CN103819523B - The synthetic method of 7-denitrification-7-halogen guanosine- - Google Patents
The synthetic method of 7-denitrification-7-halogen guanosine- Download PDFInfo
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- CN103819523B CN103819523B CN201410030095.1A CN201410030095A CN103819523B CN 103819523 B CN103819523 B CN 103819523B CN 201410030095 A CN201410030095 A CN 201410030095A CN 103819523 B CN103819523 B CN 103819523B
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- 0 O=C*(CC1)=CCC1I Chemical compound O=C*(CC1)=CCC1I 0.000 description 4
- BDNFGZJKZKCFHT-YGSFFXIESA-N CC(C)C(NC(NC1Cl)=Nc2c1c(I)c[n]2[C@@H](C1)O[C@H](COC(c(cc2)ccc2Cl)=O)/C1=[O]/C(c(cc1)ccc1Cl)=O)O Chemical compound CC(C)C(NC(NC1Cl)=Nc2c1c(I)c[n]2[C@@H](C1)O[C@H](COC(c(cc2)ccc2Cl)=O)/C1=[O]/C(c(cc1)ccc1Cl)=O)O BDNFGZJKZKCFHT-YGSFFXIESA-N 0.000 description 1
- RKXWDUWQDZVZRE-UHFFFAOYSA-N CC(C)C(Nc1nc(Cl)c(cc[nH]2)c2n1)=O Chemical compound CC(C)C(Nc1nc(Cl)c(cc[nH]2)c2n1)=O RKXWDUWQDZVZRE-UHFFFAOYSA-N 0.000 description 1
- CEODHAYOZZJGDC-UHFFFAOYSA-N Cc(cc1)ccc1C(OC(C(CNC(C(CC1)=CC=C1Cl)=O)OC1Cl)C1OC(c(cc1)ccc1Cl)=N)=O Chemical compound Cc(cc1)ccc1C(OC(C(CNC(C(CC1)=CC=C1Cl)=O)OC1Cl)C1OC(c(cc1)ccc1Cl)=N)=O CEODHAYOZZJGDC-UHFFFAOYSA-N 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention discloses a kind of synthetic method of 7-denitrification-7-halogen guanosine-; Described method comprises the steps: that formula (III) compound goes protecting group to obtain formula (IV1) or (IV2) compound in the basic conditions; Further demethyl obtains formula (I) compound, i.e. described 7-denitrification-7-halogen guanosine-;
wherein, R
1for H or OH, R
2for I, Br or Cl, R
3for H or
7-denitrification-7-halogen-the guanosine-of the present invention's synthesis is that its selling price is very high at present, and synthetic method is complicated at widely used basic raw materials of field of biology such as DNA sequencing, mark, extensions, is difficult to control; And synthetic method desired raw material of the present invention is simple and easy to get, building-up process is conventional chemical reaction, can be used for large-scale promotion and uses.
Description
Technical field
The present invention relates to chemosynthesis and biochemical field, (guanosine comprises the synthetic method of dG-X and G-X to be specifically related to a kind of 7-denitrification-7-halogen guanosine-.
Background technology
DNA sequencing technology is one of important means of modern life science and medical research.DNA sequencing from the Sanger sequencing technologies (generation order-checking) of 1977, in the time of thirties years, develop rapidly.The flux of order-checking significantly improves and cost sharply declines, and its speed of development has broken the speed of the existing Moore's Law budget of semi-conductor industry circle to have people even to think.Two generation high-throughput parallel sequencing technology appearance be the concentrated reflection of sequencing technologies develop rapidly.Adopt first-generation sequencing technologies, the Human Genome Project (HGP) cost 3,000,000,000 dollars completes the sequencing of the whole genome of people (3,000,000,000 bases).And the state-of-the-art technology of current two generations order-checking only needs about 5000 dollars just can complete the whole gene order-checking of people.
Even so, the cost of two generations order-checking and technical elements still Shortcomings, can not meet the requirement to order-checking of basic science and clinical medicine.Single-molecule sequencing technology (three generations's sequencing technologies) is arisen at the historic moment.The core of three generations's sequencing technologies directly checks order to single DNA molecules, do not do any DNA amplification reaction, thus reduce cost, improves flux.Although single-molecule sequencing technology has commercially produced product, all also there is technical difficult point, fail large-scale application.
High-flux sequence platform in the market monopolize by several external products, especially troubling is, offshore company relies on the control to sequencing reagent, almost control domestic order-checking market completely, even if we can have breakthrough on order-checking hardware, on the auxiliary products such as sequencing reagent, we are also by under one's control.Therefore, independent research is applicable to the order-checking of two generations or even three generations and checks order the sequencing reagent of platform, by changing the current market structure, the order-checking platform of setting up China autonomous has strategic meaning.For this reason, National 863,973 and the planning of " 12 " biotech development all research and development new-generation sequencing technology and the research and development of auxiliary products are classified as the object given priority to.
Reversible terminal for checking order generally all chooses U, C, A, the Nucleotide of G tetra-bases. we find the starting raw material for the synthesis of four different nucleotide bases in real work, i.e. four different base (U, C, A, G) containing substituting group nucleosides expensive, especially 7-denitrification-7-halogen guanosine-(comprising guanosine dG-X and G-X) is not only very expensive but also synthetic method is very complicated, cause a lot of research worker to avoid using guanosine (J.Org.Chem.2011 as far as possible, 76, 3457 – 3462), make perfect research work originally become some like this to regret.
Summary of the invention
The object of the present invention is to provide a kind of synthetic method of 7-denitrification-7-halogen guanosine-(guanosine dG-X and G-X); The method synthesis 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 7-denitrification-7-halogen guanosine-, described method comprises the steps:
A, formula III compound go protecting group to obtain formula (IV 1) or (IV 2) compound in the basic conditions;
B, described formula (IV 1) or (IV 2) compound in the basic conditions demethyl obtain formula I compound, i.e. described 7-denitrification-7-halogen guanosine-;
Wherein, R
1for H or OH, R
2for I, Br or Cl, R
3for H or
Preferably, in steps A, R
3during for H, production (IV 1) compound; R
3for
time, production (IV 2) compound.
Preferably, described formula III compound passes through at formula II compound
7 of purine bases connect halogen atom preparation and obtain.
Preferably, described formula II compound through type compound G007 and compound
or
there is glycosylation reaction preparation and obtain,
Preferably, described compound G007 prepares as follows and obtains:
A, compound G005
synthesis: compound S m-1
with Sm-2
reaction, obtains compound G005;
B, compound G006
synthesis: compound G005
under the effect of phosphorus oxychloride, be obtained by reacting compound G006;
C, compound G007
synthesis: compound G006
react with isobutyryl chloride in the basic conditions and obtain described compound G007.
Second aspect, the invention still further relates to a kind of synthetic method of 7-denitrification-7-propargylamine-2'-deoxidation guanylic acid, described method comprises synthesizes by aforesaid method the formula I compound obtained and synthesizes described 7-denitrification-7-propargylamine-2'-deoxidation guanylic acid further; In formula I, R1 is H.
Preferably, comprise the steps:
A, compound dG (AP
3) synthesis: at CuI, Pd (PPh
3)
4(tetrakis triphenylphosphine palladium) and TEA(triethylamine) under existent condition, trifluoroacetyl propargylamine and formula I compound
reaction, obtains compound dG (AP
3)
described formula I compound, trifluoroacetyl propargylamine, CuI, Pd (PPh
3)
4be 1:(1.5 ~ 3 with the mol ratio of TEA): (0.05 ~ 0.10): (0.02 ~ 0.05): (1.5 ~ 3);
B, compound dGTP (AP
3) synthesis: compound dG (AP
3) and tri-n-butylamine pyrophosphate salt, the chloro-4H-1 of 2-, the reaction under triethylamine and iodine exist of 3,2-benzo dioxy phosphorus-4-ketone, reaction product goes protection, obtains compound
dGTP (AP
3), i.e. described 7-denitrification-7-propargylamine-2'-deoxidation guanylic acid; Described tri-n-butylamine pyrophosphate salt, the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone and dG (AP
3) mol ratio be 2:2:1.
The third aspect, the invention still further relates to a kind of synthetic method of 7-denitrification-7-propargylamine-guanosine-, and described method comprises synthesizes by aforesaid method the formula I compound obtained and synthesize described 7-denitrification-7-propargylamine-guanosine-further; In formula I, R1 is OH.
Preferably, comprise the steps:
At CuI, Pd (PPh
3)
4with under TEA existent condition, trifluoroacetyl propargylamine and formula I compound
reaction, obtains compound G (AP
3)
i.e. described 7-denitrification-7-propargylamine-guanosine-; Described formula I compound, trifluoroacetyl propargylamine, CuI, Pd (PPh
3)
4be 1:(1.5 ~ 3 with the mol ratio of TEA): (0.05 ~ 0.10): (0.02 ~ 0.05): (1.5 ~ 3).
Fourth aspect, the invention still further relates to the purposes of 7-denitrification-7-halogen guanosine-in synthesis 7-denitrification-7-propargylamine-guanosine-that a kind of aforesaid synthetic method obtains.
The present invention has following beneficial effect:
(1) the present invention has synthesized 7-denitrification-7-halogen-2'-deoxidation guanosine-(being called for short dG-I) and 7-denitrification-7-halogen guanosine-(being called for short G-I); This compound is at widely used basic raw materials of field of biology such as DNA sequencing, mark, extensions;
(2) synthetic method provided by the invention is compared with patent CN201310489397.0, core material required in patent 201310489397.0 can only import, and raw material required for the present invention is very cheap, compared with patent 201310489350.4, the first step reaction yield of patent 201310489350.4 only has 10%, and method used in the present invention can reach 86%; Raw material used when generating G009 in patent 201310489350.4 is expensive containing the ribose of benzyl protection; and productive rate only has 45%; ribose price used herein is extremely cheap; and the experimental technique productive rate used can reach 76%; therefore synthetic method of the present invention is while substantially increasing reaction yield; also the cost reaction raw materials greatly reducing experimental raw is conventional chemical reagent, and during final step reaction treatment, the method that the present invention removes inorganic salt is more simple efficient.
(3) the final product purity that obtains of the inventive method is high, comparatively in patent, final step purifying adopts the method for organic solvent washing, is difficult to eliminate inorganic salt, and final step purifying of the present invention adopts the method for washing, inorganic salt are thoroughly washed out, thus avoid the existence of inorganic salt in product, substantially increase the purity of product, synthetic method desired raw material of the present invention is simple and easy to get, building-up process is conventional chemical reaction, can be used for large-scale promotion and uses.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious.
Fig. 1 is the building-up process schematic diagram of 7-denitrification-7-iodo-2'-deoxidation guanosine-(being called for short dG-I);
Fig. 2 is 7-denitrification-7-iodine guanosine-(being called for short G-I) building-up process schematic diagram;
Fig. 3 is that 7-denitrification-7-iodo-2'-deoxidation guanosine-dG-I is at synthesis dGTP (AP
3) in purposes;
Fig. 4 is that 7-denitrification-7-iodine guanosine-G-I is at synthesis GTP (AP
3) in purposes;
Fig. 5 is 7-denitrification-7-iodo-2'-deoxidation guanosine-dG-I's
1h-NMR;
Fig. 6 is 7-denitrification-7-propargylamine-2'-deoxidation guanylic acid dGTP (AP
3)
1h-NMR;
Fig. 7 is 7-denitrification-7-propargylamine-2'-deoxidation guanylic acid dGTP (AP
3)
31p-NMR;
Fig. 8 is 7-denitrification-7-propargylamine-2'-deoxidation guanylic acid dGTP (AP
3) HRMS spectrogram;
Fig. 9 is DNA chain extension reaction fluorescent scanning result figure; Lane1:Primer (Oligo1); Lane2: the chain extension product containing dGTP (AP3).
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make certain adjustments and improvements.These all belong to protection scope of the present invention.The present invention's raw material used, reagent are commercially available AR, CP level.Gained intermediate product of the present invention and final product adopt NMR etc. to characterize;
the synthetic method of embodiment 1,7-denitrification-7-iodo-2'-deoxidation guanosine-dG-I
In the present embodiment, as shown in Figure 1, concrete synthetic method comprises the steps: the synthesis schematic diagram of dG-I respectively
Step one,
By Sm-2(12.6g, 100mmol) be dissolved in the mixed solution of 120mLDMF and 20mL water, after stirring at room temperature 15min, add Sm-1(12.7mL, 100mmol), stirring at room temperature 46h.Screw out solvent, solid is dissolved in 2.5mL water, and filter, vacuum-drying, obtains 12.9g, productive rate 86%.
1HNMR(400MHz,DMSO-d
6):δ=10.94(s,1H),10.35(s,1H),6.58(dd,J=3.4,2.2Hz,1H),6.15(dd,J=3.4,2.1Hz,1H),6.09(s,2H).
Step 2,
By G005(10.0g, 66.6mmol) join 100mLPOCl
3in, backflow 2h, revolve after being cooled to room temperature after desolventizing, 120mL frozen water is joined in reaction, and by solid filtering, filtrate is adjusted to pH=2 with ammoniacal liquor, and throw out is filtered as after 2h in ice bath, the solid first time of filtration, with the washing of 10mL frozen water, uses 30mL ice washed with diethylether for the second time, 8.7g is obtained, productive rate 78% after draining.
1HNMR(400MHz,DMSO-d
6):δ=11.43(s,1H,NH),7.07(d,1H,NHCHCH),6.46(s,2H,NH2),6.22(d,1H,CHNH)。
Step 3,
By G006(6.7g, 39.88mmol) join in 280mL anhydrous pyridine, under ice-water bath, slow dropping isobutyryl chloride (4.6mL, 43.87mmol), stirs 20min under ice-water bath, add 600 μ L methyl alcohol cancellation, screw out solvent after continuing to stir 10min, then add 20mL ether, filter, collect solid, use 50mL80% cold methanol/20% ether, 10mL cold methanol, 10mL washed with diethylether successively, vacuum-drying, obtain 6.68g, productive rate 70%.
1HNMR(400MHz,DMSO-d
6):δ(ppm)12.34(bs,1H),10.54(bs,1H),7.49(dd,J=2.4Hz,3.2Hz,1H),6.49(dd,J=1.6Hz,3.2Hz,1H),2.78(sept,J=6.8Hz,1H),1.08(d,J=6.8Hz,6H).
Step 4,
Potassium hydroxide (3.47g, 61.8mmol) powder is dissolved in 500mL acetonitrile, slowly adds G007(6.66g, 28.0mmol), stirring at room temperature 5min, slowly adds Sm-1(14.98g, 35mmol), stirred at ambient temperature 20min.Filter, solid 10mL washed with dichloromethane, dry.Column chromatography DCM:MeOH=10:1, obtains 13.4g white solid, productive rate 76%.
1HNMR(400MHz,DMSO-d
6):δ=10.29(s,1H),8.07-7.93(m,5H),7.66-7.56(m,4H),6.62(t,J=7.2Hz,1H,),5.84-5.81(m,1H),4.71-4.67(m,1H),4.59-4.44(m,3H),3.30-3.22(m,1H),2.79-2.72(m,1H),1.06(d,J=6.8Hz,6H).
Step 5,
By G008(8.23g, 13.0mmol) be dissolved in 60mLTHF, nitrogen protection, after masking foil parcel; add NIS(3.04g, 13.51mmol) in stirred at ambient temperature 1h, add 500mLDCM, use 200mL water washing; revolve after desolventizing, column chromatography DCM:MeOH=10:1, obtains 7.98g, productive rate 81%.
1HNMR(400MHz,DMSO-d
6):δ=10.29(s,1H),8.07-7.93(m,5H),7.66-7.56(m,4H),5.84-5.81(m,1H),4.71-4.67(m,1H),4.59-4.44(m,3H),3.30-3.22(m,1H),2.79-2.72(m,1H),1.05(d,J=6.8Hz,6H).
Step 6,
By G009(1.14g, 1.5mmol) join in 0.5MMeONa/MeOH (20.0mL), be neutralized to neutral rear column chromatography DCM:MeOH=10:1 with Glacial acetic acid after backflow 3h, obtain compound dG1-D490mg, productive rate 80%.
1H-NMR(400MHz,CD
3OD)δ7.17(s,1H),6.36(dd,J=6.0,8.4Hz,1H),4.47(m,1H),3.99(s,3H),3.96(m,1H),3.77(dd,J=3.4,12.0Hz,1H),3.70(dd,J=3.7,12.0Hz,1H),2.55-2.64(ddd,J=6.0,8.4,13.4Hz,1H),2.20-2.26(ddd,J=2.4,5.9,13.4Hz,1H)。
Step 7,
By compound dG
1-D(490mg, 1.20mmol) be placed in 25mL sodium hydroxide solution (2N) backflow 5h, add acetic acid solution neutralization after cooling, filter, a small amount of water washing, dry 428mg white solid and dG-I, productive rate 91%.As shown in Figure 5:
1h-NMR (400MHz, MeOD) δ 7.09 (s, 1H), 6.35 (dd, J=6.0Hz, J=8.0Hz, 1H), 4.42-4.44 (m, 1H), 3.89-3.92 (m, 1H), 3.65-3.74 (m, 2H), 2.43 – 2.50 (m, 1H), 2.19 – 2.24 (m, 1H). note: present method is applicable to the synthesis of 7-denitrification-7-bromine and 7-denitrification-7-chloro-2'-deoxidation guanosine-dG-Br/Cl equally, when difference is the 5th step reaction, replace NIS with NBS or BCS, other all reactions steps is all identical with method.
the synthetic method of the iodo-guanosine-G-I of embodiment 2,7-denitrification-7-
In the present embodiment, as shown in Figure 2, concrete synthetic method comprises the steps: the synthesis schematic diagram of G-I respectively
Step one,
Potassium hydroxide (3.47g, 61.8mmol) powder is dissolved in 500mL acetonitrile, slowly adds G007(6.66g, 28.0mmol), stirring at room temperature 5min, slowly adds Sm-1(20.44g, 35mmol), stirred at ambient temperature 20min.Filter, solid 10mL washed with dichloromethane, dry.Column chromatography DCM:MeOH=10:1, obtains 16.77g white solid, productive rate 76%.
1H-NMR(400MHz,DMSO-d
6):δ=10.25(s,1H),8.11-7.95(m,9H),7.76-7.58(m,8H),6.64(t,J=7.2Hz,1H,),5.88-5.82(m,1H),4.74-4.66(m,1H),4.61-4.40(m,4H),1.09(d,J=6.8Hz,6H).
Step 2,
By G008(10.24g, 13.0mmol) be dissolved in 60mLTHF, nitrogen protection, after masking foil parcel; add NIS(3.04g, 13.51mmol) in stirred at ambient temperature 1h, add 500mLDCM, use 200mL water washing; revolve and desolventize rear column chromatography DCM:MeOH=10:1, obtain 9.62g, productive rate 81%.
1HNMR(400MHz,DMSO-d
6):δ=10.29(s,1H),8.19-7.96(m,9H),7.68-7.52(m,8H),5.87-5.82(m,1H),4.75-4.65(m,1H),4.58-4.35(m,4H),1.10(d,J=6.8Hz,6H).
Step 3,
By G009(1.37g, 1.5mmol) join in 0.5MMeONa/MeOH (20.0mL), be neutralized to neutral rear column chromatography DCM:MeOH=10:1 with Glacial acetic acid after backflow 3h, obtain compound G-D506mg, productive rate 80%.
1H-NMR(250MHz,DMSO-d
6):δ3.49-3.57(m,2H,H-C(5’)),3.80-3.82(m,1H,H-C(4’)),3.93(s,3H,OMe),4.01-4.03(m,1H,H-C(3’)),4.23-4.28(m,1H,H-C(2’)),5.01(t,J=5.5Hz,1H,OH-C(5’)),5.05(d,J=4.4Hz,1H,OH-C(3’)),5.25(d,J=6.2Hz,1H,OH-C(2’)),5.94(d,J=6.5Hz,1H,H-C(1’)),6.38(s,2H,NH2),7.31(s,1H,H-C(6)).
Step 4,
By compound G-D(506mg, 1.20mmol) be placed in 25mL sodium hydroxide solution (2N) backflow 5h, add acetic acid solution neutralization after cooling, filter, a small amount of water washing, dry 445mg white solid and G-I, productive rate 91%.
1hNMR (600MHz, DMSO-d
6): 4.99 (brs, 1H, 5 '-OH), 5.04 (d, 1H, J=3.2,3 '-OH), 5.26 (d, 1H, J=5.9,2 '-OH), 6.33 (s, 2H, NH2), 7.14 (s, 1H, 6-H), 10.48 (s, 1H, NH). note: present method is applicable to the synthesis of 7-denitrification-7-bromine and the chloro-guanosine-dG-Br/Cl of 7-denitrification-7-equally, when difference is that second step reacts, replace NIS with NBS or BCS, other all reactions steps is all identical with method.
the iodo-2'-deoxyguanosine dG-I of embodiment 3,7-denitrification-7-is at synthesis dGTP (AP
3
) in use
on the way
DGTP (AP in the present embodiment
3) synthesis schematic diagram as shown in Figure 3, concrete synthetic method comprises the steps: respectively
Step one,
In a single port bottle, add compound dG-I (0.25g, 0.4mmol), then take CuI (22mg; 1mmol) with Pd (PPh
3)
4(48mg; 0.04mmol) add in reaction flask, vacuumize, nitrogen protection, aluminium foil wraps up, and adds 10mlDMF, stirring and dissolving, injects TEA (0.088g; 0.8mmol) with trifluoroacetyl propargylamine (0.2g; 1.2mmol), 50 DEG C are stirred after 13 hours, reaction terminates, screw out solvent, resistates is dissolved in 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], obtains 0.1g white solid and dG (AP
3), productive rate 39%.As shown in Figure 6:
1hNMR (400MHz, MeOD) δ 7.25 (s, 1H), 6.38-6.42 (m, 1H), 4.47 – 4.50 (m, 1H), 4.33 (s, 2H), 3.96 (dd, J=3.6Hz, J=6.8Hz, 1H), 3.70 – 3.80 (m, 2H), 2.48 – 2.55 (m, 1H), 2.26 – 2.32 (m, 1H).
Step 2,
By compound dG (AP
3) vacuum-drying 12h, difference Weigh Compound dG (AP in glove box
3) (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 tri-n-butylamine that 0.3mL newly steams, after stirring at normal temperature half an hour, reaction solution is injected 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 2, stirs 1.5h.Add 1mL3% iodine (9:1Py/H2O) solution, keep iodine liquid color 15min not fade.Add 2mL water after 15min, after 2h, add 0.75mL3MNaCl solution, 20mL dehydrated alcohol ,-20 DEG C of freezing 12h, centrifugal (20min, 3200rpm).Incline supernatant liquor, after precipitation drains solvent, adds strong aqua, stirring at room temperature 5 hours.Decompression screws out solvent, and occur brown solid, RP-HPLC analyzes [condition: pillar: C18,5 μm, 4.6 × 250mm; Flow velocity: 1mL/min; Moving phase: 20mMTEAA and EtOH, 0-20%EtOH (35min), visible detection device wavelength: 650nm], retention time t=18min.RP-HPLC is separated [condition: pillar: C18,5 μm, 9.4 × 250mm; Flow velocity: 4mL/min; Moving phase: 20mMTEAA and MeOH, 0-15%MeOH (25min), UV-detector wavelength: 254nm], retention time t=15min.NaCl/EtOH removes acetic acid triethylamine salt, obtains 12mg white solid and dGTP (AP
3).Productive rate 26%.DGTP (AP
3)
1h-NMR,
31p-NMR, HRMS spectrogram is distinguished as shown in Figure 8,9,
1hNMR (400MHz, D
2o) δ 7.45 (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); As shown in Figure 7:
31pNMR (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:calcforC
14h
19n
5o
13p
3[M-H]
-558.0192, found558.0179. note: present method is applicable to 7-denitrification-7-bromine/chloro-2'-deoxyguanosine dG-Br/Cl equally at synthesis dGTP (AP
3) in purposes, when difference is that the first step is reacted, with dG-Br/Cl replace dG-I, other all reactions steps is all identical with method.
embodiment 4,7-denitrification-7-iodine guanosine-G-I are at synthesis G (AP
3
) in purposes
G (AP in the present embodiment
3) synthesis schematic diagram as shown in Figure 4, concrete synthetic method comprises the steps: respectively
In a single port bottle, add compound G-I (0.25g, 0.4mmol), then take CuI (22mg; 1mmol) with Pd (PPh
3)
4(48mg; 0.04mmol) add in reaction flask, vacuumize, nitrogen protection, aluminium foil wraps up, and adds 10mlDMF, stirring and dissolving, injects TEA (0.088g; 0.8mmol) with trifluoroacetyl propargylamine (0.2g; 1.2mmol), 50 DEG C are stirred after 13 hours, reaction terminates, screw out solvent, resistates is dissolved in 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], obtains 0.1g white solid and G (AP
3), productive rate 39%.
1hNMR (400MHz, CDCl
3) δ 7.24 (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). note: present method is applicable to 7-denitrification-7-iodine guanosine-G-I equally at synthesis G (AP
3) in purposes, when difference is that the first step is reacted, with G-Br/Cl replace G-I, other all reactions steps is all identical with method.
dGTP (the AP of embodiment 5, synthesis
3
) participate in the test of DNA chain extension reaction
Sequencing template sequence used is as follows:
5'GAGGAAAGGGAAGGGAAAGGAAGGOligo1Primer
3'CTCCTTTCCCTTCCCTTTCCTTCCCATGATCGCCATGTGCOligo2
The wherein 5' end fluorescein Dylight800 mark of Oligo1.
1) in eppendorf pipe, the DNA chain extension reaction of Reversible terminal is set up according to following system: 10 × Klenowbuffer10uL, BSA (10mg/mL) 1uL, DMSO20uL, NaCl (1M) 25uL, Klenow (exo-) pol (5U/uL) 1.32uL, dUTP (10uM) 6uL, template DNA (853ng/uL) 1.25uL, ddH
2o35.43uL, cumulative volume 100uL.
Reaction system is placed in 30 DEG C of water baths process 15 minutes, then is placed in 75 DEG C of water-baths process 10 minutes with deactivation archaeal dna polymerase.
2) separation and purification: phenol chloroform, alcohol settling simmer down to solid, adds 20uLddH
2o and 1uL0.1MNaOH, after 95 DEG C of process 5min, ice-water bath 2min cools immediately, then carries out electrophoretic analysis.
3) electrophoretic analysis: as shown in Figure 9, result as can be seen from Fig. 9, dGTP (AP3) by archaeal dna polymerase identification, can participate in the extension of DNA chain to DNA chain extension reaction fluorescent scanning result figure as its substrate.Thus the structure of dGTP (AP3) synthesized by proving further is correct.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (2)
1. a synthetic method for 7-denitrification-7-halogen guanosine-, is characterized in that, described method comprises the steps:
A, formula III compound go protecting group to obtain formula (IV 1) or (IV 2) compound in the basic conditions;
B, described formula (IV 1) or (IV 2) compound in the basic conditions demethyl obtain formula I compound, i.e. described 7-denitrification-7-halogen guanosine-;
Wherein, R
1for H or OH, R
2for I, R
3for H or
In steps A, R
3during for H, production (IV 1) compound; R
3for
time, production (IV 2) compound;
Described formula III compound obtains by connecting halogen atom preparation on 7 of formula II compound purine bases,
Described formula II compound through type G007 compound
with compound
or
there is glycosylation reaction preparation and obtain.
2. the synthetic method of 7-denitrification-7-halogen guanosine-as claimed in claim 1, it is characterized in that, described compound G007 prepares as follows and obtains:
A, compound G005
synthesis: Sm-1
with
react in acid condition, obtain compound G005;
B, compound G006
synthesis: compound G005
under the effect of phosphorus oxychloride, be obtained by reacting compound G006;
C, compound G007
synthesis: compound G006
react with isobutyryl chloride in the basic conditions and obtain described compound G007.
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|---|---|---|---|---|
| CN103087131A (en) * | 2013-01-15 | 2013-05-08 | 上海交通大学 | Reversible terminal and synthesis and use in DNA synthesis sequencing thereof |
| CN103588838A (en) * | 2013-10-30 | 2014-02-19 | 上海交通大学 | Synthesis method of base modified nucleotide and application thereof |
| CN103601778A (en) * | 2013-10-17 | 2014-02-26 | 上海交通大学 | Synthetic method of 7-denitrified-7-substituted guanosine |
| CN103601779A (en) * | 2013-10-17 | 2014-02-26 | 上海交通大学 | Synthetic method of 7-denitrified-2'-deoxidized-7-halogen substituted guanosine |
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| CN103087131A (en) * | 2013-01-15 | 2013-05-08 | 上海交通大学 | Reversible terminal and synthesis and use in DNA synthesis sequencing thereof |
| CN103601778A (en) * | 2013-10-17 | 2014-02-26 | 上海交通大学 | Synthetic method of 7-denitrified-7-substituted guanosine |
| CN103601779A (en) * | 2013-10-17 | 2014-02-26 | 上海交通大学 | Synthetic method of 7-denitrified-2'-deoxidized-7-halogen substituted guanosine |
| CN103588838A (en) * | 2013-10-30 | 2014-02-19 | 上海交通大学 | Synthesis method of base modified nucleotide and application thereof |
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