CN103588838A - Synthesis method of base modified nucleotide and application thereof - Google Patents
Synthesis method of base modified nucleotide and application thereof Download PDFInfo
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- CN103588838A CN103588838A CN201310533070.9A CN201310533070A CN103588838A CN 103588838 A CN103588838 A CN 103588838A CN 201310533070 A CN201310533070 A CN 201310533070A CN 103588838 A CN103588838 A CN 103588838A
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- 0 C*12C(CCCC3)=C3c3cc(C=CC#N)ccc3C1C2 Chemical compound C*12C(CCCC3)=C3c3cc(C=CC#N)ccc3C1C2 0.000 description 4
- SVEZGQGDAZLHQG-UHFFFAOYSA-N CCNC(C(F)(F)F)=O Chemical compound CCNC(C(F)(F)F)=O SVEZGQGDAZLHQG-UHFFFAOYSA-N 0.000 description 1
- BJDBBXYTOBMDIH-UHFFFAOYSA-N COC(c(cc1)ccc1OC)OCCO Chemical compound COC(c(cc1)ccc1OC)OCCO BJDBBXYTOBMDIH-UHFFFAOYSA-N 0.000 description 1
- LUHAHBYEHGLMSA-UHFFFAOYSA-N COc1ccc(C(OCCO)OCCO)cc1 Chemical compound COc1ccc(C(OCCO)OCCO)cc1 LUHAHBYEHGLMSA-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a synthesis method of base modified nucleotide and application thereof. According to the method, various modified nucleosides can selectively generate 5'-ribonucleoside triphosphote under the action of phosphorylation reagents. The phosphorylation reagents include annular phosphorylation agent generated in situ during reaction and a reaction product of phosphorus oxychloride and tributylammonium pyrophosphate under a synergistic effect. The base modified nucleotide synthesized by the method provided by the invention is widely used in DNA sequencing, labeling, extending and other biological fields. Current base modified nucleotide has the problems of high sale price, complex synthesis method, poor reaction selectivity, many side reactions, hard separation and purification. The synthesis method provided by the invention has the characteristics of good selectivity, fewer side reactions, easy separation and purification and easily available experimental conditions, and the synthesis processes are all conventional chemical reactions, so that the method can be popularized and applied on a large scale.
Description
Technical field
The present invention relates to chemosynthesis and biochemical field, be specifically related to a kind of preparation method and use of base modification Nucleotide.
Background technology
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 declines, and has 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 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 of left and right 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 single DNA molecules to be checked order, and does not do any DNA amplification reaction, thereby reduces cost, improves flux.Although single-molecule sequencing technology has commercially produced product, all also there is technical difficult point, fail large-scale application.
High-flux sequence platform 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 we can have breakthrough on order-checking hardware, on the auxiliary products such as sequencing reagent, we are also by under one's control.Therefore, independent research is applicable to the check order sequencing reagent of platform of two generations order-checkings or even three generations, by changing the current market structure, set up the autonomous order-checking platform of China and there is strategic meaning.For this reason, National 863,973 and " 12 " biotech development planning all classify the research and development of research and development new-generation sequencing technology and auxiliary products as give priority to object.
For the reversible terminal checking order, generally all choose U, C, A, the Nucleotide of tetra-different base modifications of G. and that traditional core thuja acid synthetic exists side reaction is always many, product is difficult to separation and purification, generally all need to be by FPLC and preparative HPLC purifying repeatedly, just can obtain the sufficiently high oligonucleotide product (Chem.Eur.J.2011 of purity, 17, 2903-2915). in the present invention, we have selected a kind of more weak ring-type phosphorylation agent, it can be effectively at 5 ' hydroxyl reaction of nucleosides, and maximum side reaction is that triphosphoric acid is connected on to 3 ' hydroxyl, other side reaction seldom, simultaneously in order further to reduce the phosphorylation reaction of 3 ' hydroxyl, when the transformation efficiency of raw material reaches 70%, reaction is stopped, because the cost ratio Nucleotide of nucleosides is much lower. synthetic method of the present invention is simple, reaction conditions is gentle, reaction process is controlled, be applicable to scale operation.And be successfully used to the synthetic of synthetic other derivatives such as reversible terminal that are applicable to DNA sequencing with our own synthetic Nucleotide.
Summary of the invention
The object of the present invention is to provide a kind of preparation method and use of base modification Nucleotide; This synthetic method raw material is simple, cheap, and reaction conditions is gentle, simple to operate, can be applicable to scale operation.
The object of the invention is to be achieved through the following technical solutions:
First aspect, the present invention relates to a kind of synthetic method of base modification Nucleotide, and described method comprises the steps:
The iodo-2 '-Deoxyribose cytidine of A, 5-
the iodo-2 '-deoxyuridine of 5-
or the iodo-2'-deoxyadenosine of 7-
with compound
under Pd/Cu catalyst action, there is Sonogashira linked reaction and obtain compound dC (AP
3)
dU (AP
3)
or dA (AP
3)
Or, the iodo-2 '-deoxyuridine of 5-
under Pd catalyst action, there is linked reaction with compound allyl amine and obtain allyl group uridine dU allyl group
B, compound dC (AP
3), dU (AP
3), dA (AP
3) or allyl group uridine dU allyl group and ring-type phosphorylation agent
there is phosphorylation reaction, then through oxidation, hydrolysis, obtain the Nucleotide dCTP (AP that propargylamine is modified
3)
dUTP (AP
3)
or dATP (AP
3)
or the Nucleotide dUTP-allyl group of allyl group modification
Second aspect, the present invention relates to synthetic 5-the propargylamine 2 '-deoxidation cytidylic acid(CMP) dCTP (AP3) obtaining of a kind of above-mentioned method in the purposes of synthesizing for the reversible terminal of DNA sequencing.
The third aspect, the present invention relates to synthetic 5-the propargylamine 2 '-deoxidation uridylate dUTP (AP3) obtaining of a kind of above-mentioned method in the purposes of synthesizing for the reversible terminal of DNA sequencing.
Fourth aspect, the present invention relates to synthetic 7-denitrification-7-propargylamine-the 2 '-deoxidation adenine nucleotide dATP (AP3) obtaining of a kind of above-mentioned method in the purposes of synthesizing for the reversible terminal of DNA sequencing.
The 5th aspect, the present invention relates to a kind of synthetic method of base modification Nucleotide, and described method comprises the steps:
A, Nucleotide dUTP react in the aqueous solution at sodium azide under NIS, NBS or NCS effect, in the 5-position of pyrimidine bases, meet I, Br or Cl, obtain compound dUTP-X
wherein, X is I, Br or Cl;
B, described compound dUTP-X obtain Nucleotide dUTP (AP with trifluoroethyl propargylamine or allyl amine generation linked reaction under Pd catalyst action
3)
or dUTP-allyl group
5-position at pyrimidine bases meets I, Br or Cl, and the reagent of employing is respectively NIS, NBS or NCS.
The 6th aspect, the present invention relates to the synthetic purposes of a 5-halogen-2 ' deoxidation uridylate dUTP-X in synthetic modification Nucleotide obtaining of a kind of above-mentioned method.
The 7th aspect, the present invention relates to a kind of synthetic method of base modification Nucleotide, and described method comprises the steps:
Compound dG (AP
3)
g (AP
3)
dG-X
or G-X
with tri-n-butylamine pyrophosphate salt, the chloro-4H-1 of 2-, the reaction under triethylamine and iodine existence of 3,2 one benzo dioxy phosphorus-4-ketone, reaction product is gone protection, obtains compound dgTP (AP
3)
gTP (AP
3)
dgTP-X
or GTP-X
wherein, X is I, Br or Cl.In reaction process, adopt NIS, NBS or NCS, other reaction reagent and reaction process are all identical can obtain corresponding dGTP-I, dGTP-Br or dGTP-Cl.
Preferably, described method also comprises the steps:
At CuI, Pd (PPh
3)
4under the condition that (tetrakis triphenylphosphine palladium) and TEA (triethylamine) exist, compound F 17-hydroxy-corticosterone
2 
there is linked reaction with dGTP-X or GTP-X, obtain compound dGTP (AP
3)
or GTP (AP
3)
described dGTP-X or GTP-X, F
2, CuI, Pd (PPh
3)
4with the mol ratio of TEA be 1: (2～3): 0.072: 0.025: (1.5～2).
Preferably, described compound G (AP
3) can be synthesized into as follows:
A, under ice-water bath agitation condition, mol ratio is 1.0: the propargylamine of (1.2～2) reacts with trifluoro-acetate, obtains compound F 17-hydroxy-corticosterone
2 
B, at CuI, Pd (PPh
3)
4under the condition that (tetrakis triphenylphosphine palladium) and TEA (triethylamine) exist, compound F 17-hydroxy-corticosterone
2and G-X
reaction, obtains compound G (AP
3)
described G-X, F
2, CuI, Pd (PPh
3)
4with the mol ratio of TEA be 1: (2～3): 0.072: 0.025: (1.5～2).
Eight aspect, the present invention relates to a kind of synthetic method of base modification Nucleotide, and described method comprises the steps:
Vinyl cyanide and compound 1
under Pd catalysis, there is linked reaction, obtain compound 2
then there is glycosylation with Hoffer ' s ' α-chlorosugar, obtain compound 3
or compound 3 '
then under alkaline condition, remove protecting group, obtain compound 4
or compound 4 '
compound 4 or 4 ' and ring-type phosphorylation agent
there is phosphorylation reaction, obtain the modified nucleotide (VIII) of expection
or Nucleotide (X)
The present invention has following beneficial effect: the present invention has synthesized the Nucleotide of serial base modification; This compound is widely used in field of biology such as DNA sequencing, mark, extensions, and its selling price is very high at present, and synthetic method is complicated, is difficult to control.Synthetic method desired raw material of the present invention is simple and easy to get, and building-up process is conventional chemical reaction, can be used for large-scale promotion and uses.
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.
Fig. 1 is total structural representation of modified nucleotide;
Fig. 2 is the building-up process schematic diagram of dCTP in embodiment 1 (AP3);
Fig. 3-A is the building-up process schematic diagram of dUTP in embodiment 3 (AP3)
Fig. 3-B is the building-up process schematic diagram of dUTP in embodiment 4 (AP3);
Fig. 4 is the building-up process schematic diagram of dATP in embodiment 5 (AP3);
Fig. 5 is the building-up process schematic diagram of dGTP in embodiment 6 (AP3);
Fig. 6 is the building-up process schematic diagram of dUTP-I in embodiment 2;
Fig. 7-A is the allylic building-up process schematic diagram of dUTP-in embodiment 7;
Fig. 7-B is the allylic building-up process schematic diagram of dUTP-in embodiment 8;
Fig. 8 is the building-up process schematic diagram of dNTP-CN in embodiment 9;
Fig. 9 is GTP (AP in embodiment 10
3) building-up process schematic diagram;
Figure 10 is the building-up process schematic diagram of NTP-CN in embodiment 11;
Figure 11-A is that dUTP in embodiment 12 (AP3) applies in synthetic reversible terminal;
Figure 11-B is the building-up process schematic diagram of connector element in reversible terminal in embodiment 12;
Figure 12-A is that dCTP in embodiment 13 (AP3) applies schematic diagram in synthetic reversible terminal;
Figure 12-B is the building-up process schematic diagram of connector element in reversible terminal in embodiment 13;
Figure 13 is that dATP in embodiment 14 (AP3) applies schematic diagram in synthetic reversible terminal;
Figure 14 is the building-up process schematic diagram of dGTP-I in embodiment 15;
Figure 15 is the building-up process schematic diagram of UTP-allyl amine in embodiment 16.
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. characterize;
The structural formula of the present embodiment Nucleotide is suc as formula shown in (II):
Its corresponding synthetic route as shown in Figure 2; Specifically comprise the steps:
The first step, compound F 17-hydroxy-corticosterone
2synthetic
Trifluoro-acetate reacts and obtains compound F 17-hydroxy-corticosterone with propargylamine in organic solvent
2, be specially: in a single port bottle, add 60ml methyl alcohol, under ice-water bath, stir, add propargylamine (60mmol, 3.3042g), stir after 15 minutes and slowly add trifluoro-acetate (86.7mmol, 11.0957g), the water-bath of 10 minutes recession deicings, reacts under room temperature 24 hours.Reaction is monitored with TLC plate, PE:EA=8:1, and baking sheet, it is product F that Rf=0.5 produces new point
2.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 adding can be the arbitrary value in 72～120mmol.
Second step, compound dC (AP
3) synthetic
In a single port bottle, add the iodo-2 '-Deoxyribose cytidine of 5-(0.70mmol, 248mg), then take 10mg CuI (25.2 μ mol) and 20mg Pd (PPh
3)
4(17.6 μ mol) adds in reaction flask, vacuumize, and nitrogen protection, aluminium foil parcel, adds 1.5ml DMF, and stirring and dissolving, adds 0.2ml TEA, takes compound F 17-hydroxy-corticosterone
2(254mg, 1.68mmol) adds in above-mentioned reaction flask after dissolving with 1ml DMF, stirring at room, and 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)：δ1.94-2.01(m，1H)，2.12-2.18(m，1H)，3.51(s，1H)，3.55-3.62(m，2H)，3.79(q，J=3.2Hz，J=6.8Hz，1H)，4.20(d，J=3.2Hz，1H)，4.28(s，1H)，5.05(t，J=4.8Hz，1H)，5.20(d，J=3.6Hz，4H)，6.10(t，J=6.4Hz，1H)，6.84(s，1H)，7.81(s，1H)，8.15(s，1H)，9.96(s，1H).
The 3rd step, compound dCTP (AP
3) synthetic
In glove box, take respectively compound dC (AP
3) 90mg (0.24mmol), tri-n-butylamine pyrophosphate salt 264mg (0.48mmol), the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone 90mg (0.48mmol) is placed in three reaction tubess.Tri-n-butylamine pyrophosphate salt is dissolved in 0.75mL dry DMF, then adds the anhydrous tri-n-butylamine of 0.9mL, stir half an hour.The chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone is dissolved in 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 to dC (AP
3) in, stir 1.5h.Add 4mL 3% iodine (9: 1Py/H2O) solution.After 15min, add 4mL water, stir 2h.Add 1mL 3M NaCl solution, then add 35mL dehydrated alcohol ,-20 ℃ of freeze overnight, centrifugal (3200r/min, 25 ℃) 20min.The supernatant liquor that inclines, obtains precipitation, drains solvent.Add 2ml strong aqua stirring at room 6h.Decompression screws out solvent, occurs brown solid, 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 separated [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 acetic acid triethylamine salt, obtains 42mg white solid, productive rate 24.5%.
1H?NMR(D
2O，400MHz)：δ2.27-2.33(m，1H)，2.44-2.50(m，1H)，4.05(s，2H)，4.19-4.31(m，3H)，4.56-4.59(m，1H)，6.21(t，J=6.0Hz，1H)，8.37(s，1H).
31P?NMR(D
2O，162MHz)：δ-22.55，-11.51，-10.31.ESI-HRMS：calc?for?C
12H
18N
4O
13P
3[M-H]
-519.0083，found519.0059.
The structural formula of the present embodiment Nucleotide dUTP-I is as shown in the formula shown in (VI):
As shown in Figure 6, concrete steps are as follows for its synthetic route: dUTP (1.0g, 2.14mmol) is joined in 20mL water and stirred under room temperature, NIS (1.44g, 6.42mmol) and NaN
3(0.83g, 12.84mmol) join in reaction and stir 15h, filter, filtrate is adjusted to pH=6.5, is loaded on diethylaminoethyl-sepharose post, and target product is used the TEAB of gradient 0-1M to do moving phase, it is concentrated after product is merged, add 3*100mL water concentration and evaporation, vacuum-drying obtains dUTP-I (1.64g, 86%)
1h NMR (D
2o, 400MHz) δ 8.26 (s, 1H), 6.27 (t, J=7.2Hz, 1H), 4.63 (m, 1H), 4.21 (m, 3H), 3.19 (q, J=7.6Hz, 18H), 2.38 (m, 2H), 1.27 (t, J=7.6Hz, 27H);
31p NMR (D
2o, 162MHz) δ-7.04 ,-10.45, if-changing NIS into NBS or NCS in 21.29. the present embodiment reaction process, other reaction reagent and reaction process are all identical, can obtain dUTP-Br and dUTP-Cl.
The structural formula of the present embodiment Nucleotide is suc as formula shown in (III):
Its corresponding synthetic route is as shown in Fig. 3-A; Specifically comprise the steps:
2.1 compound F 17-hydroxy-corticosterone
2synthetic with embodiment 1.
2.2 compound dU's (AP3) is synthetic
In a single port bottle, add the iodo-2 '-deoxyuridine of 5-
(0.7mmol, 247mg), then take 9.7mgCuI and 20.3mg Pd (PPh
3)
4add in reaction flask, vacuumize, nitrogen protection, aluminium foil parcel, adds 2.3mlDMF, and stirring and dissolving, adds 0.2ml TEA, takes compound trifluoroethyl propargylamine, i.e. F
2(254mg, 1.7mmol), adds in above-mentioned reaction flask after dissolving with DMF, stirring at room, and 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 F
3, 2 positions are very approaching.After question response finishes, evaporated under reduced pressure solvent, direct column chromatography for separation, 20: 1 DCM: MeOH is eluent, obtains 214mg, productive rate 61%.
1H?NMR(DMSO-D
6，300MHz)：δ2.11(t，J=5.1Hz，2H)，3.56-3.58(m，2H)，3.78(m，1H)，4.21(d，J=5.1Hz，3H)，5.08(t，J=5.1Hz，1H)，5.23(d，J=4.2Hz，1H)，6.09(t，J=6.6Hz，1H)，8.18(s，1H)，10.05(t，J=4.8Hz，1H)，11.63(s，1H).
In above-mentioned synthesizing, the F adding
2can be the arbitrary value in 1.4～2.1mmol, TEA can be the arbitrary value in 1.05～1.4mmol.
2.3 compound dUTP's (AP3) is synthetic
In glove box, take respectively compound dU (AP3) 60mg (0.16mmol), tri-n-butylamine pyrophosphate salt 150mg (0.32mmol), the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone 66mg (0.32mmol) is placed in three reaction tubess.Tri-n-butylamine pyrophosphate salt is dissolved in 0.5mL dry DMF, then adds the new tri-n-butylamine steaming of 0.6mL, stir half an hour.The chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone is dissolved in 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 to dU (AP3), stir 1.5h.Add 5mL 3% iodine (9: 1Py/H2O) solution.After 15min, add 4mL water, stir 2h.Add 0.5mL 3M NaCl solution, then add 30mL dehydrated alcohol ,-20 ℃ of freeze overnight, centrifugal (3200r/min, 25 ℃) 20min.The supernatant liquor that inclines, obtains precipitation, drains solvent.Add successively again TEAB solution and strong aqua, stirred overnight at room temperature., there is white solid in evaporated under reduced pressure solvent, obtains dUTP (AP3).With analysis mode, HPLC analyzes, condition: pillar: C18,10 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 20mMTEAAc and CH
3cH
2oH, gradient washing, 0%-20%CH
3cH
2oH (35min); UV-detector: 254nm.When t=13.5min, there is 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?C
12H
19N
3O
14P
3[M+H]+522.0080，found522.0070.
The structural formula of the present embodiment Nucleotide is suc as formula shown in (III):
Its corresponding synthetic route is as shown in Fig. 3-B; Specifically comprise the steps:
In a single port bottle, add dUTP-I25mg, then take 1.2mg CuI and 2.4mg Pd (PPh
3)
4add in reaction flask, vacuumize, nitrogen protection, aluminium foil parcel, adds 0.3ml DMF, and stirring and dissolving, adds 0.03ml TEA, takes compound trifluoroethyl propargylamine, i.e. F
2(25mg) with adding in above-mentioned reaction flask after DMF dissolving, stirring at room.After question response finishes, preparative HPLC separation and purification, obtains final product dUTP (AP
3) 12mg.
1HNMR(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(D20，161MHz)：δ-22.22，-11.45，-9.90。HRMS：calc?for?C
12H
19N
3O
14P
3[M+H]+522.0080，found522.0070。
The structural formula of the present embodiment Nucleotide is suc as formula shown in (IV):
As shown in Figure 4, concrete steps are as follows for its synthetic route:
The first step, compound F 17-hydroxy-corticosterone
2synthetic with embodiment 1.
Second step, compound dA (AP
3) synthetic
To adding dA-I in a single port bottle, be the iodo-2'-deoxyadenosine of 7-denitrification-7-(0.35mmol, 132mg), then take 5mg CuI (25.2 μ mol) and 10mg Pd (PPh
3)
4(8.8 μ mol) adds in reaction flask, vacuumize, and nitrogen protection, aluminium foil parcel, adds 1.5ml DMF, and stirring and dissolving, adds 0.1ml TEA, takes compound F 17-hydroxy-corticosterone
2(127mg, 0.84mmol) adds in above-mentioned reaction flask after dissolving with 1ml DMF, stirring at room, and TLC follows the tracks of reaction process.After question response is complete, screw out solvent, it is dA (AP that column chromatography for separation [V (DCM): V (MeOH)=10:1] obtains 129mg
3), productive rate 92%.
1H?NMR(DMSO-d
6，400MHz)：δ2.12-2.18(m，1H)，2.41-2.46(m，1H)，3.45-3.57(m，2H)，3.78-3.81(m，1H)，4.28-4.31(m，3H)，5.06(t，J=5.6Hz，1H)，5.25(d，J=4.0Hz，1H)，6.43-6.47(m，1H)，7.74(s，1H)，8.09(s，1H)，10.08(t，J=5.2Hz，1H).
The 3rd step, compound dATP (AP
3) synthetic
In glove box, take respectively compound dA (AP
3) 32mg (0.08mmol), tri-n-butylamine pyrophosphate salt 88mg (0.16mmol), the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone 30mg (0.16mmol) is placed in three reaction tubess.Tri-n-butylamine pyrophosphate salt is dissolved in 0.25mL dry DMF, then adds the anhydrous tri-n-butylamine of 0.3mL, stir half an hour.The chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone is dissolved in 0.25mL 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 dA (AP
3) in, stir 1.5h.Add 2mL 3% iodine (9: 1 Py/H2O) solution.After 15min, add 3mL water, stir 2h.Add 0.7mL3M NaCl solution, then add 20mL dehydrated alcohol ,-20 ℃ of freeze overnight, centrifugal (20min, 3200rpm).The supernatant liquor that inclines, obtains precipitation, drains solvent.Add 1ml 0.1M TEAB solution to dissolve, 2ml strong aqua stirred overnight at room temperature.Screw out solvent, occur brown solid, RP-HPLC analyzes [condition: pillar: C18,5 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAA and EtOH, 0-20%EtOH (40min); UV-detector wavelength: 254nm], retention time t=18.6min, RP-HPLC separated [condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: 20mM TEAA and MeOH, 0-15%MeOH (30min); UV-detector wavelength: 254nm], retention time t=25min.NaCl/EtOH removes acetic acid triethylamine salt, obtains 12.9mg white solid.Productive rate 30%.
1H?NMR(D
2O，400MHz)：δ2.46-2.59(m，2H)，4.10-4.24(m，5H)，4.70(s，1H)，6.49(t，J=6.8Hz，1H)，7.78(s，1H)，8.02(s，1H).
31P?NMR(D
2O，162MHz)：δ-22.07，-11.11，-9.29.ESI-HRMS：calc?for?C
14H
19N
5O
12P
3[M-H]
-542.0243，found542.0222.
The structural formula of the reversible terminal of the present embodiment is suc as formula shown in (XI):
As shown in Figure 5, concrete steps are as follows for its synthetic route:
In a single port bottle, add compound dGTP-I (12mg), then take CuI (2mg) and Pd (PPh
3)
4(4mg) add in reaction flask, vacuumize, nitrogen protection; aluminium foil parcel, adds 1ml DMF, stirring and dissolving; inject TEA (8mg) and trifluoroacetyl propargylamine (20mg), 50 ℃ were stirred after 13 hours, and reaction finishes; screw out solvent; resistates is dissolved in to 10ml ethyl acetate, successively with saturated sodium bicarbonate solution and saturated nacl aqueous solution washing, anhydrous sodium sulfate drying; concentrated, it is dGTP (AP that preparative HPLC separation and purification obtains 0.9mg white solid
3), productive rate 23%.
1H?NMR(400MHz，D
2O)δ7.45(s，1H)，6.34(t，J=6.8Hz，1H)，4.73(s，1H)，4.11-4.20(m，3H)，4.06(s，2H)，2.53-2.58(m，1H)，2.41-2.46(m，1H)；
31P?NMR(D
2O，162MHz)：-10.59(t，J=9.9Hz，1P)，-11.24(d，J=17.3Hz，1P)，-22.98(d，J=20.7Hz，1P).ESI-HRMS：calc?for?C
14H
19N
5O
13P
3[M-H]
-558.0192，found558.0179.
embodiment 7
The structural formula of the present embodiment Nucleotide dUTP-allyl amine is as shown in the formula shown in (VII):
Its synthetic route is as shown in Fig. 7-A, and concrete steps are as follows:
DUTP-I (1.52g, 1.67mmol) is dissolved in 50mL0.1M sodium acetate solution, and adds K
2pdCl
4(0.35g, 1.07mmol) stir 5min, the mixture of precooling (allylamine (1.8mL, 41.8mL) be dissolved in 10mL 0.1M sodium acetate with 4M acetic acid) join in reaction and stir 15h, reactant is by 0.2 μ M, the filtration unit of 1000mL filters, filtrate is adjusted to PH=6.5, be loaded on diethylaminoethyl-sepharose post, target product is used the TEAB of gradient 0-1M to do moving phase, it is concentrated after product is merged, add 3*100mL water concentration and evaporation, last triethylamine salt is dissolved in 20mL water, pour in the acetonitrile solution that 200mL contains 5g sodium perchlorate, by solid drying, obtain dUTP allyl amine (0.73g, 74%)
1h NMR (D2O, 400MHz) δ 8.18 (s, 1H), 6.58 (d, J=16.0Hz, 1H), 6.46 (dt, J=16.0,6.8Hz, 1H), 6.34 (t, J=6.8Hz, 1H), 4.70 (m, 1H), 4.29 (m, 2H), 4.23 (m, 1H), 3.74 (d, J=6.8Hz, 2H), 2.42 (m, 2H), 31P NMR (D2O, 162MHz) δ-6.91 ,-10.04.-20.98.
embodiment 8
The structural formula of the present embodiment Nucleotide dUTP-allyl amine is as shown in the formula shown in (VII):
Its synthetic route is as shown in Fig. 7-B, and concrete steps are as follows:
In a single port bottle, add compound dU-I (12mg), then take CuI (2mg) and Pd (PPh
3)
4(4mg) add in reaction flask, vacuumize, nitrogen protection; aluminium foil parcel, adds 1ml DMF, stirring and dissolving; inject TEA (8mg) and trifluoroacetyl allylamine (20mg), 50 ℃ were stirred after 13 hours, and reaction finishes; screw out solvent; resistates is dissolved in to 10ml ethyl acetate, successively with saturated sodium bicarbonate solution and saturated nacl aqueous solution washing, anhydrous sodium sulfate drying; concentrated, it is dU allyl amine that preparative HPLC separation and purification obtains 0.8mg white solid.
1H?NMR(D2O，400MHz)δ8.18(s，C6H，1H)，6.58(d，J=16.0Hz，1H)，6.46(dt，J=16.0，6.8Hz，1H)，6.34(t，J=6.8Hz，10H，1H)，4.70(m，1H)，4.29(m，2H)，4.23(m，1H)，3.74(d，J=6.8Hz，CH2，2H)，2.42(m，2H).
In glove box, take respectively above-mentioned dU allyl amine 32mg, tri-n-butylamine pyrophosphate salt 88mg (0.16mmol), the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone 30mg (0.16mmol) is placed in three reaction tubess.Tri-n-butylamine pyrophosphate salt is dissolved in 0.25mL dry DMF, then adds the anhydrous tri-n-butylamine of 0.3mL, stir half an hour.The chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone is dissolved in 0.25mL 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 dU allyl amine, stirs 1.5h.Add 2mL3% iodine (9:1Py/H2O) solution.After 15min, add 3mL water, stir 2h.Add 0.7mL 3M NaCl solution, then add 20mL dehydrated alcohol ,-20 ℃ of freeze overnight, centrifugal (20min, 3200rpm).The supernatant liquor that inclines, obtains precipitation, drains solvent.Add 1ml0.1M TEAB solution to dissolve, 2ml strong aqua stirred overnight at room temperature.Screw out solvent, occur brown solid, RP-HPLC analyzes [condition: pillar: C18,5 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAA and EtOH, 0-20% EtOH (40min); UV-detector wavelength: 254nm], retention time t=18.6min, RP-HPLC separated [condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: 20mM TEAA and MeOH, 0-15%MeOH (30min); UV-detector wavelength: 254nm], retention time t=25min.NaCl/EtOH removes acetic acid triethylamine salt, obtains white solid, dUTP allyl amine, productive rate 20%.
31P?NMR(D
2O，162MHz)δ-6.91，-10.04，-20.98；MS(m/z)：522[M-H]
+.
embodiment 9
The structural formula of the present embodiment Nucleotide is suc as formula shown in (VIII):
As shown in Figure 8, concrete steps are as follows for its synthetic route:
By compound 1 (1.0g, 3.4mmol), vinyl cyanide (0.56mL, 8.5mmol), triphenylphosphine (894mg, 3.4mmol) and tri-n-butylamine (4mL, 17mmol) are dissolved in 20mL DMF, add Pd (OAc)
2after (80mg, 0.34mmol), at 90 ℃, stir 12h, and detect and know that raw material point disappears with TLC.In reaction solution, add 250mL EA, with anhydrous magnesium sulfate drying after saturated common salt water washing and revolve and desolventize.The separated compound 2 (601mg, 81%) that obtains of column chromatography PE:EA4:1.
Three [2-(2-methoxy ethoxy) ethyl] amine (30mg, 93 μ mol) and KOH (480mg, 8.56mmol) are dissolved in 75mL acetonitrile, under room temperature, stir 20min after adding 2 (600mg, 2.75mmol).The α of Huo Fuer-chlorine sugar (1.176g, 3.02mmol) is joined in reaction and stir 2h after solvent is spin-dried for after, column chromatography DCM separated compound 3 (1.55g, 99%).
By compound 3 (1.0g, 1.75mmol) be dissolved in the anhydrous DCM of 75mL anhydrous methanol and 25mL, sodium methoxide solution (the 1.75mL that adds 0.5M methyl alcohol, 8.75mmol), and stir after 2h under room temperature, revolve and desolventize, column chromatography DCM:MeOH9: 1 obtains compound 4 (445mg, 76%).
By compound 4 (200mg, 0.6mmol), tri-n-butylamine (430 μ L, 1.8mmol) in-5 ℃, be dissolved in 10mL trimethyl phosphite 99, and add phosphorus oxychloride (55 μ L, 0.6mmol) after stirring 5min, add again phosphorus oxychloride (55 μ L, 0.6mmol) stir 25min, now compound 5 forms, in reaction, add cold tributyl ammonium pyrophosphate (262mg, 0.48mmol), tri-n-butylamine (1.14mL, 4.8mmol) and the mixture of 3mL acetonitrile, and stir 30min, in reaction, add 75mL shrend to go out and use DCM (3*20mL) extraction, water is adjusted to PH=6.5 with ammoniacal liquor, be loaded on diethylaminoethyl-sepharose post, target product is used the TEAB of gradient 0-1M to do moving phase, after being merged, product is concentrated into after the triethylamine salt of 3mL, compound 6 use 20mL acetonitrile (the containing 1g sodium perchlorate) solution that triethylamine salt form exists carry out ion-exchange, become the compound 6 (260mg of sodium-salt form, 68%)
1h NMR (D2O, 400MHz) δ 8.05 (s, 2H), 7.75 (d, J=8.4Hz, 1H), 7.66 (d, J=8.4Hz, 1H), 7.52 (m, 3H), 7.35 (t, J=7.6Hz, 1H), 6.75 (t, J=6.8Hz, 1H), 5.96 (t, J=16.4Hz, 1H), 4.82 (m, 1H), 4.37 (m, 2H), 4.26 (m, 1H), 2.98 (m, 1H), 2.31 (m, 1H),
31p NMR (D2O, 162MHz) δ 4.25 (t, J=19.8Hz, 1P), 10.02 (d, J=20.3Hz, 1P), 20.88 (t, J=20.1Hz, 1P), MS (m/z): 573[M+H]
+
embodiment 10, GTP (AP
3
) synthetic
The present embodiment Nucleotide GTP (AP
3) structural formula suc as formula shown in (IX):
As shown in Figure 9, concrete synthetic method comprises the steps: its synthetic schematic diagram
The first step:
In a single port bottle, add compound G-I (0.25g, 0.4mmol), then take CuI (22mg; 1mmol) and Pd (PPh
3)
4(tetrakis triphenylphosphine palladium) (48mg; 0.04mmol) add in reaction flask, vacuumize, nitrogen protection, aluminium foil parcel, adds 10ml DMF, and stirring and dissolving is injected TEA (triethylamine) (0.088g; 0.8mmol) with trifluoroacetyl propargylamine (0.2g; 1.2mmol), 50 ℃ were stirred after 13 hours, reaction finishes, screw out solvent, resistates is dissolved in to 100ml ethyl acetate, successively with saturated sodium bicarbonate solution and saturated nacl aqueous solution washing, anhydrous sodium sulfate drying, concentrated, column chromatography [V (ethyl acetate): V (normal hexane)=1: 3], obtaining 0.1g white solid is G (AP
3), productive rate 39%.
1H?NMR(400MHz，CDCl
3)δ7.24(s，1H)，6.38(t，J=0.8Hz，1H)，4.49-4.46(m，1H)，4.31(s，2H)，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).
Second step:
By compound G (AP
3) vacuum-drying 12h, in glove box, take respectively compound G (AP
3) (30mg, 0.072mmol), tri-n-butylamine pyrophosphate salt (E-4) (80mg, 0.145mmol), the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone (E-3) (30mg, 0.15mmol) is placed in three reaction tubess.Tri-n-butylamine pyrophosphate salt is dissolved in 0.25mL dry DMF, then adds the new tri-n-butylamine steaming of 0.3mL, stirring at normal temperature is after half an hour, reaction solution is injected to the chloro-4H-1 of 2-, in dry DMF (0.25mL) solution of 3,2-benzo dioxy phosphorus-4-ketone, stirring at normal temperature half an hour.Then this mixed solution is injected into G (AP
3) in, stir 1.5h.Add 1mL3% iodine (9: 1 Py/H2O) solution, keep iodine liquid color 15min not fade.After 15min, add 2mL water, after 2h, add 0.75mL 3M NaCl solution, 20mL dehydrated alcohol ,-20 ℃ of freezing 12h, centrifugal (20min, 3200rpm).The supernatant liquor that inclines, precipitation is drained after solvent, adds strong aqua, stirring at room 5 hours.Decompression screws out solvent, occurs brown solid, and RP-HPLC analyzes [condition: pillar: C18,5 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAA and EtOH, 0-20% EtOH (35min), visible detection device wavelength: 650nm], retention time t=18min.RP-HPLC separated [condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: 20mM TEAA and MeOH, 0-15%MeOH (25min), UV-detector wavelength: 254nm], retention time t=15min.NaCl/EtOH removes acetic acid triethylamine salt, and obtaining 12mg white solid is GTP (AP
3).Productive rate 26%.
1H?NMR(400MHz，D
2O)δ7.35(s，1H)，6.22(t，J=0.8Hz，1H)，4.59(s，2H)，4.06-3.92(m，5H)，2.48-2.41(m，1H)，2.32-2.28(m，1H)；
31P?NMR(D
2O，162MHz)：-10.65，-11.19，-22.91.ESI-HRMS：calc?for?C
22H
26N
6O
14P
3S
2[M-H]
-558.0192，found558.0179.
embodiment 11
The structural formula of the present embodiment Nucleotide is suc as formula shown in (X):
As shown in figure 10, concrete steps are as follows for its synthetic route:
By compound 1 (1.0g, 3.4mmol), vinyl cyanide (0.56mL, 8.5mmol), triphenylphosphine (894mg, 3.4mmol) and tri-n-butylamine (4mL, 17mmol) are dissolved in 20mL DMF, add Pd (OAc)
2after (80mg, 0.34mmol), at 90 ℃, stir 12h, and detect and know that raw material point disappears with TLC.In reaction solution, add 250mL EA, with anhydrous magnesium sulfate drying after saturated common salt water washing and revolve and desolventize.4: 1 separated compounds 2 (601mg, 81%) that obtain of column chromatography PE:EA.
Three [2-(2-methoxy ethoxy) ethyl] amine (30mg, 93 μ mol) and KOH (480mg, 8.56mmol) are dissolved in 75mL acetonitrile, under room temperature, stir 20min after adding compound 2 (600mg, 2.75mmol).The α of Huo Fuer-chlorine sugar (1.176g, 3.02mmol) is joined in reaction and stir 2h after solvent is spin-dried for after, column chromatography DCM separated compound 3 (1.55g, 99%).
By compound 3 (1.0g, 1.75mmol) be dissolved in the anhydrous DCM of 75mL anhydrous methanol and 25mL, sodium methoxide solution (the 1.75mL that adds 0.5M methyl alcohol, 8.75mmol), and stir after 2h under room temperature, revolve and desolventize, column chromatography DCM:MeOH9:1 obtains compound 4 (445mg, 76%).
By compound 4 (200mg, 0.6mmol), tri-n-butylamine (430 μ L, 1.8mmol) in-5 ℃, be dissolved in 10mL trimethyl phosphite 99, and add phosphorus oxychloride (55 μ L, 0.6mmol) after stirring 5min, add again phosphorus oxychloride (55 μ L, 0.6mmol) stir 25min, now compound 5 forms, in reaction, add cold tributyl ammonium pyrophosphate (262mg, 0.48mmol), tri-n-butylamine (1.14mL, 4.8mmol) and the mixture of 3mL acetonitrile, and stir 30min, in reaction, add 75mL shrend to go out and use DCM (3*20mL) extraction, water is adjusted to PH=6.5 with ammoniacal liquor, be loaded on diethylaminoethyl-sepharose post, target product is used the TEAB of gradient 0-1M to do moving phase, after being merged, product is concentrated into after the triethylamine salt of 3mL, compound 6 use 20mL acetonitrile (the containing 1g sodium perchlorate) solution that triethylamine salt form exists carry out ion-exchange, become the compound 6 of sodium-salt form.
1H-NMR(400MHz，D
2O)δ8.05(s，2H)，7.75(d，J=8.4Hz，1H)，7.66(d，J=8.4Hz，1H)，7.52(m，3H)，7.35(t，J=7.6Hz，1H)，6.75(t，J=6.8Hz，1H)，5.96(t，J=16.4Hz，1H)，4.82(m，1H)，4.37(m，2H)，4.26(m，1H)，2.98(m，1H)，2.31(m，1H)；
31P?NMR(D2O，162MHz)-4.25(t，J=19.8Hz，1P)，-10.02(d，J=20.3Hz，1P)，-20.88(t，J=20.1Hz，1P)；MS(m/z)：573[M-H]
+.
the application of embodiment 12dUTP in the synthetic reversible terminal of acid-sensitive sense
Its synthetic route is as shown in Figure 11-A, and concrete steps are as follows:
Take Y038 (6.9mg, 0.0284mmol) in single port bottle, add TAMRA (15mg, 0.0284mmol), be dissolved in 1.5mL dry DMF and dissolve, add TEA (40uL, 0.284mmol) stirring at room 4h, after revolving and desolventizing, HPLC separated 13mg.
1H?NMR(400MHz，MeOD)δ8.52(d，1H，J=1.6Hz)，8.03(dd，1H，J=1.6，7.6Hz)，7.44(d，2H，J=8.4Hz)，7.35(d，1H，J=8.0Hz)，7.26(d，2H，J=9.2Hz)，7.01(dd，2H，J=2.4，9.6Hz)，6.92～6.88(m，4H)，5.61(s，1H)，3.83～3.56(m，11H)，3.27(s，12H).
Take reactant (0.0156mmol) in single port bottle, add 1.5ml MeCN (acetonitrile), and triethylamine 22uL, stir, add to vacuumize nitrogen protection after DSC (26mg, 0.102mmol) and stir after 4h, obtain intermediate
dUTP (16mg, 0.031mmol) is dissolved in to 1.5mLNa2CO3/NaHCO3 damping fluid and joins reaction stirring 2h in intermediate, separated compound Y043, the productive rate 19% of obtaining of HPLC.HRMS：calc?for?C
50H
51N
6O
23P
3?M
+1196.2240，found1196.2249；
The synthetic schematic diagram of such connector element is as shown in Figure 11-B, and concrete steps are as follows:
The first step:
By MAG (2.72g, 20mmol) be placed in single port bottle, under ice bath, add pTSA (0.656g, 3.45mmol) and 4A (10.4g) molecular sieve stir after ten minutes, after adding p-MBA to stir 4d, add 3mL TEA, filter, and with EA, wash PE:EA:TEA8: column chromatography obtained 1.6g in 1: 1.
1H?NMR(400MHz，CDCl3)：δppm7.37(d，2H，J=8.8Hz)，6.87(d，2H，J=8.8Hz)，5.61(s，1H)，4.25～4.21(m，4H)，3.79(s，3H)，3.73～3.63(m，4H)，2.07～2.05(m，6H)；
13C?NMR(100MHz，CDCl
3)：δppm170.3，159.3，130.2，127.7，113.5，100.7，63.1，63.0，55.1，20.6
Second step, compound Y035's is synthetic
Take Y034 (600mg, 1.84mmol) in single port bottle, be dissolved in 5.26mL methyl alcohol, and add K
2cO
3(762mg, 5.52mmol) and 0.263mL water stir and spend the night under room temperature, filter after being spin-dried for solvent and are again dissolved in DCM and filter, and revolve after desolventizing to obtain 484mg.
1h NMR (400MHz, CDCl3): δ ppm7.38 (d, 2H, J=8.8Hz), 6.90 (d, 2H, J=8.8Hz), 5.55 (s, 1H), 3.81 (s, 3H), 3.78～3.74 (m, 4H), 3.69～3.66 (m, 4H);
13c NMR (100MHz, CDCl
3): δ ppm160.0,130.3,128.0,113.8,102.6,67.8,61.8.55.4.
The 3rd step, compound Y036's is synthetic
Take Y035 (420mg, 1.74mmol) in single port bottle, add 6mL DCM to dissolve and be placed in ice bath, add TEA (293mg, 2.9mmol) stir, TsCl (111mg, 0.58mmol) is dissolved in 2mL DCM and joins in reaction and stir and spend the night, after revolving and desolventizing, PE:EA:TEA2: column chromatography obtained 122mg in 1: 0.1.
1H?NMR(400MHz，MeOD)：δppm7.77(d，2H，J=8.0Hz)，7.40(d，2H，J=8.0Hz)，7.30(d，2H，J=8.4Hz)，6.88(d，2H，J=8.4Hz)，5.49(s，1H)，4.18～4.09(m，2H)，3.80(s，3H)，3.66～3.46(m，6H)，2.44(s，3H).
13C?NMR(100MHz，CDCl
3)：δppm161.3，146.4，134.4，131.4，131.0，129.1，129.0，114.4，102.8，71.0，67.9，63.7，62.2，55.7，21.6.
The 4th step, compound Y037's is synthetic
Take Y036 (100mg, 0.253mmol) in single port bottle, and be dissolved in 3mLDMF, add NaN
3(36.1mg, 0.556mmol) stirs and spends the night at 80 ℃, adds 15mL water, with merging organic phase after EA 15*3 extraction, with revolving after saturated common salt water washing, desolventizes to obtain 56mg.
1H?NMR(400MHz，MeOD)：δppm7.42(d，2H，J=8.4Hz)，6.91(d，2H，J=8.8Hz)，5.60(s，1H)，3.79(s，3H)，3.77～3.54(m，6H)，3.42(t，2H，J=4.8Hz).
13C?NMR(100MHz，CDCl
3)：δppm161.4，131.7，129.1，114.6，103.0，67.9，65.4，62.3，55.7，52.1.
The 5th step, compound Y038's is synthetic
Take Y037 (50mg, 0.187mmol) in single port bottle, add 5mL methyl alcohol and 5mg Pd/C, vacuumize rear injection hydrogen, under room temperature, stir and spend the night, filter, revolve after desolventizing to obtain 40mg.
1H?NMR(400MHz，MeOD)：δppm7.41(d，2H，J=8.4Hz)，6.91(d，2H，J=8.8Hz)，5.55(s，1H)，3.79(s，3H)，3.71～3.50(m，6H)，2.82(t，2H，J=5.6Hz).
13C?NMR(100MHz，CDCl
3)：δppm161.3，132.1，129.1，114.5，103.3，68.1，67.9，62.3，55.7，42.3.
embodiment 13, dCTP (AP
3
) application in synthetic reversible terminal dCTP-acetonylidene-FITC
Its synthetic route is as shown in Figure 12-A, and concrete steps are as follows:
The first step, compound F 17-hydroxy-corticosterone ITC-OH's is synthetic:
By Y012 (10mg, 0.061mmol) as in single port bottle, add the FITC (20mg) that is dissolved in 1.5ml dry DMF, add again TEA (anhydrous triethylamine) 80uL to stir 3.5h under room temperature, revolve to desolventize and with analysis mode HPLC, analyze afterwards: pillar: C18,5 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: A, the 0.1% TEA aqueous solution and B, CH
3oH, gradient washing, 30%～60%CH
3oH (20min), 60%～80%CH
3oH (20min), visible light detector: 546nm, preparation HPCL separation obtains 18mg.HRMS：calc?for?C
28H
28N
2O
8S
+[M]
+552.1566，found552.1572；
Second step, compound dCTP-acid labile linker-FITC's is synthetic:
Take FITC-OH (9mg) in single port bottle, add 1.5ml MeCN (acetonitrile), and triethylamine 22uL, stir, add to vacuumize nitrogen protection after DSC (26mg, 0.102mmol) and stir after 4h, obtain intermediate
by dCTP (AP
3) (16mg) be dissolved in 1.5mLNa
2cO
3/ NaHCO
3damping fluid joins reaction in intermediate and stirs 2h, and with analysis mode, HPLC analyzes: pillar: C18,5 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: A, the 0.1%TEA aqueous solution and B, CH
3oH, gradient washing, 0%～20%CH
3oH (35min), visible light detector: 546nm.When t=27.9min, there is product peak to generate, the separated compound dCTP-acid labile linker-FITC 2.8mg.HRMS:calc for C that obtains of preparative HPLC
41h
45n
6o
22p
3s[M]
+1098.1520, found1098.1531;
The synthetic schematic diagram of this connector element is as shown in Figure 12-B, and concrete steps are as follows:
The first step, compound MAG's is synthetic:
Take ethylene glycol (18.61g, 300mmol) and acetic acid (6g, 99.9mmol) in 100ml single port bottle, stir, drip the 0.112ml vitriol oil in reaction, under 25 degree, stir 24h, add 17ml saturated sodium bicarbonate solution to stir and spend the night, in reaction, add 12ml water and extract with methylene dichloride 50*8, organic layer is with after anhydrous sodium sulfate drying, revolves to desolventize with 30: 1 column chromatographies of DCM:MeOH to obtain sterling 6.3g.Productive rate 60.6%.
1H?NMR(400MHz，CDCl
3)：δppm4.20(t，2H，J=4.8Hz)，3.82(t，2H，J=4.8Hz)，2.09(s，3H)，1.93(s，1H).
Second step, compound Y008's is synthetic:
By MAG (6.3g, 60.6mmol) in 150ml single port bottle, add the anhydrous THF of 87ml, add PPTS (0.725g, 2.89mmol) to stir 15min, add 28.8g 5A molecular sieve to stir 15min, add 2-methoxyl group propylene (2.4ml, 25.9mmol) at room temperature to stir 48h, add potassium carbonate powder to make to be neutral, filtration is revolved filtrate and is obtained crude product 7.3g, PE: 3: 1 column chromatography for separation of EA obtain sterling 3.8g.Productive rate 59.4%
1h NMR (400MHz, CDCl
3): δ ppm4.20 (t, 4H, J=4.8Hz), 3.66 (t, 4H, J=4.8Hz), 2.08 (s, 6H), 1.38 (s, 6H).
The 3rd step, compound Y009's is synthetic:
Get Y008 (2g, 8.06mmol) in 100ml single port bottle, add 20ml methyl alcohol to stir, add salt of wormwood (3.339g, 24.19mmol) and 1ml water under 25 degree, stir and spend the night, by reaction solution diatomite filtration, filtrate is spin-dried for, with methylene dichloride dissolution filter, be spin-dried for to obtain product 1.23g.Productive rate 93.2%.
1H?NMR(400MHz，CDCl
3)：δppm3.72(t，4H，J=4.4Hz)，3.58(t，4H，J=4.8Hz)，2.57(bs，2H)，1.41(s，6H).
The 4th step, compound Y010's is synthetic:
Y009 (1g, 6.098mmol) is dissolved in 7.5ml DCM and is stirred, under ice bath, add 0.43ml EtN
3(triethylamine), more dropwise add under TsCl (0.291g, the 1.524mmol) room temperature being dissolved in 1.5ml DCM and stir and spend the night.Revolve and desolventize with PE: 2.5: 1 column chromatographies of EA are crossed post, obtain a sterling 380mg.Productive rate 78.4%.
1H?NMR(400MHz，CDCl
3)：δppm7.79(d，2H，J=8.0Hz)，7.34(d，2H，J=8.0Hz)，4.14(t，2H，J=4.8Hz)，3.71～3.63(m，4H)，3.49(t，2H，J=4.8Hz)，2.45(s，3H)，1.32(s，6H).
The 5th step, compound Y011's is synthetic:
Take Y010 (187mg, 0.59mmol) in single port bottle, add again NaN after adding 2.5ml DMF to stir
3(84.1mg, 1.29mmol) at 80 ℃, stir and spend the night, be cooled to and add 10ml water after room temperature and with ethyl acetate 15*4 extraction, finally merge organic phase and use again saturated common salt water washing layering, revolve except after organic layer and obtain sterling 39mg, productive rate 35% with 3: 1 column chromatographies of PE: EA.
1H?NMR(400MHz，CDCl
3)：δppm3.74(t，2H，J=4.8Hz)，3.64～3.57(m，4H)，3.37(t，2H，J=4.8Hz)，2.09(s，1H)，1.40(s，6H).
The 6th step, compound Y012's is synthetic:
Y011 (46mg, 0.243mmol) is dissolved in 3ml methyl alcohol, and adds 5mg Pd/C (10%) to vacuumize, be filled with hydrogen and stir and spend the night at 25 ℃, filter and be spin-dried for solvent, by 10: 1 column chromatography for separation of DCM: MeOH, obtain sterling 25mg.Productive rate 64%.
1H?NMR(400MHz，CDCl
3)：δppm3.73(t，2H，J=4.4Hz)，3.60～3.57(m，4H)，2.89(t，2H，J=4.8Hz)，2.83(s，1H)，1.38(s，6H).
the application of embodiment 14.dATP (AP3) in synthetic reversible terminal
As shown in figure 13, concrete steps are as follows for its synthetic route:
The first step, Compound C y5-SH's is synthetic
By cysteamine (42mg, 0.55mmol) 0.1M Na
2cO
3/ NaHCO
3(400 μ l) damping fluid adds the dry DMF of Cy5-NHS-ester (12mg, 0.02mmol) to dissolve in (1mL) after dissolving, normal temperature lucifuge stirs 1h, then adds 0.5mL 1M DTT, stirring at room 1h.Finally, 0.1% TFA (0.5ml) solution cancellation for reaction, RP-HPLC separated [condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 1mL/min; Moving phase: 0.1%TFA and CH
3cN, 5%CH
3cN (5min), 5%-60%CH
3cN (45min), 60%CH
3cN (10min), retention time t=55min; Visible detection device wavelength: 650nm], lyophilize, obtains 2.8mg blue solid, productive rate 25%.ESI-HRMS：cal?c?for?C
34H
44N
3OS[M]542.3205，Found542.3200.
Second step, dATP-SS-SPDP's is synthetic
In the single port bottle of 10mL, add Compound C y5-SH 5.5mg (0.0059mmol), then add 600 μ lNa
2cO
3/ NaHCO
3damping fluid, stirring at room, is dissolved in the anhydrous CH of 400 μ l SPDP 2.7mg (0.008mmol)
3cN, adds above-mentioned solution, adds 3 μ l Et
3n.Stirring at room reaction 2 hours.After lyophilize, thick product is used RP-HPLC separated [condition: pillar: C18,5 μ m, 9.4 * 250mm after being dissolved in 1ml TEAA damping fluid (100mM, pH7.0); Flow velocity: 4mL/min; Moving phase: 100mM TEAA and CH
3cN, 100%TEAA, 5min; 0-5%CH
3cN (5min), 5%-20%CH
3cN (30min), UV-detector wavelength: 293nm], retention time t=38min.Product lyophilize after separation, NaCl/EtOH desalination, obtains 1.5mg white solid, productive rate 34.5%.
1H?NMR(D
2O，400MHz)：δ2.46-2.51(m，1H)，2.59-2.66(m，1H)，2.74(t，J=6.4Hz，2H)，3.15(t，J=6.0Hz，2H)，4.18-4.27(m，5H)，4.71-4.74(m，1H)，6.52(t，J=6.8Hz，1H)，7.00(t，J=5.2Hz，1H)，7.55-7.60(m，3H)，8.05(s，1H)，8.10(d，J=4.0Hz，1H).ESI-HRMS：calc?for?C
22H
26N
6O
13P
3S
2[M-H]
-739.0212，Found739.0201.
The 3rd step, dATP-SS-Cy5's is synthetic
Compound dATP-SS-SPDP (3.4mg, 4.6mmol) is dissolved in to Na
3pO
4-edta buffer liquid [50mM Na
3pO
4, 10mM EDTA (pH7.4,1.5ml)] and the mixed solution of acetonitrile 0.5ml in, then it is added in the Compound C y5-SH (2.3mg, 4.2mmol) after freeze-drying, room temperature lucifuge stirs 5 hours.RP-HPLC separation and purification [condition: pillar: C18,5 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH
3cN, 5%CH
3cN (5min), 5%-60%CH
3cN (45min), retention time t=41min; Visible detection device wavelength: 650nm].Product lyophilize after separation, NaCl/EtOH desalination, obtains blue solid 0.9mg, productive rate 18%.RP-HPLC detects purity (> 95%) [condition: pillar: C18,5 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 100mM TEAA and CH
3cN, 5%CH
3cN (5min), 5%-70%CH
3cN (30min), retention time t=29min; Visible detection device wavelength: 650nm].ESI-HRMS：calc?for?C
51H
64N
8O
14P
3S
2[M-H]
2-1169.3196，Found1169.3229.
embodiment 15
Shown in the following formula V of structural formula of the present embodiment Nucleotide dGTP-I:
Its synthetic route as shown in figure 14, concrete steps are as follows: by dG-I vacuum-drying 12h, in glove box, take respectively dG-I (30mg, 0.072mmol), tri-n-butylamine pyrophosphate salt (80mg, 0.145mmol), the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone (30mg, 0.15mmol) is placed in three reaction tubess.Tri-n-butylamine pyrophosphate salt is dissolved in 0.25mL dry DMF, then adds the new tri-n-butylamine steaming of 0.3mL, stirring at normal temperature is after half an hour, reaction solution is injected to the chloro-4H-1 of 2-, in dry DMF (0.25mL) solution of 3,2-benzo dioxy phosphorus-4-ketone, stirring at normal temperature half an hour.Then this mixed solution is injected in dG-I, stirs 1.5h.Add 1mL 3% iodine (9: 1 Py/H
2o) solution, keeps iodine liquid color 15min not fade.After 15min, add 2mL water, after 2h, add 0.75mL 3M NaCl solution, 20mL dehydrated alcohol ,-20 ℃ of freezing 12h, centrifugal (20min, 3200rpm).The supernatant liquor that inclines, precipitation is drained after solvent, adds strong aqua, stirring at room 5 hours.Decompression screws out solvent, occurs brown solid, and RP-HPLC analyzes [condition: pillar: C18,5 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAA and EtOH, 0-20%EtOH (35min), visible detection device wavelength: 650nm], retention time t=18min.RP-HPLC separated [condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: 20mM TEAA and MeOH, 0-15%MeOH (25min), UV-detector wavelength: 254nm], retention time t=15min.NaCl/EtOH removes acetic acid triethylamine salt, and obtaining 12mg white solid is dGTP-I.M/z (ESI) 631.8987[M-H]
-, C
9h
13iN
2o
15p
3if requires631.8971. change NIS into NBS or NCS in the present embodiment reaction process, other reaction reagent and reaction process are all identical can obtain dGTP-Br and dGTP-Cl.
embodiment 16
The structural formula of the present embodiment Nucleotide UTP-allyl amine is as shown in the formula shown in (XI):
As shown in figure 15, concrete steps are as follows for its synthetic route:
Compound 1b (1.027g, 2.14mmol) is joined in 20mL water and stirred under room temperature, NIS (1.44g, 6.42mmol) and NaN
3(0.83g, 12.84mmol) join in reaction and stir 15h, filter, filtrate is adjusted to PH=6.5, is loaded on diethylaminoethyl-sepharose post, and target product is used the TEAB of gradient 0-1M to do moving phase, it is concentrated after product is merged, add 3*100mL water concentration and evaporation, vacuum-drying obtains compound 3b (1.66g, 86%)
Compound 3b (1.528g, 1.67mmol) is dissolved in 50mL 0.1M sodium acetate solution, and adds K
2pdCl
4(0.35g, 1.07mmol) stir 5min, the mixture of precooling (allylamine (1.8mL, 41.8mL) be dissolved in 10mL0.1M sodium acetate with 4M acetic acid) join in reaction and stir 15h, reactant is by 0.2 μ M, the filtration unit of 1000mL filters, filtrate is adjusted to PH=6.5, be loaded on diethylaminoethyl-sepharose post, target product is used the TEAB of gradient 0-1M to do moving phase, it is concentrated after product is merged, add 3*100mL water concentration and evaporation, last triethylamine salt is dissolved in 20mL water, pour in the acetonitrile solution that 200mL contains 5g sodium perchlorate, by solid drying, obtain UTP-allyl amine (0.733g, 74%).
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 (10)
1. a synthetic method for base modification Nucleotide, is characterized in that, described method comprises the steps:
The iodo-2 '-Deoxyribose cytidine of A, 5-
the iodo-2 '-deoxyuridine of 5-
or the iodo-2'-deoxyadenosine of 7-
with compound
under Pd/Cu catalyst action, there is linked reaction and obtain compound dC (AP
3)
dU (AP
3)
or dA (AP
3)
Or, the iodo-2 '-deoxyuridine of 5-
under Pd catalyst action, there is linked reaction with compound allyl amine and obtain allyl group uridine dU allyl group
B, compound dC (AP
3), dU (AP
3), dA (AP
3) or allyl group uridine dU allyl group and ring-type phosphorylation agent
there is phosphorylation reaction, then through oxidation, hydrolysis, obtain the Nucleotide dCTP (AP that propargylamine is modified
3)
dUTP (AP
3)
or dATP (AP
3)
or the Nucleotide dUTP-allyl group of allyl group modification
2. synthetic 5-the propargylamine 2 '-deoxidation cytidylic acid(CMP) dCTP (AP3) obtaining of the method for claim 1 is in the purposes of synthesizing for the reversible terminal of DNA sequencing.
3. synthetic 5-the propargylamine 2 '-deoxidation uridylate dUTP (AP3) obtaining of the method for claim 1 is in the purposes of synthesizing for the reversible terminal of DNA sequencing.
4. synthetic 7-denitrification-7-propargylamine-the 2 '-deoxidation adenine nucleotide dATP (AP3) obtaining of the method for claim 1 is in the purposes of synthesizing for the reversible terminal of DNA sequencing.
5. a synthetic method for base modification Nucleotide, is characterized in that, described method comprises the steps:
A, Nucleotide dUTP react in the aqueous solution at sodium azide under NIS, NBS or NCS effect, in the 5-position of pyrimidine bases, meet I, Br or Cl, obtain compound dUTP-X
wherein, X is I, Br or Cl;
B, described compound dUTP-X obtain Nucleotide dUTP (AP with trifluoroethyl propargylamine or allyl amine generation linked reaction under Pd catalyst action
3)
or dUTP-allyl group
6. the purposes of synthetic 5-halogen-the 2 '-deoxidation uridylate dUTP-X obtaining of a method as claimed in claim 5 in synthetic modification Nucleotide.
7. a synthetic method for base modification Nucleotide, is characterized in that, described method comprises the steps:
Compound dG (AP
3)
g (AP
3)
dG-X
or G-X
with 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 dGTP (AP
3)
gTP (AP
3)
dGTP-X
or GTP-X
wherein, X is I, Br or Cl.
8. the synthetic method of base modification Nucleotide as claimed in claim 7, is characterized in that, described method also comprises the steps:
At CuI, Pd (PPh
3)
4under the condition existing with TEA, compound F 17-hydroxy-corticosterone
2 
there is linked reaction with described dGTP-X or GTP-X, obtain described compound dGTP (AP
3) or GTP (AP
3); Described dGTP-X or GTP-X, F
2, CuI, Pd (PPh
3)
4with the mol ratio of TEA be 1: (2～3): 0.072: 0.025: (1.5～2).
9. the synthetic method of base modification Nucleotide as claimed in claim 7, is characterized in that, described compound G (AP
3) can be synthesized into as follows:
A, under ice-water bath agitation condition, mol ratio is 1.0: the propargylamine of (1.2～2) reacts with trifluoro-acetate, obtains compound
B, at CuI, Pd (PPh
3)
4under the condition existing with TEA, compound F 17-hydroxy-corticosterone
2with described G-X reaction, obtain described compound G (AP
3), described G-X, F
2, CuI, Pd (PPh
3)
4with the mol ratio of TEA be 1: (2～3): 0.072: 0.025: (1.5～2).
10. a synthetic method for base modification Nucleotide, is characterized in that, described method comprises the steps:
Vinyl cyanide and compound 1
under Pd catalysis, there is linked reaction, obtain compound 2
there is glycosylation, obtain compound 3
or compound 3 '
then under alkaline condition, remove protecting group, obtain compound 4
or compound 4 '
compound 4 or 4 ' and ring-type phosphorylation agent
reaction, obtains modified nucleotide (VIII)
or Nucleotide (X)
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| CN113980050A (en) * | 2021-10-25 | 2022-01-28 | 中元汇吉生物技术股份有限公司 | Modified nucleotide, composition and reagent |
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| CN114574495A (en) * | 2020-12-01 | 2022-06-03 | 上海交通大学医学院附属仁济医院 | Aptamer R50 modified by nucleoside derivative |
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