D-2

The mol ratio of described D-1 and HBr is 1: (4～6);

Synthesizing of D, Compound D-3: Compound D-2 and strong aqua reaction get Compound D-3 The mol ratio of described D-2 and ammoniacal liquor is 1: (50～100);

Synthesizing of E, Compound D-4: take dry DMF as solvent, under the condition that TEA exists, Compound D-3 and TAMRA (5/6)

The lucifuge reaction gets Compound D-4

The mol ratio of described TAMRA (5/6), D-3 and TEA is 1: (1～4): (10～15);

Synthesizing of F, Compound D-5: take anhydrous acetonitrile as solvent, under the condition that TEA exists, react with DSC under nitrogen protection Compound D-4, gets Compound D-5

The mol ratio of described D-4, DSC and TEA is 1: (4～6): (5～15);

Synthesizing 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 the D-5 reaction, get Compound D-6; Described D-5 and dUTP-NH ₂Mol ratio be 1: (1～2); Described Compound D-6 are the reversible terminal shown in the structure formula V.

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

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 the synthetic order-checking of DNA; Simultaneously, its synthetic desired raw material is simple and easy to, and building-up process is the conventional chemical reaction, can be used for large-scale promotion and uses.

Description of drawings

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

Fig. 1 is total synthetic process schematic diagram, the wherein R of reversible terminal of the present invention ₁Be fluorescein, R ₂Be 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 ₂The building-up process schematic diagram;

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

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 6.1 test result in embodiment 6, wherein (a) is DNA chain extension reaction PAGE electrophorogram, (b) be 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 that contains the chain extension product pH 2.0 of reversible terminal, and 4 is the fracture that contains the chain extension product pH2.2 of reversible terminal;

Figure 11 is 6.2 test result in embodiment 6, wherein (a) is DNA chain extension reaction PAGE electrophorogram, (b) be 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 that contains the chain extension product pH1.7 of reversible terminal, and 4 is the fracture that contains the chain extension product pH1.5 of reversible terminal;

Figure 12 is 6.3 test result in embodiment 6, wherein (a) is DNA chain extension reaction PAGE electrophorogram, (b) be 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 that contains the chain extension product pH1.9 of reversible terminal, and 4 is the fracture that contains the chain extension product pH1.5 of reversible terminal;

Figure 13 is 6.4 test result in embodiment 6, wherein (a) is DNA chain extension reaction PAGE electrophorogram, (b) be 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 is the fracture that contains the chain extension product 10uM DTT room temperature effect 2h of reversible terminal, 4 is the fracture that contains the chain extension product 8mM DTT room temperature effect 2h of reversible terminal, and 5-9 is respectively the fracture of the chain extension product 10mM DTT room temperature effect 10min, 20min, 30min, 1h and the 2h that contain reversible terminal;

Figure 14 be in embodiment 6 6.5 the DNA chain extension product that contains the reversible terminal of disulfide linkage in the fracture test result of 10mM DTT under different action times, wherein (a) is DNA chain extension reaction PAGE electrophorogram, (b) be 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 and contains the fracture that the reversible final link extension products of disulfide linkage 10mM DTT processes respectively 3min, 5min, 8min, 10min and 15min;

Figure 15 be in embodiment 6 6.5 the DNA chain extension product that contains the reversible terminal of disulfide linkage respectively 20, the fracture test result of 30mMDTT under different action times, wherein, M is DNA marker, 4 is contrast template, 6 is DNA chain extension reaction positive control, 1-3 is respectively and contains the fracture that the reversible final link extension products of disulfide linkage 20mM DTT processes respectively 8min, 5min and 3min, and 7-8 is respectively and contains the fracture that the reversible final link extension products of disulfide linkage 30mM DTT processes respectively 3min and 5min.

Embodiment

The present invention is described in detail below in conjunction with the drawings and specific embodiments.Following examples will help those skilled in the art further to understand the present invention, but not limit in any form the present invention.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, reagent used is commercially available AR, CP level.Gained intermediate product of the present invention and final product employing NMR etc. characterize; The total synthetic process schematic diagram of reversible terminal of the present invention as shown in Figure 1; Be adopt four kinds of different fluoresceins respectively mark contain the reversible terminal of 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):

Its corresponding synthetic route as shown in Figure 2; Specifically comprise the steps:

1.1 compound F 17-hydroxy-corticosterone ₂Synthetic

Trifluoro-acetate and propargylamine react in organic solvent and obtain compound F 17-hydroxy-corticosterone ₂, be specially: add 60ml methyl alcohol in a single port bottle, stir under ice-water bath, add propargylamine (60mmol, 3.3042g), stir and slowly add trifluoro-acetate (86.7mmol after 15 minutes, 11.0957g), the water-bath of 10 minutes recession deicings, under room temperature, reaction is 24 hours.Reaction is monitored with the TLC plate, PE: EA=8: 1, and baking sheet, Rf=0.5 produces new point and is product F2.Underpressure distillation (51 ℃ 280Pa), get 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 synthesizing, the trifluoro-acetate that adds can be the arbitrary value in 72～120mmol.

1.2 compound F 17-hydroxy-corticosterone ₃Synthetic

Add F1 (0.7mmol, 247mg) in the single port bottle, then take 9.7mgCuI and 20.3mg Pd (PPh ₃) ₄Add in reaction flask, vacuumize, nitrogen protection, the aluminium foil parcel adds 2.3ml DMF, and stirring and dissolving adds 0.2mlTEA, takes to add in above-mentioned reaction flask after F2 (254mg, 1.7mmol) dissolves with DMF, stirring at room, reaction is spent the night.The monitoring of TLC plate, EA is developping agent, and Rf=0.35 is raw material F1, and Rf=0.32 is product F3, and 2 positions are very approaching.After question response finishes, the evaporated under reduced pressure solvent, direct column chromatography for separation, 20: 1 DCM: MeOH is eluent, gets 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 synthesizing, the F that adds ₂Can be the arbitrary value in 1.4～2.1mmol, TEA can be the value of appointing in 1.05～1.4mmol.

1.3 compound dUTP-NH ₂Synthetic

Compound dUTP-NH2 synthesizes specifically as shown in Figure 3, and in Fig. 3, reaction conditions corresponding to each step is: i) DMF, tributylamine ii) DMF, F ₃Iii) I ₂, Py, H ₂O iV) NH ₃

Take respectively compound F 17-hydroxy-corticosterone in glove box ₃60mg (0.16mmol), tri-n-butylamine pyrophosphate salt 150mg (0.32mmol), 2-chloro-4H-1,3,2-benzo dioxy phosphorus-4-ketone 66mg (0.32mmol) is placed in three reaction tubess.The tri-n-butylamine pyrophosphate salt is dissolved in the 0.5mL dry DMF, then adds the new tri-n-butylamine that steams of 0.6mL, stir half an hour.2-chloro-4H-1,3,2-benzo dioxy phosphorus-4-ketone is dissolved in the 0.5mL dry DMF, adds above-mentioned tri-n-butylamine pyrophosphate salt solution by syringe under high degree of agitation, stirs 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 the 30mL dehydrated alcohol ,-20 ℃ of freeze overnight, centrifugal (3200r/min, 25 ℃) 20min.The supernatant liquor that inclines gets precipitation, drains solvent.Add successively again TEAB solution and strong aqua, stirred overnight at room temperature.White solid appears in the evaporated under reduced pressure solvent, gets dUTP-NH ₂HPLC analyzes with analysis mode, condition: pillar: C18,10 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAAc and CH ₃CH ₂OH, gradient washing, 0%-20%CH3CH2OH (35min); UV-detector: 254nm.Product peak generation is arranged 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。

1.4 compound dUTP-SPDP's is synthetic

Add dUTP (AP in the single port bottle of 10mL ₃) (be dUTP-NH ₂) 24.4mg (0.026mmol), then add 600 μ l Na ₂CO ₃/ NaHCO ₃Damping fluid, stirring at room is dissolved in the 400 anhydrous CH of μ l to SPDP12.3mg (0.039mmol) ₃CN adds mentioned solution, adds 3 μ l Et ₃N。The stirring at room reaction, analysis mode HPLC tracks to raw material and disappears.Condition: pillar: C18,10 μ m, 4.6 * 250m; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH ₃CN, 100%TEAA (5min), gradient washing 0%～10% CH ₃CN (5min), 10%～50%CH ₃CN (50min); UV-detector: 293nm; Product peak generation is arranged in the time of 27.55min.After 8 hours, stopped reaction, preparation HPLC separation and purification product gets 5mg.Condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: 100mM TEAA and CH ₃CN, 100%TEAA (5min), gradient washing 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 synthesizing, the SPDP that adds can be the arbitrary value in 0.039～0.078mmol.

1.5 fluorescein (rhodamine TAMRA (5/6)) mercaptan

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

Get cysteamine (73.5mg, 0.95mmol) in 10mL single port bottle, add 400 μ l Na ₂CO ₃/ NaHCO ₃The damping fluid stirring and dissolving, the aluminium foil parcel; Get TAMRA (5/6) (10mg, 0.019mmol), with adding in above-mentioned reaction flask after the dissolving of 0.95mL dry DMF, after lucifuge stirring at room 1h, add 1.33mL 1M DTT, stirring at room 2.5h.TLC plate monitoring: DCM: MeOH=5: there is the product dot generation at 1, Rf=0.7 place.Separate and adopt preparation HPLC to purify, get 8.8mg, productive rate 94.6%.Condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: 0.1%TFA and CH ₃CN, 100%TFA (5min), gradient washing 0%-8% CH ₃CN (5min), 8%-50%CH ₃CN (50min); UV-detector: 293nm and 546nm collect 41min and go out the 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 synthesizing, the cysteamine that adds can be the arbitrary value in 0.19～0.95mmol, and DTT can be the arbitrary value in 0.76～1.33mmol.

1.6 compound dUTP-T's is synthetic

Get 5mg RDM-SH (5mg, 0.01mmol) in 10ml single port bottle, vacuumize, nitrogen protection, aluminium foil parcel; 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 the RDM-SH reaction flask after stirring and dissolving; Stirring at room, analysis mode HPLC detection reaction.Condition: pillar: C18,10 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH ₃CN, 5% CH ₃CN (5min), gradient washing 5%-35% CH3CN (60min); UV-detector: 293nm and 546nm; Product peak generation is arranged when 49.74min.Stopped reaction after 10h, the preparation HPLC separation and purification.Get the 2.76mg. condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: 100mM TEAA and CH ₃CN, 5% TEAA (5min), gradient washing 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 synthesizing, the dUTP-SPDP that adds can be arbitrary value in 0.01～0.02mmol.

Embodiment 2

Its corresponding synthetic route as shown in Figure 5; Specifically comprise the steps:

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

2.2 compound G1's is synthetic

Get 23mg F ₃(0.06mmol) in the single port bottle of 10mL, add the 1mL dissolve with methanol, add 0.1mL strong aqua (6mmol), stirred overnight at room temperature.TLC plate monitoring: DCM: MeOH=3: 1, product G1 Rf=0.15.Separate and adopt the 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 synthesizing, the ammoniacal liquor that adds can be arbitrary value in 3～6mmol.

2.3 compound G2's is synthetic

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

In above-mentioned synthesizing, the SPDP that adds can be arbitrary value in 0.03～0.06mmol.

2.4 compound G3's is synthetic

Get 6.8mg RDM-SH (0.013mmol, it is synthetic with embodiment 1) and in 10mL single port bottle, vacuumize nitrogen protection, the aluminium foil parcel; Separately get 9mg G ₂(0.019mmol) in 10ml single port bottle, use 0.5ml CH ₃CN, not dissolving fully, then add 1ml methyl alcohol, dissolve complete is injected into it in above-mentioned reaction flask stirring at room 9h.Analysis mode HPLC detection reaction, condition: pillar: C18,10 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: H ₂O and CH ₃OH, gradient washing 0%-10% CH ₃OH (5min), 10%-70% CH ₃OH (55min); UV-detector: 293nm and 546nm; Product peak generation is arranged when 49min.Preparative HPLC separates to get the 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 synthesizing, the G that adds ₂Can be arbitrary value in 0.016～0.026mmol.

2.5 compound dUTP-T's is synthetic

Compound dUTP-T synthesizes specifically as shown in Figure 6, and in Fig. 6, reaction conditions corresponding to each step is: i) DMF, tributylamine ii) DMF, G ₃Iii) I ₂, Py, H2O iV) and NH3.

Take respectively compound G in glove box ₃6mg (0.007mmol), tri-n-butylamine pyrophosphate salt 7.7mg (0.014mmol), 2-chloro-4-H-1,3,2-benzo dioxy phosphorus-4-ketone 2.8mg (0.014mmol) is placed in three reaction tubess.The tri-n-butylamine pyrophosphate salt is dissolved in the 0.15mL dry DMF, then adds the new tri-n-butylamine that steams of 0.15mL, stir half an hour.2-chloro-4H-1,3,2-benzo dioxy phosphorus-4-ketone is dissolved in the 0.15mL dry DMF, adds above-mentioned tri-n-butylamine pyrophosphate salt solution by syringe under high degree of agitation, stirs 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 the 9mL dehydrated alcohol ,-20 ℃ of freeze overnight, centrifugal (3200r/min, 25 ℃) 20min.The supernatant liquor that inclines gets precipitation, drains solvent, gets dUTP-T.HPLC analyzes with analysis mode, condition: pillar: C18,10 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH ₃CN, 5% CH3CN (5min), gradient washing 5%-35% CH3CN (60min); UV-detector: 293nm and 546nm; Product peak generation is arranged when 49.7min.Stopped reaction after 10h, the preparation HPLC separation and purification.Condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: 100mM TEAA and CH ₃CN, 5%TEAA (5min), gradient washing 5%-25% CH ₃CN (45min); 80% CH ₃CN (10min), UV-detector: 293nm and 546nm.Collect product peak, 51min place.

By

embodiment

1,2 as can be known,

embodiment

1,2 correspondences the reversible terminal of same, be all select a kind of fluorescein respectively mark contain the reversible terminal of a kind of Nucleotide (U); Particularly, from chemical structure and synthetic method aspect, at first use the reversible terminal of fluorescein TAMRA mark dUTP, synthesized this reversible terminal with two kinds of different synthetic routes.Wherein, the synthetic method of embodiment 1 is just introduced 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, efficient is low; And the synthetic method of embodiment 2 first connects fluorescein, and in the end step reaction just connects triphosphoric acid, and in the end the reaction product in a step just must use HPLC separation and purification so only have.

Embodiment 3

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

Its corresponding synthetic route as shown in Figure 7, concrete steps are as follows:

3.1 compound N-1 is synthetic

Get a 100ml single port bottle, add Mercaptamine 0.75g (6.6mmol), use the 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.TLC follows the tracks of reaction process, and stopped reaction after 24h screws out solvent, plate layer chromatography, and MeOH: EA=1: 1, get the 44mg product, be 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 synthesizing, the 2-HEDS that adds can be arbitrary value in 6.6～13.2mmol, and TEA can be arbitrary value in 13.2～19.8mmol.

3.2 compound N-2 is synthetic

Add the 2mL dry DMF in the single port bottle of 10mL, then add 22mg (84 μ mol) compound N-1, lucifuge, stir under room temperature, with 20mg (38 μ mol) TAMRA (5/6)) be dissolved in the 4mL dry DMF, inject, then add 80 μ L (570 μ mol) triethylamine.Stirring reaction under room temperature, TLC track to raw material and disappear.Question response is removed DMF under decompression after finishing, and take 3: 1 DCM/MeOH as developping agent, the separation and purification of TLC plate gets product 20mg.ESI-HRMS：cals?forC ₂₉H ₃₁N ₃O ₅S ₂[M]565.1705，found?565.1717.

In above-mentioned synthesizing, the N-1 that adds can be arbitrary value in 38～152 μ mol, and triethylamine can be arbitrary value in 380～570 μ mol.

3.3 compound N-3 is synthetic

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

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

3.4 compound N-4 is synthetic

Compound dUTP-NH ₂Synthetic with embodiment 1.

With 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 the reaction solution (starting material compound N-3 9.6mg (0.014mmol)) of N-3 is joined in above-mentioned buffered soln stirring reaction under room temperature.Analysis mode HPLC detection reaction k.Condition: pillar: C18,10 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH ₃CN, 5% CH3CN (5min), gradient washing 5%-35% CH3CN (60min); UV-detector: 293nm and 546nm; Product peak generation is arranged when 49.1min.Condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: 100mMTEAA and CH ₃CN, 5% TEAA (5min), gradient washing 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 synthesizing, the dUTP-NH that adds ₂Can be arbitrary value in 0.014～0.028mmol.

In the present embodiment, at first synthetic connector element N-2, then N-2 directly with the fluorescein active ester reaction of buying, avoided using price higher and the SPDP of side reaction easily occurs, more then and DSC and new synthetic dUTP-NH ₂Reaction can obtain expecting product; The reaction product that this method obtains is structurally structurally different with the product that

embodiment

1,2 obtains, and contains through these two kinds of biological assessments in the evaluation of reversible terminal at DNA sequencing of different structure of disulfide linkage to be advantageously applied to DNA sequencing.

Embodiment 4

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

Its synthetic route as shown in Figure 9, concrete steps are as follows:

4.1 Na ₂Se ₂Synthesizing 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 the 0.2gNaOH solid, add the 5ml water dissolution under ice bath is cooling, under the N2 protection, this solution under agitation is added drop-wise in above-mentioned selenium solution, room temperature reaction 1h, reaction makes the reaction trend fully half an hour under 90 ℃ afterwards, obtains having the henna Na of feature ₂Se ₂Alkaline aqueous solution, this solution need not processed and namely can be used for the synthetic of next step diselenide.

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

4.2 dihydroxy ethyl diselenide (Compound D-1) is synthetic

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), 50 ℃ of stirrings of oil bath.TLC follows the tracks of reaction process, and stirring is spent the night.After question response finishes, screw out solvent, column chromatography, PE: EA=1: 1, get the 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 synthesizing, the Na that adds ₂Se ₂Can be arbitrary value in 2.0～4.0mmol.

4.3 Compound D-2, D-3's is synthetic

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

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

4.4 Compound D-4 is synthetic

Add the 2mL dry DMF in the single port bottle of 10mL, then add 26mg Compound D-3 (104 μ mol), lucifuge, stir under room temperature, with 20mg (38 μ mol) TAMRA (5/6)) be dissolved in the 4mL dry DMF, inject, then add 80 μ L (570 μ mol) triethylamine.Stirring reaction under room temperature, TLC track to raw material and disappear.Question response is removed DMF under decompression after finishing, and take 3: 1 DCM/MeOH as developping agent, the separation and purification of TLC plate gets product 21mg.ESI-HRMS：cals?forC ₂₉H ₃₁N ₃O ₅Se ₂?[M+H]662.0594，found?662.0582.

In above-mentioned synthesizing, the D-3 that adds can be arbitrary value in 38～152 μ mol, and triethylamine can be arbitrary value in 380～570 μ mol.

4.5 Compound D-5 is synthetic

Add Compound D-4 11mg (17 μ mol) in reaction flask, inject 0.5mL anhydrous acetonitrile and 20 μ L triethylamines (143 μ mol) under nitrogen protection, stir under room temperature.Add N under nitrogen protection in another reaction flask, N-succinimidyl carbonate (DSC) 22mg (85 μ mol), more above-mentioned mixed solution is merged stirring reaction under room temperature.TLC follows the tracks of to react to raw material and disappears.After raw material disappears, stopped reaction, reaction product is directly used in next step reaction.

In above-mentioned synthesizing, the DSC that adds can be arbitrary value in 68～102 μ mol, and triethylamine can be arbitrary value in 85～255 μ mol.

4.6 Compound D-6 is synthetic

With compound 19mg dUTP-NH ₂(20 μ mol) is dissolved in 0.5mL NaHCO ₃/ Na ₂CO ₃In the buffered soln of (pH is 8.73), the reaction solution (starting material compound D-4 11mg (17 μ mol)) of more above-mentioned synthetic whole D-5 is joined in above-mentioned buffered soln stirring reaction under room temperature.Analysis mode HPLC detection reaction.Condition: pillar: C18,10 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH ₃CN, 5% CH3CN (5min), gradient washing 5%-35% CH3CN (60min); UV-detector: 293nm and 546nm; Product peak generation is arranged when 49.1min.Condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: 100mMTEAA and CH ₃CN, 5% TEAA (5min), gradient washing 5%-25% CH ₃CN (45min); 80% CH ₃CN (10min), UV-detector: 293nm and 546nm.Collection product peak.ESI-HRMS：cals?for?C ₄₂H ₄₃N ₆O ₂₀P ₃Se ₂ ^4-[M+H]1202.95，found?1202.87.

In above-mentioned synthesizing, the dUTP-NH that adds ₂Can be arbitrary value in 17～34 μ mol.

Embodiment 5

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

Its synthetic route as shown in Figure 8, concrete steps are as follows:

5.1 compound T-1's is synthetic

Take Pidolidone as raw material, add hydrochloric acid, under stirring, ice bath drips sodium nitrite solution, after dropwising, continue to react 3～5h under ice bath, then move under room temperature and spend the night; After reaction finished, pressure reducing and steaming water added acetic acid ethyl dissolution, filtered, and filtrate is used anhydrous sodium sulfate drying, filtered, and was spin-dried for solvent and got T-1, and described step is specially:

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

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

5.2 compound T-2's is synthetic

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

Get 9.52g (68mmol) compound T1-1 (crude product) and be placed in the 500mL two-mouth bottle, inject the 150mL anhydrous tetrahydro furan under nitrogen protection, under room temperature, stirring is dissolved T1-1 fully.Under ice-water bath, slowly drip 9mL 10M borine/dimethyl sulphide solution in reaction system in 4h.After dropwising, continue at room temperature to stir 4h, then add 100mL methyl alcohol cancellation reaction.The pressure reducing and steaming solvent, then add 100mL methyl alcohol, revolve desolventizing and get 8.30g yellow oily liquid.Take a morsel, silica gel column chromatography separates, and DCM/MeOH was eluent in 20: 1, got sterling and was used 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 synthesizing, the borine that adds can be arbitrary value in 102～340mmol.

5.3 compound T-3's is synthetic

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

Get compound T-2 crude product 8.30g (68mmol) and be placed in the 250mL two-mouth bottle, add 5.90g (86mmol) imidazoles.Inject the 100mL methylene dichloride under nitrogen protection.Dimethyl tertiary butyl chloride silane (TBSCl) 16.51g (110mmol) is dissolved in the 50mL methylene dichloride, is expelled in above-mentioned system, stirring reaction 17h under room temperature.React and add the methylene dichloride dilution after complete, use successively 2M HCl, water and saturated NaHCO ₃Solution washing, anhydrous sodium sulfate drying filters, and the evaporated under reduced pressure solvent gets 12.49g yellow oily liquid.Silica gel column chromatography separates, and petrol ether/ethyl acetate drip washing in 15: 1 gets 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 synthesizing, the TBSCl that adds can be arbitrary value in 102～170mmol.

5.4 compound T-4's is synthetic

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 finishes; add 15% sodium hydroxide solution cancellation reaction; anhydrous sodium sulfate drying filters the rotary evaporation desolventizing and gets T-4, and described step is specially:

Get compound T-3 4.32g (19.0mmol) in two mouthfuls of flasks, N ₂The lower 45mL methylene dichloride that injects of protection, stirring and dissolving, under cryosel is bathed (15 ℃), (1M/L is at toluene, 30.0mmol) slowly to inject 30.0mL diisobutyl aluminium hydride (DIBAL-H).After stirring 30min, the TLC monitoring shows that raw material disappears.Stop stirring, add 120mL 0.2M HCl cancellation reaction, dichloromethane extraction three times merges the saturated NaHCO of organic phase ₃Solution is washed once, and anhydrous sodium sulfate drying filters, and the evaporated under reduced pressure solvent gets 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 synthesizing, the DIBAL-H that adds can be arbitrary value in 28.5～47.5mmol.

5.5 compound T-6's is synthetic

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

Get compound T-4 1.07g (4.6mmol) in the single port flask, add bromoethanol 1.15g (9.2mmol), A-15 catalyzer 230mg, reflux stirs, and after 2h, the TLC detection display disappeared originally.Stop stirring, remove by filter A-15, screw out solvent, column chromatography gets 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 synthesizing, the bromoethanol that adds can be arbitrary value in 6.9～13.8mmol.

5.6.1 compound T-7-1's is synthetic

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

Get compound T-6-1 110mg obtained in the previous step (0.32mmol), add 5mL THF, stir 10min, then add 0.64mL (0.64mmol, 1M in THF) tetrabutyl ammonium fluoride (TBAF) solution.Stir 60min under room temperature, TLC follows the tracks of reaction process, after question response finishes, and the evaporated under reduced pressure solvent, directly silica gel column chromatography separates, and 5: 1PE/EA is eluent, obtains oily 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 synthesizing, the tetrabutyl fluoride amine that adds can be arbitrary value in 0.48～0.64mmol.

5.6.2 compound T-7-2's is synthetic

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

Get compound T-6-2 70mg obtained in the previous step (0.21mmol), add 5mL THF, stir 10min, then add 0.41mL (0.41mmol, 1M in THF) tetrabutyl ammonium fluoride (TBAF) solution.Stir 40min under room temperature, TLC follows the tracks of reaction process, after question response finishes, and the evaporated under reduced pressure solvent, directly silica gel column chromatography separates, 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 synthesizing, the tetrabutyl fluoride amine that adds can be arbitrary value in 0.32～0.42mmol.

5.7.1 compound T-8-1's is synthetic

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

Get compound T-7-1 40mg obtained in the previous step (0.18mmol), be dissolved in 2mL ammoniacal liquor, stir 40h under room temperature, TLC follows the tracks of reaction process, and question response adds appropriate ethanol after finishing, and the evaporated under reduced pressure solvent gets 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 synthesizing, the ammoniacal liquor that adds can be arbitrary value in 9～18mmol.

5.7.2 compound T-8-2's is synthetic

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

Get compound T-7-2 136mg obtained in the previous step (0.60mmol), be dissolved in 5mL ammoniacal liquor, react 60h under room temperature, TLC follows the tracks of reaction process, and question response adds appropriate ethanol after finishing, and the evaporated under reduced pressure solvent gets 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 synthesizing, the ammoniacal liquor that adds can be arbitrary value in 30～60mmol.

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

5.8 compound T-9-2's is synthetic

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

Add the 2mL dry DMF in the single port bottle of 10mL, add again 30mg (84 μ mol) compound T-8-2, lucifuge, stir under room temperature, 20mg (38 μ mol) TAMRA (5/6) is dissolved in the 4mL dry DMF, inject, then add 80 μ L (570 μ mol) triethylamine (TEA).Stirring reaction under room temperature, TLC track to raw material and disappear.Question response is removed DMF under decompression after finishing, and take 3: 1 DCM/MeOH as developping agent, the separation and purification of TLC plate gets 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 synthesizing, the T-8 that adds can be arbitrary value in 114～228 μ mol, and TEA can be arbitrary value in 190～760 μ mol.

5.9 compound F 17-hydroxy-corticosterone-2 is synthetic

Trifluoro-acetate and propargylamine react in organic solvent and obtain compound F 17-hydroxy-corticosterone-2, and described step is specially:

Add 15ml methyl alcohol in a single port bottle, stir under ice-water bath, add propargylamine E-2 (30mmol, 1.65g), stir and slowly add trifluoro-acetate E-1 (39mmol, 4.99g) after 20 minutes, the water-bath of 10 minutes recession deicings, under room temperature, reaction is 24 hours.Reaction is monitored with the TLC plate.After reaction finished, the evaporated under reduced pressure solvent added the 30ml chloroform, saturated NaHCO ₃Twice of solution washing (2 * 30ml), saturated NaCl solution washing (30ml), anhydrous sodium sulfate drying filters, the evaporated under reduced pressure solvent, underpressure distillation gets 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)。

5.10 compound F 17-hydroxy-corticosterone-3 is synthetic

Linked reaction occurs with 5-iodo-2 '-deoxyuridine F1 and obtains compound F 17-hydroxy-corticosterone-3 in compound F 17-hydroxy-corticosterone-2 under the cuprous iodide effect, described step is specially:

Add 5-iodo-2 '-deoxyuridine F-1 (0.7mmol, 247.9mg) in a single port bottle, 8mL DMF, stirring and dissolving, lucifuge.Add cuprous iodide (0.14mmol, 26.7mg), nitrogen protection is stirred and cuprous iodide was fully dissolved in 20 minutes.Add successively 0.25mL TEA, trifluoroacetyl propargylamine F-2 (2.8mmol, 423.0mg), Pd (PPh ₃) ₄(0.07mmol, 80.9mg), under room temperature, reaction is spent the night.Reaction is monitored with the TLC plate.The evaporated under reduced pressure solvent, column chromatography, DCM: MeOH=20: 1 is eluent, gets 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 ₂Synthetic

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

Take compound F 17-hydroxy-corticosterone-3 (one) 60mg (0.16mmol) in glove box, tri-n-butylamine pyrophosphate salt E-4 (two) 150mg (0.32mmol), 2-chloro-4H-1,3,2-benzo dioxy phosphorus-4-ketone E-3 (three) 66mg (0.32mmol) is placed in respectively three reaction tubess.(1) is dissolved in the 0.5ml dry DMF, then adds the new tri-n-butylamine that steams of 0.6ml, stir half an hour.(2) are dissolved in the 0.5ml dry DMF, under high degree of agitation, (one) are injected in (two), stir half an hour.Above-mentioned mixed solution is injected (three), stir 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 the 30ml dehydrated alcohol, subzero 20 ℃ of freeze overnight, centrifugal (3200r/min, 25 ℃) 20min.The supernatant liquor that inclines is drained solvent.Add again the TEAB solution of 1ml 1M, add the 4ml strong aqua, stirred overnight at room temperature.White solid appears in the evaporated under reduced pressure solvent.First analyze condition with analysis mode HPLC: pillar: C18,5 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAAc and ethanol, 0min 20mM TEAAc, 35min 20% ethanol; UV-detector: 260nm.Preparative HPLC separates, and gets the 26mg white solid, productive rate 18%.Pillar: C18,5 μ m, 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 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 compound T-10-2's is synthetic

Compound T-9-2 and DSC react to get compound T-10-2 under alkaline condition, described step is specially:

Add compound T-9-2 10mg (0.017mmol) in a reaction flask, vacuumize, nitrogen protection is injected the 0.5ml anhydrous acetonitrile with syringe, adds 20 μ l triethylamines, stirs under room temperature.Add N in another reaction flask, N-succinimidyl carbonate (DSC) 27mg (0.105mmol) vacuumizes, and stirring reaction under room temperature is injected above-mentioned mixed solution in nitrogen protection.TLC follows the tracks of to react to raw material and disappears.After raw material disappeared, stopped reaction was directly used in next step.

In above-mentioned synthesizing, the DSC that adds can be arbitrary value in 0.085～0.136mmol.

5.13 compound T-11-2's is synthetic

Compound T-10-2 and compound dUTP-NH ₂Substitution reaction occurs under alkaline condition get 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, stirring reaction under room temperature.At first separate unreacted compound T-10-2 with the large plate of TLC after reaction finishes, then separate with preparative HPLC, get compound T-11-2 sterling 4.5mg, productive rate 33.3%.TR is the 28.5min. condition: pillar: C18,5 μ m, 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 μ m, 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 synthesizing, the dUTP-NH that adds ₂Can be arbitrary value in 0.018～0.036mmol.

Embodiment 6, to the biological assessment of synthetic reversible terminal

Whether can be applied to DNA sequencing in order to detect the reversible terminal that the present invention synthesizes, the present embodiment has detected the characteristic of reversible terminal two aspects of embodiment 1～5:

1) whether can be identified by archaeal dna polymerase, participate in the extension of DNA as the substrate of archaeal dna polymerase;

2) participate in to remove the entrained fluorophor of this reversible terminal after the DNA chain extension, so that the extension of next round.

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

6.1 the fracture test (the reversible terminal of embodiment 5) of the reversible terminal of acid-sensitive sense under DNA chain extension reaction and pH2.0 condition thereof

1) set up the DNA chain extension reaction of reversible terminal according to following system in the eppendorf pipe: 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 ℃ of water baths processed 15 minutes, then be placed in 75 ℃ of water-baths and process 10 minutes with the deactivation archaeal dna polymerase.The cleavage reaction that reaction product is used for follow-up reversible terminal fluorophor.

2) cleavage reaction of the reversible terminal fluorophor of acid-sensitive sense

Add 13.5uL 0.24M HCl in DNA chain extension reaction system, regulate pH to 2.0, room temperature treatment 30 minutes, regulate pH to 8.0 with 1M Tris again, get the cleavage reaction product and carry out the 12%PAGE electrophoretic analysis, as shown in figure 10, as shown in Figure 10, the reversible terminal of acid-sensitive sense can be identified by archaeal dna polymerase, participate in the extension of DNA chain as its substrate, but under the acidic conditions of pH2.0 and pH2.2, the fluorophor fracture poor effect that reversible terminal is entrained also needs further to adjust failure condition.

6.2 contain the fracture test (the reversible terminal of embodiment 5) of DNA chain extension product under the pH1.7 acidic conditions of the reversible terminal of acid-sensitive sense

Set up the DNA chain extension reaction according to the method in 6.1, add 9uL 0.48M HCl in DNA chain extension reaction system, regulate pH to 1.7, room temperature treatment 30 minutes, regulate pH to 8.0 with 1M Tris again, get the cleavage reaction product and carry out the 12%PAGE electrophoretic analysis, as shown in figure 11, as shown in Figure 11, the DNA chain extension product that contains reversible terminal, the fracture effect is better than pH2.0 under the pH1.7 condition, and approximately the extension products fluorophor of 50% left and right is ruptured, but still have not fracture of considerable part, thereby await further optimization.

6.3 contain the fracture test (the reversible terminal of embodiment 5) of DNA chain extension product under Dowex 50Wx8 environment of the reversible terminal of acid-sensitive sense

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

6.4 the reversible terminal of disulfide linkage is at DNA chain extension reaction and the fracture under different DTT concentration thereof test (

embodiment

1,2,3 reversible terminal)

1) set up the DNA chain extension reaction that contains the reversible terminal of disulfide linkage according to following system in the eppendorf pipe: 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 the reversible terminal fluorophor of disulfide linkage

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

6.5 contain the DNA chain extension product test of the fracture under action times different from 30mM DTT at 10mM, 20mM respectively of the reversible terminal of disulfide linkage

Test be

embodiment

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

In order further to optimize the failure condition of the DNA chain extension product that contains the reversible terminal of disulfide linkage, shorten rupture time, tested respectively different concns DTT in different treatment the fracture effect under the time:

1) act on respectively 3 minutes to 15 minutes under 10mM DTT room temperature, and detection of broken effect: set up the DNA chain extension reaction according to the method in 6.4, adding final concentration in DNA chain extension reaction system is that the DTT of 10mM processes respectively different time, get the cleavage reaction product and carry out the 12%PAGE electrophoretic analysis, as shown in figure 14, as shown in Figure 14, the DNA chain extension product fluorescent scanning result demonstration after the DTT of 10mM room temperature effect 3min, 5min, 8min that contains the reversible terminal of disulfide linkage still has fluorescent signal, illustrates that under this concentration, DTT can not be fully with disulfide bonds; After effect 10min, faint fluorescent signal is arranged, after 15min, fluorescent signal can't detect substantially, and when the DTT of demonstration 10mM processes 15 minutes, fracture disulfide linkage effect is better.

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

The biological assessment of embodiment 4 reversible terminals 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 equally its synthetic reversible terminal can be applied to DNA sequencing.

In sum, the present invention is for adopting the fluorescein-labelled reversible terminal that contains Nucleotide U.The test result of embodiment 6 has verified that further reversible terminal of the present invention completed the biochemical reaction requirement of satisfying high-flux sequence, possesses practical prospect preferably.

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

Claims

1. reversible terminal, its structural formula is suc as formula shown in (I):

R wherein ₁Be fluorescein, R ₂Be connector element.

2. reversible terminal as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is suc as formula shown in (II):

3. reversible terminal as claimed in claim 2, is characterized in that, described reversible terminal is synthesized as follows:

B, compound F 17-hydroxy-corticosterone ₃Synthetic: at CuI, Pd (PPh ₃) ₄Under the condition that exists with TEA, compound F 17-hydroxy-corticosterone ₂And F ₁

Reaction gets compound F 17-hydroxy-corticosterone ₃

Described F ₁, F ₂, CuI, Pd (PPh ₃) ₄With the mol ratio of TEA be 1: (2～3): 0.072: 0.025: (1.5～2);

C, compound dUTP-NH ₂Synthetic: compound F 17-hydroxy-corticosterone ₃With tri-n-butylamine pyrophosphate salt, 2-chloro-4H-1, the reaction under triethylamine and iodine existence of 3,2-benzo dioxy phosphorus-4-ketone, reaction product is gone protection, gets compound dUTP-NH ₂

Described tri-n-butylamine pyrophosphate salt, 2-chloro-4H-1,3,2-benzo dioxy phosphorus-4-ketone and F ₃Mol ratio be 2: 2: 1;

D, compound dUTP-SPDP's is synthetic: under the condition that TEA exists, and compound dUTP-NH ₂In sodium carbonate sodium bicarbonate buffer liquid, with the SPDP take anhydrous acetonitrile as solvent

Reaction gets compound dUTP-SPDP Described dUTP-NH ₂With the mol ratio of SPDP be 1: (1.5～3);

The lucifuge reaction gets compound R DM-SH

F, compound dUTP-T's is synthetic: with Na ₃PO ₄-edta buffer liquid and acetonitrile are solvent, and compound dUTP-SPDP and RDM-SH reaction get compound dUTP-T; The mol ratio of described RDM-SH, dUTP-SPDP is 1: (1～2); Described compound dUTP-T namely has the reversible terminal of eliminant shown in formula (II).

4. reversible terminal as claimed in claim 2, is characterized in that, described reversible terminal is synthesized as follows:

And F ₂Reaction gets compound F 17-hydroxy-corticosterone ₃ Described F ₁, F ₂, CuI, Pd (PPh ₃) ₄With the mol ratio of TEA be 1: (2～3): 0.072: 0.025: (1.5～2);

C, compound G ₁Synthetic: take methyl alcohol as solvent, compound F 17-hydroxy-corticosterone ₃With the strong aqua reaction, get compound Described F ₃With the mol ratio of ammoniacal liquor be 1: (50～100);

Reaction gets compound

Described G ₁With the mol ratio of SPDP be 1: (1～2);

The lucifuge reaction gets compound R DM-SH

Described RDM-SH and G ₂Mol ratio be 1: (1.2～2);

G, compound dUTP-T's is synthetic: compound G ₃With tri-n-butylamine pyrophosphate salt, 2-chloro-4H-1, the reaction under triethylamine and iodine existence of 3,2-benzo dioxy phosphorus-4-ketone, reaction product is gone protection, gets compound dUTP-T; Described tri-n-butylamine pyrophosphate salt, 2-chloro-4H-1,3,2-benzo dioxy phosphorus-4-ketone and G ₃Mol ratio be 2: 2: 1; Described compound dUTP-T namely has the reversible terminal of eliminant shown in formula (II).

5. reversible terminal as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is suc as formula shown in (III):

6. reversible terminal as claimed in claim 5, is characterized in that, described reversible terminal is synthesized as follows:

Reaction gets compound N-1

The mol ratio of described Mercaptamine, 2-HEDS and TEA is 1: (1～2): (2～3);

Synthesizing of B, compound N-2: take dry DMF as solvent, under the condition that TEA exists, compound N-1 and TAMRA (5/6)

The lucifuge reaction gets compound N-2

The mol ratio of described TAMRA (5/6), N-1 and TEA is 1: (1～4): (10～15);

Synthesizing of C, compound N-3: take anhydrous acetonitrile as solvent, under the condition that TEA exists, react with DSC under the nitrogen protection condition compound N-2, gets compound N-3

The mol ratio of described N-2, DSC and TEA is 1: (4～6): (5～15);

Synthesizing of D, compound N-4: with NaHCO ₃/ Na ₂CO ₃Buffered soln be solvent, compound dUTP-NH ₂

7. reversible terminal as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is suc as formula shown in (IV):

8. reversible terminal as claimed in claim 7, is characterized in that, described reversible terminal is synthesized as follows:

A, compound T-1's is synthetic: under the condition that hydrochloric acid exists, Pidolidone reacts with Sodium Nitrite under the ice bath agitation condition, gets compound T-1

The mol ratio of described T-1 and borine is 1: (1.5～5);

C, compound T-3's is synthetic: make solvent with methylene dichloride, under the condition that imidazoles exists, compound T-2 reacts with TBSCl under nitrogen protection, gets compound T-3

The mol ratio of described T-2 and TBSCl is 1: (1.5～2.5);

D, compound T-4's is synthetic: make solvent with methylene dichloride, compound T-3 reacts with DIBAL-H under nitrogen protection and cryosel bath, gets compound T-4

The mol ratio of described T-3 and DIBAL-H is 1: (1.5～2.5);

E, compound T-6's is synthetic: under the condition that the A-15 catalyzer exists, compound T-4 and the reaction of bromoethanol amine get compound T-6

The mol ratio of described T-4 and bromoethanol amine is 1: (1.5～3);

F, compound T-7's is synthetic: under the condition that tetrabutyl fluoride amine exists, at room temperature dehydroxylation protection of compound T-6 gets compound T-7

The mol ratio of described T-6 and tetrabutyl fluoride amine is 1: (1.5～2);

G, compound T-8's is synthetic: compound T-7 and the reaction of excess of ammonia water get compound

The mol ratio of described T-7 and ammoniacal liquor is 1: (50～100);

Reaction gets compound T-9 The mol ratio of described TAMRA (5/6), T-8 and TEA is 1: (3～6): (5～20);

I, compound T-10's is synthetic: compound T-9 gets compound T-10 under the condition that DSC exists

The mol ratio of described T-9 and DSC is 1: (5～8);

J, compound T-11's is synthetic: compound T-10 and dUTP-NH ₂

9. reversible terminal as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is as shown in formula V:

10. reversible terminal as claimed in claim 9, is characterized in that, described reversible terminal is synthesized as follows:

Synthesizing of B, Compound D-1: take THF as solvent, bromoethanol and Na under nitrogen protection ₂Se ₂45～55 ℃ of stirring reactions of alkaline aqueous solution oil bath get Compound D-1

Described bromoethanol and Na ₂Se ₂Mol ratio be 1: (1～2);

Synthesizing of C, Compound D-2: take dimethylbenzene as solvent, Compound D-1 and HBr reaction get compound

The mol ratio of described D-1 and HBr is 1: (4～6);

Synthesizing of D, Compound D-3: Compound D-2 and strong aqua reaction get Compound D-3

The mol ratio of described D-2 and ammoniacal liquor is 1: (50～100);

Synthesizing of E, Compound D-4: take dry DMF as solvent, under the condition that TEA exists, Compound D-3 and TAMRA (5/6) The lucifuge reaction gets Compound D-4

The mol ratio of described TAMRA (5/6), D-3 and TEA is 1: (1～4): (10～15);

The mol ratio of described D-4, DSC and TEA is 1: (4～6): (5～15);

Synthesizing of G, Compound D-6: with NaHCO ₃/ Na ₂CO ₃Buffered soln be solvent, compound dUTP-NH ₂

11. the purposes of a reversible terminal as described in claim 1～10 in the synthetic order-checking of DNA.