CN103484106A - Four-color fluorescence labeling reversible terminal and use thereof in DNA (Deoxyribonucleic Acid) sequencing - Google Patents

Four-color fluorescence labeling reversible terminal and use thereof in DNA (Deoxyribonucleic Acid) sequencing Download PDF

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CN103484106A
CN103484106A CN201310401580.0A CN201310401580A CN103484106A CN 103484106 A CN103484106 A CN 103484106A CN 201310401580 A CN201310401580 A CN 201310401580A CN 103484106 A CN103484106 A CN 103484106A
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reversible terminal
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CN103484106B (en
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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
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Shanghai Jiaotong University
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Abstract

The invention discloses a four-color fluorescence labeling reversible terminal and a use thereof in DNA (Deoxyribonucleic Acid) sequencing. The structural formula of the reversible terminal is as shown in a formula (I) in the specification, wherein R1 is triphosphate; R2 is H or OH; the basic group is U, C, A, G or derivatives thereof; the connecting unit is a bifunctional compound which is breakable under a mild condition; the fluorescence group is one selected from a combination of BODIPY, fluorescein, rhodamine, coumarin, xanthene, cyanin, pyrene, phthalocyanine, alexa, squarene dye and an energy transferring dye, and derivatives thereof. The reversible terminal provided by the invention can be used for DNA single-molecule sequencing; simultaneously, raw materials required by the synthesis of the reversible terminal provided by the invention are simple and easy to get and the synthesis process of the reversible terminal is completely involved with conventional chemical reactions, so that the four-color fluorescence labeling reversible terminal can be popularized and utilized to a large scale; biological evaluation results indicate that the reversible terminal is capable of completely meeting the biochemical reaction requirements of high-flux sequencing and has a good practical prospect.

Description

The reversible terminal of four look fluorescent marks and the purposes in DNA sequencing thereof
Technical field
The present invention relates to chemosynthesis and biochemical field, be specifically related to the class reversible terminal of four look fluorescent marks and the purposes in DNA sequencing thereof.
Background technology
The DNA sequencing technology is one of important means of modern life science and medical research.DNA sequencing is since the Sanger sequencing technologies (generation order-checking) of 1977, in the time of thirties years, and develop rapidly.The flux of order-checking significantly improves and cost sharply descends, and has the people even to think that its speed of development broken the speed of the existing Moore's Law budget of semi-conductor industry circle.Two generation the high-throughput parallel sequencing technology appearance be the concentrated reflection of sequencing technologies develop rapidly.Adopt first-generation sequencing technologies, 3,000,000,000 dollars of sequencings that complete the whole genome of people (3,000,000,000 bases) of the Human Genome Project (HGP) cost.And the state-of-the-art technology of current two generations order-checking only needs 5000 dollars just can complete the whole gene order-checking of people.
Even so, the cost of two generations order-checking and technical elements be Shortcomings still, 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 is directly checked order to single DNA molecules, does not do any DNA amplification reaction, thereby Cost reduction improves flux.Although the single-molecule sequencing technology has commercially produced product, all also there is technical difficult point, fail large-scale application.
High-flux sequence platform is in the market monopolized by several external products, especially troubling is, offshore company relies on the control to sequencing reagent, almost completely controlled domestic order-checking market, even if on order-checking hardware, we can have breakthrough, on the auxiliary products such as sequencing reagent, we are also by under one's control.Therefore, independent research is applicable to the check order sequencing reagent of platform of two generations order-checkings or even three generations, will be to changing the current market structure, set up the autonomous order-checking platform of China and have strategic meaning.For this reason, National 863,973 and " 12 " biotech development planning research and development that all will research and develop new-generation sequencing technology and auxiliary products classify the object of giving priority to as.
Summary of the invention
The object of the present invention is to provide the class reversible terminal of four look fluorescent marks and the purposes in DNA sequencing thereof; This reversible terminal has the isotope of redox-sensitive characteristic.The present invention is in harness basis, by multidisciplinary intersection, at first novel four look fluorescent mark Nucleotide have been synthesized, and the DNA extension of development based on this nucleus thuja acid, realize single fluorescence molecule hypersensitive optical detective technology the final three generations's sequencing technologies prototype with single-molecule sequencing ability of realizing.
The objective of the invention is to be achieved through the following technical solutions:
The present invention relates to the reversible terminal of a kind of four look fluorescent mark, its structural formula is suc as formula shown in (I):
Figure BDA0000377748020000021
Wherein, R 1for triphosphate; R 2for H or OH; Base is U, C, A, G or derivatives thereof; The bifunctional compound of connector element for rupturing under mild conditions; The combination that fluorescence group is selected from BODIPY, fluorescein, rhodamine, tonka bean camphor, xanthene, cyanine, pyrene, phthalocyanine, alexa, squarene dyestuff, generate energy transferred dyes with and derivative in a kind of.
Preferably, the structural formula of described reversible terminal is suc as formula shown in (II):
Figure BDA0000377748020000022
Preferably, described reversible terminal (II) is synthesized as follows:
A, compound F 17-hydroxy-corticosterone 2synthetic: under the ice-water bath agitation condition, mol ratio is 1.0:(1.2～2) propargylamine with trifluoro-acetate, react, obtain compound F 17-hydroxy-corticosterone 2
Figure BDA0000377748020000023
B, compound F 17-hydroxy-corticosterone 3synthetic: at CuI, Pd (PPh 3) 4under the condition existed with TEA, compound F 17-hydroxy-corticosterone 2and F 1
Figure BDA0000377748020000024
(be for No. CAS: 172163-62-1, name is called: mix-the iodo-2 '-pancreatic desoxyribonuclease of 7-of 7-denitrification) reaction obtains compound F 17-hydroxy-corticosterone 3
Figure BDA0000377748020000031
described compound F 17-hydroxy-corticosterone 1, compound F 17-hydroxy-corticosterone 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, the chloro-4H-1 of 2-, the reaction under triethylamine and iodine existence of 3,2-benzo dioxy phosphorus-4-ketone, reaction product is gone protection, obtains compound dUTP-NH 2
Figure BDA0000377748020000032
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;
D, compound dUTP-SPDP's is synthetic: under the condition existed at TEA, and compound dUTP-NH 2in sodium carbonate sodium bicarbonate buffer liquid, and take the SPDP that anhydrous acetonitrile is solvent (3-(2-pyridine dimercapto) propionic acid N-hydroxy-succinamide ester) reaction, obtain compound dUTP-SPDP
Figure BDA0000377748020000034
described compound dUTP-NH 2with the mol ratio of SPDP be 1:(1.5～3);
E, compound R DM-SH's is synthetic: under the condition existed at DTT, cysteamine in sodium carbonate sodium bicarbonate buffer liquid with compound TAMRA (5/6) the lucifuge reaction, obtain compound R DM-SH
Figure BDA0000377748020000036
the mol ratio of described compound 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 and acetonitrile are solvent, and compound dUTP-SPDP reacts with RDM-SH, obtain compound dUTP-T; The mol ratio of described compound R DM-SH, compound dUTP-SPDP is 1:(1～2); Described compound dUTP-T has the reversible terminal of eliminant shown in formula (II).
Preferably, described reversible terminal (II) is synthesized as follows:
A, compound F 17-hydroxy-corticosterone 2synthetic: under the ice-water bath agitation condition, mol ratio is 1.0:(1.2～2) propargylamine with trifluoro-acetate, react, obtain compound F 17-hydroxy-corticosterone 2
Figure BDA0000377748020000041
B, compound F 17-hydroxy-corticosterone 3synthetic: at CuI, Pd (PPh 3) 4under the condition existed with TEA, compound F 17-hydroxy-corticosterone 1
Figure BDA0000377748020000042
and F 2reaction, obtain compound F 17-hydroxy-corticosterone 3 described compound F 17-hydroxy-corticosterone 1, compound F 17-hydroxy-corticosterone 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 3react with strong aqua, obtain compound
Figure BDA0000377748020000044
described F 3with the mol ratio of strong aqua be 1:(50～100);
D, compound G 2synthetic: make solvent with methyl alcohol and anhydrous acetonitrile, compound G 1with SPDP reaction, obtain compound G 2
Figure BDA0000377748020000046
described compound G 1with the mol ratio of SPDP be 1:(1～2);
E, compound R DM-SH's is synthetic: under the condition existed at DTT, cysteamine in sodium carbonate sodium bicarbonate buffer liquid with compound TAMRA (5/6)
Figure BDA0000377748020000051
the lucifuge reaction, obtain compound R DM-SH
Figure BDA0000377748020000052
the mol ratio of described compound TAMRA (5/6), cysteamine and DTT is 1:(10～50): (40～70);
F, compound G 3synthetic: using methyl alcohol and acetonitrile as solvent, compound R DM-SH under the nitrogen protection of aluminium foil parcel with compound G 2reaction, obtain compound G 3
Figure BDA0000377748020000053
described compound R DM-SH and compound G 2mol ratio be 1:(1.2～2);
G, compound dUTP-T's is synthetic: compound G 3with tri-n-butylamine pyrophosphate salt, the chloro-4H-1 of 2-, the reaction under triethylamine and iodine existence of 3,2-benzo dioxy phosphorus-4-ketone, reaction product is gone 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; Described compound dUTP-T has the reversible terminal of eliminant shown in formula (II).
Preferably, the structural formula of described reversible terminal is suc as formula shown in (III):
Figure BDA0000377748020000054
Preferably, described reversible terminal (III) is synthesized as follows:
A, compound N-1 synthetic: take methyl alcohol as solvent, under the condition existed at TEA, Mercaptamine under the ice bath agitation condition with the 2-HEDS
Figure BDA0000377748020000055
reaction, obtain compound N-1
Figure BDA0000377748020000056
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 existed at TEA, compound N-1 and TAMRA (5/6)
Figure BDA0000377748020000061
the lucifuge reaction, obtain compound N-2
Figure BDA0000377748020000062
the mol ratio of described TAMRA (5/6), compound N-1 and TEA is 1:(1～4): (10～15);
Synthesizing of C, compound N-3: take anhydrous acetonitrile as solvent, under the condition existed at TEA, react with DSC under the nitrogen protection condition compound N-2, obtains compound N-3
Figure BDA0000377748020000063
the mol ratio of described compound 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
Figure BDA0000377748020000064
react with compound N-3, obtain compound N-4; Described compound N-3 and dUTP-NH 2mol ratio be 1:(1～2); Described compound N-4 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):
Figure BDA0000377748020000065
Preferably, described reversible terminal (IV) is synthesized as follows:
A, Na 2se 2the preparation of alkaline aqueous solution: under ice bath is cooling, by NaBH 4solid is dissolved in water and forms NaBH 4solution; Add selenium powder and cetyl trimethylammonium bromide after the NaOH solid is water-soluble, at N 2under protection, then add described NaBH 4solution reacts 0.5～1h after room temperature reaction 0.5～1.5h under 85～95 ℃, obtains Na 2se 2alkaline aqueous solution; Described NaBH 4, selenium powder and Na0H 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, obtain Compound D-1
Figure BDA0000377748020000071
described bromoethanol and Na 2se 2mol ratio be 1:(1～2);
Synthesizing of C, Compound D-2: take dimethylbenzene as solvent, react with HBr Compound D-1, obtains compound the mol ratio of described Compound D-1 and HBr is 1:(4～6);
Synthesizing of D, Compound D-3: react with strong aqua Compound D-2, obtains Compound D-3
Figure BDA0000377748020000073
the mol ratio of described Compound D-2 and strong aqua is 1:(50～100);
Synthesizing of E, Compound D-4: take dry DMF as solvent, under the condition existed at TEA, Compound D-3 and TAMRA (5/6)
Figure BDA0000377748020000074
the lucifuge reaction, obtain Compound D-4
Figure BDA0000377748020000075
the mol ratio of described compound TAMRA (5/6), Compound D-3 and TEA is 1:(1～4): (10～15);
Synthesizing of F, Compound D-5: take anhydrous acetonitrile as solvent, under the condition existed at TEA, react with DSC under nitrogen protection Compound D-4, obtains Compound D-5
Figure BDA0000377748020000076
the mol ratio of described Compound 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
Figure BDA0000377748020000077
with the 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 are the reversible terminal shown in structural formula (IV).
Preferably, the structural formula of described reversible terminal is as shown in formula V:
Figure BDA0000377748020000081
Preferably, described reversible terminal (V) is synthesized as follows:
Synthetic (the NH of A, compound 1 2cH 2cH 2sTrity): Mercaptamine adds the triphenylmethyl chloride reaction in organic solvent, and recrystallization obtains white solid, and compound 1;
Synthetic (FITC-S-Trity) of B, compound 2: compound 1 reacts to obtain compound 2 with fluorescein FITC (fluorescein isothiocyanate) under alkaline condition;
Synthetic (FITC-SH) of C, compound 3: compound 2 is removed to protect and obtains compound 3;
Synthetic (CF3CONHCH2CCH) of D, compound 7: propargylamine reacts to obtain compound 7 with trifluoro-acetate;
Synthetic [dC (AP3)] of E, compound 8: linked reaction occurs and obtains compound 8 in the iodo-2 '-Deoxyribose cytidine of 5-and compound 7 under the Pd catalyst action;
Synthetic [dCTP (AP3)] of F, compound 4: compound 8 connects triphosphoric acid and obtains compound 4; Gained compound 4 needs the preparative HPLC purifying;
Synthetic [dCTP-S-S-SPDP] of G, compound 5: compound 4 reacts with SPDP and obtains compound 5, thereby introduces disulfide linkage in compound 5;
Synthetic (the end product dCTP-S-S-FITC) of H, compound 6: compound 5 reacts with compound 3 generation disulfide exchange and obtains end product compound 6; Described compound 6 has the reversible terminal shown in formula (V).Compound 6 needs the preparative HPLC purifying equally.
Preferably, described reversible terminal (V) is synthesized as follows:
A, compound F 17-hydroxy-corticosterone 2synthetic: under the ice-water bath agitation condition, mol ratio is 1.0:(1.2～2) propargylamine with trifluoro-acetate, react, obtain compound F 17-hydroxy-corticosterone 2
Figure BDA0000377748020000082
B, compound F 17-hydroxy-corticosterone 3synthetic: at CuI, Pd (PPh 3) 4under the condition existed with TEA, compound F 17-hydroxy-corticosterone 1
Figure BDA0000377748020000083
and F 2reaction, obtain compound F 17-hydroxy-corticosterone 3
Figure BDA0000377748020000091
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 3react with strong aqua, obtain compound G 1 described F 3with the mol ratio of strong aqua be 1: (50～100);
D, compound G 2synthetic: make solvent with methyl alcohol and anhydrous acetonitrile, compound G 1with SPDP
Figure BDA0000377748020000093
reaction, obtain compound G 2
Figure BDA0000377748020000094
described G 1with the mol ratio of SPDP be 1: (1～2);
E, compound F 17-hydroxy-corticosterone ITC-SH's is synthetic: under the condition existed at DTT, 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
Figure BDA0000377748020000095
the mol ratio of described FITC, cysteamine and DTT is 1: (10～50): (40～70);
F, compound G 3synthetic: 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, obtain compound G 3
Figure BDA0000377748020000101
described FITC-SH and G 2mol ratio be 1: (1.2～2);
G, compound dCTP-T's is synthetic: compound G 3with tri-n-butylamine pyrophosphate salt (E-4), the chloro-4H-1 of 2-, the reaction under triethylamine and iodine existence of 3,2-benzo dioxy phosphorus-4-ketone (E-3), reaction product is gone protection, obtains compound dUTP-T; Described E-4, E-3 and G 3mol ratio be 2: 2: 1; Described compound dCTP-T has the reversible terminal shown in formula (V).
Preferably, the structural formula of described reversible terminal is suc as formula shown in (VI):
Figure BDA0000377748020000102
Preferably, described reversible terminal (VI) is synthesized as follows:
A, Compound C y5-SH's is synthetic
Figure BDA0000377748020000103
By fluorescein Cy5 active ester (molecular formula: C 33h 39kN 2o 8s 2, molecular weight: 694.90) with cysteamine, react and obtain Compound C y5-SH;
B, compound dATP (AP3)-SPDP's is synthetic
Figure BDA0000377748020000111
Compound dATP (AP3) is reacted to obtain to compound dATP (AP3)-SPDP with SPDP, thereby introduce disulfide linkage; Product needs the preparative HPLC purifying;
C, compound dATP-SS-Cy5's is synthetic
Figure BDA0000377748020000112
Compound dATP-SPDP and Cy5-SH are at Na 3pO 4the mercaptan permutoid reaction occurs in-edta buffer solution obtain compound dATP-SS-Cy5; This compound dATP-SS-Cy5 has the reversible terminal shown in formula (VI).This compound needs the preparative HPLC purifying.
Preferably, the structural formula of described reversible terminal is suc as formula shown in (VII):
Figure BDA0000377748020000113
Preferably, described reversible terminal (VII) is synthesized as follows:
Synthesizing of A, compound a 2: compound a 1
Figure BDA0000377748020000114
react to obtain compound a 2 with the sec.-propyl formyl chloride under alkaline condition.
Synthesizing of B, compound a 3: compound a 2 reacts to obtain compound a 3 with N-iodosuccimide (NIS) in anhydrous solvent;
Synthesizing of C, compound a 4: compound a 3 removes protecting group and obtains compound a 4 under alkaline condition;
Synthesizing of D, compound a 5: compound a 4 is demethyl under alkaline condition, obtains compound a 5;
Synthesizing of E, compound a 6: linked reaction occurs with the propargylamine trifluoro-acetate and obtains compound a 6 in compound a 5 under the Pd catalyst action;
Synthesizing of F, compound a 7: the above-mentioned same method of compound a 6 use connects triphosphoric acid and obtains compound a 7;
Synthesizing of G, compound a 8: compound a 7 reacts to obtain compound a 8 with SPDP, thereby introduces disulfide linkage;
H, compound Texas-red-NH 2cH 2cH 2sH's (a9) is synthetic: fluorescein Texas-red and amineothiot generation substitution reaction obtain compound a 9;
Synthesizing of I, compound a 10: disulfide exchange occur with compound a 9 and react in compound a 8, obtain compound a 10, and described compound a 10 has the reversible terminal shown in formula (VII).This compound needs the preparative HPLC purifying.
Preferably, the structural formula of described reversible terminal is suc as formula shown in (VIII):
Preferably, described reversible terminal (VIII) is synthesized as follows:
A, compound
Figure BDA0000377748020000122
synthetic: take methyl alcohol as solvent, under the condition existed at TEA, Mercaptamine under the ice bath agitation condition with the 2-HEDS
Figure BDA0000377748020000123
reaction, obtain compound
Figure BDA0000377748020000124
the mol ratio of described Mercaptamine, 2-HEDS and TEA is 1: (1～2): (2～3);
B, compound Texas Red1's is synthetic: the dry DMF (DMF) of take is solvent, under the condition existed at TEA, and compound
Figure BDA0000377748020000125
react with Texas Red-X lucifuge, obtain compound Texas Red1; Described Texas Red-X,
Figure BDA0000377748020000126
with the mol ratio of TEA be 1: (1～4): (10～15);
C, compound dGTP-S-S-Texas Red2's is synthetic: 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); 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 suc as formula shown in (IX):
Figure BDA0000377748020000131
Preferably, described reversible terminal (IX) is synthesized as follows:
A, compound
Figure BDA0000377748020000132
synthetic: take methyl alcohol as solvent, under the condition existed at TEA, Mercaptamine under the ice bath agitation condition with the 2-HEDS
Figure BDA0000377748020000133
reaction, obtain compound
Figure BDA0000377748020000134
the mol ratio of described Mercaptamine, 2-HEDS and TEA is 1: (1～2): (2～3);
B, compound F 17-hydroxy-corticosterone ITC1's is synthetic: the dry DMF (DMF) of take is solvent, under the condition existed at TEA, and compound
Figure BDA0000377748020000135
react with the FITC lucifuge, obtain compound F 17-hydroxy-corticosterone ITC1; Described FITC,
Figure BDA0000377748020000136
with the mol ratio of TEA be 1: (1～4): (10～15);
C, compound dCTP-S-S-FITC's is synthetic: 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); 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 suc as formula shown in (X):
Preferably, described reversible terminal (X) is synthesized as follows:
A, compound
Figure BDA0000377748020000138
synthetic: take methyl alcohol as solvent, under the condition existed at TEA, Mercaptamine under the ice bath agitation condition with the 2-HEDS
Figure BDA0000377748020000139
reaction, obtain compound
Figure BDA00003777480200001310
the mol ratio of described Mercaptamine, 2-HEDS and TEA is 1: (1～2): (2～3);
B, Compound C y51's is synthetic: the dry DMF (DMF) of take is solvent, under the condition existed at TEA, and compound
Figure BDA0000377748020000141
react with the Cy5 lucifuge, obtain Compound C y51; Described Cy5,
Figure BDA0000377748020000142
with the mol ratio of TEA be 1: (1～4): (10～15);
C, compound dATP-S-S-Cy5's is synthetic: 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); 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 the reversible terminal of a kind of aforesaid four look fluorescent mark 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; Simultaneously, its synthetic desired raw material is simple and easy to get, and building-up process is the conventional chemical reaction, can be used for large-scale promotion and uses.
The accompanying drawing explanation
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.
The total synthetic process schematic diagram that Fig. 1 is reversible terminal of the present invention, wherein, R 1for triphosphate; R 2for H or OH; Base is U, C, A, G or derivatives thereof; The bifunctional compound of connector element for rupturing under mild conditions; The combination that fluorescence group is selected from BODIPY, fluorescein, rhodamine, tonka bean camphor, xanthene, cyanine, pyrene, phthalocyanine, alexa, squarene dyestuff, generate energy transferred dyes with and derivative in a kind of;
The building-up process schematic diagram of the reversible terminal that Fig. 2 is embodiment 1;
Fig. 3 is compound dUTP-NH in embodiment 1 2the building-up process schematic diagram;
The process schematic diagram that Fig. 4 is TAMRA in embodiment 1 (5/6) mercaptan;
The building-up process schematic diagram of the reversible terminal that Fig. 5 is embodiment 2;
The building-up process schematic diagram that Fig. 6 is compound dUTP-T in embodiment 2;
The building-up process schematic diagram of the reversible terminal that Fig. 7 is embodiment 3;
Fig. 8 is the building-up process schematic diagram of embodiment 4 containing the reversible terminal of two selenium keys;
The building-up process schematic diagram of the reversible terminal that Fig. 9 is embodiment 5dCTP-S-S-FITC;
Figure 10 is compound dUTP (AP in embodiment 5 3) the building-up process schematic diagram;
The another kind of synthetic method schematic diagram of the reversible terminal that Figure 11 is embodiment 6dCTP-S-S-FITC;
The building-up process schematic diagram of the reversible terminal that Figure 12 is embodiment 7dATP-S-S-Cy5;
The building-up process schematic diagram of the reversible terminal that Figure 13 is embodiment 8dGTP-S-S-Texas Red-X;
The building-up process schematic diagram of the reversible terminal that Figure 14 is embodiment 9 (structural formula VIII) dGTP-S-S-Texas Red-X;
The building-up process schematic diagram of the reversible terminal that Figure 15 is embodiment 10 (structural formula IX) dCTP-S-S-FITC;
The building-up process schematic diagram of the reversible terminal that Figure 16 is embodiment 11 (structural formula X) dATP-S-S-Cy5;
Figure 17 (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 fractures for the chain extension product 10uM DTT room temperature effect 2h containing reversible terminal, 4 fractures for the chain extension product 8mM DTT room temperature effect 2h containing reversible terminal, 5-9 is respectively the fracture containing chain extension product 10mM DTT room temperature effect 10min, 20min, 30min, 1h and the 2h of reversible terminal;
Figure 18 be the DNA chain extension product that contains the reversible terminal of disulfide linkage at 10mM DTT the fracture test result under different action time, 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 19 be contain the reversible terminal of disulfide linkage DNA chain extension product respectively 20, the fracture test result of 30mM DTT under different action time, wherein (a) is the PAGE electrophorogram, (b) be the fluorescent scanning result schematic diagram, 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;
The DNA chain extension product that Figure 20 is reversible terminal II, III, IV, V, VI, VII, VIII, IX, X is the fracture test result under different action time at 20mM DTT respectively, wherein (a) is the fluorescent scanning result schematic diagram, (b) be 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 further to understand the present invention, but not limit in any form the present invention.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, reagent used is commercially available AR, CP level.Gained intermediate product of the present invention and final product employing NMR etc. are characterized; The total synthetic process schematic diagram of reversible terminal of the present invention as shown in Figure 1; To adopt the reversible terminal of four kinds of different fluoresceins difference marks 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):
Figure BDA0000377748020000161
Its corresponding synthetic route as shown in Figure 2; Specifically comprise the steps:
1.1 compound F 17-hydroxy-corticosterone 2synthetic
Trifluoro-acetate reacts and obtains compound F 17-hydroxy-corticosterone with propargylamine in organic solvent 2, be specially: add 60ml methyl alcohol in a single port bottle, 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, under room temperature, reaction is 24 hours.Reaction is monitored with the TLC plate, PE:EA=8:1, and baking sheet, it is product F2 that Rf=0.5 produces new point.Underpressure distillation (51 ℃ 280Pa), obtain 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 added can be the arbitrary value in 72～120mmol.
1.2 compound F 17-hydroxy-corticosterone 3synthetic
Add F1 (0.7mmol, 247mg) in a single port bottle, then take 9.7mgCuI and 20.3mg Pd (PPh 3) 4add in reaction flask, vacuumize, nitrogen protection, the aluminium foil parcel, add 2.3ml DMF, and stirring and dissolving, add 0.2ml TEA, and take after compound F 17-hydroxy-corticosterone 2 (254mg, 1.7mmol) dissolves with DMF and add in above-mentioned reaction flask, 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, 20:1DCM: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 synthesizing, 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 2synthetic
Compound dUTP-NH 2synthetic specifically as shown in Figure 3, 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), the chloro-4H-1 of 2-, 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 steamed of 0.6mL, stir half an hour.The chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone is dissolved in the 0.5mL dry DMF, under high degree of agitation, by syringe, adds above-mentioned tri-n-butylamine pyrophosphate salt solution, stirs half an hour.Then this mixed solution is injected into to 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 the 30mL dehydrated alcohol ,-20 ℃ of freeze overnight, centrifugal (3200r/min, 25 ℃) 20min.The supernatant liquor that inclines, obtain precipitation, drains solvent.Add successively again TEAB solution and strong aqua, stirred overnight at room temperature.White solid, appear in the evaporated under reduced pressure solvent, obtains dUTP-NH 2.With analysis mode, HPLC is analyzed, 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.When t=13.5min, there is the product peak to generate.
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?for?C12H18N3O14P3Na[M+Na]+543.9899，found543.9883。
1.4 compound dUTP-SPDP's is synthetic
Add dUTP-NH in the single port bottle of 10mL 224.4mg (0.026mmol), then add 600 μ l Na 2cO 3/ NaHCO 3damping fluid, stirring at room, be dissolved in the anhydrous CH of 400 μ l to SPDP12.3mg (0.039mmol) 3cN, add above-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 * 250mm; 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; In the time of 27.55min, there is the product peak to generate.After 8 hours, stopped reaction, preparation HPLC separation and purification product, obtain 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 added can be the arbitrary value in 0.039～0.078mmol.
1.5 fluorescein (rhodamine TAMRA (5/6)) mercaptan
Fluorescein (rhodamine TAMRA (5/6)) mercaptan 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 3the damping fluid stirring and dissolving, the aluminium foil parcel; Get TAMRA (5/6) (10mg, 0.019mmol), add in above-mentioned reaction flask after dissolving by the 0.95mL dry DMF, after lucifuge stirring at room 1h, add 1.33mL1M DTT, stirring at room 2.5h.The monitoring of TLC plate: DCM:MeOH=5:1, there is the product dot generation at the Rf=0.7 place.Separate and adopt preparation HPLC to purify, obtain 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 added 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; When 49.74min, there is the product peak to generate.Stopped reaction after 10h, the preparation HPLC separation and purification.Obtain 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，found1121.1655。
In above-mentioned synthesizing, the dUTP-SPDP added can be arbitrary value in 0.01～0.02mmol.
embodiment 2
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 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 G1Rf=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?forC 12H 15N 3O 5[M]281.1012，found281.1015.
In above-mentioned synthesizing, the ammoniacal liquor added 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), add the methanol solution of above-mentioned G1 after dissolving with the 0.5mL anhydrous acetonitrile, stirring at room 10h.TLC plate monitoring: MeOH:EA=1:6, product Rf=0.55.Stopped reaction, screw out solvent, and plate layer chromatography obtains the 9.5mg product.ESI-HRMS：cals?for?C 20H 22N 4O 6S 2[M]478.0981，found478.0974.
In above-mentioned synthesizing, the SPDP added 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, do not dissolve fully, then add 1ml methyl alcohol, and dissolve complete, be 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; When 49min, there is the product peak to generate.Preparative HPLC separates to obtain 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 added 2it can 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), the chloro-4-H-1 of 2-, 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 steamed of 0.15mL, stir half an hour.The chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone is dissolved in the 0.15mL dry DMF, under high degree of agitation, by syringe, adds above-mentioned tri-n-butylamine pyrophosphate salt solution, stirs 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 the 9mL dehydrated alcohol ,-20 ℃ of freeze overnight, centrifugal (3200r/min, 25 ℃) 20min.The supernatant liquor that inclines, obtain precipitation, drains solvent, obtains dUTP-T.With analysis mode, HPLC is analyzed, 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; When 49.7min, there is the product peak to generate.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.
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。
From embodiment 1,2, embodiment 1,2 correspondences the reversible terminal of same, are all to select the reversible terminal of a kind of fluorescein difference mark containing a kind of Nucleotide (U); Particularly, from chemical structure and synthetic method aspect, at first use the reversible terminal of fluorescein TAMRA mark dUTP, by two kinds of different synthetic routes, synthesized this reversible terminal.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, 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 in the end reaction product of a step just must use the HPLC separation and purification.
embodiment 3
The structural formula of the reversible terminal of the present embodiment is suc as formula shown in (III):
As shown in Figure 7, concrete steps are as follows for its corresponding synthetic route:
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, obtain the 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 synthesizing, the 2-HEDS added 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, under room temperature, stir, by 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 tracks to raw material and disappears.Question response is removed DMF under decompression after finishing, and take 3:1DCM/MeOH as developping agent, and the separation and purification of TLC plate obtains product 20mg.ESI-HRMS：cals?for?C 29H 31N 3O 5S 2[M]565.1705，found565.1717.
In above-mentioned synthesizing, the N-1 added 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-29.6mg (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 to stirring reaction under room temperature.TLC follows the tracks of to react to raw material and disappears.After raw material disappears, stopped reaction, not treatedly be directly used in next step reaction.ESI-HRMS：cals?for?C 34H 34N 4O 9S 2[M]706.1767，found706.1761.
In above-mentioned synthesizing, the DSC added 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.
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 the reaction solution of N-3 (starting material compound N-39.6mg (0.014mmol)) is joined in above-mentioned buffered soln to 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; When 49.1min, there is the product peak to generate.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, 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 added 2it can be arbitrary value in 0.014～0.028mmol.
In the present embodiment, at first synthetic connector element N-2, then N-2 directly react with the fluorescein active ester of buying, and has avoided using price higher and the SPDP of side reaction easily occurs, then follows and DSC and newly 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 in these the two kinds evaluations of reversible terminal at DNA sequencing containing the different structure of disulfide linkage of biological assessment, can be advantageously applied to DNA sequencing.
embodiment 4
The structural formula of the reversible terminal of the present embodiment is as shown in the formula shown in (IV):
Figure BDA0000377748020000231
As shown in Figure 8, concrete steps are as follows for its synthetic route:
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 N2 protection, this solution under agitation is added drop-wise in above-mentioned selenium solution, room temperature reaction 1h, after under 90 ℃ reaction make the reaction trend fully half an hour, obtain having the henna Na of feature 2se 2alkaline aqueous solution, this solution does not need to process and can be used for the synthetic of next step diselenide.
In above-mentioned synthesizing, the selenium powder added 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, obtain 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 added 2se 2it can 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, obtain Compound D-2, then add excessive concentrated ammonia (2.3ml, 30mmol), stirring at room, the silica-gel plate separation and purification, obtain yellow liquid D-3.
In above-mentioned synthesizing, the HBr added 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, under room temperature, stir, by 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 tracks to raw material and disappears.Question response is removed DMF under decompression after finishing, and take 3:1DCM/MeOH as developping agent, and the separation and purification of TLC plate obtains product 21mg.ESI-HRMS：cals?for?C 29H 31N 3O 5Se 2[M+H]662.0594，found662.0582.
In above-mentioned synthesizing, the D-3 added 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-411mg (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 to 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 added 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
By 19mg compound dUTP-NH 2(20 μ mol) 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 D-411mg (17 μ mol)) of above-mentioned synthetic whole Compound D-5 is joined in above-mentioned buffered soln to 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; When 49.1min, there is the product peak to generate.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 the product peak.ESI-HRMS：cals?for?C 42H 43N 6O 20P 3Se 2 4-[M+H]1202.95，found1202.87.
In above-mentioned synthesizing, the dUTP-NH added 2it can be arbitrary value in 17～34 μ mol.
embodiment 5
Shown in the following formula V of the structural formula of the reversible terminal of the present embodiment, its corresponding synthetic route is as shown in Fig. 9,10:
5.1 the synthetic (NH of compound 1 2cH 2cH 2sTrity)
Take 1g Mercaptamine (8.8mmol) in 250ml single port bottle, add 40ml DCM, under stirring at room, add 1.3ml TFA (17.6mmol), add subsequently 2.45g Trt-Cl (8.8mmol), after 23h, stopped reaction, with 1mol/L NaOH solution (25ml) cancellation reaction, organic phase is stayed in extraction, with saturated aqueous common salt (20ml), wash, anhydrous sodium sulfate drying, filter, and screws out solvent, the EA recrystallization, obtain the 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).
5.2 synthetic (FITC-S-Trity) of compound 2
16mg compound 1 (51.4 μ mol) is dissolved in to 2.8ml CH 3cN/CHCl 3(9:5) in, add 18 μ l DIPEA, add subsequently 20mg FITC (51.4 μ mol), stirring at room 36h.Take 9:1DCM/MeOH as developping agent, and the separation and purification of TLC plate obtains the 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).
5.3 synthetic (FITC-SH) of compound 3
23mg compound 2 (0.032mmol) is dissolved in 400 μ l TFA, adds 8 μ l triethyl silicanes, stirring at room, TLC follows the tracks of reaction process, stopped reaction after 1h, toluene (0.5ml*4) revolves the band solvent.Preparation HPLC separates, and gets the 12mg yellow solid, productive rate 80%.Condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: the 0.1%TFA aqueous solution and CH 3cN, 5%CH 3cN (5min), gradient washing, 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.
5.4 the synthetic (CF of compound 7 3cONHCH 2cCH)
Add 60ml methyl alcohol in a single port bottle, 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, under room temperature, reaction is 24 hours.Decompression screws out solvent, adds CHCl 3(60ml), use respectively saturated sodium bicarbonate solution (60ml), each extraction of saturated aqueous common salt (60m1) once.The organic phase anhydrous sodium sulfate drying, decompression screws out solvent, and (51 ℃ 280Pa), obtain the 3.53g colourless liquid, productive rate 39% in the resistates underpressure distillation. 1H?NMR(CDCl 3，300MHz)：δ?2.32(t，J=4.0Hz，1H)，4.13-4.15(m，2H)，6.92(s，1H).
5.5 synthetic [dC (AP3)] of compound 8
Add the iodo-2 '-Deoxyribose cytidine of 5-(0.70mmol, 248mg) in a single port bottle, then take 10mg CuI (25.2 μ mol) and 20mg Pd (PPh 3) 4(17.6 μ mol) adds in reaction flask, vacuumizes nitrogen protection, aluminium foil parcel; add 1.5ml DMF, stirring and dissolving, add 0.2ml TEA, takes compound 7 (254mg; 1.68mmol) with adding in above-mentioned reaction flask after 1ml DMF dissolving, stirring at room, 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).
5.6 synthetic [the synthesis of dCTP (AP3)] of compound 4
Take respectively 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.The tri-n-butylamine pyrophosphate salt is dissolved in the 0.75mL dry DMF, then adds the anhydrous tri-n-butylamine of 0.9mL, stir half an hour.The chloro-4H-l of 2-, 3,2-benzo dioxy phosphorus-4-ketone is dissolved in the 0.75mL dry DMF, under high degree of agitation, by syringe, adds above-mentioned tri-n-butylamine pyrophosphate salt solution, stirs 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 the 35mL dehydrated alcohol ,-20 ℃ of freeze overnight, centrifugal (3200r/min, 25 ℃) 20min.The supernatant liquor that inclines, obtain precipitation, drains solvent.Add 2ml strong aqua stirring at room 6h.Decompression screws out solvent, brown solid occurs, and RP-HPLC analyzes [condition: pillar: C18,10 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAA and EtOH, gradient washing, 0%-20%EtOH (35min); UV-detector: 254nm], retention time t=11min.RP-HPLC separates [condition: pillar: C18,5 μ m, 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 the acetic acid triethylamine salt, obtains the 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.
5.7 synthetic [dCTP-S-S-SPDP] of compound 5
Add compound 4 18mg (0.019mmol) in the single port bottle of 10mL, then add 600 μ l Na 2cO 3/ NaHCO 3damping fluid, stirring at room, be dissolved in the anhydrous CH of 400 μ l to SPDP6mg (0.019mmol) 3cN, add above-mentioned solution, adds 3 μ l Et 3n.Stirring at room reaction 3 hours.Freeze-drying, dissolve with 1ml TEAA buffer (100mM, pH7.0), and RP-HPLC separates to obtain compound 5 (retention time t=34min).NaCl/EtOH removes the acetic acid triethylamine salt, obtains the 1.5mg white solid.Productive rate 11%.RP-HPLC separation 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%-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.
5.8 synthetic (the end product dCTP-S-S-FITC) of compound 6
Get compound 5 (3mg, 2.7 μ mol) and, in 10mL single port bottle, add 0.4ml Na 3pO 4-edta buffer liquid dissolves, and gets 1.9mg compound 3 (4.0mmol) with after the dissolving of 0.1ml acetonitrile, joins above-mentioned solution, aluminium foil parcel, stirring at room reaction 6h.With after TEAA damping fluid (100mM, pH7.0) dilution, reaction product is separated [condition: pillar: C18,5 μ m, 9.4 * 250mn by RP-HPLC; Flow velocity: 4mL/min; Moving phase: 20mM TEAA and CH 3cN, 5%TEAA (5min), gradient washing 5%-17.5%CH 3cN (25min); UV-vis detector: 254nm and 495nm.Retention time t=27min].Lyophilize, NaCl/EtOH removes the acetic acid triethylamine salt, obtains the 1mg yellow solid.Productive rate 35%.RP-HPLC detects purity (>99%) [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, visible light detects 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 the structural formula of the reversible terminal of the present embodiment:
Figure BDA0000377748020000281
Its corresponding synthetic route as shown in figure 11; Specifically comprise the steps:
6.1 compound F 17-hydroxy-corticosterone 2, F 3synthetic with embodiment 1
6.2 compound G 1synthetic
Get the 23mg compound F 17-hydroxy-corticosterone 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 G1Rf=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-MS：cals?for?C 12H 17N 4O 4[M]281.10，found281.12.
In above-mentioned synthesizing, the ammoniacal liquor added can be arbitrary value in 3～6mmol.
6.3 compound G 2(FITC-SH) synthesize
Get 8.5mg G 1(0.03mmol), use the 0.5mL dissolve with methanol; Get 9.4mg SPDP (0.03mmol), add the methanol solution of above-mentioned G1 after dissolving with the 0.5mL anhydrous acetonitrile, stirring at room 10h.TLC plate monitoring: MeOH:EA=1:6, product Rf=0.55.Stopped reaction, screw out solvent, and plate layer chromatography obtains the 9.5mg product.ESI-MS：cals?for?C 20H 24N 5O 5S 2[M]478.10，found478.11.
In above-mentioned synthesizing, the SPDP added can be arbitrary value in 0.03～0.06mmol.
6.4 compound G3's is synthetic
Get 6.8mg G 2be that FITC-SH (0.013mmol, it is synthetic with embodiment 1) in 10mL single port bottle, vacuumizes nitrogen protection, the aluminium foil parcel; Separately get 9mg G 2(0.019mmol) in 10ml single port bottle, use 0.5ml CH 3cN, do not dissolve fully, then add 1ml methyl alcohol, and dissolve complete, be 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; When 49min, there is the product peak to generate.Preparative HPLC separates to obtain the 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).
In above-mentioned synthesizing, the G added 2it can be arbitrary value in 0.016～0.026mmol.
6.5 compound dCTP-T is the synthetic of G4
Compound dCTP-T synthesizes specifically 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.
Take respectively 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.The tri-n-butylamine pyrophosphate salt is dissolved in the 0.15mL dry DMF, then adds the new tri-n-butylamine steamed of 0.15mL, stir half an hour.The chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone is dissolved in the 0.15mL dry DMF, under high degree of agitation, by syringe, adds above-mentioned tri-n-butylamine pyrophosphate salt solution, stirs 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 the 9mL dehydrated alcohol ,-20 ℃ of freeze overnight, centrifugal (3200r/min, 25 ℃) 20min.The supernatant liquor that inclines, obtain precipitation, drains solvent, obtains dCTP-T.RP-HPLC detects purity (>99%) [condition: pillar: C18,10 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH3CN, 5%CH 3cN (5min), gradient washing 5%-35%CH3CN (60min); UV, visible light detects 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?for?C 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:
7.1 Compound C y5-SH's is synthetic
By 28mg cysteamine (0.363mmol) (400 μ l Na 2cO 3/ NaHCO 3damping fluid dissolves) add in Cy5-NHS-ester (5mg, 0.008mmol) (dissolving of 1mL dry DMF), the normal temperature lucifuge stirs 1h, then adds 0.5mL1M DTT, stirring at room 1h.Finally, 0.1%TFA (0.5ml) solution cancellation for reaction, after lyophilize, RP-HPLC separates (retention time is about 54min).Obtain 1.2mg.ESI-HRMS：calc?for?C 34H 44N 3OS[M]542.3205，Found542.3200.
7.2 compound dATP (AP3)-SPDP's is synthetic
Add dATP (AP3) 9mg (0.010mmol) in the single port bottle of 10mL, then add 800 μ l Na 2cO 3/ NaHCO 3damping fluid, stirring at room, be dissolved in the anhydrous CH of 700 μ l to SPDP5.6mg (0.015mmol) 3cN, add above-mentioned solution, adds 4 μ l Et 3n.The stirring at room reaction, after 4 hours, stopped reaction, separation and purification obtains 2.9mgdATP (AP3)-SPDP.
7.3 compound dATP-SS-Cy5's is synthetic
DATP-SPDP (2.9mg) is dissolved in to Na 3pO 4-edta buffer liquid [50mMNa 3pO 4, 10mM EDTA (pH7.4,1.5ml)] and the mixed solution of acetonitrile 0.5ml in, then it is added in the Cy5-SH (2.3mg) after freeze-drying, the room temperature lucifuge stirs 5 hours.The 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 μ m, 4.6 * 250mn; 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; When 49.1min, there is the product peak to generate.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, 53min place.HPLC purity 95%, the 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):
Figure BDA0000377748020000311
As shown in figure 13, concrete steps are as follows for its synthetic route:
8.1 compound a 2 is synthetic
Compound a 1 (0.20g; 0.714mmol) be dissolved in the 3mL anhydrous pyridine and slowly add own propyl group to add acyl chlorides (0.75mL; 7.14mmol), stir 1h in ice-water bath, the stopped reaction aftertreatment obtains white solid 0.39g; 92% productive rate.
8.2 compound a 3 is synthetic
Compound 2 (0.42g; 0.84mmol) be dissolved in dry DMF (5mL), then add N-iodosuccimide (NIS) (220mg; 0.9mmol) stir.Stirring at room 24h, aftertreatment silicagel column purifying obtains compound a 30.5g, productive rate 91%.
8.3 compound a 4 is synthetic
Compound a 3 (0.5g; 0.95mmol) be dissolved in 65 ℃ of stirring 12h of 0.5M10mL sodium methoxide solution, then with conventional post-treating method and column chromatography, obtain white solid compound a 40.24g; Productive rate 74%.
8.4 compound a 5 is synthetic
Compound a 4 (270mg, 0.663mmol) is dissolved in the NaOH solution of 12N, backflow 4h.Cooling rear with HCl neutralization pH=6 only, use DCM:CH after concentrated 3oH (1:1,100mL) washs to obtain white solid 255mg, and 98%, 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).
8.5 compound a 6 is synthetic
Compound a 5 (0.25g; 0.4mmol) be dissolved in DMF (10mL) and add Pd (0) (48mg; 0.04mmol) and CuI (22mg; 1mmol), under room temperature, stir 10min, then add trifluoroacetyl propargylamine (0.2g; 1.2mmol) and triethylamine (0.088g; 0.8mmol), 50 ℃ are continued reaction 13h, and the silicagel column purifying obtains white solid 0.1g; 39% productive rate, 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).
8.6 compound a 7 is synthetic
Take respectively compound a 6 (30mg, 0.072mmol), 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.The tri-n-butylamine pyrophosphate salt is dissolved in the 0.75mL dry DMF, then adds the anhydrous tri-n-butylamine of 0.9mL, stir half an hour.The chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone is dissolved in the 0.75mL dry DMF, under high degree of agitation, by syringe, adds above-mentioned tri-n-butylamine pyrophosphate salt solution, stirs 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 the 35mL dehydrated alcohol ,-20 ℃ of freeze overnight, centrifugal (3200r/min, 25 ℃) 20min.The supernatant liquor that inclines, obtain precipitation, drains solvent.Add 2ml strong aqua stirring at room 6h.Decompression screws out solvent, brown solid occurs, and the RP-HPLC purifying obtains the 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)-SPDP a8's is synthetic
Add compound a 718mg (0.019mmol) in the single port bottle of 10mL, then add 600 μ lNa 2cO 3/ NaHCO 3damping fluid, stirring at room, be dissolved in the anhydrous CH of 400 μ l to SPDP6mg (0.019mmol) 3cN, add above-mentioned solution, adds 3 μ l Et 3n.Stirring at room reaction 3 hours.Freeze-drying, dissolve with 1ml TEAA buffer (100mM, pH7.0), and RP-HPLC separates to obtain compound a 8.NaCl/EtOH removes the acetic acid triethylamine salt, obtains the 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 2synthesizing SH)
23mg compound Texas-red is dissolved in 400 μ l TFA, adds 8 μ l triethyl silicanes, stirring at room, TLC follows the tracks of reaction process, stopped reaction after 1h, toluene (0.5ml*4) revolves the band solvent.Preparation HPLC separates, and gets the 12mg yellow solid, productive rate 80%.Condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: the 0.1%TFA aqueous solution and CH 3cN, 5%CH 3cN (5min), gradient washing, 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.
8.9 compound 10dGTP-SS-Texas Red-X's (end product dGTP-S-S-Texas Red) is synthetic
Get compound a 8 (2.7 μ mol) and, in 10mL single port bottle, add 0.4ml Na 3pO 4-edta buffer liquid dissolves, and gets 1.9mg compound a 9 (4.0mmol) with after the dissolving of 0.1ml acetonitrile, joins above-mentioned solution, aluminium foil parcel, stirring at room reaction 6h.With after TEAA damping fluid (100mM, pH7.0) dilution, reaction product is separated [condition: pillar: C18,5 μ m, 9.4 * 250mm by RP-HPLC; Flow velocity: 4mL/min; Moving phase: 20mM TEAA and CH 3cN, 5%TEAA (5min), gradient washing 5%-17.5%CH 3cN (25min); UV-vis detector: 254nm and 495nm.Retention time t=27min].Lyophilize, NaCl/EtOH removes the acetic acid triethylamine salt, obtains the 1mg yellow solid.Productive rate 35%.RP-HPLC detects purity (>99%) [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, visible light detects wavelength: 293nm and 495nm.Retention time t=37min].HRMS：caic?for?C56H67N9021P3S4[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:
9.1 compound Texas Red1's is synthetic
1, 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 1.85ml TEA (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:FA=1:1, obtain the 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 synthesizing, the 2-HEDS added can be arbitrary value in 6.6～13.2mmol, and TEA can be arbitrary value in 13.2～19.8mmol.
2, add the 2mL dry DMF in the single port bottle of 10mL, then add 22mg (84 μ mol) above-claimed cpd, lucifuge, stir under room temperature, and 20mg Texas Red 1 is dissolved in to the 4mL dry DMF, injects, then add 80 μ L (570 μ mol) triethylamine.Stirring reaction under room temperature, TLC tracks to raw material and disappears.Question response is removed DMF under decompression after finishing, and take 3:1DCM/MeOH as developping agent, and the separation and purification of TLC plate obtains product 23mg.
9.2 compound dGTP-S-S-Texas Red's is synthetic
Add above-claimed cpd Texas Red19.6mg 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 to stirring reaction under room temperature.TLC follows the tracks of to react to raw material and disappears.After raw material disappears, stopped reaction, not treatedly be directly used in next step reaction.In above-mentioned synthesizing, 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 reaction solution that will be reacted with DSC (starting material compound N-39.6mg (0.014mmol)) joins 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; When 49.1min, there is the product peak to generate.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.HRMS：caic?for?C56H67N9021P3S4[M-H] -1422.2544，found1422.2546.HPLC?purity93％.
In above-mentioned synthesizing, 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):
Figure BDA0000377748020000341
As shown in figure 15, concrete steps are as follows for its synthetic route:
10.1 compound F 17-hydroxy-corticosterone ITC1's is synthetic
1. 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 1.85ml TEA (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:FA=1:1, obtain the 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 synthesizing, the 2-HEDS added can be arbitrary value in 6.6～13.2mmol, and TEA can be arbitrary value in 13.2～19.8mmol.
2. add the 2mL dry DMF in the single port bottle of 10mL, then add 22mg (84 μ mol) above-claimed cpd, lucifuge, stir under room temperature, and 20mg FITC is dissolved in to the 4mL dry DMF, injects, then add 80 μ L (570 μ mol) triethylamine.Stirring reaction under room temperature, TLC tracks to raw material and disappears.Question response is removed DMF under decompression after finishing, and 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.
10.2 compound dCTP-S-S-FITC's is synthetic
Add compound above-claimed cpd FITC19.6mg 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 to stirring reaction under room temperature.TLC follows the tracks of to react to raw material and disappears.After raw material disappears, stopped reaction, not treatedly be directly used in next step reaction.In above-mentioned synthesizing, 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 reaction solution that will be reacted with DSC (starting material compound N-39.6mg (0.014mmol)) joins 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; When 49.1min, there is the product peak to generate.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-MS：cals?for?C 42H 45N 7O 19P 3S 2 4-[M+2H]1110.12，found1110.12.
In above-mentioned synthesizing, 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):
Figure BDA0000377748020000361
As shown in figure 16, concrete steps are as follows for its synthetic route:
11.1 Compound C y5 derivative is synthetic
1. 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 1.85ml TEA (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:FA=1:1, obtain the 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 synthesizing, the 2-HEDS added can be arbitrary value in 6.6～13.2mmol, and TEA can be arbitrary value in 13.2～19.8mmol.
2. add the 2mL dry DMF in the single port bottle of 10mL, then add 22mg (84 μ mol) above-claimed cpd, lucifuge, stir under room temperature, and 20mg Cy5 is dissolved in to the 4mL dry DMF, injects, then add 80 μ L (570 μ mol) triethylamine.Stirring reaction under room temperature, TLC tracks to raw material and disappears.Question response is removed DMF under decompression after finishing, and take 3:1DCM/MeOH as developping agent, and the separation and purification of TLC plate obtains product 23mg.
11.2 compound dATP-S-S-Cy5's is synthetic
Add compound above-claimed cpd Cy5 derivative 9.6mg 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 (0.105mmo1), more above-mentioned mixed solution is merged to stirring reaction under room temperature.TLC follows the tracks of to react to raw material and disappears.After raw material disappears, stopped reaction, not treatedly be directly used in next step reaction.In above-mentioned synthesizing, 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 reaction solution that will be reacted with DSC (starting material compound N-39.6mg (0.014mmol)) joins 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; When 49.1min, there is the product peak to generate.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.The sign of this compound needs to upgrade.
In above-mentioned synthesizing, the dATP-NH added 2it can be arbitrary value in 0.014～0.028mmol.
embodiment 12, to the biological assessment of synthetic reversible terminal
For whether the reversible terminal that detects synthesized of the present invention can be applied to DNA sequencing, the present embodiment has detected the characteristic of two aspects of reversible terminal of embodiment 1～11:
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 can removing 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, then for the reversible terminal of disulfide linkage, detected respectively these the two kinds entrained fluorophors of dissimilar reversible terminal under different concns reductive agent condition and whether can rupture for 75 ℃.Specific as follows:
12.1 the reversible terminal of disulfide linkage is in DNA chain extension reaction and the test of the fracture under different DTT concentration (the reversible terminal of embodiment 1,2,3,5,6,7,8,9,10,11) thereof
1) set up the DNA chain extension reaction containing the reversible terminal of disulfide linkage according to following system in the eppendorf pipe: 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 to 30 ℃ of water baths and processes 15 minutes, then be placed in 75 ℃ of water-baths and process 10 minutes with the deactivation archaeal dna polymerase.Cleavage reaction by reaction product for follow-up reversible terminal fluorophor.
2) cleavage reaction of the reversible terminal fluorophor of disulfide linkage
At room temperature process with the DTT of 10uM, 8mM and 10mM the DNA chain extension reaction product that contains the reversible terminal of disulfide linkage respectively, 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 17, as shown in Figure 17, 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, illustrate that it can be applied to the high-flux sequence reaction fully.
12.2 the DNA chain extension product that contains the reversible terminal of the disulfide linkage test of the fracture under action time different from 30mM DTT at 10mM, 20mM respectively
What test is the reversible terminal of embodiment 1,2,3,5,6,7,8,9,10,11, and evaluation method and the effect of the reversible terminal of this class formation 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: add the DTT that final concentration is 10mM to process respectively different time in DNA chain extension reaction system, get the cleavage reaction product and carry out the 12%PAGE electrophoretic analysis, as shown in figure 18, as shown in Figure 18, the DNA chain extension product that contains the reversible terminal of disulfide linkage fluorescent scanning result demonstration after the DTT of 10mM room temperature effect 3min, 5min, 8min still has fluorescent signal, illustrates that under this concentration, DTT can not be fully by 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, in DNA chain extension reaction system, add respectively the DTT that final concentration is 20mM and 30mM to process respectively different time, get the cleavage reaction product and carry out the 12%PAGE electrophoretic analysis, as shown in figure 19, as shown in Figure 19, the DNA chain extension product that contains the reversible terminal of disulfide linkage is 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 by the disulfide bonds of reversible terminal.For the reversible terminal of two selenium keys, can under reductive agent DTT effect, rupture equally, the DTT concentration that just fracture needs is larger, and the time also can be longer.
The biological assessment of embodiment 4 reversible terminals is identical with embodiment 1,2,3, and just the used in amounts of reductive agent DTT will be increased to 10 times; Its result illustrates that its synthetic reversible terminal can be applied to DNA sequencing equally.
embodiment 13, to the biological assessment of synthetic reversible terminal
For whether the reversible terminal that detects synthesized of the present invention can be applied to DNA sequencing, it is the characteristic of two aspects of reversible terminal of Compound I I, III, IV, V, VI, VII, VIII, IX, X that the present embodiment has further detected embodiment 5～11:
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 can removing 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 detects the effect that under different DTT concentration, the entrained fluorophor of (20mM) reversible terminal ruptures.Specific as follows:
1) set up the DNA chain extension reaction of reversible terminal according to following system in the eppendorf pipe: 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, ddH 2o35.43uL, cumulative volume 100uL.
Reaction system is placed in to 30 ℃ of water baths and processes 15 minutes, then be placed in 75 ℃ of water-baths and process 10 minutes with the deactivation archaeal dna polymerase.Cleavage reaction by reaction product for follow-up reversible terminal fluorophor.
2) cleavage reaction of reversible terminal fluorophor
Add (20mM) solution under DTT concentration in DNA chain extension reaction system, get the cleavage reaction product and carry out the 12%PAGE electrophoretic analysis, as shown in figure 20, as shown in Figure 20, reversible terminal can be identified by archaeal dna polymerase, participate in the extension of DNA chain as its substrate, and the entrained fluorophor of reversible terminal ruptures fully, effect is fine.Fully can be for order-checking.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, without fluorescent signal, 20mM DTT is described, can makes the reversible terminal of Compound I I, III, IV, V, VI, VII, VIII, IX, X rupture fully in 1min.
In sum, the present invention adopts the reversible terminal of four kinds of different fluoresceins difference marks containing four kinds of different IPs thuja acids (A, G, C, U).Secondly, in the biochemical reaction of high-flux sequence, with only by a kind of fluorescein-labeled four kinds of Nucleotide reaction systems, compare, use respectively four kinds of fluorescein-labeled four kinds of Nucleotide reaction systems, can be by 4 times of the time shortens of biochemical reaction under same condition, this accuracy rate for the high-flux sequence result is extremely important, because the prolongation along with the reaction times, template DNA meeting Partial digestion in the order-checking system, cause reacting the increase of noise, thereby reduce the order-checking accuracy rate.Therefore, by four kinds of fluorescein-labeled systems, can greatly provide the accuracy rate of order-checking.The test result of embodiment 6 has further verified that reversible terminal of the present invention has completed the biochemical reaction requirement that meets 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 (18)

1. the reversible terminal of look fluorescent mark, its structural formula is suc as formula shown in (I):
Figure FDA0000377748010000011
Wherein, R 1for triphosphate; R 2for H or OH; Base is U, C, A, G or derivatives thereof; The bifunctional compound of connector element for rupturing under mild conditions; The combination that fluorophor is selected from BODIPY, fluorescein, rhodamine, tonka bean camphor, xanthene, cyanine, pyrene, phthalocyanine, alexa, squarene dyestuff, generate energy transferred dyes with and derivative in a kind of.
2. the reversible terminal of four look fluorescent mark as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is suc as formula shown in (II):
Figure FDA0000377748010000012
3. the reversible terminal of four look fluorescent mark as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is suc as formula shown in (III):
Figure FDA0000377748010000013
4. the reversible terminal of four look fluorescent mark as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is suc as formula shown in (IV):
Figure FDA0000377748010000021
5. the reversible terminal of four look fluorescent mark as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is as shown in formula V:
Figure FDA0000377748010000022
6. the reversible terminal of four look fluorescent mark as claimed in claim 5, is characterized in that, described reversible terminal is synthesized as follows:
Synthesizing of A, compound 1: Mercaptamine adds the triphenylmethyl chloride reaction in organic solvent, and recrystallization obtains white solid, and compound 1;
Synthesizing of B, compound 2: compound 1 reacts to obtain compound 2 under alkaline condition with fluorescein FTTC;
Synthesizing of C, compound 3: compound 2 is removed to protect and obtains compound 3;
Synthesizing of D, compound 7: propargylamine reacts to obtain compound 7 with trifluoro-acetate;
Synthesizing of E, compound 8: linked reaction occurs and obtains compound 8 in the iodo-2 '-Deoxyribose cytidine of 5-and compound 7 under the Pd catalyst action;
Synthesizing of F, compound 4: compound 8 connects triphosphoric acid and obtains compound 4;
G, compound 5 synthetic: compound 4 reacts with SPDP and obtains compound 5;
H, compound 6 synthetic: compound 5 reacts with compound 3 generation disulfide exchange and obtains compound 6; Described compound 6 has the reversible terminal shown in formula (V).
7. the reversible terminal of four look fluorescent mark as claimed in claim 5, is characterized in that, described reversible terminal is synthesized as follows:
A, compound F 17-hydroxy-corticosterone 2synthetic: under the ice-water bath agitation condition, mol ratio is 1.0:(1.2～2) propargylamine with trifluoro-acetate, react, obtain compound F 17-hydroxy-corticosterone 2
Figure FDA0000377748010000023
B, compound F 17-hydroxy-corticosterone 3synthetic: at CuI, Pd (PPh 3) 4under the condition existed with TEA, compound F 17-hydroxy-corticosterone 1
Figure FDA0000377748010000031
and F 2reaction, obtain compound F 17-hydroxy-corticosterone 3
Figure FDA0000377748010000032
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 3react with strong aqua, obtain compound G 1
Figure FDA0000377748010000033
described F 3with the mol ratio of strong aqua be 1:(50～100);
D, compound G 2synthetic: make solvent with methyl alcohol and anhydrous acetonitrile, compound G 1with SPDP
Figure FDA0000377748010000034
reaction, obtain compound G 2
Figure FDA0000377748010000035
, described G 1with the mol ratio of SPDP be 1:(1～2):
E, compound F 17-hydroxy-corticosterone ITC-SH's is synthetic: under the condition existed at DTT, cysteamine reacts with compound F 17-hydroxy-corticosterone ITC lucifuge in sodium carbonate sodium bicarbonate buffer liquid, obtains compound F 17-hydroxy-corticosterone ITC-SH
Figure FDA0000377748010000036
the mol ratio of described FITC, cysteamine and DTT is 1:(10～50): (40～70);
F, compound G 3synthetic: 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, obtain compound G 3 described FITC-SH and G 2mol ratio be 1:(1.2～2);
G, compound dCTP-T's is synthetic: compound G 3with tri-n-butylamine pyrophosphate salt, the chloro-4H-1 of 2-, the reaction under triethylamine and iodine existence of 3,2-benzo dioxy phosphorus-4-ketone, reaction product is gone 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 G 3mol ratio be 2:2:1; Described compound dCTP-T has the reversible terminal shown in formula (V).
8. the reversible terminal of four look fluorescent mark as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is suc as formula shown in (VI):
9. the reversible terminal of four look fluorescent mark as claimed in claim 8, is characterized in that, described reversible terminal (VI) is synthesized as follows:
A, Compound C y5-SH's is synthetic: fluorescein Cy5 active ester is reacted with cysteamine and obtain Compound C y5-SH;
B, compound dATP (AP3)-SPDP's is synthetic: compound dATP (AP3) is reacted to obtain to compound dATP (AP3)-SPDP with SPDP:
C, compound dATP-SS-Cy5's is synthetic: compound dATP-SPDP and Cy5-SH are at Na 3pO 4the mercaptan permutoid reaction occurs in-edta buffer solution obtain compound dATP-SS-Cy5; This compound dATP-SS-Cy5 has the reversible terminal shown in formula (VI).
10. the reversible terminal of four look fluorescent mark as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is suc as formula shown in (VII):
Figure FDA0000377748010000051
11. the reversible terminal of four look fluorescent mark as claimed in claim 10, is characterized in that, described reversible terminal is synthesized as follows:
Synthesizing of A, compound a 2: compound a 1
Figure FDA0000377748010000052
react to obtain compound a 2 with the sec.-propyl formyl chloride under alkaline condition.
Synthesizing of B, compound a 3: compound a 2 reacts to obtain compound a 3 in anhydrous solvent with N-iodosuccimide:
Synthesizing of C, compound a 4: compound a 3 removes protecting group and obtains compound a 4 under alkaline condition;
Synthesizing of D, compound a 5: compound a 4 is demethyl under alkaline condition, obtains compound a 5;
Synthesizing of E, compound a 6: linked reaction occurs with the propargylamine trifluoro-acetate and obtains compound a 6 in compound a 5 under the Pd catalyst action;
Synthesizing of F, compound a 7: compound a 6 connects triphosphoric acid and obtains compound a 7;
Synthesizing of G, compound a 8: compound a 7 reacts to obtain compound a 8 with SPDP;
Synthesizing of H, compound a 9: fluorescein Texas-red and amineothiot generation substitution reaction obtain compound a 9;
Synthesizing of I, compound a 10: disulfide exchange occur with compound a 9 and react in compound a 8, obtain compound a 10; Described compound a 10 has the reversible terminal shown in formula (VII).
12. the reversible terminal of four look fluorescent mark as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is suc as formula shown in (VIII):
13. the reversible terminal of four look fluorescent mark as claimed in claim 12, is characterized in that, described reversible terminal is synthesized as follows:
A, compound
Figure FDA0000377748010000061
synthetic: take methyl alcohol as solvent, under the condition existed at TEA, Mercaptamine under the ice bath agitation condition with the 2-HEDS
Figure FDA0000377748010000062
reaction, obtain compound
Figure FDA0000377748010000063
the mol ratio of described Mercaptamine, 2-HEDS and TEA is 1:(1～2): (2～3):
B, compound Texas Red1's is synthetic: take dry DMF as solvent, and under the condition existed at TEA, compound
Figure FDA0000377748010000064
react with Texas Red-X lucifuge, obtain compound Texas Red1; Described Texas Red-X,
Figure FDA0000377748010000065
with the mol ratio of TEA be 1:(1～4): (10～15);
C, compound dGTP-S-S-Texas Red2's is synthetic: with NaHCO 3/ Na 2cO 3buffered soln be solvent, compound dGTP-NH 2react with Texas Red1, obtain compound dGTP-S-S-Texas Red2; Described Texas Red1 and dGTP-NH 2mol ratio be 1:(1～2); Described compound dGTP-S-S-Texas Red2 has the reversible terminal of structural formula shown in formula (VIII).
14. the reversible terminal of four look fluorescent mark as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is suc as formula shown in (IX):
Figure FDA0000377748010000066
15. the reversible terminal of four look fluorescent mark as claimed in claim 14, is characterized in that, described reversible terminal is synthesized as follows:
A, compound
Figure FDA0000377748010000067
synthetic: take methyl alcohol as solvent, under the condition existed at TEA, Mercaptamine under the ice bath agitation condition with the 2-HEDS
Figure FDA0000377748010000068
reaction, obtain compound
Figure FDA0000377748010000069
the mol ratio of described Mercaptamine, 2-HEDS and TEA is 1:(1～2): (2～3):
B, compound F 17-hydroxy-corticosterone ITC1's is synthetic: take dry DMF as solvent, and under the condition existed at TEA, compound
Figure FDA0000377748010000071
react with the FITC lucifuge, obtain compound F 17-hydroxy-corticosterone ITC1; Described FITC,
Figure FDA0000377748010000072
with the mol ratio of TEA be 1:(1～4): (10～15);
C, compound dCTP-S-S-FITC's is synthetic: with NaHCO 3/ Na 2cO 3buffered soln be solvent, compound dCTP-NH 2react with FITC1, obtain compound dGTP-S-S-FITC; Described FITC1 and dCTP-NH 2mol ratio be 1:(1～2); Described compound dCTP-S-S-FITC has the reversible terminal of structural formula shown in formula (IX).
16. the reversible terminal of four look fluorescent mark as claimed in claim 1, is characterized in that, the structural formula of described reversible terminal is suc as formula shown in (X):
Figure FDA0000377748010000073
17. the reversible terminal of four look fluorescent mark as claimed in claim 16, is characterized in that, described reversible terminal is synthesized as follows:
A, compound
Figure FDA0000377748010000074
synthetic: take methyl alcohol as solvent, under the condition existed at TEA, Mercaptamine under the ice bath agitation condition with the 2-HEDS
Figure FDA0000377748010000075
reaction, obtain compound
Figure FDA0000377748010000076
the mol ratio of described Mercaptamine, 2-HEDS and TEA is 1:(1～2): (2～3):
B, Compound C y51's is synthetic: take dry DMF as solvent, and under the condition existed at TEA, compound
Figure FDA0000377748010000077
react with the Cy5 lucifuge, obtain Compound C y51; Described Cy5, with the mol ratio of TEA be 1:(1～4): (10～15);
C, compound dATP-S-S-Cy5's is synthetic: with NaHCO 3/ Na 2cO 3buffered soln be solvent, compound dATP-NH 2react with Cy51, obtain compound dATP-S-S-Cy5; Described Cy51 and dATP-NH 2mol ratio be 1:(1～2); Described compound dATP-S-S-Cy5 has the reversible terminal of eliminant shown in formula (X).
18. the purposes of the four reversible terminals of look fluorescent mark as described as any one in claim 1～17 in DNA sequencing.
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