CN103601778A - Synthetic method of 7-denitrified-7-substituted guanosine - Google Patents
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Abstract
The invention discloses a synthetic method of 7-denitrified-7-substituted guanosine; the method comprise following steps: a compound with formula (IV 1) or (IV 2) is obtained from a compound with formula (III) by removing protective groups under a alkaline condition; further a compound with formula (I) is obtained by demethylation, that is the 7-denitrified-7-substituted guanosine; wherein, R1 is H or H, R2 is I, Br or Cl, R3 is H or OBz. The synthetic 7-denitrified-7-halogen substituted guanosine is a basic raw material which is widely applied to DNA sequencing, marking, extension and other biological fields, and is very expensive at market with complex synthetic method and difficult control; the synthetic method provided by the invention has the advantages that the raw materials are simple and easily available, and the synthetic process is a routine chemical reaction, and the method can be widely used in a large scale.
Description
Technical field
The present invention relates to chemosynthesis and biochemical field, be specifically related to the synthetic method of a kind of 7-denitrification-7-9 substituted guanine nucleosides (guanosine comprises dG-X and G-X).
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-bases of G. 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-7-9 substituted guanine nucleosides (comprising guanosine dG-X and G-X) is not only very expensive but also synthetic method is very complicated, causes a lot of research workers to avoid using guanosine (J.Org.Chem.2011,76 as far as possible, 3457-3462), some is sorry to make so originally perfectly research work become.If the in the situation that of necessary four bases, the flower of having to pays a high price for.In the present invention, we have synthesized a series of guanosine by two kinds of synthetic methods and have comprised that dG-X and G-X and synthetic method are simple, and reaction conditions is gentle, and reaction process is controlled, are applicable to scale operation.And successfully synthesized 7-denitrification-7-propargylamine-2 '-deoxyguanytic acid dGTP (AP with our own synthetic guanosine
3) and 7-denitrification-7-propargylamine guanosine-G (AP
3).
Summary of the invention
The object of the present invention is to provide the synthetic method of a kind of 7-denitrification-7-9 substituted guanine nucleosides (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-7-9 substituted guanine nucleosides, and described method comprises the steps:
A, formula (III) compound obtain formula (IV1) or (IV2) compound in the alkaline condition protecting group of going down;
B, described formula (IV1) or (IV2) compound demethyl under alkaline condition obtain formula (I) compound, i.e. described 7-denitrification-7-9 substituted guanine nucleosides;
Wherein, R
1for H or OH, R
2for I, Br or C1, R
3for H or OBz.
Preferably, in steps A, R
3during for H, production (IV1) compound; R
3during for OBz, production (IV2) compound.
Preferably, described formula (III) compound through type (II) compound and compound
or
there is glycosylation reaction preparation and obtain,
Preferably, described formula (II) compound passes through at compound G007
on 7 of purine bases, connect halogen atom preparation and obtain.
Preferably, described compound G007 be prepare as follows and:
A, compound G005
synthetic: Sm-1
with Sm-2
under acidic conditions, react, obtain compound G005;
B, compound G006
synthetic: compound G005
under the effect of phosphorus oxychloride, reaction obtains compound G006;
C, compound G007
synthetic: compound G006
under alkaline condition, react with pivalyl chloride 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 by synthetic formula (I) compound obtaining of aforesaid method further synthesizes described 7-denitrification-7-propargylamine-2 '-deoxidation guanylic acid; R in formula (I)
1for H.
Preferably, comprise the steps:
A, compound dG (AP
3) synthetic: at CuI, Pd (PPh
3)
4under the condition that (tetrakis triphenylphosphine palladium) and TEA (triethylamine) exist, trifluoroacetyl propargylamine and formula (I) compound
reaction, obtains compound dG (AP
3)
described formula (I) compound, trifluoroacetyl propargylamine, CuI, Pd (PPh
3)
4with the mol ratio of TEA be 1:(2～3): 0.072:0.025:(1.5～2);
B, compound dGTP (AP
3) synthetic: compound dG (AP
3) with 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 is gone 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 by the further synthetic described 7-denitrification-7-propargylamine-guanosine-of synthetic formula (I) compound obtaining of aforesaid method; R in formula (I)
1for OH.
Preferably, comprise the steps:
At GuI, Pd (PPh
3)
4under the condition existing with TEA, trifluoroacetyl propargylamine and formula (I) compound
reaction, obtains compound G (AP
3)
it is described 7-denitrification-7-propargylamine-guanosine-; Described formula (I) compound, trifluoroacetyl propargylamine, GuI, Pd (PPh
3)
4with the mol ratio of TEA be 1:(2～3): 0.072:0.025:(1.5～2).
Fourth aspect, the invention still further relates to 7-denitrification-7-9 substituted guanine nucleosides that a kind of aforesaid synthetic method makes purposes in synthetic 7-denitrification-7-propargylamine-guanosine-.
The present invention has following beneficial effect: the present invention has synthesized the iodo-2 '-deoxidation guanosine-of 7-denitrification-7-(being called for short dG-I) and 7-denitrification-7-iodine guanosine-(being called for short G-I); This compound is that its selling price is very high at present at widely used basic raw materials of field of biology such as DNA sequencing, mark, extensions, 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 the building-up process schematic diagram of the synthetic method 1 of the iodo-2 '-deoxidation guanosine-of 7-denitrification-7-(being called for short dG-I).
Fig. 2 is the building-up process schematic diagram of the synthetic method 2 of the iodo-2 '-deoxidation guanosine-of 7-denitrification-7-(being called for short dG-I).
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 (AP
3) in purposes.
Fig. 6 is that 7-denitrification-7-iodine guanosine-G-I is at synthetic GTP (AP
3) in purposes.
Fig. 7 is the iodo-2 '-deoxidation guanosine-of 7-denitrification-7-dG-I's
1h-NMR.
Fig. 8 is 7-denitrification-7-propargylamine-2 '-deoxidation guanylic acid dGTP (AP
3)
1h-NMR.
Fig. 9 is 7-denitrification-7-propargylamine-2 '-deoxidation guanylic acid dGTP (AP
3)
31p-NMR.
Figure 10 is 7-denitrification-7-propargylamine-2 '-deoxidation guanylic acid dGTP (AP
3) HRMS spectrogram.
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;
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
By compound dG
1-A (0.20g; 0.714mmol) be dissolved in anhydrous pyridine, slowly drip pivalyl chloride (0.75mL at 0 ℃; 7.14mmol), at 0 ℃, stir after 1h, add 2ml methyl alcohol, stir 10min, screw out solvent, add ethyl acetate (200ml) and saturated sodium bicarbonate solution (50ml) extraction, separated organic phase, adds saturated sodium bicarbonate solution and saturated common salt water washing successively, anhydrous sodium sulfate drying, screw out solvent, silica gel column chromatography [V (ethyl acetate): V (sherwood oil)=1:1], obtaining 0.39g white solid is compound dG
1-B, productive rate 92%.
1H?NMR(400MHz，CD
3OD)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).
By compound dG
1-B (0.42g; 0.84mmol) be dissolved in dry DMF, under vigorous stirring, add 4-N-iodosuccinimide (220mg; 0.9mmol), stirring at room 22h, screw out solvent, add 100ml ether and the extraction of 50ml sodium hydrogen carbonate solution, isolate organic phase, saturated sodium-chloride washing, anhydrous sodium sulfate drying, screw out solvent, silica gel column chromatography [V (ethyl acetate): V (sherwood oil)=1:1], obtaining 0.5g white solid is compound dG
1-C, productive rate 91%.
1H-NMR(400MHz，CD
3OD)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 dG
1-C is dissolved in methyl alcohol/sodium methylate (10ml) of 0.5M, at 65 ℃, stir 12h, add again 10ml saturated sodium bicarbonate solution, continue to stir 10min, screw out methyl alcohol, add the extraction of 50ml ethyl acetate, organic layer is respectively with saturated sodium bicarbonate solution and saturated nacl aqueous solution washing, and anhydrous sodium sulfate drying, concentrates, resistates silica gel column chromatography [V (methyl alcohol): V (methylene dichloride)=1:10], obtaining 0.24g white solid is compound dG
1-D, productive rate 74%.
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 4,
By compound dG
1-D is placed in sodium hydroxide solution (2N) backflow 4h, adds 2N hydrochloric acid soln after cooling, and regulator solution pH is 6.Concentrated, add 100ml methylene dichloride and methyl alcohol mixed liquor (V:V=1:1) washing, merge organic phase, concentrated that 255mg white solid is dG-I, productive rate 98%.DG-I's
1h-NMR spectrogram as shown in Figure 1,
1hNMR (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 synthetic of 7-denitrification-7-bromine and chloro-2 '-deoxidation guanosine-dG-Br/C1 equally, when difference is second step reaction, with NBS or BCS, replace 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
After Sm-1 (27.3g, 138mmol) is joined in 70mL water, then add 3.0mL concentrated hydrochloric acid to stir 0.5h at 90 ℃, after being cooled to room temperature, add sodium-acetate (13.6g, 165mmol) stir, by Sm-2 (20.0g, 159mmol) and sodium-acetate (7.0g, 85.4mmol) be dissolved in 150mL water and join in reaction, stir 2h at 80 ℃ after, move under zero degrees celsius and stir 1.5h, filter, and with frozen water and washing with acetone, drain to obtain 15.4g, productive rate 74%.
1H?NMR(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).
G005 (10.0g, 66.6mmol) is joined to 100mL POCl
3in, backflow 2h, after being cooled to and revolving after room temperature and to desolventize, 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 after as for 2h in ice bath, the solid of filtration is for the first time with the washing of 10mL frozen water, for the second time with the washing of 30mL ice ether, after draining, obtain 8.7g, productive rate 78%.
1H?NMR(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,
By G006 (8.5g, 50.42mmol) join in 120mL anhydrous pyridine, add again pivalyl chloride (21.68mL, 176.20mmol) and stir 2h under room temperature after revolve and desolventize, be dissolved in 1.7L methylene dichloride, organic phase, with after 0.1M hydrochloric acid soln (2*350mL) washing, is revolved and is desolventized 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,
By G007 (3.10g; 12.27mmol) be dissolved in 60mL THF; nitrogen protection, after masking foil parcel, adds NIS (3.04g; 13.51mmol) under room temperature, stir 1h; add 500mL DCM, use 200mL water washing, after revolving and desolventizing; column chromatography DCM:MeOH99:1 obtains 3.76g, productive rate 81%.
1H?NMR(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(CH
3)
3)。
Step 5,
By G008 (1.5g; 4.0mmol) and ammonium sulfate (15mg; 0.11mmol) at hexamethyldisilazane (15mL; 72.76mmol), backflow 20h is in the protection of argon gas; revolve after desolventizing and add 40mL ethylene dichloride; add Sm-1 (2.304; 6.0mmol) and TMSOTf (1.25mL; 6.47mmol) and after stirring to clarify under 50 degrees Celsius, stir 24h under room temperature; add 60mL DCM, and with 30mL saturated sodium bicarbonate and saturated common salt water washing, revolve except after organic phase; 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, with Glacial acetic acid, be neutralized to neutral rear column chromatography DCM:MeOH5:1 and obtain compound dG
1-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 is placed in sodium hydroxide solution (2N) backflow 4h, adds 2N hydrochloric acid soln after cooling, and regulator solution pH is 6.Concentrated, add 100ml methylene dichloride and methyl alcohol mixed liquor (V:V=1:1) washing, merge organic phase, concentrated that 255mg white solid is dG-I, productive rate 98%.
1h NMR (400MHz, Me0D) 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 synthetic of 7-denitrification-7-bromine and chloro-2 '-deoxidation guanosine-dG-Br/C1 equally, when difference is four-step reaction, with NBS or BCS, replace 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
By G008 (1.5g; 4.0mmol) and ammonium sulfate (15mg; 0.11mmol) at hexamethyldisilazane (15mL; 72.76mmol), backflow 20h is in the protection of argon gas; revolve after desolventizing and add 40mL ethylene dichloride; add G-I-O (6.0mmol) and TMSOTf (1.25mL; 6.47mmol) and after stirring to clarify under 50 degrees Celsius, stir 24h under room temperature; add 60mL DCM; and with 30mL saturated sodium bicarbonate and saturated common salt water washing; revolve except after organic phase, column chromatography obtains G-I-B compound 1.6g.1H?NMR(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)。
G-I-B (1.056g, 1.5mmol) is joined in 0.5MMeONa/MeOH (20.0mL), after backflow 3h, with Glacial acetic acid, be neutralized to neutral rear column chromatography DCM:MeOH5:1 and obtain 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，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 3,
Compound G-I-A is placed in to sodium hydroxide solution (2N) backflow 4h, adds 2N hydrochloric acid soln after cooling, regulator solution pH is 6.Concentrated, add 100ml methylene dichloride and methyl alcohol mixed liquor (V:V=1:1) washing, merge organic phase, concentrated that 255mg white solid is G-I, productive rate 98%.1H NMR (600MHz, DMSO): 4.99 (br s, 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 synthetic of 7-denitrification-7-bromine and chlorine guanine nucleosides G-Br/C1 equally, difference is that it is bromine or chlorine that the first step is reacted 7 of guanine base of raw material G008 used, and other all reactions steps and method are all identical.
one of synthetic method of embodiment 4,7-denitrification-7-bromine/chloro-2 '-deoxyguanosine 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
By compound dG
1-A (0.20g; 0.714mmol) be dissolved in anhydrous pyridine, slowly drip pivalyl chloride (0.75mL at 0 ℃; 7.14mmol), at 0 ℃, stir after 1h, add 2ml methyl alcohol, stir 10min, screw out solvent, add ethyl acetate (200ml) and saturated sodium bicarbonate solution (50ml) extraction, separated organic phase, adds saturated sodium bicarbonate solution and saturated common salt water washing successively, anhydrous sodium sulfate drying, screw out solvent, silica gel column chromatography [V (ethyl acetate): V (sherwood oil)=1:1], obtaining 0.39g white solid is compound dG
1-B, productive rate 92%.
1H?NMR(400MHz，CD
3OD)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).
By compound dG
1-B (0.42g; 0.84mmol) be dissolved in dry DMF, under vigorous stirring, add 4-bromo-succinimide or 4-chlorosuccinimide (220mg; 0.9mmol), stirring at room 22h, screw out solvent, add 100ml ether and the extraction of 50ml sodium hydrogen carbonate solution, isolate organic phase, saturated sodium-chloride washing, anhydrous sodium sulfate drying, screw out solvent, silica gel column chromatography [V (ethyl acetate): V (sherwood oil)=1:1], obtaining 0.5g white solid is compound dG
1-E, productive rate 91%.
1H-NMR(400MHz，CD
3OD)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 dG
1-E is dissolved in methyl alcohol/sodium methylate (10ml) of 0.5M, at 65 ℃, stir 12h, add again 10ml saturated sodium bicarbonate solution, continue to stir 10min, screw out methyl alcohol, add the extraction of 50ml ethyl acetate, organic layer is respectively with saturated sodium bicarbonate solution and saturated nacl aqueous solution washing, and anhydrous sodium sulfate drying, concentrates, resistates silica gel column chromatography [V (methyl alcohol): V (methylene dichloride)=1:10], obtaining 0.24g white solid is compound dG
1-F, productive rate 74%.
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 4,
By compound dG
1-F is placed in sodium hydroxide solution (2N) backflow 4h, adds 2N hydrochloric acid soln after cooling, and regulator solution pH is 6.Concentrated, add 100ml methylene dichloride and methyl alcohol mixed liquor (V:V=1:1) washing, merge organic phase, concentrated that 255mg white solid is dG
1-G, productive rate 98%.
1H?NMR(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 '-deoxyguanosine dG-I of embodiment 5,7-denitrification-7-is at synthetic dGTP (AP
3
) in purposes
DGTP (AP in the present embodiment
3) synthetic schematic diagram as shown in Figure 5, concrete synthetic method comprises the steps: respectively
In a single port bottle, add compound dG-I (0.25g, 0.4mmol), then take CuI (22mg; 1mmol) and Pd (PPh
3)
4(48mg; 0.04mmol) add in reaction flask, vacuumize, nitrogen protection, aluminium foil parcel, adds 10ml DMF, and stirring and dissolving is injected TEA (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 dG (AP
3), productive rate 39%.
1H?NMR(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).
By compound dG (AP
3) vacuum-drying 12h, in glove box, take respectively compound dG (AP
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 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 2, stirs 1.5h.Add 1mL3% iodine (9:1Py/H2O) solution, keep iodine liquid color 15min not fade.After 15min, add 2mL water, after 2h, add 0.75mL3M 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 (AP
3).Productive rate 26%.DGTP (AP
3)
1h-NMR,
31p-NMR, HRMS spectrogram are distinguished as shown in Figure 8,9, 10,
1h NMR (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);
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. notes: present method is applicable to 7-denitrification-7-bromine/chloro-2 '-deoxyguanosine dG-Br/C1 equally at synthetic dGTP (AP
3) in purposes, difference is the first step when reaction to replace dG-I with dG-Br/C1, 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 embodiment
3) 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) and Pd (PPh
3)
4(48mg; 0.04mmol) add in reaction flask, vacuumize, nitrogen protection, aluminium foil parcel, adds 10ml DMF, and stirring and dissolving is injected TEA (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) 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 synthetic G (AP
3) in purposes, difference is the first step when reaction to replace G-I with G-Br/C1, other all reactions steps and method are all identical.
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 7-denitrification-7-9 substituted guanine nucleosides, is characterized in that, described method comprises the steps:
A, formula (III) compound obtain formula (IV1) or (IV2) compound in the alkaline condition protecting group of going down;
B, described formula (IV1) or (IV2) compound demethyl under alkaline condition obtain formula (I) compound, i.e. described 7-denitrification-7-9 substituted guanine nucleosides;
Wherein, R
1for H or OH, R
2for I, Br or C1, R
3for H or OBz.
2. the synthetic method of 7-denitrification-7-9 substituted guanine nucleosides as claimed in claim 1, is characterized in that, in steps A, and R
3during for H, production (IV1) compound; R
3during for OBz, production (IV2) compound.
3. the synthetic method of 7-denitrification-7-9 substituted guanine nucleosides as claimed in claim 2, is characterized in that, described formula (III) compound through type (II) compound and compound
or
there is glycosylation reaction preparation and obtain,
4. the synthetic method of 7-denitrification-7-9 substituted guanine nucleosides as claimed in claim 3, is characterized in that, described formula (II) compound passes through at compound G007
on 7 of purine bases, connect halogen atom preparation and obtain.
5. the synthetic method of 7-denitrification-7-9 substituted guanine nucleosides as claimed in claim 4, is characterized in that, described compound G007 be prepare as follows and:
A, compound G005
synthetic: Sm-1
with Sm-2
under acidic conditions, react, obtain compound G005;
B, compound G006
synthetic: compound G005
under the effect of phosphorus oxychloride, reaction obtains compound G006;
C, compound G007
synthetic: compound G006
under alkaline condition, react with pivalyl chloride and obtain described compound G007.
6. the synthetic method of 7-denitrification-7-propargylamine-2 '-deoxidation guanylic acid, it is characterized in that, described method comprises by synthetic formula (I) compound obtaining of the method for claim 1 further synthesizes described 7-denitrification-7-propargylamine-2 '-deoxidation guanylic acid; R in formula (I)
1for H.
7. the synthetic method of 7-denitrification-7-propargylamine-2 '-deoxidation guanylic acid as claimed in claim 6, is characterized in that, comprises the steps:
A, compound dG (AP
3) synthetic: at CuI, Pd (PPh
3)
4under the condition existing with TEA, trifluoroacetyl propargylamine and formula (I) compound
reaction, obtains compound dG (AP
3)
described formula (I) compound, trifluoroacetyl propargylamine, CuI, Pd (PPh
3)
4with the mol ratio of TEA be 1:(2～3): 0.072:0.025:(1.5～2);
B, compound dGTP (AP
3) synthetic: compound dG (AP
3) with 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 is gone 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.
8. a synthetic method for 7-denitrification-7-propargylamine-guanosine-, is characterized in that, described method comprises by synthetic formula (I) compound obtaining of the method for claim 1 further synthesizes described 7-denitrification-7-propargylamine-guanosine-; R in formula (I)
1for 0H.
9. the synthetic method of 7-denitrification-7-propargylamine-guanosine-as claimed in claim 8, is characterized in that, comprises the steps:
At CuI, Pd (PPh
3)
4under the condition existing with TEA, trifluoroacetyl propargylamine and formula (I) compound
reaction, obtains compound G (AP
3)
it is described 7-denitrification-7-propargylamine-guanosine-; Described formula (I) compound, trifluoroacetyl propargylamine, CuI, Pd (PPh
3)
4with the mol ratio of TEA be 1:(2～3): 0.072:0.025:(1.5～2).
10. the purposes of 7-denitrification-7-9 substituted guanine nucleosides that a synthetic method as claimed in claim 1 makes in synthetic 7-denitrification-7-propargylamine-guanosine-.
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