CN102199183B - C-di-GMP, analogues thereof and preparation method thereof - Google Patents
C-di-GMP, analogues thereof and preparation method thereof Download PDFInfo
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- CN102199183B CN102199183B CN 201010135002 CN201010135002A CN102199183B CN 102199183 B CN102199183 B CN 102199183B CN 201010135002 CN201010135002 CN 201010135002 CN 201010135002 A CN201010135002 A CN 201010135002A CN 102199183 B CN102199183 B CN 102199183B
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Abstract
The invention discloses a c-di-GMP and analogues thereof which have structures of a general formula I. The invention also discloses a novel preparation method-a kettle phosphoramidite method which can be used for rapidly, simply and conveniently preparing c-di-GMP compounds with high yield and low cost on a large scale and at mild conditions. The c-di-GMP is prevalent in bacteria and is a novel second messenger molecular which takes part in regulating multiple physiological functions. The research shows that the c-di-GMP and the analogues thereof can inhibit the formation of bacterium biological membranes and the multiplication of eukaryotic cells; and thereof the c-di-GMP and the analogues thereof have good medicine development prospect.
Description
Technical field
The present invention relates to encircle the dinucleotides compounds, relate in particular to c-di-GMP (c-di-GMP) and analogue thereof, also relate to the preparation method of this compounds, be specifically related to apply the synthetic c-di-GMP of a still phosphoramidite method and analogue thereof.
Background technology
C-di-GMP (c-di-GMP) is ubiquity in bacterium, participates in regulating the novel second messenger molecule of different physiological roles, not only has important physiologically active, also participates in many important biological respinses.Research in recent years shows, c-di-GMP and the similar thing of ring dinucleotides thereof can the biomembranous formation of anti-bacteria and eukaryotic propagation, therefore have good drug development prospect.
Yet up to the present the signal transduction mechanism of c-di-GMP is still unknown, this is mainly because its action target is unknown, and the c-di-GMP that obtains q.s just becomes one of prerequisite of further research c-di-GMP biological action.Because c-di-GMP is few at the content of occurring in nature, being difficult to obtain by natural mode can be for enough c-di-GMP of research, and the chemical synthesis process that therefore develops efficient c-di-GMP and analogue thereof is very important.
The synthetic method of having reported at present be mainly the guanosine of protecting be initial phosphotriester method or hydrogen-phosphonate method, also have that to take comparatively simple protection sugar be initial method.But there is obvious defect mostly in these methods: the violent unstable products of the cyclization productive rate had condition lower, that have, the route had deprotection long, that have can not carry out well, and these problems have all limited a large amount of c-di-GMP of chemosynthesis.
Summary of the invention
In order to overcome the defect of above-mentioned report method, the purpose of this invention is to provide class c-di-GMP and an analogue thereof, and extensive, high yield, low cost, mild condition, the fast and convenient method for preparing c-di-GMP and analogue thereof.
For achieving the above object, the present invention adopts following technical scheme:
One class c-di-GMP and analogue thereof, two ribose unit are connected by 3 '-5 ' phosphodiester bond in two molecules, and form a twelve-ring shape sugar-phosphoric acid skeleton, have the structure meaned as general formula I:
Wherein, B
1, B
2be respectively natural base A, C, G, T, U and any manually modified base;
X
1, X
2be respectively OH, H, OR, R, halogen, SH, SR, NH
2, NHR, NR or CN;
Y
1, Y
2be respectively O, S or Se;
Z
1, Z
2be respectively OH, SH, SeH, BR or OR;
Wherein said R is C1-C4 alkyl or ternary-seven-membered ring; Described halogen is F, Cl, Br or I.
Preferably, B
1, B
2for natural base A, C, G, T or U, in the two, at least one is G; X
1, X
2be respectively OH, H, OR or halogen; Y
1, Y
2be respectively O; Z
1, Z
2be respectively OH; Wherein, described R is the C1-C4 alkyl, preferable methyl; Described halogen is F, Cl, Br or I, preferably F.
In preferred embodiment of the present invention, described c-di-GMP and analogue thereof are:
C-di-GMP (c-di-GMP, B in general formula I
1, B
2=G, X
1, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH);
Ring 2 '-fluorine cytidylic acid-(3 '-5 ')-guanylic acid (c (F) CpGp, B in general formula I
1=C, B
2=G, X
1=F, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH);
Ring 2 '-methoxyl group guanylic acid-(3 '-5 ')-guanylic acid (c (MeO) GpGp, B in general formula I
1, B
2=G, X
1=OMe, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH);
Ring 2 '-fluorouridylic acid-(3 '-5 ')-guanylic acid (c (F) UpGp, B in general formula I
1=U, B
2=G, X
1=F, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH);
Ring 2 '-deoxythymidylic acid-(3 '-5 ')-guanylic acid (cdTpGp, B in general formula I
1=T, B
2=G, X
1=H, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH).
The present invention also provides a kind of phosphoramidite reagent that utilizes, and efficiently prepares in large quantities the method for c-di-GMP and analogue thereof, i.e. a still phosphoramidite method.
The c-di-GMP that general formula I means and the preparation method of analogue thereof comprise the steps:
(1) by N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine; 5 '-((4; 4 '-dimethoxy)-trityl)-3 '-((2-cyanoethyl-N; the N-di-isopropyl)-phosphoramidite base)-nucleosides and 1H-tetrazole under protection of inert gas in anhydrous solvent room temperature reaction 4-6 hour; evaporate to dryness after organic solvent extraction adds peroxy tert-butyl alcohol, trifluoroacetic acid room temperature reaction rear pillar separates to obtain P-(2-cyanoethyl)-nucleosides-(3 '-5 ')-N in solvent
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine;
(2) by P-(2-cyanoethyl)-nucleosides-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine, the 1H-tetrazole is placed in anhydrous solvent under protection of inert gas, drip 2-cyanoethoxyl-N, N, N ', N '-tetra-isopropylamine base phosphoramidite room temperature reaction 4-6 hour, evaporate to dryness after organic solvent extraction adds after the peroxy tert-butyl alcohol stirring reaction to obtain crude product cyclization product in solvent;
(3) crude product cyclization product is dissolved in the tetrahydrofuran solution of 1M tetrabutyl ammonium fluoride, adds after strong aqua stirs and extract, partly prepare liquid phase separation, obtain c-di-GMP (c-di-GMP) and analogue thereof.
Wherein said nucleosides be ethanoyl protection or unprotected 2 '-t-Butyldimethylsilyl guanosine, 2 '-fluorine cytidine, 2 '-methoxyl group guanosine, 2 '-floxuridine, 2 '-deoxythymidine etc.; Described solvent is selected from acetonitrile, methylene dichloride, chloroform, tetrahydrofuran (THF) etc.
Wherein the described post separation condition of step (1) is preferably the normal pressure column chromatography, and eluent is 15: 1 methylene chloride/methanol.
Wherein the described condition optimization that partly prepares liquid phase separation of step (2) is: mobile phase A=0.05M acetic acid triethylamine buffer solution, B=acetonitrile; Gradient 0-60min, A 100% to A 80%/B 20%; Detect wavelength 254nm, flow velocity 5ml/min.
Wherein said N
2prepared according to the following steps by-ethanoyl-2 '-t-Butyldimethylsilyl guanosine:
5 '-(4,4 '-dimethoxy)-trityl-N
2add sour organic solution in-ethanoyl-2 '-t-Butyldimethylsilyl guanosine, stirring at room 15 minutes, decompression post sharp separation, obtain N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine.Wherein said acid is selected from trifluoracetic acid, acetic acid etc., and organic solvent is selected from methylene dichloride, chloroform, tetrahydrofuran (THF) etc.
Wherein said crude product cyclization product also can be made c-di-GMP and analogue thereof according to the following steps:
Crude product cyclization product is dissolved in fluorine-containing desiliconization protection reagent, and stirring at room 12 hours, add alkali, stirring at room 40 hours, and evaporate to dryness alkali, residuum is soluble in water, chloroform extraction three times, the evaporate to dryness water, partly prepare liquid phase separation, obtains c-di-GMP and analogue thereof.Wherein said fluorine-containing desiliconization protection reagent is selected from triethylamine trihydrofluoride, triethylamine hydrofluoride, hydrogen fluoride pyridine etc., and alkali is selected from strong aqua, methylamine alcohol solution etc.
The invention provides a class ring dinucleotides compounds, be specifically related to class c-di-GMP and an analogue thereof, this compounds can the biomembranous formation of anti-bacteria and eukaryotic propagation, therefore has good drug development prospect.The present invention also provides a kind of method of utilizing the synthetic ring of phosphoramidite reagent dinucleotides compounds.With other report method compare, this method has that productive rate is high, route is short, easy and simple to handle, reaction conditions is gentle, reagent is cheap and easy to get and protecting group removes the advantage that waits fully, is conducive to prepare on a large scale c-di-GMP (c-di-GMP) and analogue thereof.Utilize this method partly to be modified sugared loop section and the phosphoric acid of ring dinucleotides compounds easily, other similar thing of ring dinucleotides of difficult preparation of report method is applied simultaneously.
Embodiment
All solvents, raw material and reagent, if do not specialized, are analytical pure or chemical pure.The Non-aqueous processing of solvent carries out according to ordinary method.
The instrument of product separation, evaluation and method: tlc silica gel GF254 (60 type) produces for Qingdao Haiyang Chemical Industry Group Corp., column layer chromatography silicone rubber (200-300 order) is that Industrial Co., Ltd. of upper marine nation produces, and the import column layer chromatography silicone rubber is that German Merck company produces; TLC is by the 254nm ultraviolet detection; HR-FAB is used Bruker BIFLEXTMIII mass spectrograph to measure; Nuclear magnetic resonance spectrum is used Varian VXR-500, Bruker400, JOEL AL300 nmr determination; Hydrogen spectrum, carbon spectrum be take TMS as interior mark, and phosphorus is composed with 85%H
3pO
4for external standard; HPLC is used Gilson high performance liquid phase instrument, uses Angela Venusil XBP C-18 semipreparative column to separate.
Embodiment 1 c-di-GMP (c-di-GMP, B in general formula I
1, B
2=G, X
1, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH)
Reagent and reaction conditions: trifluoracetic acid i.3%; The ii.1H-tetrazole, acetonitrile; Iii. peroxy tert-butyl alcohol; The iv.3% trifluoracetic acid; V.2-cyanoethoxyl-N, N, N ', N '-tetra-isopropylamine base phosphoramidite, 1H-tetrazole, acetonitrile; Vi. peroxy tert-butyl alcohol; The tetrahydrofuran solution of vii.1M tetrabutyl ammonium fluoride; Viii. strong aqua.
1. synthesize N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine
5 '-(4,4 '-dimethoxy)-trityl)-N
2add 3%CF in-ethanoyl-2 '-t-Butyldimethylsilyl guanosine (buying 122mg, 0.164mmol from Shanghai JiMa pharmacy Technology Co., Ltd)
3the CH of COOH
2c1
2solution 5ml, stirring at room 15 minutes, decompression post sharp separation, obtain N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine.
1H?NMR(DMSO-d
6，500MHz)δ12.04(s，1H，H-1)，11.69(s，1H，2-NH)，8.26(s，1H，H-8)，5.83(d，1H，H-1’)，5.55-5.57(dd，1H，H-2’)，5.03(brs，1H，5-OH)，4.08-4.10(m，1H，H-4’)，3.94-3.96(m，1H，H-3’)，3.60-3.67(dd，1H，H-5’)，3.54-3.59(dd，1H，H-5’)，2.17(s，3H，-COCH
3)，0.71(s，9H，-C(CH
3)
3)，-0.08(s，3H，-SiCH
3)，-0.19(s，3H，-SiCH
3)；
13C?NMR(DMSO-d
6，125MHz)δ173.5，154.8，149.0，148.0，137.6，120.0，86.2，86.0，76.2，70.7，61.4，40.0，39.9，39.7，39.5，39.3，39.2，39.0，25.5，23.8，17.7，-5.0，-5.4；HRMS(ESI-TOF
+)calcd.for?C
18H
29N
5O
6Si(M+H)440.19599，found?440.19696，calcd.for?C
18H
29N
5O
6Si(M+Na)462.17793，found?462.17850.
Data show that synthetic product is correct.
2. synthesize N
2-ethanoyl-P-(2-cyanoethyl)-2 '-t-Butyldimethylsilyl guanosine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine
N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine (70mg, 0.159mmol), 5 '-((4,4 '-dimethoxy)-trityl)-N
2-ethanoyl-3 '-((2-cyanoethyl-N; the N-di-isopropyl)-phosphoramidite base)-2 '-t-Butyldimethylsilyl guanosine (buying from Shanghai JiMa pharmacy Technology Co., Ltd) (170mg; 0.180mmol) and 1H-tetrazole (34mg; 0.485mmol) the pump drainage argon shield; add anhydrous acetonitrile 4ml, room temperature reaction 5 hours, solvent evaporated; the dissolution sample that adds methylene chloride, 5%NaHCO
3extraction, the methylene dichloride strip aqueous, merge organic phase, anhydrous Na
2sO
4drying, filter, solvent evaporated, and the methylene dichloride dissolution sample, add peroxy tert-butyl alcohol 0.1ml, and stirring at room 10 minutes, add 3%CF
3the CH of COOH
2cl
2solution (5ml) stirring at room approximately 10 minutes, the normal pressure post separates, and obtains N
2-ethanoyl-P-(2-cyanoethyl)-2 '-t-Butyldimethylsilyl guanosine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine.
1H?NMR(DMSO-d
6，500MHz)δ12.05(s，1H，H-1)，12.03(s，1H，H-1)，11.73(s，1H，2-NH)，11.59(s，1H，2-NH)，8.26(s，1H，H-8)，8.23(s，1H，H-8)，5.85(dd，2H，H-1’)，5.30(d，1H，J＝5.5Hz，H-3’)，4.77-4.82(m，2H，H-4’)，4.61(t，1H，J＝5.5Hz，5.5Hz，H-3’)，4.32-4.33(m，2H，H-2’)，4.15-4.20(dd，4H，H-5’)，3.59(d，1H，J＝2.5Hz，OH-3’)，2.89(t，1H，J＝5.8Hz，5.8Hz，OH-5’)，2.49(t，4H，J＝1.8Hz，1.8Hz，-CH
2CH
2CN)，2.17(s，3H，-COCH
3)，2.16(s，3H，-COCH
3)，0.742(s，9H，-C(CH
3)
3)，0.655(s，9H，-C(CH
3)
3)，-0.026(s，3H，-SiCH
3)，-0.098(s，3H，-SiCH
3)，-0.147(s，3H，-SiCH
3)，-0.264(s，3H，-SiCH
3)；
13C?NMR(DMSO-d
6，125MHz)δ173.5，154.7，149.1，148.7，148.2，148.0，138.0，137.1，120.4，120.0，118.0，87.0，85.0，84.5，82.9，78.2，75.1，74.7，69.8，67.8，62.6，60.8，54.9，39.5，29.0，25.5，25.2，23.8，19.0，17.7，17.5，13.9，-4.9，-5.3，-5.4，-5.9；
31P?NMR(DMSO-d
6，121.5MHz)δ-0.58687(s)；HRMS(ESI-TOF
+)calcd.for?C
39H
60N
11O
14P
1Si
2(M+H)994.36701，found?994.36649，calcd.for?C
39H
60N
11O
14PSi
2(M+Na)1016.34896，found?1016.34631.
Data show that synthetic product is correct.
3. synthesize c-di-GMP
N
2-ethanoyl-P-(2-cyanoethyl)-2 '-t-Butyldimethylsilyl guanosine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine (35mg, 0.035mmol), 1H-tetrazole (15mg; 0.214mmol) predrying, the pump drainage argon shield, add anhydrous acetonitrile 4ml; stirring at room half an hour, drip 2-cyanoethoxyl-N, N; N '; N '-tetra-isopropylamine base phosphoramidite (0.05ml, 0.157mmol), room temperature reaction 5 hours; add a small amount of water termination reaction, 5%NaHCO
3extraction, the methylene dichloride strip aqueous, merge organic phase, anhydrous Na
2sO
4drying, filter solvent evaporated, the methylene dichloride dissolution sample, add peroxy tert-butyl alcohol 0.1ml, stirring at room 10 minutes, solvent evaporated, obtain crude product cyclization product 5, is dissolved in the tetrahydrofuran solution 0.8ml of 1M tetrabutyl ammonium fluoride, stirring at room 4 hours, add the 4ml strong aqua, stirring at room 40 hours, solvent evaporated, chloroform extraction three times, evaporate to dryness water, partly prepare liquid phase separation, obtain c-di-GMP (c-di-GMP).
1H?NMR(D
2O，500MHz)δ8.02(s，2H，H-8)，5.94(s，2H，H-1’)，5.00(m，2H，H-3’)，4.80(d，J＝5.0Hz，2H，H-2’)，4.39-4.43(m，4H，H-5’)，4.10(m，1H，H-4’)；
13C?NMR(D
2O，125MHz)δ158.7，154.3，150.8，137.6，117.0，90.7，80.8，74.0，71.1，63.8，59.7，47.4，43.0，11.2，8.9，8.2；
31P?NMR(D
2O，121.5MHz)δ2.50(s)；HRMS(ESI-TOF
-)calcd.for?C
20H
24N
10O
14P
2(M-H)689.0876，found?689.0876.
Data show that synthetic product is correct.
Embodiment 2 ring 2 '-fluorine cytidylic acids-(3 '-5 ')-guanylic acid (c (F) CpGp, B in general formula I
1=C, B
2=G, X
1=F, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH)
5 '-[(4,4 '-dimethoxy)-trityl]-N
4-ethanoyl-3 '-[(2-cyanoethyl-N, N-di-isopropyl)-phosphoramidite base]-2 '-fluorine cytidine (buying from Shanghai JiMa pharmacy Technology Co., Ltd) (142mg, 0.180mmol), N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine (70mg, 0.159mmol), and 1H-tetrazole (34mg; 0.485mmol) the pump drainage argon shield, add anhydrous acetonitrile 4ml, room temperature reaction 5 hours; solvent evaporated, the dissolution sample that adds methylene chloride, 5%NaHCO
3extraction, the methylene dichloride strip aqueous, merge organic phase, anhydrous Na
2sO
4drying, filter, solvent evaporated, and the methylene dichloride dissolution sample, add peroxy tert-butyl alcohol 0.1ml, and stirring at room 10 minutes, add 5ml 3%CF
3the CH of COOH
2cl
2solution room temperature stir about 10 minutes, the normal pressure post separates, and obtains N
4-ethanoyl-P-(2-cyanoethyl)-2 '-fluorine cytidine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine.
1H?NMR(DMSO-d
6，500MHz)δ12.04(s，1H，H-1)，11.62(s，1H，2-NH)，10.94(s，1H，4-NH)，8.31(s，1H，H-8)，8.19(d，1H，H-6)，7.20(d，1H，H-5)，5.97(d，1H，H-1’)，5.42(d，1H，H-1’)，5.30(d，1H，J＝5.5Hz，H-3’)，4.91-5.02(m，1H，H-4’)，4.59(t，1H，J＝5.5Hz，5.5Hz，H-3’)，4.32-4.33(m，2H，H-2’)，4.08-4.24(m，6H)，3.78(dd，1H，H-5’)，3.62(d，1H，OH-3’)，2.89(t，1H，J＝5.8Hz，5.8Hz，OH-5’)，2.17(s，3H，-COCH
3)，2.09(s，3H，-COCH
3)，1.22(s，3H，-SiCH
3)，0.86(s，3H，-SiCH
3)，0.73(s，9H，-C(CH
3)
3)；
31P?NMR(DMSO-d
6，121.5MHz)δ-1.82093(s)；HRMS(ESI-TOF+)calcd.for?C
32H
45FN
9O
13PSi(M+H)842.27005，found842.26861，calcd.for?C
32H
45FN
9O
13PSi(M+Na)864.25200，found?864.25201.
Data show that synthetic product is correct.
Other step, with example 1, obtains c (F) CpGp (B in general formula I
1=C, B
2=G, X
1=F, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH).
1H?NMR(D
2O，400MHz)δ7.92(s，1H，H-8)，7.90(d，1H，J＝7.6Hz，H-6)，5.93(s，1H，H-1’)，5.87(s，1H，H-1’)，5.81(d，1H，J＝7.6Hz，H-5)，5.12(dd，1H，J＝4.0Hz，52.4Hz，4.0Hz，H-2’-C)，4.23-4.31(m，5H，H-5’，H-2’-G)，3.94-4.00(m，2H，H-4’)，3.57(d，1H，J＝37.6Hz，H-3’-C)；HRMS(ESI-TOF-)calcd.forC
19H
22FN
8O
13P
2(M-H)651.07711，found?651.07749.
Data show that synthetic product is correct.
Embodiment 3 ring 2 '-methoxyl group guanylic acids-(3 '-5 ')-guanylic acid (c (MeO) GpGp, B in general formula I
1, B
2=G, X
1=OMe, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH)
5 '-((4,4 '-dimethoxy)-trityl)-N
2-ethanoyl-3 '-((2-cyanoethyl-N, N-di-isopropyl)-phosphoramidite base)-2 '-methoxyl group guanosine (buying from Shanghai JiMa pharmacy Technology Co., Ltd) (151mg, 0.180mmol), N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine (70mg, 0.159mmol), and 1H-tetrazole (34mg; 0.485mmol) the pump drainage argon shield, add anhydrous acetonitrile 4ml, room temperature reaction 5 hours; solvent evaporated, the dissolution sample that adds methylene chloride, 5%NaHCO
3extraction, the methylene dichloride strip aqueous, merge organic phase, anhydrous Na
2sO
4drying, filter, solvent evaporated, and the methylene dichloride dissolution sample, add peroxy tert-butyl alcohol 0.1ml, and stirring at room 10 minutes, add 3%CF
3the CH of COOH
2cl
2solution (5ml) stirring at room approximately 10 minutes, the normal pressure post separates, and obtains N
2-ethanoyl-P-(2-cyanoethyl)-2 '-methoxyl group guanosine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine.
1H?NMR(DMSO-d
6，500MHz)δ12.03(s，2H，H-1)，11.68(s，1H，2-NH)，11.61(s，1H，2-NH)，8.30(s，1H，H-8)，8.23(s，1H，H-8)，5.85(dd，2H，H-1’)，5.32(d，1H，J＝5.5Hz，H-3’)，5.03-5.11(m，1H，H-3’)，4.58-4.62(m，2H，H-4’)，3.58(dd，2H，-CH
2CH
2CN)，3.36(d，1H，OH-3’)，2.91(t，1H，J＝5.8Hz，5.8Hz，OH-5’)，2.18(s，6H，-COCH
3)，0.91(s，3H，-SiCH
3)，0.74(s，3H，-SiCH
3)；
31P?NMR(DMSO-d
6，121.5MHz)δ-1.10193(s)；HRMS(ESI-TOF
+)calcd.forC
34H
48N
11O
14PSi(M+H)894.29619，found?894.29512，calcd.for?C
34H
48N
11O
14PSi(M+Na)916.27813，found?916.27729.
Data show that synthetic product is correct.
Other step, with example 1, obtains c (MeO) GpGp (B in general formula I
1, B
2=G, X
1=OMe, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH).
1H?NMR(D
2O，400MHz)δ7.91(s，1H，H-8)，7.89(s，1H，H-8)，5.84(s，1H，H-1’)，5.75(s，1H，H-1’)，4.88-4.97(m，2H，H-3’)，4.34(d，1H，J＝5.2Hz，H-2’)，4.20-2.28(m，4H，H-4’，H-5’)，3.59(s，3H，-CH
3)；
31P?NMR(D
2O，600MHz)δ-2.11(s)，-2.38(s)；HRMS(ESI-TOF)calcd.for?C
21H
25N
10O
14P
2(M-H)703.10324，found?703.10070.
Data show that synthetic product is correct.
Embodiment 4 ring 2 '-fluorouridylic acid-(3 '-5 ')-guanylic acid (c (F) UpGp, B in general formula I
1=U, B
2=G, X
1=F, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH)
5 '-((4,4 '-dimethoxy)-trityl)-3 '-((2-cyanoethyl-N, the N-di-isopropyl)-phosphoramidite base)-2 '-floxuridine (buying from Shanghai JiMa pharmacy Technology Co., Ltd) (135mg, 0.180mmol), N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine (70mg, 0.159mmol), and 1H-tetrazole (34mg; 0.485mmol) the pump drainage argon shield, add anhydrous acetonitrile 4ml, room temperature reaction 5 hours; solvent evaporated, the dissolution sample that adds methylene chloride, 5%NaHCO
3extraction, the methylene dichloride strip aqueous, merge organic phase, anhydrous Na
2sO
4drying, filter, solvent evaporated, and the methylene dichloride dissolution sample, add peroxy tert-butyl alcohol (0.1ml), and stirring at room 10 minutes, add 3%CF
3the CH of COOH
2cl
2solution (5ml) stirring at room approximately 10 minutes, the normal pressure post separates, and obtains P-(2-cyanoethyl)-2 '-floxuridine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine.
Other step, with example 1, obtains c (F) UpGp (B in general formula I
1=U, B
2=G, X
1=F, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH).
1H?NMR(D
2O，400MHz)δ7.97(s，1H，H-8)，7.75(d，1H，J＝8.4Hz，H-6)，5.88(s，1H，H-1’)，5.84(s，1H，H-1’)，5.64(d，1H，J＝8.4Hz，H-5)，5.20(dd，1H，J＝4.4Hz，52.4Hz，4.4Hz，H-2’-U)，4.19-4.26(m，4H，H-5’)，3.91-3.96(m，2H，H-4’)，3.55(d，1H，J＝33.2Hz，H-3’-U)；
31P?NMR(D
2O，600MHz)δ-2.11(s)，-2.22(s)；HRMS(ESI-TO?F
-)calcd.for?C
19H
21FN
7O
14P
2(M-H)652.06113，found652.06296.
Data show that synthetic product is correct.
Embodiment 5 ring 2 '-deoxythymidylic acid-(3 '-5 ')-guanylic acid (cdTpGp, B in general formula I
1=T, B
2=G, X
1=H, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH)
5 '-((4,4 '-dimethoxy)-trityl)-3 '-((2-cyanoethyl-N, the N-di-isopropyl)-phosphoramidite base)-2 '-deoxythymidine (buying from Shanghai JiMa pharmacy Technology Co., Ltd) (134mg, 0.180mmol), N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine (70mg, 0.159mmol), and 1H-tetrazole (34mg; 0.485mmol) the pump drainage argon shield, add anhydrous acetonitrile 4ml, room temperature reaction 5 hours; solvent evaporated, the dissolution sample that adds methylene chloride, 5%NaHCO
3extraction, the methylene dichloride strip aqueous, merge organic phase, anhydrous Na
2sO
4drying, filter, solvent evaporated, and the methylene dichloride dissolution sample, add peroxy tert-butyl alcohol (0.1ml), and stirring at room 10 minutes, add 3%CF
3the CH of COOH
2cl
2solution (5ml) stirring at room approximately 10 minutes, the normal pressure post separates, and obtains P-(2-cyanoethyl)-2 '-deoxythymidine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine.
1H?NMR(DMSO-d
6，400MHz)δ8.19(s，1H，H-8)，7.62(d，1H，H-6)，6.14(dd，1H，J＝6.0Hz，2.8Hz，6.0Hz，H-1’-T)，5.84(d，1H，J＝6.4Hz，H-1’-G)，4.92-4.95(m，1H，H-3’-T)，4.60(t，1H，J＝5.4Hz，5.4Hz，H-3’-G)，4.28-4.31(m，2H，H-4’)，4.13-4.20(m，4H，H-5’)，3.52(d，1H，J＝3.2Hz，OH-3’)，2.90(t，1H，J＝5.8Hz，5.8Hz，OH-5’)，2.16(s，3H，-COCH
3)，1.20(s，3H，-SiCH
3)，0.88(s，3H，-SiCH
3)，0.71(s，9H，-C(CH
3)
3)；
31P?NMR(DMSO-d
6，600MHz)δ-2.71(s)；HRMS(ESI-TOF
+)calcd.for?C
31H
45N
8O
13PSi?(M+H)797.26858，found?797.26847，calcd.forC
31H
45N
8O
13PSi(M+Na)916.27813，found?916.27729.
Data show that synthetic product is correct.
Other step, with example 1, obtains cdTpGp (B in general formula I
1=T, B
2=G, X
1=H, X
2=OH, Y
1, Y
2=O, Z
1, Z
2=OH).
1H?NMR(D
2O，400MHz)δ7.97(s，1H，H-8)，7.75(d，1H，J＝8.4Hz，H-6)，5.88(s，1H，H-1’)，5.84(s，1H，H-1’)，5.64(d，1H，J＝8.4Hz，H-5)，5.20(dd，1H，J＝4.4Hz，52.4Hz，4.4Hz，H-2’-U)，4.19-4.26(m，4H，H-5’)，3.91-3.96(m，2H，H-4’)，3.55(d，1H，J＝33.2Hz，H-3’-U)；
31P?NMR(D
2O，600MHz)δ-1.52(s)，-1.95(s)；HRMS(ESI-TOF
-)calcd.for?C
20H
25N
7O
14P
2(M-H)648.08620，found648.08500.
Data show that synthetic product is correct.
Embodiment 6 c-di-GMPs (c-di-GMP, B1 in general formula I, B2=G, X1, X2=OH, Y1, Y2=O, Z1, Z2=OH).
Adopt the synthetic N of method in embodiment 1
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine, and N
2-ethanoyl-P-(2-cyanoethyl)-2 '-t-Butyldimethylsilyl guanosine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine.
N
2-ethanoyl-P-(2-cyanoethyl)-2 '-t-Butyldimethylsilyl guanosine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine (35mg, 0.035mmol), 1H-tetrazole (15mg; 0.214mmol) predrying, the pump drainage argon shield, add anhydrous acetonitrile 4ml; stirring at room half an hour, drip 2-cyanoethoxyl-N, N; N '; N '-tetra-isopropylamine base phosphoramidite (0.05ml, 0.157mmol), room temperature reaction 5 hours; add a small amount of water termination reaction, 5%NaHCO
3extraction, the methylene dichloride strip aqueous, merge organic phase, anhydrous Na
2sO
4drying, filter, solvent evaporated, and the methylene dichloride dissolution sample, add peroxy tert-butyl alcohol 0.1ml, stirring at room 10 minutes, solvent evaporated, obtain crude product cyclization product 5.By crude product cyclization product 5 (39mg, 0.035mmol) be dissolved in triethylamine trihydrofluoride (0.58ml, 3.56mmol) in, 25 ℃ are stirred 12 hours, add the 7.8ml strong aqua, stirring at room 40 hours, solvent evaporated, chloroform extraction three times, evaporate to dryness water, partly prepare liquid phase separation, obtain c-di-GMP (c-di-GMP).
1H?NMR(D
2O，500MHz)δ8.02(s，2H，H-8)，5.94(s，2H，H-1’)，5.00(m，2H，H-3’)，4.80(d，J＝5.0Hz，2H，H-2’)，4.39-4.43(m，4H，H-5’)，4.10(m，1H，H-4’)；
13C?NMR(D
2O，125MHz)δ158.7，154.3，150.8，137.6，117.0，90.7，80.8，74.0，71.1，63.8，59.7，47.4，43.0，11.2，8.9，8.2；
31P?NMR(D
2O，121.5MHz)δ2.50(s)；HRMS(ESI-TOF
-)calcd.for?C
20H
24N
10O
14P
2(M-H)689.0876，found?689.0876.
Data show that synthetic product is correct.
The foregoing is only preferred embodiment of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive.Those skilled in the art is understood, and in the spirit and scope that limit in the claims in the present invention, can carry out many changes to it, revise, and even equivalence, but all will fall within the scope of protection of the present invention.
Claims (2)
1. the synthetic method of c-di-GMP, comprise the steps:
(1) synthetic N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine
122mg5 '-(4,4 '-dimethoxy)-trityl)-N
2add 3%CF in-ethanoyl-2 '-t-Butyldimethylsilyl guanosine
3the CH of COOH
2cl
2solution 5ml, stirring at room 15 minutes, decompression post sharp separation, obtain N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine;
(2) synthetic N
2-ethanoyl-P-(2-cyanoethyl)-2 '-t-Butyldimethylsilyl guanosine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine
70mg N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine, 170mg5 '-((4,4 '-dimethoxy)-trityl)-N
2-ethanoyl-3 '-((2-cyanoethyl-N, N-di-isopropyl)-phosphoramidite base)-2 '-t-Butyldimethylsilyl guanosine and argon shield of 34mg1H-tetrazole pump drainage, add the 4ml anhydrous acetonitrile; room temperature reaction 5 hours; solvent evaporated, the dissolution sample that adds methylene chloride, 5%NaHCO
3extraction, the methylene dichloride strip aqueous, merge organic phase, anhydrous Na
2sO
4drying, filter, solvent evaporated, and the methylene dichloride dissolution sample, add the 0.1ml peroxy tert-butyl alcohol, and stirring at room 10 minutes, add 5ml3%CF
3the CH of COOH
2cl
2solution stirring at room 10 minutes, the normal pressure post separates, and obtains N
2-ethanoyl-P-(2-cyanoethyl)-2 '-t-Butyldimethylsilyl guanosine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine;
(3) synthetic c-di-GMP
35mgN
2-ethanoyl-P-(2-cyanoethyl)-2 '-t-Butyldimethylsilyl guanosine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine; the 15mg1H-tetrazole is predrying, and the pump drainage argon shield adds the 4ml anhydrous acetonitrile; stirring at room half an hour; drip 0.05ml2-cyanoethoxyl-N, N, N '; N '-tetra-isopropylamine base phosphoramidite; room temperature reaction 5 hours, add a small amount of water termination reaction, 5%NaHCO
3extraction, the methylene dichloride strip aqueous, merge organic phase, anhydrous Na
2sO
4drying, filter solvent evaporated, the methylene dichloride dissolution sample, add the 0.1ml peroxy tert-butyl alcohol, stirring at room 10 minutes, solvent evaporated, obtain crude product cyclization product, and it is dissolved in to 0.8ml, in the tetrahydrofuran solution of 1M tetrabutyl ammonium fluoride, stirring at room 4 hours, add the 4ml strong aqua, stirring at room 40 hours, solvent evaporated, chloroform extraction three times, the evaporate to dryness water, partly prepare liquid phase separation, obtains c-di-GMP;
Wherein, the structural formula of described c-di-GMP is as follows:
2. the synthetic method of c-di-GMP comprises the following steps:
(1) synthetic N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine
122mg5 '-(4,4 '-dimethoxy)-trityl)-N
2add 5ml, 3%CF in-ethanoyl-2 '-t-Butyldimethylsilyl guanosine
3the CH of COOH
2cl
2solution, stirring at room 15 minutes, decompression post sharp separation, obtain N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine;
(2) synthetic N
2-ethanoyl-P-(2-cyanoethyl)-2 '-t-Butyldimethylsilyl guanosine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine
70mgN
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine, 170mg5 '-((4,4 '-dimethoxy)-trityl)-N
2-ethanoyl-3 '-((2-cyanoethyl-N, N-di-isopropyl)-phosphoramidite base)-2 '-t-Butyldimethylsilyl guanosine and argon shield of 34mg1H-tetrazole pump drainage, add the 4ml anhydrous acetonitrile; room temperature reaction 5 hours; solvent evaporated, the dissolution sample that adds methylene chloride, 5%NaHCO
3extraction, the methylene dichloride strip aqueous, merge organic phase, anhydrous Na
2sO
4drying, filter, solvent evaporated, and the methylene dichloride dissolution sample, add the 0.1ml peroxy tert-butyl alcohol, and stirring at room 10 minutes, add 5ml3%CF
3the CH of COOH
2cl
2solution stirring at room 10 minutes, the normal pressure post separates, and obtains N
2-ethanoyl-P-(2-cyanoethyl)-2 '-t-Butyldimethylsilyl guanosine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine;
(3) synthetic c-di-GMP
35mgN
2-ethanoyl-P-(2-cyanoethyl)-2 '-t-Butyldimethylsilyl guanosine-(3 '-5 ')-N
2-ethanoyl-2 '-t-Butyldimethylsilyl guanosine; the 15mg1H-tetrazole is predrying, and the pump drainage argon shield adds the 4ml anhydrous acetonitrile; stirring at room half an hour; drip 0.05ml2-cyanoethoxyl-N, N, N '; N '-tetra-isopropylamine base phosphoramidite; room temperature reaction 5 hours, add a small amount of water termination reaction, 5%NaHCO
3extraction, the methylene dichloride strip aqueous, merge organic phase, anhydrous Na
2sO
4drying, filter, solvent evaporated, and the methylene dichloride dissolution sample, add the 0.1ml peroxy tert-butyl alcohol, stirring at room 10 minutes, solvent evaporated, obtain crude product cyclization product; 39mg crude product cyclization product is dissolved in the 0.58ml triethylamine trihydrofluoride, and 25 ℃ are stirred 12 hours, add the 7.8ml strong aqua, stirring at room 40 hours, and solvent evaporated, chloroform extraction three times, the evaporate to dryness water, partly prepare liquid phase separation, obtains c-di-GMP;
Wherein, the structure of c-di-GMP is as follows:
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