CN103601779B - The synthetic method of 7-denitrification-2 '-deoxidation-7-halogen 9 substituted guanine nucleosides - Google Patents

The synthetic method of 7-denitrification-2 '-deoxidation-7-halogen 9 substituted guanine nucleosides Download PDF

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CN103601779B
CN103601779B CN201310489397.0A CN201310489397A CN103601779B CN 103601779 B CN103601779 B CN 103601779B CN 201310489397 A CN201310489397 A CN 201310489397A CN 103601779 B CN103601779 B CN 103601779B
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denitrification
deoxidation
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CN103601779A (en
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沈玉梅
邵志峰
龚兵
刘亚智
赵小东
江敏
李小卫
汤道年
伍新燕
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Shanghai Jiaotong University
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Abstract

The invention discloses a kind of synthetic method of 7-denitrification-2 '-deoxidation-7-halogen 9 substituted guanine nucleosides; Described method comprises and following formula (V) compound demethyl under alkali condition is obtained to following formula (I) compound, i.e. described 7-denitrification-2 '-deoxidation-7-halogen 9 substituted guanine nucleosides;(I)，(V), R wherein2For I, Br or Cl. The synthetic iodo-2 '-NSC 22837 of 7-denitrification-7-of the present invention is that its selling price is very high at present, and synthetic method complexity at widely used base stocks of field of biology such as DNA sequencing, mark, extensions; And synthetic method needed raw material provided by the invention is simple and easy to get, building-up process is conventional chemical reaction, can be used for large-scale promotion and uses.

Description

The synthetic method of 7-denitrification-2 '-deoxidation-7-halogen 9 substituted guanine nucleosides
Technical field
The present invention relates to chemical synthesis and biochemical field, be specifically related to a kind of 7-denitrification-2 '-deoxidation-7-halogen and replaceThe synthetic method of guanosine (guanosine dG-X).
Background technology
DNA sequencing technology is one of important means of modern life science and medical research. DNA sequencing was from 1977 yearsSanger sequencing technologies (generation order-checking) starts, in the time of thirties years, and develop rapidly. The flux of order-checking is largeWidth improves and cost sharply declines, and has people even to think that its speed of development has been broken existing mole of semi-conductor industry circle fixedThe speed of rule budget. Two generation high flux parallel sequencing technology appearance be the concentrated reflection of sequencing technologies develop rapidly. AdoptFirst generation sequencing technologies, 3,000,000,000 dollars of the Human Genome Project (HGP) costs complete (3,000,000,000 of the whole genomes of peopleBase) sequencing. And that the state-of-the-art technology of current two generations order-checking only needs 5000 dollars of left and right just can complete people is wholeGene order-checking.
Even so, cost and the still Shortcomings of technical elements of the order-checking of two generations, can not meet basic science and clinical doctorLearn the requirement to order-checking. Single-molecule sequencing technology (three generations's sequencing technologies) is arisen at the historic moment. The core of three generations's sequencing technologies isDirectly single DNA molecules is checked order, do not do any DNA amplification reaction, thereby reduce cost, improve 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, stateOuter company relies on control to sequencing reagent, has almost controlled domestic order-checking market completely, even if on order-checking hardware ICan have breakthrough, on the auxiliary products such as sequencing reagent, we are also by under one's control. Therefore, independent research applicable toThe check order sequencing reagent of platform of two generations order-checkings or even three generations, by changing the current market structure, to set up China autonomousOrder-checking platform has strategic meaning. For this reason, National 863,973 and " 12 " biotech development plan allClassify the research and development of research and development new-generation sequencing technology and auxiliary products as give priority to object.
For the reversible terminal that checks order, one all chooses U, C, A, the nucleotides of tetra-bases of G. and we send out in real workNow for the synthesis of the initiation material of four different nucleotide bases, four different bases (U, C, A, G) containing substituting group coreGlycosides is expensive, and especially 7-denitrification-2 '-deoxidation-7-halogen 9 substituted guanine nucleosides (guanosine dG-X) not only veryCostliness and synthetic method are very complicated, cause a lot of research workers to avoid using guanosine (J.Org.Chem. as far as possible2011,76,3457-3462), make so originally perfectly research work become some and regret. If necessary fourIn the situation of individual base, the flower of having to pays a high price for. By one, method is synthetic has simply and easily obtained such change in the present inventionCompound and synthetic method are simple, reaction condition gentleness, and course of reaction is controlled, is applicable to large-scale production. And with we fromOneself has successfully synthesized 7-denitrification-7-propargylamine-2 '-deoxyguanytic acid dGTP (AP by synthetic guanosine3)。
Summary of the invention
The object of the present invention is to provide a kind of 7-denitrification-2 '-deoxidation-7-halogen 9 substituted guanine nucleosides (guanosineDG-X) synthetic method; The method synthesis material is simple, cheap, and reaction condition gentleness is simple to operate, can be applicable to largeLarge-scale production.
The object of the invention is to be achieved through the following technical solutions:
The present invention relates to a kind of synthetic method of 7-denitrification-2 '-deoxidation-7-halogen 9 substituted guanine nucleosides, described method bagDraw together following formula (V) compound demethyl under alkali condition is obtained to following formula (I) compound, i.e. described 7-denitrification-2 '-Deoxidation-7-halogen 9 substituted guanine nucleosides;
(I)，
(V), R wherein2For I, Br or Cl.
Preferably, described formula (V) compound by removing formula (IV) compound protecting group preparation under alkali condition:
(IV), wherein, R is protection hydroxyl or amino protecting group.
Preferably, described protecting group is valeryl or isobutyryl.
Preferably, by the purine bases at formula (III) compound 7 of described formula (IV) compound connect halogen systemStandby and obtain:
(III). Can adopt and connect iodine 7 of purine bases with NIS or other iodination reagent; Also canAdopt the reagent such as NBS or NCS to connect bromine or chlorine 7 of purine bases.
Preferably, described formula (III) compound by using two of protecting group protection (II) compound under alkali conditionIndividual hydroxyl and an amino are prepared and obtain:
(II). This formula (II) compound name is called 7-Deaza-2 '-deoxy-6-methoxyguanosine,No. CAS: 86392-74-7, molecular formula: C12H16N404, molecular weight: 280.29.
The invention still further relates to a kind of synthetic method of 7-denitrification-7-propargylamine-2 '-deoxidation guanylic acid, described methodComprise by described in further synthesizing of the synthetic 7-denitrification-2 '-deoxidation-7-halogen 9 substituted guanine nucleosides obtaining of aforesaid method7-denitrification-7-propargylamine-2 '-deoxidation guanylic acid.
Preferably, comprise the steps:
A, compound dG (AP3) synthetic: at CuI, Pd (PPh3)4(tetrakis triphenylphosphine palladium) and TEA (three secondAmine) exist condition under, trifluoroacetyl propargylamine and formula (I) compoundReaction, obtains compounddG(AP3)
B, compound dGTP (AP3) synthetic: compound dG (AP3) with tri-n-butylamine pyrophosphate (E-4), 2-chlorine-4H-1,3,2-benzo dioxy phosphorus-4-ketone (E-3) reaction under triethylamine and iodine existence, product is gone protection, obtainingCompounddGTP(AP3), i.e. described 7-denitrification-7-propargylamine-2 '-deoxidation guanosintThuja acid.
Preferably, in steps A, described formula (I) compound, trifluoroacetyl propargylamine, CuI, Pd (PPh3)4And TEAMol ratio be 1:(2～3): 0.072:0.025:(1.5～2).
Preferably, in step B, described tri-n-butylamine pyrophosphate, the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone anddG(AP3) mol ratio be 2:2:1.
The invention still further relates to 7-denitrification-7-halogen 9 substituted guanine nucleosides that a kind of aforesaid synthetic method makes at synthetic 7-Purposes in denitrification-7-propargylamine-2 '-deoxidation guanylic acid.
The present invention has following beneficial effect: the present invention has synthesized 7-denitrification-7-halogen-2 '-deoxidation guanosine and (has been called for shortDG-X); This compound is at widely used base stocks of field of biology such as DNA sequencing, mark, extensions, at presentIts selling price is very high, and synthetic method complexity, is difficult to control. Synthetic method needed 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.
Brief description of the drawings
By reading the detailed description of non-limiting example being done with reference to the following drawings, other spy of the present inventionLevy, object and advantage will become more obvious:
Fig. 1 is the building-up process signal of the synthetic method 1 of the iodo-2 '-deoxidation guanosine of 7-denitrification-7-(being called for short dG-I)Figure.
Fig. 2 is the building-up process signal of the synthetic method 2 of the iodo-2 '-deoxidation guanosine of 7-denitrification-7-(being called for short dG-I)Figure.
Fig. 3 is 7-denitrification-7-iodine guanosine (being called for short G-I) building-up process schematic diagram.
Fig. 4 is 7-denitrification-7-bromine/chloro-2 '-deoxidation guanosine (being called for short dG-G) building-up process schematic diagram.
Fig. 5 is that the iodo-2 '-deoxidation guanosine of 7-denitrification-7-dG-I is at synthetic dGTP (AP3) in purposes.
Fig. 6 is that 7-denitrification-7-iodine guanosine G-I is at synthetic GTP (AP3) in purposes.
Fig. 7 is the 1H-NMR of the iodo-2 '-deoxidation guanosine of 7-denitrification-7-dG-I.
Fig. 8 is 7-denitrification-7-propargylamine-2 '-deoxidation guanylic acid dGTP (AP3)1H-NMR。
Fig. 9 is 7-denitrification-7-propargylamine-2 '-deoxidation guanylic acid dGTP (AP3)31P-NMR。
Figure 10 is 7-denitrification-7-propargylamine-2 '-deoxidation guanylic acid dGTP (AP3) HRMS spectrogram.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail. Following examples will contribute to this areaTechnical staff further understands the present invention, but does not limit in any form the present invention. It should be pointed out that this areaThose of ordinary skill, without departing from the inventive concept of the premise, can also make certain adjustments and improvements. TheseAll belong to protection scope of the present invention. The present invention's raw material, reagent used is commercially available AR, CP level. Gained of the present inventionIntermediate product and end product employing NMR etc. characterize;
One of synthetic method of embodiment 1, the iodo-2 '-deoxidation guanosine of 7-denitrification-7-dG-I
In the present embodiment, as shown in Figure 1, concrete synthetic method comprises the steps: respectively the synthetic schematic diagram of dG-I
Step 1,
By compound dG1-A (0.20g; 0.714mmol) be dissolved in anhydrous pyridine, slowly drip pivalyl chloride at 0 DEG C(0.75mL; 7.14mmol), stir after 1h at 0 DEG C, add 2ml methyl alcohol, stir 10min, screw out solvent, addEthyl acetate (200ml) and saturated sodium bicarbonate solution (50ml) extraction, separate organic phase, adds successively saturated carbonAcid hydrogen sodium solution and saturated common salt water washing, anhydrous sodium sulfate drying, screws out solvent, silica gel column chromatography [V (acetic acid secondEster): V (benzinum)=1:1], obtaining 0.39g white solid is compound dG1-B, productive rate 92%.1HNMR(400MHz，CD3OD)δ7.28(d，J=3.7Hz，1H)，6.66(dd，J=5.9，8.6Hz，1H)，6.51(d，J=3.7Hz，1H)，5.41(m，1H)，4.33-4.36(m，2H)，4.22(m，1H)，4.08(s，3H)，2.83-2.96(m，2H)，2.54-2.70(m，2H)，2.48-2.54(ddd，J=2.0，5.9，14.2Hz，1H)，1.15-1.23(m，27H).
Step 2,
By compound dG1-B (0.42g; 0.84mmol) be dissolved in dry DMF, under vigorous stirring, add 4-iodo fourthImidodicarbonic diamide (220mg; 0.9mmol), stirring at room temperature 22h, screws out solvent, adds 100ml ether and 50ml bicarbonateSodium solution extraction, isolates organic phase, saturated sodium-chloride washing, and anhydrous sodium sulfate drying, screws out solvent, silica gel column layerAnalyse [V (ethyl acetate): V (benzinum)=1:1], obtaining 0.5g white solid is compound dG1-C, productive rate 91%.1H-NMR(400MHz，CD3OD)δ7.43(s，1H)，6.63(dd，J=6.0，8.2Hz，1H)，5.41(m，1H)，4.33-4.36(m，2H)，4.23(m，1H)，4.09(s，3H)，2.78-2.94(m，2H)，2.57-2.70(m，2H)，2.50-2.57(ddd，J=2.3，6.0，14.2Hz，1H)，1.17-1.24(m，27H).
Step 3,
By compound dG1-C is dissolved in methyl alcohol/sodium methoxide (10ml) of 0.5M, stirs 12h at 65 DEG C, then adds 10mlSaturated sodium bicarbonate solution, continues to stir 10min, screws out methyl alcohol, adds the extraction of 50ml ethyl acetate, and organic layer respectivelyWith saturated sodium bicarbonate solution and saturated nacl aqueous solution washing, anhydrous sodium sulfate drying, concentrated, residue silica gel column layerAnalyse [V (methyl alcohol): V (carrene)=1:10], obtaining 0.24g white solid is compound dG1-D, productive rate 74%.1H-NMR(400MHz，CD3OD)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 4,
By compound dG1-D is placed in sodium hydroxide solution (2N) backflow 4h, adds 2N hydrochloric acid solution after cooling,Regulator solution pH is 6. Concentrated, add 100ml carrene and methyl alcohol mixed liquor (V:V=1:1) washing, be associated withMachine phase, concentrated that 255mg white solid is dG-I, productive rate 98%. DG-I's1H-NMR as shown in Figure 7,1HNMR(400MHz，MeOD)δ7.09(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: it is fast that this method is applicable to 7-denitrification-7-bromine and chloro-2 '-deoxidation bird equallyPurine nucleosides dG-Br/Cl's is synthetic, when difference is second step reaction, and with NBS or BCS replacement NIS,Other all reactions steps and method are all identical.
Two of the synthetic method of embodiment 2, the iodo-2 '-deoxidation guanosine of 7-denitrification-7-dG-I
In the present embodiment, as shown in Figure 2, concrete synthetic method comprises the steps: respectively the synthetic schematic diagram of dG-I
Step 1,
After Sm-1 (27.3g, 138mmol) is joined in 70mL water, then add 3.0mL concentrated hydrochloric acid to stir at 90 DEG C0.5h, is cooled to after room temperature and adds sodium acetate (13.6g, 165mmol) to stir, by Sm-2 (20.0g, 159mmol)With sodium acetate (7.0g, 85.4mmol) is dissolved in 150mL water and joins in reaction, move to stir 2h at 80 DEG C afterUnder zero degrees celsius, stir 1.5h, filter, and with frozen water and acetone washing, drain to obtain 15.4g, productive rate 74%.1HNMR(400MHz，DMSO)：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,
G005 (10.0g, 66.6mmol) is joined to 100mLPOCl3In, backflow 2h, is cooled to after room temperature and revolves and removeAfter solvent, 120mL frozen water is joined in reaction, and by solid filtering, filtrate is adjusted to PH=2 with ammoniacal liquor, and willSediment filters after as for 2h in ice bath, and the solid of filtration with the washing of 10mL frozen water, is used 30mL ice for the first time for the second timeEther washing, obtains 8.7g, productive rate 78% after draining.1HNMR(400MHz，DMSO)：δ=11.43(s，1H，NH)，7.07(d，1H，NHCHCH)，6.46(s，2H，NH2)，6.22(d，1H，CHNH)。
Step 3,
G006 (8.5g, 50.42mmol) is joined in 120mL anhydrous pyridine, then add pivalyl chloride (21.68mL,176.20mmol) and stir 2h under room temperature after revolve and desolventize, be dissolved in 1.7L carrene, organic phase 0.1M saltAfter acid solution (2*350mL) washing, revolve and desolventize rear column chromatography DCM:MeOH10:1 and obtain 8.15g, productive rate 64%.1HNMR(400MHz，[D6]-DMSO)：δ=9.98(s，1H，NHC(0))，7.50(d，J=3.6Hz，1H，NHCHCH)，6.49(d，J=3.6Hz，1H，CHNH)，1.20(s，9H，C(CH3)3)。
Step 4,
G007 (3.10g, 12.27mmol) is dissolved in 60mLTHF, and nitrogen protection, after masking foil parcel, addsNIS (3.04g, 13.51mmol) stirs 1h under room temperature, adds 500mLDCM, uses 200mL water washing, revolves and removesAfter solvent, column chromatography DCM:Me0H99:1 obtains 3.76g, productive rate 81%.1HNMR(400MHz，DMSO)：δ=12.65(s，1H，CHNH)，10.06(s，1H，NHC(0))，7.73(d，J=2.4Hz，1H，CH)，1.19(s，9H，C(CH3)3)。
Step 5,
By G008 (1.5g, 4.0mmol) and ammonium sulfate (15mg, 0.11mmol) HMDS (15mL,72.76mmol), backflow 20h, in the protection of argon gas, revolves after desolventizing and adds 40mL dichloroethanes, adds Sm-1(2.304,6.0mmol) and TMSOTf (1.25mL, 6.47mmol) and under room temperature, stir to clarify after take the photograph in 50Under family name's degree, stir 24h, add 60mLDCM, and with 30mL saturated sodium bicarbonate and saturated common salt water washing, revolve apart fromMachine mutually after, column chromatography obtains 1.48g, productive rate 45%.1HNMR(400MHz，D6-DMSO)：δ=10.29(s，1H)，8.02(s，1H，)，7.90-7.41(m，10H)，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)，1.15(s，9H).
Step 6,
G009 (1.056g, 1.5mmol) is joined in 0.5MMeONa/MeOH (20.0mL), after backflow 3h, use iceAcetic acid is neutralized to neutral rear column chromatography DCM:MeOH5:1 and obtains compound dG1-D490mg, productive rate 80%.1H-NMR(400MHz，CD3OD)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 dG1-D is placed in sodium hydroxide solution (2N) backflow 4h, adds 2N hydrochloric acid solution after cooling,Regulator solution pH is 6. Concentrated, add 100ml carrene and methyl alcohol mixed liquor (V:V=1:1) washing, be associated withMachine phase, concentrated that 255mg white solid is dG-I, productive rate 98%.1HNMR(400MHz，MeOD)δ7.09(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: this method is applicable to the synthetic of 7-denitrification-7-bromine and chloro-2 '-deoxidation guanosine dG-Br/Cl, difference equallyBe in the time of four-step reaction, with NBS or BCS replacement NIS, other all reactions steps and method are all identical.
The synthetic method of embodiment 3, the iodo-guanosine G-I of 7-denitrification-7-
In the present embodiment, as shown in Figure 3, concrete synthetic method comprises the steps: respectively the synthetic schematic diagram of G-I
Step 1,
By G008 (1.5g, 4.0mmol) and ammonium sulfate (15mg, 0.11mmo1) HMDS (15mL,72.76mmol), backflow 20h, in the protection of argon gas, revolves after desolventizing and adds 40mL dichloroethanes, adds G-I-0(6.0mmol) and TMSOTf (1.25mL, 6.47mmol) and under room temperature, stir to clarify after under 50 degrees Celsius, stirMix 24h, add 60mLDCM, and with 30mL saturated sodium bicarbonate and saturated common salt water washing, revolve except after organic phase postChromatography obtains G-I-B compound 1.6g. 1HNMR (600MHz, DMSO): δ=10.29 (s, 1H), 8.02 (s,1H)，7.91-7.85(m，6H)，7.64-7.58(m，3H)，7.46-7.39(m，6H)，6.48(d，J=3.9Hz，1H)，6.41(t，J=6.1，6.1Hz，1H)，6.32(dd，J=6.0，4.0Hz，1H)，4.82(dd，J=10.7，5.0Hz，1H)，4.75(dd，J=11.8，4.4Hz，1H)，4.63(dd，J=11.8，5.7Hz，1H)，1.15(s，9H)。
Step 2,
G-I-B (1.056g, 1.5mmol) is joined in 0.5MMeONa/MeOH (20.0mL), after backflow 3h, use iceAcetic acid is neutralized to neutral rear column chromatography DCM:MeOH5:1 and obtains G-I-A compound 490mg, productive rate 80%. 1H-NMR (DMSO-d6,250MHz)：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，0H-C(5’))，5.05(d，J=4.4Hz，1H，0H-C(3’))，5.25(d，J=6.2Hz，1H，0H-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 3,
Compound G-I-A is placed in to sodium hydroxide solution (2N) backflow 4h, adds 2N hydrochloric acid solution after cooling, adjustJoint pH value of solution is 6. Concentrated, add 100ml carrene and methyl alcohol mixed liquor (V:V=1:1) washing, merge organic phase,Concentrated that 255mg white solid is G-I, productive rate 98%. 1HNMR (600MHz, DMSO): 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: this method is applicable to 7-denitrification-7-bromine and chlorine guanine equallyNucleosides G-Br/Cl's is synthetic, and difference is that it is bromine that the first step is reacted 7 of guanine base of raw material G008 usedOr chlorine, other all reactions steps and method are all identical.
One of synthetic method of embodiment 4,7-denitrification-7-bromine/chloro-2-NSC 22837 dG-I
In the present embodiment, as shown in Figure 4, concrete synthetic method comprises the steps: respectively the synthetic schematic diagram of dG-G
Step 1,
By compound dG1-A (0.20g; 0.714mmol) be dissolved in anhydrous pyridine, slowly drip pivalyl chloride at 0 DEG C(0.75mL; 7.14mmol), stir after 1h at 0 DEG C, add 2ml methyl alcohol, stir 10min, screw out solvent, addEthyl acetate (200ml) and saturated sodium bicarbonate solution (50ml) extraction, separate organic phase, adds successively saturated carbonAcid hydrogen sodium solution and saturated common salt water washing, anhydrous sodium sulfate drying, screws out solvent, silica gel column chromatography [V (acetic acid secondEster): V (benzinum)=1:1], obtaining 0.39g white solid is compound dG1-B, productive rate 92%.1HNMR(400MHz，CD3OD)δ7.28(d，J=3.7Hz，1H)，6.66(dd，J=5.9，8.6Hz，1H)，6.51(d，J=3.7Hz，1H)，5.41(m，1H)，4.33-4.36(m，2H)，4.22(m，1H)，4.08(s，3H)，2.83-2.96(m，2H)，2.54-2.70(m，2H)，2.48-2.54(ddd，J=2.0，5.9，14.2Hz，1H)，1.15-1.23(m，27H).
Step 2,
By compound dG1-B (0.42g; 0.84mmol) be dissolved in dry DMF, under vigorous stirring, add 4-bromo fourthImidodicarbonic diamide or 4-chlorosuccinimide (220mg; 0.9mmol), stirring at room temperature 22h, screws out solvent, adds100ml ether and the extraction of 50ml sodium bicarbonate solution, isolate organic phase, saturated sodium-chloride washing, and anhydrous sodium sulfate is dryDry, screw out solvent, silica gel column chromatography [V (ethyl acetate): V (benzinum)=1:1], obtaining 0.5g white solid is compounddG1-E, productive rate 91%.1H-NMR(400MHz，CD3OD)δ7.43(s，1H)，6.63(dd，J=6.0，8.2Hz，1H)，5.41(m，1H)，4.33-4.36(m，2H)，4.23(m，1H)，4.09(s，3H)，2.78-2.94(m，2H)，2.57-2.70(m，2H)，2.50-2.57(ddd，J=2.3，6.0，14.2Hz，1H)，1.17-1.24(m，27H).
Step 3,
Compound dG1-E is dissolved in methyl alcohol/sodium methoxide (10ml) of 0.5M, stirs 12h at 65 DEG C, then add 10mlSaturated sodium bicarbonate solution, continues to stir 10min, screws out methyl alcohol, adds the extraction of 50ml ethyl acetate, and organic layer respectivelyWith saturated sodium bicarbonate solution and saturated nacl aqueous solution washing, anhydrous sodium sulfate drying, concentrated, residue silica gel column layerAnalyse [V (methyl alcohol): V (carrene)=1:10], obtaining 0.24g white solid is compound dG1-F, productive rate 74%.1H-NMR(400MHz，CD3OD)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 4,
By compound dG1-F is placed in sodium hydroxide solution (2N) backflow 4h, adds 2N hydrochloric acid solution after cooling,Regulator solution pH is 6. Concentrated, add 100ml carrene and methyl alcohol mixed liquor (V:V=1:1) washing, be associated withMachine phase, concentrated that 255mg white solid is dG1-G, productive rate 98%.1HNMR(400MHz，MeOD)δ7.09(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).
The iodo-2 '-NSC 22837 dG-I of embodiment 5,7-denitrification-7-is at synthetic dGTP (AP 3 ) in purposes
As shown in Figure 5, concrete synthetic method comprises the steps: respectively the synthetic schematic diagram of dGTP in the present embodiment (AP3)
Step 1,
In a single port bottle, add compound dG-I (0.25g, 0.4mmol), then take CuI (22mg; 1mmol)And Pd (PPh3) 4 (48mg; 0.04mmol) add in reaction bulb, vacuumize, nitrogen protection, aluminium foil parcel, adds10mlDMF, stirring and dissolving, injects TEA (0.088g; 0.8mmol) with trifluoroacetyl propargylamine (0.2g; 1.2Mmol), 50 DEG C were stirred after 13 hours, and reaction finishes, and screws out solvent, and residue is dissolved in to 100ml ethyl acetate, complies withInferior to saturated sodium bicarbonate solution and saturated nacl aqueous solution washing, anhydrous sodium sulfate drying, concentrated, column chromatography [V (secondAcetoacetic ester): V (n-hexane)=1:3], obtaining 0.1g white solid is dG (AP3), productive rate 39%.1HNMR(400MHz，MeOD)δ7.25(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 (AP3) vacuum drying 12h, in glove box, take respectively compound dG (AP3)(30mg，0.072Mmol), tri-n-butylamine pyrophosphate (80mg, 0.145mmol), the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone (30Mg, 0.15mmol) be placed in three reaction tubes. Tri-n-butylamine pyrophosphate is dissolved in 0.25mL dry DMF, thenAdd the new tri-n-butylamine steaming of 0.3mL, after stirring at normal temperature half an hour, reactant liquor is injected to the chloro-4H-1 of 2-, 3,2-benzoIn dry DMF (0.25mL) solution of dioxy phosphorus-4-ketone, stirring at normal temperature half an hour. Then this mixed liquor is injected intoIn 2, stir 1.5h. Add 1mL3% iodine (9:1Py/H20) solution, keep iodine liquid color 15min not fade.After 15min, add 2mL water, after 2h, add 0.75mL3MNaCl solution, 20mL absolute ethyl alcohol ,-20 DEG CFreezing 12h, centrifugal (20min, 3200rpm). The supernatant that inclines, precipitation is drained after solvent, adds concentrated ammonia liquor, chamberTemperature stirs 5 hours. Decompression screws out solvent, occurs brown solid, and RP-HPLC analyzes [condition: pillar: C18,5 μM, 4.6 × 250mm; Flow velocity: 1mL/min; Mobile phase: 20mMTEAA and EtOH, 0-20%EtOH (35min),Visible detection device wavelength: 650nm], retention time t=18min. RP-HPLC separates [condition: pillar: C18,5μ m, 9.4 × 250mm; Flow velocity: 4mL/min; Mobile phase: 20mMTEAA and MeOH, 0-15%MeOH (25Min), UV-detector wavelength: 254nm], retention time t=15min. NaCl/EtOH removes acetic acid triethylamineSalt, obtaining 12mg white solid is dGTP (AP3). Productive rate 26%. DGTP's (AP3)1H-NMR、31P-NMR、HRMSSpectrogram is distinguished as shown in Figure 8,9, 10,1HNMR(400MHz，D2O)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)；31PNMR(D2O，162MHz)：-10.59(t，J=9.9Hz，1P)，-11.24(d，J=17.3Hz，1P)，-22.98(d，J=20.7Hz，1P).ESI-HRMS：calcforC14H19N5O13P3[M-H]-558.0192, found558.0179. note: this method is applicable to 7-denitrification equallyThe purposes of-7-bromine/chloro-2 '-NSC 22837 dG-Br/Cl in synthetic dGTP (AP3), difference is firstWhen step reaction, with dG-Br/Cl replacement dG-I, other all reactions steps and method are all identical.
Embodiment 6,7-denitrification-7-iodine guanosine G-I is at synthetic G (AP 3 ) in purposes
G (AP in the present embodiment3) synthetic schematic diagram as shown in Figure 6, 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) andPd(PPh3)4(48mg; 0.04mmol) add in reaction bulb, vacuumize, nitrogen protection, aluminium foil parcel, adds 10mlDMF, stirring and dissolving, injects TEA (0.088g; 0.8mmol) with trifluoroacetyl propargylamine (0.2g; 1.2mmol),50 DEG C were stirred after 13 hours, and reaction finishes, and screws out solvent, and residue is dissolved in to 100ml ethyl acetate, successively with fullWith sodium bicarbonate solution and saturated nacl aqueous solution washing, anhydrous sodium sulfate drying, concentrated, column chromatography [V (acetic acid secondEster): V (n-hexane)=1:3], obtaining 0.1g white solid is G (AP3), productive rate 39%.1HNMR(400MHz，CDCl3)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). note: this method is applicable to 7-denitrification-7-iodine guanosine G-I equally at synthetic G (AP3)In purposes, difference is first step when reaction, replaces G-I, other all reactions steps with G-Br/ClAll identical with method.
Above specific embodiments of the invention are described. It will be appreciated that, the present invention is not limited to above-mentioned spyDetermine embodiment, those skilled in the art can make various distortion or amendment within the scope of the claims, and this is shadow notRing flesh and blood of the present invention.

Claims (4)

1. a synthetic method for 7-denitrification-2'-deoxidation-7-halogen 9 substituted guanine nucleotides, is characterized in that, described inMethod comprises the steps:
Step 1,
In a single port bottle, add compound dG-I0.4mmol, then take CuI1mmol and Pd (PPh3)40.04mmolAdd in reaction bulb, vacuumize, nitrogen protection, aluminium foil parcel, adds 10mlDMF, and stirring and dissolving is injected TEA0.8Mmol and trifluoroacetyl propargylamine 1.2mmol, 50 DEG C were stirred after 13 hours, and reaction finishes, and screws out solvent, by remnantsThing is dissolved in 100ml ethyl acetate, successively with saturated sodium bicarbonate solution and saturated nacl aqueous solution washing, anhydrous sodium sulfateDry, concentrated, the ethyl acetate-n-hexane mixed solvent that is 1:3 by volume ratio carries out column chromatography, obtains 0.1g white solidBody is dG (AP3), productive rate 39%;
Step 2,
By compound dG (AP3) vacuum drying 12h, in glove box, take respectively compound dG (AP3)0.072mmol、Tri-n-butylamine pyrophosphate 0.145mmol, the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone 0.15mmol is placed in threeIn reaction tube; Tri-n-butylamine pyrophosphate is dissolved in 0.25mL dry DMF, then add 0.3mL new steam three justButylamine, after stirring at normal temperature half an hour, injects the chloro-4H-1 of 2-reactant liquor, 3,2-benzo dioxy phosphorus-4-ketone anhydrousIn DMF0.25mL solution, stirring at normal temperature half an hour; Then this mixed liquor is injected into dG (AP3) in, stir 1.5h;Add the Py-H of the iodine of 1mL3%2In O solution, keep iodine liquid color 15min not fade, described Py-H2In O solution,Py-H2The volume ratio of O is 9:1; After 15min, add 2mL water, after 2h, add 0.75mL3MNaCl solution,20mL absolute ethyl alcohol ,-20 DEG C of freezing 12h, centrifugal 20min under 3200rpm; The supernatant that inclines, precipitation is drained solventAfter, add concentrated ammonia liquor, stirring at room temperature 5 hours; Decompression screws out solvent, occurs brown solid, carries out RP-HPLC analysis,The condition of this step RP-HPLC is: pillar: C18,5 μ m, 4.6 × 250mm; Flow velocity: 1mL/min; Mobile phase:20mMTEAA and EtOH, 0-20%EtOH35min, visible detection device wavelength: 650nm, retention time t=18min;Carry out RP-HPLC separation, the condition of this step RP-HPLC is: pillar: C18,5 μ m, 9.4 × 250mm; Flow velocity:4mL/min; Mobile phase: 20mMTEAA and MeOH, 0-15%MeOH25min, UV-detector wavelength: 254nm,Retention time t=15min, NaCl/EtOH removes acetic acid triethylamine salt, and obtaining 12mg white solid is dGTP (AP3)，Productive rate 26%.
2. the synthetic method of 7-denitrification-2'-deoxidation-7-halogen 9 substituted guanine nucleotides as claimed in claim 1, itsBe characterised in that, the preparation concrete steps of described compound dG-I are: following formula (V) compound is gone down at alkali conditionMethyl obtains down, i.e. described 7-denitrification-2'-deoxidation-7-halogen 9 substituted guanine nucleosides;
Wherein R2For I;
Described formula (V) compound by removing formula (IV) compound protecting group preparation to obtain under alkali condition:
Wherein, R is protection hydroxyl or amino protecting group;
Described protecting group is valeryl or isobutyryl;
By the purine bases at formula (III) compound 7 of described formula (IV) compound connect halogen preparation and obtain:
Described formula (III) compound by under alkali condition with two hydroxyls of protecting group protection (II) compound andAn amino is prepared and obtains:
3. the synthetic method of 7-denitrification-7-propargylamine-2'-deoxidation guanylic acid as claimed in claim 1, its spyLevy and be, in step 1, described dG-I compound, trifluoroacetyl propargylamine, CuI, Pd (PPh3)4With TEA moleThan being 1:(2～3): 0.072:0.025:(1.5～2).
4. the synthetic method of 7-denitrification-7-propargylamine-2'-deoxidation guanylic acid as claimed in claim 1, its spyLevy and be, in step 2, described tri-n-butylamine pyrophosphate, the chloro-4H-1 of 2-, 3,2-benzo dioxy phosphorus-4-ketone and dG (AP3)Mol ratio be 2:2:1.
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