CN103484106B

CN103484106B - Four look fluorescent mark Reversible terminal and the purposes in DNA sequencing thereof

Info

Publication number: CN103484106B
Application number: CN201310401580.0A
Authority: CN
Inventors: 沈玉梅; 邵志峰; 赵小东; 汤道年; 龚兵; 李小卫; 江敏; 刘亚智; 魏晓飞; 伍新燕
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2013-09-05
Filing date: 2013-09-05
Publication date: 2015-08-19
Anticipated expiration: 2033-09-05
Also published as: CN103484106A

Abstract

The invention discloses a kind of four look fluorescent mark Reversible terminal and the purposes in DNA sequencing thereof; The structural formula of described Reversible terminal is such as formula shown in (I): (I); Wherein, R ₁for triphosphate; R ₂for H or OH; Base is U, C, A, G or derivatives thereof; Connector element is the bifunctional compound that can rupture in a mild condition; Fluorophor is selected from the one in BODIPY, fluorescein, rhodamine, tonka bean camphor, xanthene, cyanine, pyrene, phthalocyanine, alexa, squarene dyestuff, the combination of generate energy transferred dyes and its derivative.Reversible terminal of the present invention can be used for the order-checking of DNA single molecule; Simultaneously, the synthesis desired raw material of Reversible terminal of the present invention is simple and easy to get, and building-up process is conventional chemical reaction, can be used for large-scale promotion and uses, and biological evaluation result shows that such Reversible terminal can meet the biochemical reaction requirement of high-flux sequence completely, possesses good practical prospect.

Description

Four look fluorescent mark Reversible terminal and the purposes in DNA sequencing thereof

Technical field

The present invention relates to chemosynthesis and biochemical field, be specifically related to class four look fluorescent mark Reversible terminal and the purposes in DNA sequencing thereof.

Background technology

DNA sequencing technology is one of important means of modern life science and medical research.DNA sequencing from the Sanger sequencing technologies (generation order-checking) of 1977, in the time of thirties years, develop rapidly.The flux of order-checking significantly improves and cost sharply declines, and its speed of development has broken the speed of the existing Moore's Law budget of semi-conductor industry circle to have people even to think.Two generation high-throughput parallel sequencing technology appearance be the concentrated reflection of sequencing technologies develop rapidly.Adopt first-generation sequencing technologies, the Human Genome Project (HGP) cost 3,000,000,000 dollars completes the sequencing of the whole genome of people (3,000,000,000 bases).And the state-of-the-art technology of current two generations order-checking only needs about 5000 dollars just can complete the whole gene order-checking of people.

Even so, the cost of two generations order-checking and technical elements still Shortcomings, can not meet the requirement to order-checking of basic science and clinical medicine.Single-molecule sequencing technology (three generations's sequencing technologies) is arisen at the historic moment.The core of three generations's sequencing technologies directly checks order to single DNA molecules, do not do any DNA amplification reaction, thus reduce cost, improves flux.Although single-molecule sequencing technology has commercially produced product, all also there is technical difficult point, fail large-scale application.

High-flux sequence platform in the market monopolize by several external products, especially troubling is, offshore company relies on the control to sequencing reagent, almost control domestic order-checking market completely, even if we can have breakthrough on order-checking hardware, on the auxiliary products such as sequencing reagent, we are also by under one's control.Therefore, independent research is applicable to the order-checking of two generations or even three generations and checks order the sequencing reagent of platform, by changing the current market structure, the order-checking platform of setting up China autonomous has strategic meaning.For this reason, National 863,973 and the planning of " 12 " biotech development all research and development new-generation sequencing technology and the research and development of auxiliary products are classified as the object given priority to.

Summary of the invention

The object of the present invention is to provide class four look fluorescent mark Reversible terminal and the purposes in DNA sequencing thereof; This Reversible terminal has isotope of redox-sensitive characteristic.The present invention is in harness basis, pass through multi-crossed disciplines, first novel four look fluorescence-labeled nucleotides have been synthesized, and development is based on the DNA extension of this nucleus thuja acid, realize single fluorescence molecule hypersensitive optical detective technology and the final three generations's sequencing technologies prototype realizing having single-molecule sequencing ability.

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

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

Wherein, R ₁for triphosphate; R ₂for H or OH; Base is U, C, A, G or derivatives thereof; Connector element is the bifunctional compound that can rupture in a mild condition; Fluorescence group is selected from the one in BODIPY, fluorescein, rhodamine, tonka bean camphor, xanthene, cyanine, pyrene, phthalocyanine, alexa, squarene dyestuff, the combination of generate energy transferred dyes and its derivative.

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

Preferably, described Reversible terminal (II) is synthesized as follows:

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

B, compound F 17-hydroxy-corticosterone ₃synthesis: at CuI, Pd (PPh ₃) ₄with under TEA existent condition, compound F 17-hydroxy-corticosterone ₂and F ₁ (No. CAS is: 172163-62-1, and name is called: 7-denitrification mix the iodo-2 '-pancreatic desoxyribonuclease of-7-) reaction, obtains compound F 17-hydroxy-corticosterone ₃ described compound F 17-hydroxy-corticosterone ₁, compound F 17-hydroxy-corticosterone ₂, CuI, Pd (PPh ₃) ₄be 1:(2 ~ 3 with the mol ratio of TEA): 0.072:0.025:(1.5 ~ 2);

C, compound dUTP-NH ₂synthesis: compound F 17-hydroxy-corticosterone ₃with tri-n-butylamine pyrophosphate salt, the chloro-4H-1 of 2-, the reaction under triethylamine and iodine exist of 3,2-benzo dioxy phosphorus-4-ketone, reaction product goes protection, obtains compound dUTP-NH ₂ described tri-n-butylamine pyrophosphate salt, the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone and compound F 17-hydroxy-corticosterone 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 (3-(2-pyridine dimercapto) propionic acid N-hydroxy-succinamide ester) reacts, and obtains compound dUTP-SPDP described compound dUTP-NH ₂be 1:(1.5 ~ 3 with the mol ratio of SPDP);

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 compound 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 and acetonitrile are solvent, and compound dUTP-SPDP and RDM-SH reacts, and obtains compound dUTP-T; The mol ratio of described compound R DM-SH, compound dUTP-SPDP is 1:(1 ~ 2); Namely described compound dUTP-T has the Reversible terminal of eliminant shown in formula (II).

Preferably, described Reversible terminal (II) is synthesized 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 compound F 17-hydroxy-corticosterone ₁, compound F 17-hydroxy-corticosterone ₂, CuI, Pd (PPh ₃) ₄be 1:(2 ~ 3 with the mol ratio of TEA): 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:(50 ~ 100 with the mol ratio of strong aqua);

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

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

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

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

Preferably, described Reversible terminal (III) 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), compound 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 under nitrogen protection condition, obtains compound N-3 the mol ratio of described compound N-2, DSC 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 compound N-3, obtain compound N-4; Described compound 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 (IV) 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 Na0H 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 the mol ratio of described Compound 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 Compound D-2 and strong aqua 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 compound TAMRA (5/6), Compound 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 Compound D-4, DSC 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 Compound D-5 and compound dUTP-NH ₂mol ratio be 1:(1 ~ 2); Described Compound D-6 is the Reversible terminal shown in structural formula (IV).

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

Preferably, described Reversible terminal (V) is synthesized as follows:

Synthesis (the NH of A, compound 1 ₂cH ₂cH ₂sTrity): Mercaptamine adds triphenylmethyl chloride reaction in organic solvent, and recrystallization obtains white solid, i.e. compound 1;

The synthesis (FITC-S-Trity) of B, compound 2: compound 1 reacts to obtain compound 2 with fluorescein FITC (fluorescein isothiocyanate) in the basic conditions;

The synthesis (FITC-SH) of C, compound 3: compound 2 is gone to protect and obtain compound 3;

The synthesis (CF3CONHCH2CCH) of D, compound 7: propargylamine and trifluoro-acetate react to obtain compound 7;

The synthesis [dC (AP3)] of E, compound 8: the iodo-2 '-Deoxyribose cytidine of 5-and compound 7 linked reaction occur under Pd catalyst action and obtain compound 8;

The synthesis [dCTP (AP3)] of F, compound 4: compound 8 connects triphosphoric acid and namely obtains compound 4; Gained compound 4 needs preparative HPLC purifying;

The synthesis [dCTP-S-S-SPDP] of G, compound 5: compound 4 and SPDP react and obtain compound 5, thus introduce disulfide linkage in compound 5;

The synthesis (end product dCTP-S-S-FITC) of H, compound 6: compound 5 and compound 3 disulfide exchange occur and react and obtain final product compound 6; Namely described compound 6 has the Reversible terminal shown in formula (V).Compound 6 needs preparative HPLC purifying equally.

Preferably, described Reversible terminal (V) is synthesized 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 G ₁ described F ₃be 1 with the mol ratio of strong aqua: (50 ~ 100);

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

The synthesis of E, compound F 17-hydroxy-corticosterone ITC-SH: under DTT existent condition, cysteamine reacts with compound F 17-hydroxy-corticosterone ITC (fluorescein isothiocyanate) lucifuge in sodium carbonate sodium bicarbonate buffer liquid, obtains compound F 17-hydroxy-corticosterone ITC-SH the mol ratio of described FITC, cysteamine and DTT is 1: (10 ~ 50): (40 ~ 70);

F, compound G ₃synthesis: using methyl alcohol and acetonitrile as solvent, compound F 17-hydroxy-corticosterone ITC-SH under the nitrogen protection of aluminium foil parcel with G ₂reaction, obtains compound G ₃ described FITC-SH and G ₂mol ratio be 1: (1.2 ~ 2);

The synthesis of G, compound dCTP-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 dCTP-T has the Reversible terminal shown in formula (V).

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

Preferably, described Reversible terminal (VI) is synthesized as follows:

The synthesis of A, Compound C y5-SH

By fluorescein Cy5 active ester (molecular formula: C ₃₃h ₃₉kN ₂o ₈s ₂, molecular weight: 694.90) react with cysteamine and obtain Compound C y5-SH;

The synthesis of B, compound dATP (AP3)-SPDP

Compound dATP (AP3) and SPDP are reacted to obtain compound dATP (AP3)-SPDP, thus introduces disulfide linkage; Product needs preparative HPLC purifying;

The synthesis of C, compound dATP-SS-Cy5

Compound dATP-SPDP and Cy5-SH is at Na ₃pO ₄there is mercaptan permutoid reaction in-edta buffer solution and namely obtain compound dATP-SS-Cy5; Namely this compound dATP-SS-Cy5 has the Reversible terminal shown in formula (VI).This compound needs preparative HPLC purifying.

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

Preferably, described Reversible terminal (VII) is synthesized as follows:

The synthesis of A, compound a 2: compound a 1 compound a 2 is reacted to obtain in the basic conditions with sec.-propyl formyl chloride.

The synthesis of B, compound a 3: compound a 2 reacts to obtain compound a 3 with N-iodosuccimide (NIS) in anhydrous solvent;

The synthesis of C, compound a 4: compound a 3 removes protecting group in the basic conditions and obtains compound a 4;

The synthesis of D, compound a 5: compound a 4 demethyl in the basic conditions, obtains compound a 5;

The synthesis of E, compound a 6: compound a 5 and propargylamine trifluoro-acetate linked reaction occur under Pd catalyst action and namely obtain compound a 6;

The synthesis of F, compound a 7: compound a 6 connects triphosphoric acid by above-mentioned same method and namely obtains compound a 7;

The synthesis of G, compound a 8: compound a 7 and SPDP react to obtain compound a 8, thus introduce disulfide linkage;

H, compound Texas-red-NH ₂cH ₂cH ₂the synthesis of SH (a9): fluorescein Texas-red and amineothiot generation substitution reaction, obtain compound a 9;

The synthesis of I, compound a 10: compound a 8 and compound a 9 disulfide exchange occur and react, and obtain compound a 10, namely described compound a 10 has the Reversible terminal shown in formula (VII).This compound needs preparative HPLC purifying.

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

Preferably, described Reversible terminal (VIII) is synthesized as follows:

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

The synthesis of B, compound Texas Red1: with dry DMF (DMF) for solvent, under TEA existent condition, compound react with Texas Red-X lucifuge, obtain compound Texas Red1; Described Texas Red-X, be 1 with the mol ratio of TEA: (1 ~ 4): (10 ~ 15);

The synthesis of C, compound dGTP-S-S-Texas Red2: with NaHCO ₃/ Na ₂cO ₃the buffered soln of (pH is 8.73) is solvent, compound d6TP-NH ₂react with Texas Red1, obtain compound dGTP-S-S-Texas Red2; Described Texas Red1 and dGTP-NH ₂mol ratio be 1: (1 ~ 2); Namely described compound dGTP-S-S-Texas Red2 has the Reversible terminal of structural formula shown in formula (VIII).

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

Preferably, described Reversible terminal (IX) is synthesized as follows:

The synthesis of B, compound F 17-hydroxy-corticosterone ITC1: with dry DMF (DMF) for solvent, under TEA existent condition, compound react with FITC lucifuge, obtain compound F 17-hydroxy-corticosterone ITC1; Described FITC, be 1 with the mol ratio of TEA: (1 ~ 4): (10 ~ 15);

The synthesis of C, compound dCTP-S-S-FITC: with NaHCO ₃/ Na ₂cO ₃the buffered soln of (pH is 8.73) is solvent, compound dCTP-NH ₂react with FITC1, obtain compound dGTP-S-S-FITC; Described FITC1 and dCTP-NH ₂mol ratio be 1: (1 ~ 2); Namely described compound dCTP-S-S-FITC has the Reversible terminal of structural formula shown in formula (IX).

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

Preferably, described Reversible terminal (X) is synthesized as follows:

The synthesis of B, Compound C y51: with dry DMF (DMF) for solvent, under TEA existent condition, compound react with Cy5 lucifuge, obtain Compound C y51; Described Cy5, be 1 with the mol ratio of TEA: (1 ~ 4): (10 ~ 15);

The synthesis of C, compound dATP-S-S-Cy5: with NaHCO ₃/ Na ₂cO ₃the buffered soln of (pH is 8.73) is solvent, compound dATP-NH ₂react with Cy51, obtain compound dATP-S-S-Cy5; Described Cy51 and dATP-NH ₂mol ratio be 1: (1 ~ 2); Namely described compound dATP-S-S-Cy5 has the Reversible terminal of eliminant shown in formula (X).

The invention still further relates to the purposes of a kind of aforesaid four look fluorescent mark Reversible terminal in DNA sequencing.

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 order-checking of DNA single molecule; 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, and R ₁for triphosphate; R ₂for H or OH; Base is U, C, A, G or derivatives thereof; Connector element is the bifunctional compound that can rupture in a mild condition; Fluorescence group is selected from the one in BODIPY, fluorescein, rhodamine, tonka bean camphor, xanthene, cyanine, pyrene, phthalocyanine, alexa, squarene dyestuff, the combination of generate energy transferred dyes and its derivative;

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 embodiment 4 containing two selenium key Reversible terminal;

Fig. 9 is the building-up process schematic diagram of the Reversible terminal of embodiment 5dCTP-S-S-FITC;

Figure 10 is compound dUTP (AP in embodiment 5 ₃) building-up process schematic diagram;

Figure 11 is the another kind of synthetic method schematic diagram of the Reversible terminal of embodiment 6dCTP-S-S-FITC;

Figure 12 is the building-up process schematic diagram of the Reversible terminal of embodiment 7dATP-S-S-Cy5;

Figure 13 is the building-up process schematic diagram of the Reversible terminal of embodiment 8dGTP-S-S-Texas Red-X;

Figure 14 is the building-up process schematic diagram of the Reversible terminal of embodiment 9 (structural formula VIII) dGTP-S-S-Texas Red-X;

Figure 15 is the building-up process schematic diagram of the Reversible terminal of embodiment 10 (structural formula IX) dCTP-S-S-FITC;

Figure 16 is the building-up process schematic diagram of the Reversible terminal of embodiment 11 (structural formula X) dATP-S-S-Cy5;

Figure 17 (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;

The rupture test result that Figure 18 is DNA chain extension product containing disulfide linkage Reversible terminal under 10mM DTT different action time, 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 19 is DNA chain extension product containing the disulfide linkage Reversible terminal rupture test result respectively 20, under 30mM DTT different action time, wherein (a) is PAGE electrophorogram, b () is fluorescent scanning result schematic diagram, M is DNAmarker, 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;

Figure 20 is the rupture test result of DNA chain extension product respectively under 20mM DTT different action time of Reversible terminal II, III, IV, V, VI, VII, VIII, IX, X, wherein (a) is fluorescent scanning result schematic diagram, b () is GR dyeing schematic diagram, M:20bp.

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, PE:EA=8:1, baking sheet, and 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 a 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 Weigh Compound F2 (254mg, 1.7mmol) adds in above-mentioned reaction flask after dissolving with DMF, and 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, 20:1DCM: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 arbitrary value in 1.05 ~ 1.4mmol.

1.3 compound dUTP-NH ₂synthesis

Compound dUTP-NH ₂synthesis concrete as shown in Figure 3, 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 5mL3% iodine (9:1Py/H2O) solution.Add 4mL water after 15min, stir 2h.Add 0.5mL3M 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，found522.0070；calc forC12H18N3O14P3Na[M+Na]+543.9899，found543.9883。

The synthesis of 1.4 compound dUTP-SPDP

DUTP-NH is added in the single port bottle of 10mL ₂24.4mg (0.026mmol), then add 600 μ lNa 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 × 250mm; 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 1.33mL1M 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，found1121.1655。

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 G1Rf=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 forC ₁₂H ₁₅N ₃O ₅[M]281.1012，found281.1015.

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，found478.0974.

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 1mL3% iodine (9:1Py/H2O) solution.Add 1mL water after 15min, stir 2h.Add 0.5mL3M 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 single 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, obtains 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, removing DMF under decompression, take 3:1DCM/MeOH as developping agent, and the separation and purification of TLC plate obtains product 20mg.ESI-HRMS：cals forC ₂₉H ₃₁N ₃O ₅S ₂[M]565.1705，found565.1717.

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-29.6mg (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.Raw material disappear after, stopped reaction, not treated be directly used in next step reaction.ESI-HRMS：calsfor C ₃₄H ₃₄N ₄O ₉S ₂[M]706.1767，found706.1761.

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-39.6mg (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

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:

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, obtains 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

Solvent is made with dimethylbenzene, compound 100mg D-1 (0.40mmol) and excessive HBr (162mg, 2.0mmol) at room temperature stir 6h, obtain Compound D-2, then excessive concentrated ammonia (2.3ml, 30mmol) is added, stirring at room temperature, silica-gel plate separation and purification, obtains yellow liquid D-3.

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, removing DMF under decompression, take 3:1DCM/MeOH as developping agent, and the separation and purification of TLC plate obtains product 21mg.ESI-HRMS：cals forC ₂₉H ₃₁N ₃O ₅Se ₂[M+H]662.0594，found662.0582.

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-411mg (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 19mg compound 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-411mg (17 μm of ol)) of whole Compound 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: 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.ESI-HRMS：cals for C ₄₂H ₄₃N ₆O ₂₀P ₃Se ₂ ^4-[M+H]1202.95，found1202.87.

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

embodiment 5

Shown in the following formula V of structural formula of the Reversible terminal of the present embodiment, the synthetic route of its correspondence is as shown in Fig. 9,10:

Synthesis (the NH of 5.1 compounds 1 ₂cH ₂cH ₂sTrity)

Take 1g Mercaptamine (8.8mmol) in 250ml single port bottle, add 40ml DCM, under stirring at room temperature, add 1.3ml TFA (17.6mmol), add 2.45g Trt-Cl (8.8mmol) subsequently, after 23h, stopped reaction, with 1mol/L NaOH solution (25ml) cancellation reaction, organic phase is stayed in extraction, wash with saturated aqueous common salt (20ml), anhydrous sodium sulfate drying, filters, and screws out solvent, EA recrystallization, obtains 577mg white solid.Productive rate 20.5%. ¹H NMR(MeOD，400MHz)：δ 2.49-2.52(m，2H)，2.58-2.61(m，2H)，7.62(t，J=7.2Hz，3H)，7.33(t，J=7.2Hz，6H)，7.45(d，J=7.6Hz，6H).

The synthesis (FITC-S-Trity) of 5.2 compounds 2

16mg compound 1 (51.4 μm of ol) is dissolved in 2.8ml CH ₃cN/CHCl ₃(9:5) in, add 18 μ l DIPEA, add 20mg FITC (51.4 μm of ol) subsequently, stirring at room temperature 36h.Take 9:1DCM/MeOH as developping agent, the separation and purification of TLC plate obtains 31.2mg yellow solid, productive rate 85.7%. ¹H NMR(MeOD，400MHz)：δ2.42(t，J=6.4Hz，2H)，3.34(t，J=5.2Hz，2H)，6.52(dd，J=2.4Hz，J=8.8Hz，2H)，6.63-6.65(m，4H)，6.91(d，J=8.8Hz，2H)，7.12(t，J=7.2Hz，3H)，7.20(t，J=7.6Hz，6H)，7.31(d，J=7.6Hz，6H)，7.45(s，1H)，7.76(dd，J=1.6Hz，J=8.4Hz，1H)，7.85(d，J=8.4Hz，1H).

The synthesis (FITC-SH) of 5.3 compounds 3

Be dissolved in by 23mg compound 2 (0.032mmol) in 400 μ l TFA, add 8 μ l triethyl silicanes, stirring at room temperature, TLC follows the tracks of reaction process, stopped reaction after 1h, and toluene (0.5ml*4) revolves band solvent.Preparation HPLC is separated, and gets 12mg yellow solid, productive rate 80%.Condition: pillar: C18,5 μm, 9.4 × 250mm; Flow velocity: 4mL/min; Moving phase: the 0.1%TFA aqueous solution and CH ₃cN, 5%CH ₃cN (5min), gradient wash, 5%-45%CH ₃cN (40min), retention time t=44min, UV-detector: 254nm. ¹H NMR(MeOD，400MHz)：δ 2.69(t，J=6.4Hz，2H)，3.69(t，J=6.8Hz，2H)，6.69-6.72(m，2H)，6.84(d，J=2.0Hz，2H)，6.91(d，J=8.8Hz，2H)，7.56(s，1H)，7.76-7.83(m，1H)，7.99(t，J=8.4Hz，1H).calc for C ₂₃H ₁₇N ₂O ₅S ₂[M-H] ^-465.0579，found465.0565.

Synthesis (the CF of 5.4 compounds 7 ₃cONHCH ₂cCH)

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.Decompression screws out solvent, adds CHCl ₃(60ml), use saturated sodium bicarbonate solution (60ml) respectively, saturated aqueous common salt (60m1) respectively extracts once.Organic phase anhydrous sodium sulfate drying, decompression screws out solvent, and resistates underpressure distillation (51 DEG C, 280Pa), obtains 3.53g colourless liquid, productive rate 39%. ¹H NMR(CDCl ₃，300MHz)：δ 2.32(t，J=4.0Hz，1H)，4.13-4.15(m，2H)，6.92(s，1H).

The synthesis [dC (AP3)] of 5.5 compounds 8

In a single port bottle, add the iodo-2 '-Deoxyribose cytidine (0.70mmol, 248mg) of 5-, then take 10mg CuI (25.2 μm of ol) and 20mg Pd (PPh ₃) ₄(17.6 μm of ol) adds in reaction flask, vacuumizes, nitrogen protection, and aluminium foil wraps up; add 1.5ml DMF, stirring and dissolving, add 0.2ml TEA, Weigh Compound 7 (254mg; add in above-mentioned reaction flask after 1.68mmol) dissolving with 1ml DMF, stirring at room temperature, reaction is spent the night.Screw out solvent, take DCM:MeOH=5:1 as developping agent, the separation and purification of TLC plate must obtain 153mg, productive rate 58%. ¹H NMR(DMSO-D ₆，400MHz)：δ 1.94-2.01(m，1H)，2.12-2.18(m，1H)，3.51(s，1H)，3.55-3.62(m，2H)，3.79(q，J=3.2Hz，J=6.8Hz，1H)，4.20(d，J=3.2Hz，1H)，4.28(s，1H)，5.05(t，J=4.8Hz，1H)，5.20(d，J=3.6Hz，4H)，6.10(t，J=6.4Hz，1H)，6.84(s，1H)，7.81(s，1H)，8.15(s，1H)，9.96(s，1H).

The synthesis [synthesis of dCTP (AP3)] of 5.6 compounds 4

Difference Weigh Compound 890mg (0.24mmol), tri-n-butylamine pyrophosphate salt 264mg (0.48mmol), the chloro-4H-1 of 2-in glove box, 3,2-benzo dioxy phosphorus-4-ketone 90mg (0.48mmol) is placed in three reaction tubess.Tri-n-butylamine pyrophosphate salt is dissolved in 0.75mL dry DMF, then adds the anhydrous tri-n-butylamine of 0.9mL, stir half an hour.Chloro-for 2-4H-l, 3,2-benzo dioxy phosphorus-4-ketone is dissolved in 0.75mL 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 8, stirs 1.5h.Add 4mL3% iodine (9:1 Py/H2O) solution.Add 4mL water after 15min, stir 2h.Add 1 mL3MNaCl solution, then add 35mL dehydrated alcohol ,-20 DEG C of freeze overnight, centrifugal (3200r/min, 25 DEG C) 20min.Incline supernatant liquor, obtains precipitation, drains solvent.Add 2ml strong aqua stirring at room temperature 6h.Decompression screws out solvent, and occur brown solid, RP-HPLC analyzes [condition: pillar: C18,10 μm, 4.6 × 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAA and EtOH, gradient wash, 0%-20%EtOH (35min); UV-detector: 254nm], retention time t=11min.RP-HPLC is separated [condition: pillar: C18,5 μm, 9.4 × 250mm; Flow velocity: 4mL/min; Moving phase: 20mM TEAA and MeOH, 0%-20%MeOH (35min), retention time t=16min; UV-vis detector: 254nm], NaCl/EtOH removes acetic acid triethylamine salt, obtains 42mg white solid, productive rate 24.5%. ¹H NMR(D ₂O，400MHz)：δ 2.27-2.33(m，1H)，2.44-2.50(m，1H)，4.05(s，2H)，4.19-4.31(m，3H)，4.56-4.59(m，1H)，6.21(t，J=6.0Hz，1H)，8.37(s，1H). ³¹P NMR(D ₂O，162MHz)：δ-22.55，-11.51，-10.31.ESI-HRMS：calc for C ₁₂H ₁₈N ₄O ₁₃P ₃[M-H] ^-519.0083，found519.0059.

The synthesis [dCTP-S-S-SPDP] of 5.7 compounds 5

In the single port bottle of 10mL, add compound 4 18mg (0.019mmol), 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 SPDP6mg (0.019mmol) ₃cN, adds above-mentioned solution, adds 3 μ l Et ₃n.Stirring at room temperature reacts 3 hours.Freeze-drying, dissolve with 1ml TEAA buffer (100mM, pH7.0), RP-HPLC is separated to obtain compound 5 (retention time t=34min).NaCl/EtOH removes acetic acid triethylamine salt, obtains 1.5mg white solid.Productive rate 11%.RP-HPLC separation 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%-10%CH3CN (5min), 10%-50%CH3CN (50min); UV-detector: 293nm and 254nm. ¹H NMR(D ₂O，400MHz)：δ 2.18-2.26(m，1H)，2.36-2.43(m，1H)，2.66(t，J=6.4Hz，2H)，3.08(t，J=6.8Hz，2H)，4.08(s，1H)，4.17(s，1H)，6.17(t，J=6.4Hz，1H)，7.18-7.21(m，1H)，7.75(d，J=6.0Hz，2H)，8.03(s，1H)，8.29(d，J=4.8Hz，1H).ESI-HRMS：calc for C ₂₀H ₂₅N ₅O ₁₄P ₃S ₂[M-H] ^-716.0052，found716.0052.

The synthesis (end product dCTP-S-S-FITC) of 5.8 compounds 6

Get compound 5 (3mg, 2.7 μm of ol) and, in 10mL single port bottle, add 0.4ml Na ₃pO ₄-edta buffer liquid dissolves, and after getting the dissolving of 1.9mg compound 3 (4.0mmol) 0.1ml acetonitrile, join above-mentioned solution, aluminium foil wraps up, stirring at room temperature reaction 6h.After TEAA damping fluid (100mM, pH7.0) dilution, reaction product is separated [condition: pillar: C18,5 μm, 9.4 × 250mn by RP-HPLC; Flow velocity: 4mL/min; Moving phase: 20mM TEAA and CH ₃cN, 5%TEAA (5min), gradient wash 5%-17.5%CH ₃cN (25min); UV-vis detector: 254nm and 495nm.Retention time t=27min].Lyophilize, NaCl/EtOH removes acetic acid triethylamine salt, obtains 1mg yellow solid.Productive rate 35%.RP-HPLC detects purity (> 99%) [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, visible light determined wavelength: 293nm and 495nm.Retention time t=37min]. ¹h NMR (D ₂o, 400MHz): δ 2.01-2.15 (m, 1H), 2.28-2.39 (m, 1H), 2.55-2.70 (m, 2H), 2.85-3.01 (m, 4H), 3.73-3.82 (m, 1H), 3.85-4.26 (m, 6H), 4.52 (s, 1H), 5.92 (t, J=4.0Hz, 1H), 6.72-6.79 (m, 4H), 7.31 (dd, J=2.8Hz, J=8.8Hz, 2H), 7.37 (s, 1H), 7.62 (d, J=8.8Hz, 1H), 7.79 (s, 1H), 7.92 (d, J=8.0Hz, 1H). ³¹p NMR (D ₂o, 162MHz): δ-21.22 ,-11.07 ,-8.27.ESI-HRMS:calc for C ₃₈h ₃₈n ₆o ₁₉p ₃s ₃[M-H] ^-1071.0567, found1071.0604; Calc for C ₃₈h ₃₇n ₆o ₁₆p ₂s ₃[M-H ₂pO ₃] ^-991.0903, found991.0912.HPLC purity 99%.

embodiment 6

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

The synthetic route of its correspondence as shown in figure 11; Specifically comprise the steps:

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

6.2 compound G ₁synthesis

Get 23mg compound F 17-hydroxy-corticosterone 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 G1Rf=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-MS：cals forC ₁₂H ₁₇N ₄O ₄[M]281.10，found281.12.

6.3 compound G ₂(FITC-SH) synthesis

Get 8.5mg G ₁(0.03mmol) 0.5mL dissolve with methanol, is used; 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-MS：cals for C ₂₀H ₂₄N ₅O ₅S ₂[M]478.10，found478.11.

The synthesis of 6.4 compound G3

Get 6.8mg G ₂namely FITC-SH (0.013mmol, its synthesis is with embodiment 1), in 10mL single port bottle, vacuumizes nitrogen protection, and 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(D ₂O，400MHz)：δ 2.01-2.15(m，1H)，2.28-2.39(m，1H)，2.55-2.70(m，2H)，2.85-3.01(m，4H)，3.73-3.82(m，1H)，3.85-4.26(m，6H)，4.52(s，1H)，5.92(t，J=4.0Hz，1H)，6.72-6.79(m，4H)，7.31(dd，J=2.8Hz，J=8.8Hz，2H)，7.37(s，1H)，7.62(d，J=8.8Hz，1H)，7.79(s，1H)，7.92(d，J=8.0Hz，1H).

The synthesis of 6.5 compound dCTP-T and G4

The synthesis of compound dCTP-T is concrete as shown in figure 11, and the reaction conditions that in Figure 11, final step is corresponding is: i) DMF, tributylamine ii) DMF, G ₃iii) I ₂, Py, H ₂o iV) NH ₃.

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 1mL3% iodine (9:1Py/H2O) solution.Add 1mL water after 15min, stir 2h.Add 0.5mL3M 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 dCTP-T.RP-HPLC detects purity (> 99%) [condition: pillar: C18,10 μm, 4.6 × 250mm; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH3CN, 5%CH ₃cN (5min), gradient wash 5%-35%CH3CN (60min); UV, visible light determined wavelength: 293nm and 495nm.Retention time t=37min]. ¹H NMR(D ₂O，400MHz)：δ 2.01-2.15(m，1H)，2.28-2.39(m，1H)，2.55-2.70(m，2H)，2.85-3.01(m，4H)，3.73-3.82(m，1H)，3.85-4.26(m，6H)，4.52(s，1H)，5.92(t，J=4.0Hz，1H)，6.72-6.79(m，4H)，7.31(dd，J=2.8Hz，J=8.8Hz，2H)，7.37(s，1H)，7.62(d，J=8.8Hz，1H)，7.79(s，1H)，7.92(d，J=8.0Hz，1H). ³¹P NMR(D ₂O，162MHz)：δ-21.22，-11.07，-8.27.ESI-HRMS：calc for C ₃₈H ₃₈N ₆O ₁₉P ₃S ₃[M-H] ^-1071.0567，found 1071.0604；calc forC ₃₈H ₃₇N ₆O ₁₆P ₂S ₃[M-H ₂PO ₃] ^-991.0903，found991.0912.

embodiment 7

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

As shown in figure 12, concrete steps are as follows for its synthetic route:

The synthesis of 7.1 Compound C y5-SH

By 28mg cysteamine (0.363mmol) (400 μ l Na ₂cO ₃/ NaHCO ₃buffer solution) add in Cy5-NHS-ester (5mg, 0.008mmol) (dissolving of 1mL dry DMF), normal temperature lucifuge stirs 1h, then adds 0.5mL1M DTT, stirring at room temperature 1h.Finally, reaction 0.1%TFA (0.5ml) solution cancellation, after lyophilize, RP-HPLC is separated (retention time is about 54min).Obtain 1.2mg.ESI-HRMS：calc for C ₃₄H ₄₄N ₃OS[M]542.3205，Found542.3200.

The synthesis of 7.2 compound dATP (AP3)-SPDP

In the single port bottle of 10mL, add dATP (AP3) 9mg (0.010mmol), then add 800 μ l Na ₂cO ₃/ NaHCO ₃damping fluid, stirring at room temperature, is dissolved in the anhydrous CH of 700 μ l SPDP5.6mg (0.015mmol) ₃cN, adds above-mentioned solution, adds 4 μ l Et ₃n.Stirring at room temperature is reacted, and after 4 hours, stopped reaction, separation and purification obtains 2.9mgdATP (AP3)-SPDP.

The synthesis of 7.3 compound dATP-SS-Cy5

DATP-SPDP (2.9mg) is dissolved in Na ₃pO ₄-edta buffer liquid [50mMNa ₃pO ₄, 10mM EDTA (pH7.4,1.5ml)] and acetonitrile 0.5ml mixed solution in, then it added in the Cy5-SH after freeze-drying (2.3mg), room temperature lucifuge stirs 5 hours.Reversed-phase HPLC separation and purification, retention time is about 41min.Freeze-drying.Target compound detects purity (> 95%) through RP-HPLC.Condition: pillar: C18,10 μm, 4.6 × 250mn; 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: 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, 53min place.HPLC purity 95%, HRMS test shows that product structure is correct.

embodiment 8

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

As shown in figure 13, concrete steps are as follows for its synthetic route:

The synthesis of 8.1 compound as 2

Compound a 1 (0.20g; 0.714mmol) be dissolved in 3mL anhydrous pyridine and slowly add own propyl group and add acyl chlorides (0.75mL; 7.14mmol), stir 1h in ice-water bath, stopped reaction aftertreatment obtains white solid 0.39g; 92% productive rate.

The synthesis of 8.2 compound as 3

Compound 2 (0.42g; 0.84mmol) be dissolved in dry DMF (5mL), then add N-iodosuccimide (NIS) (220mg; 0.9mmol) stir.Stirring at room temperature 24h, aftertreatment silica column purification obtains compound a 30.5g, productive rate 91%.

The synthesis of 8.3 compound as 4

Compound a 3 (0.5g; 0.95mmol) be dissolved in 0.5M10mL sodium methoxide solution 65 DEG C and stir 12h, then obtain compound as white solid a40.24g with conventional post-treating method and column chromatography; Productive rate 74%.

The synthesis of 8.4 compound as 5

Compound a 4 (270mg, 0.663mmol) is dissolved in the NaOH solution of 12N, backflow 4h.With HCl neutralization only pH=6 after cooling, after concentrated, use DCM:CH ₃oH (1:1,100mL) washs to obtain white solid 255mg, and 98%, i.e. compound a 4. ¹H NMR(400MHz，DMSO)δ 10.48(s，1H)，7.12(s，1H)，6.35(s，1H)，6.26(d，J=0.8Hz，1H)，4.27(s，1H)，3.75(s，1H)，3.49(t，J=0.8Hz，1H)，2.35-2.28(m，1H)，2.09-2.01(m，1H).

The synthesis of 8.5 compound as 6

Compound a 5 (0.25g; 0.4mmol) be dissolved in DMF (10mL) and add Pd (0) (48mg; 0.04mmol) with CuI (22mg; 1mmol), stirred at ambient temperature 10min, then adds trifluoroacetyl propargylamine (0.2g; 1.2mmol) with triethylamine (0.088g; 0.8mmol), 50 DEG C are continued reaction 13h, and silica column purification obtains white solid 0.1g; 39% productive rate, i.e. compound a 6. ¹H NMR(400MHz，CDCl ₃)67.24(s，1H)，6.38(t，J=0.8Hz，1H)，4.49-4.46(m，1H)，4.31(s，1H)，3.94(d，J=1.6Hz，1H)，3.78-3.68(m，1H)，3.54-2.47(m，1H)，2.3-2.24(m，1H).

The synthesis of 8.6 compound as 7

Difference Weigh Compound a6 (30mg in glove box, 0.072mmol), tri-n-butylamine pyrophosphate salt 264mg (0.48mmol), the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone 90mg (0.48mmol) is placed in three reaction tubess.Tri-n-butylamine pyrophosphate salt is dissolved in 0.75mL dry DMF, then adds the anhydrous tri-n-butylamine of 0.9mL, stir half an hour.Chloro-for 2-4H-1,3,2-benzo dioxy phosphorus-4-ketone is dissolved in 0.75mL 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 8, stirs 1.5h.Add 4mL3% iodine (9:1Py/H2O) solution.Add 4mL water after 15min, stir 2h.Add 1mL3M NaCl solution, then add 35mL dehydrated alcohol ,-20 DEG C of freeze overnight, centrifugal (3200r/min, 25 DEG C) 20min.Incline supernatant liquor, obtains precipitation, drains solvent.Add 2ml strong aqua stirring at room temperature 6h.Decompression screws out solvent, and occur brown solid, RP-HPLC purifying obtains 42mg white solid. ¹H NMR(400MHz，D2O)67.35(s，1H)，6.22(t，J=0.8Hz，1H)，4.59(s，1H)，4.06-3.92(m，5H)，2.48-2.41(m，1H)，2.32—2.28(m，1H)；31P NMR(D20，162MHz)：8.93，11.11，22.46.

8.7 compound dGTP (AP ₃) synthesis of-SPDP a8

In the single port bottle of 10mL, add compound a 718mg (0.019mmol), then add 600 μ lNa ₂cO ₃/ NaHCO ₃damping fluid, stirring at room temperature, is dissolved in the anhydrous CH of 400 μ l SPDP6mg (0.019mmol) ₃cN, adds above-mentioned solution, adds 3 μ l Et ₃n.Stirring at room temperature reacts 3 hours.Freeze-drying, dissolve with 1ml TEAA buffer (100mM, pH7.0), RP-HPLC is separated to obtain compound a 8.NaCl/EtOH removes acetic acid triethylamine salt, obtains 1.5mg white solid.Productive rate 11%. ¹H NMR(400MHz，D2O)δ 8.10(s，1H)，7.54(s，1H)，7.08(s，1H)，6.99(s，1H)，6.14(s，1H)，4.48(s，1H)，3.99-3.94(m，5H)，2.89(t，J=0.8Hz，2H)，2.48(t，J=0.8Hz，2H)；31P NMR(D ₂O，162MHz)：9.7，11.10，22.52；HRMS：calc for C22H26N6014P3S2[M-H] ^-755.0166，found755.0166.

8.8 compound a 9 (Texas-red-NH ₂cH ₂cH ₂sH) synthesis

Be dissolved in by 23mg compound Texas-red in 400 μ l TFA, add 8 μ l triethyl silicanes, stirring at room temperature, TLC follows the tracks of reaction process, stopped reaction after 1h, and toluene (0.5ml*4) revolves band solvent.Preparation HPLC is separated, and gets 12mg yellow solid, productive rate 80%.Condition: pillar: C18,5 μm, 9.4 × 250mm; Flow velocity: 4mL/min; Moving phase: the 0.1%TFA aqueous solution and CH ₃cN, 5%CH ₃cN (5min), gradient wash, 5%-45%CH ₃cN (40min), retention time t=44min, UV-detector: 254nm. ¹H NMR(400MHz，DMSO)68.60(s，1H)，8.19(d，J=0.8Hz，1H)，7.46(d，J=0.8Hz，1H)，6.63(s，1H)，3.6-3.52(m，4H)，3.1(s，3H)，2.79-2.59(m，6H)，2.16-2.03(m，3H)，1.55-1.24(m，5H)；HRMS：calc for C39H4N407S3[M-H] ^-777.2483，found777.2450.

The synthesis of 8.9 compound 10dGTP-SS-Texas Red-X (end product dGTP-S-S-Texas Red)

Get compound a 8 (2.7 μm of ol) and, in 10mL single port bottle, add 0.4ml Na ₃pO ₄-edta buffer liquid dissolves, and after getting the dissolving of 1.9mg compound a 9 (4.0mmol) 0.1ml acetonitrile, join above-mentioned solution, aluminium foil wraps up, stirring at room temperature reaction 6h.After TEAA damping fluid (100mM, pH7.0) dilution, reaction product is separated [condition: pillar: C18,5 μm, 9.4 × 250mm by RP-HPLC; Flow velocity: 4mL/min; Moving phase: 20mM TEAA and CH ₃cN, 5%TEAA (5min), gradient wash 5%-17.5%CH ₃cN (25min); UV-vis detector: 254nm and 495nm.Retention time t=27min].Lyophilize, NaCl/EtOH removes acetic acid triethylamine salt, obtains 1mg yellow solid.Productive rate 35%.RP-HPLC detects purity (>99%) [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, visible light determined wavelength: 293nm and 495nm.Retention time t=37min].HRMS：caic forC56H67N9021P3S4[M-H] ^-1422.2544，found1422.2546.HPLC purity93％.

embodiment 9

The structural formula of the Reversible terminal of the present embodiment is as shown in VIII:

As shown in figure 14, concrete steps are as follows for its synthetic route:

The synthesis of 9.1 compound Texas Red1

1, a 100ml single port bottle is got, 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 1.85ml TEA (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:FA=1:1, obtains 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)。

2, in the single port bottle of 10mL, add 2mL dry DMF, then add 22mg (84 μm of ol) above-claimed cpd, lucifuge, stirred at ambient temperature, 20mg Texas Red 1 is dissolved in 4mL dry DMF, injects, 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, removing DMF under decompression, take 3:1DCM/MeOH as developping agent, and the separation and purification of TLC plate obtains product 23mg.

The synthesis of 9.2 compound dGTP-S-S-Texas Red

In reaction flask, add above-claimed cpd Texas Red19.6mg, 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.Raw material disappear after, stopped reaction, not treated be directly used in next step reaction.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.

By compound dGTP-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-39.6mg (0.014mmol)) reacted with DSC, 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, 53min place.HRMS：caic for C56H67N9021P3S4[M-H] ^-1422.2544，found1422.2546.HPLC purity93％.

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

embodiment 10

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

As shown in figure 15, concrete steps are as follows for its synthetic route:

The synthesis of 10.1 compound F 17-hydroxy-corticosterone ITC1

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 1.85ml TEA (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:FA=1:1, obtains 44mg product, is yellow oily liquid.

2. in the single port bottle of 10mL, add 2mL dry DMF, then add 22mg (84 μm of ol) above-claimed cpd, lucifuge, stirred at ambient temperature, is dissolved in 4mL dry DMF by 20mg FITC, injects, then adds 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, removing DMF under decompression, take 3:1DCM/MeOH as developping agent, and the separation and purification of TLC plate obtains product 20mg.ESI-MS：cais for C ₂₉H ₃₃N ₄O ₄S ₂[M]565.17，found565.17.

The synthesis of 10.2 compound dCTP-S-S-FITC

In reaction flask, add compound above-claimed cpd FITC19.6mg, 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.Raw material disappear after, stopped reaction, not treated be directly used in next step reaction.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.

By compound dCTP-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-39.6mg (0.014mmol)) reacted with DSC, 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-MS：cals for C ₄₂H ₄₅N ₇O ₁₉P ₃S ₂ ^4-[M+2H]1110.12，found1110.12.

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

embodiment 11

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

As shown in figure 16, concrete steps are as follows for its synthetic route:

The synthesis of 11.1 Compound C y5 derivatives

2. in the single port bottle of 10mL, add 2mL dry DMF, then add 22mg (84 μm of ol) above-claimed cpd, lucifuge, stirred at ambient temperature, is dissolved in 4mL dry DMF by 20mg Cy5, injects, then adds 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, removing DMF under decompression, take 3:1DCM/MeOH as developping agent, and the separation and purification of TLC plate obtains product 23mg.

The synthesis of 11.2 compound dATP-S-S-Cy5

In reaction flask, add compound above-claimed cpd Cy5 derivative 9.6mg, 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 (0.105mmo1), then merged by above-mentioned mixed solution, stirred at ambient temperature reacts.TLC follows the tracks of reaction to raw material and disappears.Raw material disappear after, stopped reaction, not treated be directly used in next step reaction.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.

By compound dATP-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-39.6mg (0.014mmol)) reacted with DSC, 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, 53min place.The sign of this compound needs to upgrade.

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

embodiment 12, 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 ~ 11:

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 whether the fluorophor that then to have detected respectively under different concns reductive agent condition entrained by these two kinds dissimilar Reversible terminal for disulfide linkage Reversible terminal can rupture.Specific as follows:

12.1 disulfide linkage Reversible terminal are in DNA chain extension reaction and the test of the fracture under different DTT concentration (Reversible terminal of embodiment 1,2,3,5,6,7,8,9,10,11) 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, DMSO20uL, NaCl (1M) 25uL, Klenow (exo-) pol (5U/uL) 1.32uL, dUTP (10uM) 6uL, template DNA (853ng/uL) 1.25uL, ddH2O35.43uL, 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 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 17, as shown in Figure 17, 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.

12.2 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 1,2,3,5,6,7,8,9,10,11, evaluation method and the effect of the Reversible terminal of this class formation 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: 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 18, as shown in Figure 18, the fluorescent scanning result display after the effect of DTT room temperature 3min, 5min, 8min of 10mM of DNA chain extension product containing disulfide linkage Reversible terminal still has fluorescent signal, and 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 19, as shown in Figure 19, 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.

embodiment 13, 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 5 ~ 11 i.e. Compound II per, III, IV, V, VI, VII, VIII, IX, X further:

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, then 75 DEG C of process detect the effect of the fluorophor fracture under different DTT concentration entrained by (20mM) Reversible terminal with deactivation klenow DNA polymerase activity in 10 minutes for acid-sensitive Reversible terminal.Specific as follows:

1) in eppendorf pipe, the DNA chain extension reaction of Reversible terminal is set up according to following system: 10 × Klenowbuffer10uL, BSA (10mg/mL) 1uL, DMSO20uL, NaCl (1M) 25uL, Klenow (exo-) pol (5U/uL) 1.32uL, dUTP (10uM) 6uL, template DNA (853ng/uL) 1.25uL, ddH ₂o35.43uL, cumulative volume 100uL.

2) cleavage reaction of Reversible terminal fluorophor

(20mM) solution add DTT concentration in DNA chain extension reaction system under, get cleavage reaction product and carry out 12%PAGE electrophoretic analysis, as shown in figure 20, as shown in Figure 20, Reversible terminal can by archaeal dna polymerase identification, participate in the extension of DNA chain as its substrate, and the fluorophor entrained by Reversible terminal ruptures completely, effect is fine.May be used for order-checking completely.In Figure 20, each implication indicated is as follows:

Lane1:dUTP (dUTP-SS-T) inserts

Lane2:dUTP (dUTP-SS-T) inserts, and adds DTT, C=20mM, 1min

Lane3:dUTP (dUTP-SS-T) inserts, and adds DTT, C=20mM, 3min

Lane4:dUTP (dUTP-SS-T) inserts, and adds DTT, C=20mM, 5min

Lane5:dUTP (dUTP-SS-T) inserts, and adds DTT, C=20mM, 10min

Conclusion: after adding reductive agent DTT, during 1min, unstressed configuration signal, illustrate 20mM DTT, can make Compound II per, the Reversible terminal of III, IV, V, VI, VII, VIII, IX, X ruptures completely in 1min.

In sum, the present invention adopts four kinds of different fluoresceins to mark Reversible terminal containing four kinds of different IPs thuja acids (A, G, C, U) respectively.Secondly, in the biochemical reaction of high-flux sequence, with only with compared with a kind of fluorescein-labeled four kinds of Nucleotide reaction systems, use four kinds of fluorescein-labeled four kinds of Nucleotide reaction systems respectively, under identical condition can by the time shorten 4 times of biochemical reaction, this accuracy rate for high-flux sequence result is extremely important, because along with the prolongation in reaction times, template DNA meeting Partial digestion in order-checking system, causes the increase of reacting noise, thus reduces order-checking accuracy rate.Therefore, by four kinds of fluorescein-labeled systems, the accuracy rate of order-checking can greatly be provided.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 four look fluorescent mark Reversible terminal, it is characterized in that, the structural formula of described Reversible terminal is as shown in formula III:

Described method comprises the steps:

The synthesis of D, compound N-4: with NaHCO ₃/ Na ₂cO ₃buffered soln be solvent, compound dUTP-NH ₂ react with compound N-3, obtain compound N-4; Described compound 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 preparation method for four look fluorescent mark Reversible terminal, is characterized in that, the structural formula of described Reversible terminal is such as formula shown in (VIII):

Described method comprises the steps:

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

The synthesis of B, compound Texas Red 1: take dry DMF as solvent, under TEA existent condition, compound react with Texas Red-X lucifuge, obtain compound Texas Red 1; Described Texas Red-X, be 1:(1 ~ 4 with the mol ratio of TEA): (10 ~ 15);

The synthesis of C, compound dGTP-S-S-Texas Red 2: with NaHCO ₃/ Na ₂cO ₃buffered soln be solvent, compound dGTP-NH ₂ react with Texas Red 1, obtain compound dGTP-S-S-Texas Red 2; Described Texas Red 1 and dGTP-NH ₂mol ratio be 1:(1 ~ 2); Namely described compound dGTP-S-S-Texas Red 2 has the Reversible terminal of structural formula shown in formula (VIII).

3. a preparation method for four look fluorescent mark Reversible terminal, is characterized in that, the structural formula of described Reversible terminal is such as formula shown in (IX):

Described method comprises the steps:

The synthesis of B, compound F 17-hydroxy-corticosterone ITC 1: take dry DMF as solvent, under TEA existent condition, compound react with FITC lucifuge, obtain compound F 17-hydroxy-corticosterone ITC 1; Described FITC, be 1:(1 ~ 4 with the mol ratio of TEA): (10 ~ 15);

The synthesis of C, compound dCTP-S-S-FITC: with NaHCO ₃/ Na ₂cO ₃buffered soln be solvent, compound dCTP-NH ₂ react with FITC 1, obtain compound dGTP-S-S-FITC; Described FITC 1 and dCTP-NH ₂mol ratio be 1:(1 ~ 2); Namely described compound dCTP-S-S-FITC has the Reversible terminal of structural formula shown in formula (Ⅸ).

4. a preparation method for four look fluorescent mark Reversible terminal, is characterized in that, the structural formula of described Reversible terminal is such as formula shown in (X):

Described method comprises the steps:

The synthesis of B, Compound C y51: take dry DMF as solvent, under TEA existent condition, compound react with Cy5 lucifuge, obtain Compound C y51; Described Cy5, be 1:(1 ~ 4 with the mol ratio of TEA): (10 ~ 15);

The synthesis of C, compound dATP-S-S-Cy5: with NaHCO ₃/ Na ₂cO ₃buffered soln be solvent, compound dATP-NH ₂ react with Cy51, obtain compound dATP-S-S-Cy5; Described Cy51 and dATP-NH ₂mol ratio be 1:(1 ~ 2); Namely described compound dATP-S-S-Cy5 has the Reversible terminal of eliminant shown in formula (X).

5. the purposes of Nucleotide reaction system in DNA sequencing that the four look fluorescent mark Reversible terminal that method obtains as described in Claims 1 to 4 are formed, it is characterized in that, described DNA sequencing is high-flux sequence.