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CN104628574A - Carbonic ester photosensitive reagent as well as preparation method thereof and method for preparing 5'-photosensitive protected nucleoside - Google Patents

Carbonic ester photosensitive reagent as well as preparation method thereof and method for preparing 5'-photosensitive protected nucleoside Download PDF

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CN104628574A
CN104628574A CN201510075776.4A CN201510075776A CN104628574A CN 104628574 A CN104628574 A CN 104628574A CN 201510075776 A CN201510075776 A CN 201510075776A CN 104628574 A CN104628574 A CN 104628574A
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nitrophenyl
preparation
ortho
photosensitive
nucleosides
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石平
林金来
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C201/00—Preparation of esters of nitric or nitrous acid or of compounds containing nitro or nitroso groups bound to a carbon skeleton
    • C07C201/06—Preparation of nitro compounds
    • C07C201/12—Preparation of nitro compounds by reactions not involving the formation of nitro groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/63—One oxygen atom
    • C07D213/64—One oxygen atom attached in position 2 or 6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/56—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical

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Abstract

The invention relates to a carbonic ester photosensitive reagent as well as a preparation method thereof and a method for preparing the 5'-photosensitive protected nucleoside. The structural formula of the carbonic ester photosensitive reagent is shown in the description, wherein X is -OX', imidazole group and substituted imidazole group; the method for preparing the 5'-photosensitive protected nucleoside comprises the following steps: processing the 2'-deoxynucleoside, 2-o-nitrophenyl-1-propyl carbonic ester photosensitive reagent and the triethylamine to obtain the 5'-photosensitive protected nucleoside crude product, purifying the crude product to obtain the 5'-photosensitive protected nucleoside. The method for preparing the 5'-photosensitive protected nucleoside is capable of average increasing the selectivity for 3.4 times, reducing the purifying process, increasing the yield, greatly reducing the production cost and avoiding the use of virulent diphosgene.

Description

The preparation method of a kind of carbonates photosensitive reagents and preparation method thereof, 5 '-protection against light sensitivity nucleosides
Technical field
The invention belongs to technical field of organic synthesis, be specifically related to the preparation method of a kind of carbonates photosensitive reagents and preparation method thereof, 5'-protection against light sensitivity nucleosides.
Background technology
Biochip technology is a kind of High biotechnology utilizing making nucleic acid molecular hybridization principle to combine with microelectronics and formed.Medical diagnosis on disease, drug screening, bio-science research can be widely used in, many fields such as preferred good child-rearing, judicial expertise, Food Hygiene Surveillance, environment measuring of farm crop.Because original position photoetching technique can prepare high density oligonucleotide gene chip; at present; gene chip based on original position photoetching technique is widely adopted, and 5 '-protection against light sensitivity nucleosides and derivative 5 '-photosensitive phosphoramidite monomer thereof are one of critical materialses of such gene chip of preparation.But, in the method (following graphic shown) of existing preparation 5 '-protection against light sensitivity nucleosides,
There is the difficult problem that two common: (1), when introducing 5 '-photosensitive group, owing to using acyl chloride photosensitive reagents, the selectivity that reaction lack is enough, particularly react on a large scale, the more difficult control of selective reaction condition of acyl chloride photosensitive reagents, while generation 5 '-protection against light sensitivity nucleosides (5 '-Photolabile dN), there is by product 3 '-protection against light sensitivity nucleosides (3 '-Photolabile dN) and 3 ' of a great deal of, 5 '-bis-protection against light sensitivity nucleosides (3 ', 5 '-diPhotolabile dN) generate, different according to base, the principal product 5 '-protection against light sensitivity nucleosides generated and by product (3 '-protection against light sensitivity nucleosides and 3 ', 5 '-bis-protection against light sensitivity nucleosides etc.) ratio be generally 5:1 to 2:1 (reference: Sigrid Buhler, Irene Lagoja etc., Helvetica Chimica Acta, 2004, v87 (3), 620-659).Because 3 '-protection against light sensitivity nucleosides is very close with 5 '-protection against light sensitivity nucleosides separating property, make separation 5 '-protection against light sensitivity nucleosides and 3 '-protection against light sensitivity nucleosides quite difficult.Even if be separated, because both easily intersect mutually, cause 5 '-protection against light sensitivity nucleoside product to lose comparatively large, productive rate is on the low side.Therefore, find the reagent primary (1 °) alcohol and secondary (2 °) alcohol to high selectivity, improve the extremely important (reference: Abbas-Alli G.Shaikh of selectivity of reaction, Chem Review, 1996, v96,951-976); (2) trichloromethylchloroformate (diphosgene) (reference: Woffgang Pfleiderer & Heiner Giegrich, must be used when preparing acyl chloride photosensitive reagents, 1998, United States Patent (USP), US5763599), trichloromethylchloroformate toxicity is very large, operate very inconvenient, and serious environment pollution, should avoid as far as possible aborning using.
Summary of the invention
For the deficiencies in the prior art; the invention provides the preparation method of a kind of carbonates photosensitive reagents and preparation method thereof, 5'-protection against light sensitivity nucleosides; the present invention synthesizes a kind of new carbonates photosensitive reagents; it has height reaction preference with to primary (1 °) alcohol and secondary (2 °) alcohol; photosensitive group selectivity is introduced 5 '-position of nucleosides, the productive rate of 5'-protection against light sensitivity nucleosides is significantly improved.
The technical solution used in the present invention is:
A kind of carbonates photosensitive reagents, its structural formula is:
Wherein X is-OX ', imidazolyl, substituted imidazole base, and described substituted imidazole base is glyoxal ethyline base, 1,2,4-triazol radical; In-OX ', X ' is p-nitrophenyl (4-nitrophenyl), pentafluorophenyl group (pentafluorophenyl), 2-pyridyl (2-pyridyl), and succinimido (succinimidyl).
A preparation method for carbonates photosensitive reagents, step comprises:
A, 2-ortho-nitrophenyl-1-propyl alcohol is dissolved in organic solvent, adds carbonic diester class reagent or carbonyl diurethane azole reagent, then add triethylamine, stirred at ambient temperature reaction 6-10h;
B, by reacted solution priority water and saturated sodium-chloride water solution extraction, retain organic solution layer, after organic solution anhydrous sodium sulfate drying, filter, filtrate evaporate to dryness, vacuum-drying, obtains 2-ortho-nitrophenyl-1-propyl carbonate class photosensitive reagents.
In described steps A, organic solvent is selected from methylene dichloride, trichloromethane, the one in ethyl acetate, toluene;
In described steps A, 2-ortho-nitrophenyl-1-propyl alcohol, carbonic diester class reagent or carbonyl diurethane azole reagent, triethylamine concentration are in organic solvent respectively 0.40-0.65mol/L, 0.50-0.75mol/L, 0.40-0.65mol/L.
Described steps A carbonic diester class reagent is selected from two (p-nitrophenyl) carbonic ether, two (penta fluoro benzene) carbonic ether, carbonic acid two-2-pyridine ester, N, N ' one in-succinimidyl carbonate; Carbonyl diurethane azole reagent is selected from N'N-carbonyl dimidazoles, N'N-carbonyl diurethane (glyoxal ethyline), the one in N'N-carbonyl diurethane (1,2,4-triazole);
Described steps A ambient temperature is 10-30 DEG C.
A preparation method for 5'-protection against light sensitivity nucleosides, step comprises:
A, 2 ' of drying-deoxynucleoside is dissolved in the mixed solvent of anhydrous pyridine and methylene dichloride, be cooled to-3 DEG C-3 DEG C, add 2-ortho-nitrophenyl-1-propyl carbonate class photosensitive reagents and triethylamine successively, at room temperature stirring reaction 14-22h, pressure reducing and steaming reaction solvent, obtain 5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-2 '-deoxynucleoside crude product, i.e. 5'-protection against light sensitivity nucleosides crude product;
B, by the extraction of the mixed solution of 5'-protection against light sensitivity nucleosides crude product ethyl acetate and sodium bicarbonate aqueous solution; retain ethyl acetate layer; ethyl acetate layer priority water and saturated sodium-chloride water solution washing; then ethyl acetate layer anhydrous sodium sulfate drying; filter; filtrate evaporate to dryness; product silica gel column chromatography after evaporate to dryness is purified; take methylene chloride/methanol as eluting solvent; collect product component, concentrated to obtain 5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-2 '-deoxynucleoside and 5'-protection against light sensitivity nucleosides.
In described step a, 2 '-deoxynucleoside is selected from 2 '-deoxythymidine, 2 '-deoxyuridine, N-ethanoyl-2 '-Deoxyribose cytidine, N-benzoyl-2'-deoxyadenosine, one in N-isobutyryl-2'-pancreatic desoxyribonuclease;
In described step a, 2 '-deoxynucleoside, 2-ortho-nitrophenyl-1-propyl carbonate class photosensitive reagents and the triethylamine concentration in the mixed solvent of anhydrous pyridine and methylene dichloride is respectively 0.25-0.35mol/L, 0.29-0.39mol/L, 0.26-0.36mol/L.
In described step a, the volume ratio of anhydrous pyridine and methylene dichloride is 4-6:1;
In described step a, ambient temperature is 10-30 DEG C;
In described step b, ethyl acetate and sodium bicarbonate aqueous solution volume ratio are 1-2:1; Sodium bicarbonate aqueous solution mass percentage concentration is 3-5%;
In described step b, in eluting solvent, methylene dichloride and methyl alcohol volume ratio are 20-50:1.
The reaction principle of the preparation method of 5'-protection against light sensitivity nucleosides is as follows:
In formula, B rfor thymus pyrimidine (Thymine is called for short T), N4-acetylcytosine (N 4-Acetyl Cytosine, is called for short Ac-C), N6-benzoyl adenine (N 6-Benzoyl-Adenine, is called for short Bz-A), and N2-isobutyryl guanine (N 2-ibu-Guanine, is called for short ibu-G) etc.Because the carbon atom be connected with ortho-nitrophenyl in 5 '-(2-ortho-nitrophenyl-1-propyl carbonate) has chirality, gained 5'-protection against light sensitivity nucleoside product should be the mixture that ratio is about two diastereomers of 1:1, to product 1h HMR and HPLC clearly can observe the existence of two diastereomers in analyzing.
The present invention uses the carbonates photosensitive reagents primary (1 °) alcohol and secondary (2 °) alcohol to height reaction preference, primary (1 °) alcohol 5 '-OH in the deoxynucleoside optionally protected with deoxynucleoside or base reacts, selectivity generates 5'-protection against light sensitivity nucleosides, required product (5 '-protection against light sensitivity nucleosides) and by product (3 '-protection against light sensitivity nucleosides and 3 ' will be generated, 5 '-bis-protection against light sensitivity nucleosides) ratio from 5:1 to 2:1, (average out to 3.5:1) brings up to 14:1 to 10:1 (average out to 12:1), selectivity on average improves 3.4 times, simplify purification process, improve productive rate, greatly reduce production cost, avoid the trichloromethylchloroformate using severe toxicity simultaneously, for preparation is widely used in the new process that the 5'-protection against light sensitivity nucleosides of biochip technology and derivative thereof provide a kind of highly effective, there is very high using value.
Embodiment
Embodiment 1:
5 '-NPPOC-dT ( 5) preparation
The preparation feedback step of 1a. photosensitive reagents:
Room temperature 25 DEG C, in reaction vessel, by 2-ortho-nitrophenyl-1-propyl alcohol 1[2-(2-nitrophenyl) propan-1-ol, 54.4g, 0.30mol] is dissolved in methylene dichloride (544ml), adds two (p-nitrophenyl) carbonic ether 2[Bis (4-nitrophenyl) carbonate, 0.33mol], then add triethylamine (0.303mol); Stirring at room temperature reaction system, after 6 hours, HPLC shows to react completely, stopped reaction, (2x 300ml, represents with after the extraction of 300ml water reaction soln priority use water, extract once with 300ml water again, coextraction 2 times) and saturated sodium-chloride water solution (300ml) extraction, retain organic (methylene dichloride) layer, after dichloromethane layer anhydrous sodium sulfate drying, filter, filtrate evaporate to dryness, vacuum-drying, obtains p-nitrophenyl carbonic acid (2-ortho-nitrophenyl)-1-propyl ester 3, 87.3g, HPLC purity is 96%, and productive rate is 84%.
1b. selects linked reaction step:
By 2 ' of drying-deoxythymidine 4(Thymidine, 0.20mol) is dissolved in anhydrous pyridine (485ml) and methylene dichloride (97ml), and cooling, adds oil of mirbane carbonic acid (2-ortho-nitrophenyl)-1-propyl ester successively 0 DEG C of priority 3(0.224mol) with triethylamine (0.204mol), finish, in room temperature 25 DEG C of stirring reaction systems, after 14 hours, HPLC shows to react completely, stopped reaction, pressure reducing and steaming reaction solvent, obtains 5 '-[p-nitrophenyl carbonic acid (2-ortho-nitrophenyl)-1-propyl carbonate]-2 '-deoxythymidine 5(5 '-NPPOC-dT) reacting coarse product.Now, HPLC shows that product (5 '-protection against light sensitivity nucleosides) is 14:1 with the ratio of by product (3 '-protection against light sensitivity nucleosides and 3 ', 5 '-bis-protection against light sensitivity nucleosides).
1c, purification step:
By above-mentioned 5 '-NPPOC-dT reacting coarse product ethyl acetate (500ml) and 3% sodium bicarbonate aqueous solution (300ml) extraction, retain ethyl acetate layer, ethyl acetate layer is use water (300ml) and saturated sodium-chloride water solution (300ml) washing successively, then ethyl acetate layer anhydrous sodium sulfate drying, filter, filtrate evaporate to dryness, obtains 5 '-NPPOC-dT crude product.5 '-NPPOC-dT crude product purified by silica gel column chromatography is purified, and take methylene chloride/methanol as eluting solvent, collects product component, concentrates to obtain 5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-2 '-deoxythymidine 5(5 '-NPPOC-dT), 73.7g, HPLC purity is 98.3%, and productive rate is 82%.5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-2 '-deoxythymidine 5the chemical structure of (5 '-NPPOC-dT) is passed through 1h NMR and LC-MS (M +) confirm.
5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-2 '-deoxythymidine 5(5 '-NPPOC-dT): 1h NMR (CDCl 3, 400MHz) δ (ppm) 8.65 (s, NH, diastereomer), 8.63 (s, NH, diastereomer), 7.76 (m, 1-Ar-H, H-NPPOC), 7.57 (t, 1-Ar-H, H-NPPOC), 7.40 (m, 2 Ar-H, H-NPPOC), 7.31 (s, H-6, diastereomer), 7.28 (s, H-6, diastereomer), 6.33 (t, H-1 ', diastereomer), 6.29 (t, H-1 ', diastereomer), 4.31-3.87 (m, 6H, H-3 ', H-4 ', 2H-5 ',-the CH2 of NPPOC), 3.76 (m, 1H,-the CH of NPPOC), 2.61-2.58 (dd, 1H, 3 '-OH), 2.39-2.18 (m, 2H, H-2 '), 1.85, 1.74 (2s, 3H, 5-CH3), 1.38, 1.35 (2d, 3H,-the CH3 of NPPOC), LC-MS:m/z for (M+H) +, calculated value C 20h 24n 3o 9, 450.41., measured value 450.4.
Embodiment 2:
5 '-NPPOC-N-Ac-dC ( 9) preparation
The preparation feedback step of 2a. photosensitive reagents:
At room temperature 20 DEG C, in reaction vessel, by 2-ortho-nitrophenyl-1-propyl alcohol 1[2-(2-nitrophenyl) propan-1-ol, 0.30mol] is dissolved in trichloromethane (544ml), adds two (penta fluoro benzene) carbonic ether 6[Bis (pentafluorophenyl) carbonate0.33mol], then add triethylamine (0.303mol).Stirring at room temperature reaction system, after 9 hours, HPLC shows to react completely, stopped reaction, and reaction soln is use water (2x 300ml) and saturated sodium-chloride water solution (300ml) extraction successively, retains organic (trichloromethane) layer; After trichloromethane layer anhydrous sodium sulfate drying, filter, evaporate to dryness, vacuum-drying, obtains penta fluoro benzene carbonic acid (2-ortho-nitrophenyl)-1-propyl ester 7, 95.1g, HPLC purity is 97%, and productive rate is 81%.
2b. selects linked reaction step:
N-ethanoyl-2 by drying '-Deoxyribose cytidine 8(N-Ac-dC, 0.20mol) is dissolved in anhydrous pyridine (540ml) and methylene dichloride (108ml), and cooling, adds penta fluoro benzene carbonic acid (2-ortho-nitrophenyl)-1-propyl ester successively 0 DEG C of priority 7(0.224mol) with triethylamine (0.204mol); finish; in room temperature 20 DEG C of stirring reaction systems; after 18 hours; HPLC shows to react completely; stopped reaction, pressure reducing and steaming reaction solvent, obtains 5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-N-ethanoyl-2 '-Deoxyribose cytidine 9(5 '-NPPOC-N-Ac-dC) reacting coarse product.Now, HPLC shows that product (5 '-protection against light sensitivity nucleosides) is 12:1 with the ratio of by product (3 '-protection against light sensitivity nucleosides and 3 ', 5 '-bis-protection against light sensitivity nucleosides).
2c. purification step:
Extract by above-mentioned 5 '-NPPOC-N-Ac-dC reacting coarse product ethyl acetate (500ml) with 3% sodium bicarbonate aqueous solution (300ml), retain ethyl acetate layer, ethyl acetate layer is use water (300ml) and saturated sodium-chloride water solution (300ml) washing successively, then ethyl acetate layer anhydrous sodium sulfate drying, filter, filtrate evaporate to dryness, obtains 5 '-NPPOC-N-Ac-dC crude product.5 '-NPPOC-N-Ac-dC crude product purified by silica gel column chromatography is purified, and take methylene chloride/methanol as elute soln, collects product component, concentrates to obtain 5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-N-ethanoyl-2 '-Deoxyribose cytidine 9(5 '-NPPOC-N-Ac-dC), 75.3g, HPLC purity is 98.1%, and productive rate is 79%.5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-N-ethanoyl-2 '-Deoxyribose cytidine 9the chemical structure of (5 '-NPPOC-N-Ac-dC) is passed through 1h NMR and LC-MS (M +) confirm.
5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-N-ethanoyl-2 '-Deoxyribose cytidine 9(5 '-NPPOC-N-Ac-dC): 1h NMR (CDCl 3, 400MHz) δ (ppm) 8.92 (br, ethanoyl-NH, diastereomer), 8.89 (br, ethanoyl-NH, diastereomer), 7.92 (d, H-6, diastereomer), 7.89 (d, H-6, diastereomer), 7.74 (m, 1-Ar-H, H-NPPOC), 7.55 (t, 1-Ar-H, H-NPPOC), 7.38 (m, 2 Ar-H, H-NPPOC), 7.29 (d, H-5, diastereomer), 7.26 (d, H-5 diastereomer), 6.24 (t, H-1 ', diastereomer), 6.19 (t, H-1 ', diastereomer), 4.32-3.89 (m, 6H, H-3 ', H-4 ', 2H-5 ',-the CH2 of NPPOC), 3.75 (m, 1H,-the CH of NPPOC), 2.62-2.59 (dd, 1H, 3 '-OH), 2.38-2.17 (m, 2H, H-2 '), 2.14, 2.11 (2s, 3H, CH3C=O), 1.40, 1.37 (2d, 3H,-the CH3 of NPPOC), LC-MS:m/z for (M+H) +, calculated value C 21h 25n 4o 9, 477.44., measured value 477.5.
Embodiment 3:
5 '-NPPOC-N-Bz-dA ( 13) preparation
The preparation feedback step of 3a. photosensitive reagents:
At room temperature 25 DEG C, in reaction vessel, by 2-ortho-nitrophenyl-1-propyl alcohol 1[2-(2-nitrophenyl) propan-1-ol, 0.30mol] is dissolved in ethyl acetate (540ml), adds N'N-carbonyl dimidazoles 10(1,1 '-Carbonyldiimidazole, 0.33mol), then add triethylamine (0.303mol).Stirring at room temperature reaction system, after 7 hours, HPLC shows to react completely, stopped reaction, and reaction soln is use water (2x 300ml) and saturated sodium-chloride water solution (300ml) extraction successively, retains organic (ethyl acetate) layer; After ethyl acetate layer anhydrous sodium sulfate drying, filter, evaporate to dryness, vacuum-drying, obtains TMSIM N imidazole carbonic acid (2-ortho-nitrophenyl)-1-propyl ester 11, 69.4g, HPLC purity is 96%, and productive rate is 84%.
3b. selects linked reaction step:
By the N-of drying benzoyl-2'-deoxyadenosine 12(N-Bz-dA, 71.1g, 0.20mol) is dissolved in anhydrous pyridine (710ml) and methylene dichloride (142ml), and cooling, adds TMSIM N imidazole carbonic acid (2-ortho-nitrophenyl)-1-propyl ester successively 0 DEG C of priority 11(0.224mol) with triethylamine (0.204mol); finish; in room temperature 25 DEG C of stirring reaction systems; after 16 hours; HPLC shows to react completely; stopped reaction, pressure reducing and steaming reaction solvent, obtains 5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-N-benzoyl-2'-deoxyadenosine 13(5 '-NPPOC-N-Bz-dA) reacting coarse product.Now, HPLC shows that product (5 '-protection against light sensitivity nucleosides) is 11:1 with the ratio of by product (3 '-protection against light sensitivity nucleosides and 3 ', 5 '-bis-protection against light sensitivity nucleosides).
3c. purification step:
Extract by above-mentioned 5 '-NPPOC-N-Bz-dA reacting coarse product ethyl acetate (600ml) with 3% sodium bicarbonate aqueous solution (400ml), retain ethyl acetate layer, ethyl acetate layer is use water (400ml) and saturated sodium-chloride water solution (400ml) washing successively, then ethyl acetate layer anhydrous sodium sulfate drying, filter, filtrate evaporate to dryness, obtains 5 '-NPPOC-N-Bz-dA crude product.5 '-NPPOC-N-Bz-dA crude product purified by silica gel column chromatography is purified, and take methylene chloride/methanol as eluting solvent, collects product component, concentrates to obtain 5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-N-benzoyl-2'-deoxyadenosine 13(5 '-NPPOC-N-Bz-dA), 90.1g, HPLC purity is 98.2%, and productive rate is 80%.5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-N-benzoyl-2'-deoxyadenosine 13the chemical structure of (5 '-NPPOC-N-Bz-dA) is passed through 1h NMR and LC-MS (M +) confirm.
5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-N-benzoyl-2'-deoxyadenosine 13(5 '-NPPOC-N-Bz-dA): 1h NMR (CDCl 3, 400MHz) δ (ppm) 9.29 (br, benzoyl-N H, diastereomer), 9.25 (br, benzoyl-N H, diastereomer), 8.61 (s, H-2, diastereomer), 8.58 (s, H-2, diastereomer), 8.19 (s, H-8, diastereomer), 8.16 (s, H-8, diastereomer), 7.86 (m, 2H, the Ar-H of benzoyl), 7.75 (m, 1-Ar-H, H-NPPOC), 7.56 (t, 1-Ar-H, H-NPPOC), 7.45-7.44 (m, 3H, the Ar-H of benzoyl), 7.37 (m, 2 Ar-H, H-NPPOC), 6.35 (t, H-1 ', diastereomer), 6.31 (t, H-1 ', diastereomer), 4.31-3.88 (m, 6H, H-3 ', H-4 ', 2H-5 ',-the CH2 of NPPOC), 3.74 (m, 1H,-the CH of NPPOC), 2.83-2.80 (dd, 1H, 3 '-OH), 2.39-2.17 (m, 2H, H-2 '), 1.41, 1.38 (2d, 3H,-the CH3 of NPPOC), LC-MS:m/z for (M+H) +, calculated value C 27h 27n 6o 8, 563.53., measured value 563.5.
Embodiment 4:
5 '-NPPOC-dT ( 5) preparation
The preparation feedback step of 4a. photosensitive reagents:
At room temperature 26 DEG C, in reaction vessel, by 2-ortho-nitrophenyl-1-propyl alcohol 1[2-(2-nitrophenyl) propan-1-ol, 54.4g, 0.30mol] is dissolved in methylene dichloride (540ml), adds carbonic acid two-2-pyridine ester 14(Di-2-pyridyl carbonate, 0.33mol), then add triethylamine (0.303mol).Stirring at room temperature reaction system, after 10 hours, HPLC shows to react completely, stopped reaction, and reaction soln is use water (2x 300ml) and saturated sodium-chloride water solution (300ml) extraction successively, retains organic (methylene dichloride) layer; After dichloromethane layer anhydrous sodium sulfate drying, filter, evaporate to dryness, vacuum-drying, obtain (2-pyridine) carbonic acid-2-ortho-nitrophenyl-1-propyl ester 15, 74.4g, HPLC purity is 97%, and productive rate is 82%.
4b. selects linked reaction step:
By the N-isobutyryl-2'-pancreatic desoxyribonuclease of drying 16(N-ibu-dG, 0.20mol) is dissolved in anhydrous pyridine (670ml) and methylene dichloride (134ml), and cooling, adds (2-pyridine) carbonic acid-2-ortho-nitrophenyl-1-propyl ester successively 0 DEG C of priority 15(0.224mol) with triethylamine (0.204mol); finish; in room temperature 26 DEG C of stirring reaction systems; after 22 hours; HPLC shows to react completely; stopped reaction, pressure reducing and steaming reaction solvent, obtains 5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-N-isobutyryl-2'-pancreatic desoxyribonuclease 17(5 '-NPPOC-N-ibu-dG) reacting coarse product.Now, HPLC shows that product (5 '-protection against light sensitivity nucleosides) is 10:1 with the ratio of by product (3 '-protection against light sensitivity nucleosides and 3 ', 5 '-bis-protection against light sensitivity nucleosides).
4c. purification step:
Extract by above-mentioned 5 '-NPPOC-N-ibu-dG reacting coarse product ethyl acetate (600ml) with 3% sodium bicarbonate aqueous solution (400ml), retain ethyl acetate layer, ethyl acetate layer is use water (400ml) and saturated sodium-chloride water solution (400ml) washing successively, then ethyl acetate layer anhydrous sodium sulfate drying, filter, filtrate evaporate to dryness, obtains 5 '-NPPOC-N-ibu-dG crude product.5 '-NPPOC-N-ibu-dG crude product purified by silica gel column chromatography is purified, and take methylene chloride/methanol as eluting solvent, collects product component, concentrates to obtain 5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-N-isobutyryl-2'-pancreatic desoxyribonuclease 17(5 '-NPPOC-N-ibu-dG), 82.8g, HPLC purity is 98.0%, and productive rate is 76%.5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-N-isobutyryl-2'-pancreatic desoxyribonuclease 17the chemical structure of (5 '-NPPOC-N-ibu-dG) is passed through 1h NMR and LC-MS (M +) confirm.
5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-N-isobutyryl-2'-pancreatic desoxyribonuclease 17(5 '-NPPOC-N-ibu-dG): 1h NMR (CDCl 3, 400MHz) δ (ppm) 10.25 (s, guanine-NH, diastereomer), 10.21 (s, guanine-NH, diastereomer), 9.28 (br, isobutyryl-NH, diastereomer), 9.24 (br, isobutyryl-NH, diastereomer), 7.97 (s, H-8, diastereomer), 7.94 (s, H-8, diastereomer), 7.74 (m, 1-Ar-H, H-NPPOC), 7.57 (t, 1-Ar-H, H-NPPOC), 7.38 (m, 2 Ar-H, H-NPPOC), 6.19 (t, H-1 ', diastereomer), 6.16 (t, H-1 ', diastereomer), 4.33-3.90 (m, 6H, H-3 ', H-4 ', 2H-5 ',-the CH2 of NPPOC), 3.73 (m, 1H,-the CH of NPPOC), 2.77-2.74 (m, 1H,-the CH of isobutyryl), 2.61-2.58 (dd, 1H, 3 '-OH), 2.39-2.17 (m, 2H, H-2 '), 1.42, 1.39 (2d, 3H,-the CH3 of NPPOC), 1.21, 1.18 (dd, 6H,-the CH3 of isobutyryl), LC-MS:m/z for (M+H) +, calculated value C 24h 29n 6o 9, 545.51, measured value 545.5.

Claims (10)

1. a carbonates photosensitive reagents, its structural formula is:
Wherein X is-OX ', imidazolyl, substituted imidazole base; The middle X ' of-OX ' is p-nitrophenyl, pentafluorophenyl group, 2-pyridyl and succinimido.
2. a preparation method for carbonates photosensitive reagents, step comprises:
A, 2-ortho-nitrophenyl-1-propyl alcohol is dissolved in organic solvent, adds carbonic diester class reagent or carbonyl diurethane azole reagent, then add triethylamine, stirred at ambient temperature reaction 6-10h;
B, by reacted solution priority water and saturated sodium-chloride water solution extraction, retain organic solution layer, after organic solution anhydrous sodium sulfate drying, filter, filtrate evaporate to dryness, vacuum-drying, obtains 2-ortho-nitrophenyl-1-propyl carbonate class photosensitive reagents.
3. preparation method as claimed in claim 2, is characterized in that: in described steps A, organic solvent is selected from methylene dichloride, trichloromethane, the one in ethyl acetate or toluene.
4. preparation method as claimed in claim 2, is characterized in that: in described steps A, 2-ortho-nitrophenyl-1-propyl alcohol, carbonic diester class reagent or carbonyl diurethane azole reagent, triethylamine concentration are in organic solvent respectively 0.40-0.65mol/L, 0.50-0.75mol/L, 0.40-0.65mol/L.
5. preparation method as claimed in claim 2, it is characterized in that: described steps A carbonic diester class reagent is selected from two (p-nitrophenyl) carbonic ether, two (penta fluoro benzene) carbonic ether, carbonic acid two-2-pyridine ester, N, N ' one in-succinimidyl carbonate; Carbonyl diurethane azole reagent is selected from N'N-carbonyl dimidazoles, N'N-carbonyl diurethane (glyoxal ethyline), the one in N'N-carbonyl diurethane (1,2,4-triazole).
6. a preparation method for 5'-protection against light sensitivity nucleosides, step comprises:
A, 2 ' of drying-deoxynucleoside is dissolved in the mixed solvent of anhydrous pyridine and methylene dichloride, be cooled to-3 DEG C-3 DEG C, add 2-ortho-nitrophenyl-1-propyl carbonate class photosensitive reagents and triethylamine successively, at room temperature stirring reaction 14-22h, pressure reducing and steaming reaction solvent, obtain 5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-2 '-deoxynucleoside crude product, i.e. 5'-protection against light sensitivity nucleosides crude product;
B, by 5'-protection against light sensitivity nucleosides crude product ethyl acetate and sodium bicarbonate aqueous solution extraction; retain ethyl acetate layer; ethyl acetate layer priority water and saturated sodium-chloride water solution washing; then ethyl acetate layer anhydrous sodium sulfate drying; filter; filtrate evaporate to dryness; product silica gel column chromatography after evaporate to dryness is purified; take methylene chloride/methanol as eluting solvent; collect product component, concentrated to obtain 5 '-(2-ortho-nitrophenyl-1-propyl carbonate)-2 '-deoxynucleoside and 5'-protection against light sensitivity nucleosides.
7. preparation method as claimed in claim 6; it is characterized in that: in described step a, 2 '-deoxynucleoside is selected from 2 '-deoxythymidine, 2 '-deoxyuridine, N-ethanoyl-2 '-Deoxyribose cytidine, N-benzoyl-2'-deoxyadenosine, one in N-isobutyryl-2'-pancreatic desoxyribonuclease.
8. preparation method as claimed in claim 6, is characterized in that: in described step a, 2 '-deoxynucleoside, 2-ortho-nitrophenyl-1-propyl carbonate class photosensitive reagents and the triethylamine concentration in the mixed solvent of anhydrous pyridine and methylene dichloride is respectively 0.25-0.35mol/L, 0.29-0.39mol/L, 0.26-0.36mol/L.
9. preparation method as claimed in claim 6, is characterized in that: in described step a, the volume ratio of anhydrous pyridine and methylene dichloride is 4-6:1.
10. preparation method as claimed in claim 6, is characterized in that: in described step b, ethyl acetate and sodium bicarbonate aqueous solution volume ratio are 1-2:1; Sodium bicarbonate aqueous solution mass percentage concentration is 3-5%; In eluting solvent, methylene dichloride and methyl alcohol volume ratio are 20-50:1.
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