CN104211741A - Synthesis method of deuterium-substituted nucleoside phosphoramidite monomer - Google Patents
Synthesis method of deuterium-substituted nucleoside phosphoramidite monomer Download PDFInfo
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- CN104211741A CN104211741A CN201410450722.7A CN201410450722A CN104211741A CN 104211741 A CN104211741 A CN 104211741A CN 201410450722 A CN201410450722 A CN 201410450722A CN 104211741 A CN104211741 A CN 104211741A
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- thymidine
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- 0 C*C1=CN(*(C2)OCC2(C)O)C(*)N*1=O Chemical compound C*C1=CN(*(C2)OCC2(C)O)C(*)N*1=O 0.000 description 1
Abstract
The invention discloses a synthesis method of a deuterium-substituted nucleoside phosphoramidite monomer. According to the technical scheme, the synthesis method of the deuterium-substituted nucleoside phosphoramidite monomer comprises the following steps of oxidization of thymidine 3'-hydroxy, deuterium-substituted reduction of 5'-DMT-3'-carbonyl-thymidine and sub-phosphorylation of 5'-DMT-3'-deuterium-substituted thymidine. According to the synthesis method, a deuterium-substituted nucleoside phosphoramidite monomer is synthesized by using 5'-DMT thymidine raw materials, the raw materials are easily obtained, the synthesis steps are simplified, and the synthesis method is suitable for large-scale production; a solid-phase oxidizing agent is adopted to oxidize 5'-DMT-3'-thymidine to obtain a key intermediate, namely 5'-DMT-3'-carbonyl-thymidine with high product purity, so that a column chromatography separation process is omitted, the purity of the product is improved, and the production cost is reduced; and 5'-DMT-3'-carbonyl-thymidine is reduced at a low temperature by using sodium borodeuteride to stereoselectively generate 5'-DMT-3'-deuterium-substituted thymidine, so that the high deuterium-substituted yield is obtained in the reaction process, and 5'-DMT-3'-deuterium-substituted thymidine can be directly used for the next-step reaction.
Description
Technical field
The invention belongs to the chemosynthesis technical field of Nucleotide, be specifically related to a kind of synthetic method of deuterated nucleoside phosphoramidites monomer.
Background technology
Deuterated nucleosides is the important structural unit preparing preformed oligonucleotide acid of a class.The Nucleotide of deuterate and nucleotide units are used to RNA and DNA synthesizing chain.In nuclear magnetic resonance experiment research, the non-deuterate of one of them unit, adjacent cells is deuterated, deuterate is enough high makes the nonoverlapping non-deuterate unit resonance of the resonance of deuterate unit, therefore higher structure (the Manalo MN of bioactive DNA and RNA can be had by the technique study mixing deuterated nucleosides, P é rez LM, LiWang A Hydrogen-bonding and pi-pi base-stacking interactions are coupled in DNA, as suggested by calculated and experimental trans-Hbond deuterium isotope shifts.
j Am Chem Soc.2007,129,11298-11299.).On the other hand, C-D key stretching vibration in infrared spectra, be in 2200cm
-1be different from the infrared absorption displacement of other functional group in biomolecules, therefore dynamics research probe (the L.B. Sagle of the biomolecules such as protein, nucleic acid is successfully used to, J. Zimmermann, P.E. Dawson, F.E. Romesberg, A high-resolution probe of protein folding.
j. Am. Chem. Soc.2004,126,3384-3385.; (b) J. Zimmermann, F.E. Romesberg, Carbon-deuterium bonds as non-perturbative infrared probes of protein dynamics, electrostatics, heterogeneity, and folding.
methods Mol. Biol., 2014,1084,101-119.).Deuterated Nucleotide and natural nucleotide have intimate identical space structure, therefore deuterated Nucleotide is mixed the nucleic acid with physiological function, the space structure of nucleic acid can not be destroyed, and the special infrared absorption wave number of C-D key can be utilized to study the dynamic process of functional nucleic acid.
Summary of the invention
The technical problem that the present invention solves there is provided a kind of simple to operate and with low cost with the synthetic method of the natural thymidine deuterated nucleoside phosphoramidites monomer that is raw material.
Technical scheme of the present invention is: a kind of synthetic method of deuterated nucleoside phosphoramidites monomer, is characterized in that comprising the following steps:
(1) oxidation of thymidine 3 '-hydroxyl
Drop into methylene chloride, 5 '-DMT-thymidine and phase oxidative agent in a kettle., stirring at room temperature reaction 12h, cross and filter phase oxidative agent, solvent evaporated obtains crude product, crude product is dissolved in ethyl acetate, uses saturated common salt water washing, after organic phase uses anhydrous sodium sulfate drying, solvent evaporated obtains product 5 '-DMT-3 '-carbonyl-thymidine, and wherein phase oxidative agent is the column chromatography silica gel that load has pyridinium chlorochromate drone salt PCC;
The deuterated reduction of (2) 5 '-DMT-3 '-carbonyl-thymidine
Drop into solvent deuterated methanol, 5 '-DMT-3 '-carbonyl-thymidine and boron deuterate sodium in a kettle., in-10 DEG C of temperature stirring reaction 10h, cross and filter insolubles, solvent evaporated obtains crude product, and crude product uses petroleum ether-ethyl acetate column chromatography for separation to obtain product 5 '-DMT-3 '-deuterated-thymidine;
(3) 5 '-DMT-3 '-deuterated-thymidine phosphorus acylated
Methylene chloride, 5 '-DMT-3 '-deuterated-thymidine, N is added under nitrogen protection in reactor; N-diisopropyl ethyl amine and 2-cyanoethyl N; N-di-isopropyl chloro phosphoramidite; room temperature reaction 2h; add solvent evaporated, under nitrogen protection, use n-hexane-ethyl acetate column chromatography to obtain the deuterated nucleoside phosphoramidites monomer of product.
The preparation method of the phase oxidative agent described in step of the present invention (1) is: add methylene chloride, pyridinium chlorochromate drone salt PCC and 200-300 order column chromatography silica gel in a kettle., stirred at ambient temperature 3h, underpressure distillation removing methylene chloride, obtains solid oxidizing agent.
Principal reaction equation in the synthetic method of deuterated nucleoside phosphoramidites monomer of the present invention is:
。
The present invention has following beneficial effect:
(1) use the deuterated nucleoside phosphoramidites monomer of 5 '-DMT-thymidine Material synthesis, raw material is easy to get, and simplifies synthesis step, is applicable to scale production;
(2) column chromatography for separation 5 '-DMT-3 '-carbonyl-thymidine can cause Disassembling Products, product purity reduces, the present invention uses phase oxidative agent to be oxidized 5 '-DMT-thymidine, obtain key intermediate 5 '-DMT-3 '-carbonyl-thymidine product purity high, avoid column chromatography for separation process, improve product purity, reduce production cost;
(3) use boron deuterate sodium to reduce 5 '-DMT-3 '-carbonyl-thymidine at low temperatures and can generate 5 '-DMT-3 '-deuterated-thymidine by Stereoselective, reaction has high deuterated yield, can be directly used in next step reaction;
(4) use the deuterated yield of method detection reaction at 1H nuclear magnetic resonance characteristic peak, detection method simple and fast, is convenient to the real-time monitoring of reaction process.
Embodiment
Be described in further details foregoing of the present invention by the following examples, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment, all technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1
The synthesis of 5 '-DMT-3 '-carbonyl-thymidine
In 200mL reactor, drop into 100mL methylene chloride, then add the agent of 10g phase oxidative, 5.5g 5 '-DMT thymidine, under the condition of stirring at room temperature, react 12h.Solids removed by filtration insolubles, solvent evaporated obtains crude product.Crude product is dissolved in 100mL ethyl acetate, uses 50mL saturated common salt water washing 3 times.Organic phase uses anhydrous sodium sulfate drying, cross and filter siccative, evaporate to dryness organic phase, obtains 5 '-DMT-3 '-carbonyl-thymidine 5g, and yield is 91%, product purity is greater than 96%, be directly used in next step reaction, in building-up process, the preparation method of solid oxidizing agent is: in 500mL reactor, drop into 300mL methylene chloride, 2.6g pyridinium chlorochromate drone salt PCC and 7.4g 200-300 order column chromatography silica gel, stirring at room temperature 3h, underpressure distillation removing methylene chloride, obtains 10g solid oxidizing agent.
5 '-DMT-3 '-carbonyl-thymidine 1H NMR 9.05 (s, 1H), 7.74 (s, 1H), 7.34-7.31 (m, 11H), 6.92-6.90 (m, 4 H), 6.67 (t, J=7 Hz, 1H), 4.25 (s, 1H), 3.87 (s, 6H), 3.73-3.71 (m, 1H), 3.52-3.50 (m, 1H), 3.18-3.14 (m, 1H), 2.86-2.80 (m, 1H), 1.40 (s, 3H). ESI-MS:543.1 (M+1)+.
Embodiment 2
The synthesis of 5 '-DMT-3 '-deuterated-thymidine
1.5g 5 '-DMT-3 '-carbonyl-thymidine, 1g boron deuterate sodium and 10mL solvent deuterated methanol is dropped into, in-10 DEG C of thermotonus 10h in 50mL reactor.Cross and filter insolubles, Distillation recovery deuterated methanol.Crude product uses petroleum ether-ethyl acetate column chromatography for separation, obtains 5 '-DMT-3 '-deuterated-thymidine 0.8g, and deuterated yield, by the ratio calculation of 1 '-hydrogen and 3 '-position remaining hydrogen in proton nmr spectra, is 96%.
5 '-DMT-3 '-deuterated-thymidine Rf=0.5 (5% MeOH/CH2Cl2). 1H NMR 7.67-7.68 (m, 1H), 7.48-7.29 (m, 10H), 6.92-6.90 (m, 4H), 6.52-6.49 (m, 1H), 4.15 (t, J=5 Hz, 1H), 3.86 (s, 6H), 3.35-3.45 (m, 2H), 2.52-2.48 (m, 1H), 2.40-2.36 (m, 1H), 1.54 (s, 3H). ESI-MS:546.5 (M+1)+.
Embodiment 3
The preparation of 5 '-DMT-3 '-deuterated-thymidine phosphoramidite and oligonucleotide
In 100mL reactor, drop into 60mL methylene chloride, then add 3.2g 5 '-DMT-3 '-deuterated-thymidine, 2.6mL N, N-diisopropyl ethyl amine and 3mL 2-cyanoethyl N, N-di-isopropyl chloro phosphoramidite, room temperature reaction 2h, adds solvent evaporated.Under nitrogen protection, use n-hexane-ethyl acetate column chromatography, obtaining product is white blister solid 6.5g.White blister the solid 5 '-DMT-3 '-deuterated-thymidine phosphoramidite obtained is directly used in DNA solid phase synthesis oligonucleotide.
5 '-DMT-3 '-deuterated-thymidine phosphoramidite Rf=0.65 (6:3:1 n-C6H14/EtOAc/Et3N). 1H NMR (400 MHz, CDCl3) δ 7.67 (br, 0.6 H, disastereomer 1), 7.62 (br, 0.4 H, disastereomer 2) 7.48-7.41 (m, 2H), 7.36-7.30 (m, 7H), 6.87 (m, 4H), 6.44 (m, 1H), 4.21 (s, 0.6 H, disastereomer 1), 4.16 (s, 0.4 H, disastereomer 1), 3.83 (s, 3H), 3.82 (s, 3H), 3.68-3.50 (m, 5H), 3.48 (m, 1H), 3.35 (m, 1H), 2.65 (m, 1H), 2.45 (m, 1H), 2.35 (m, 1H), 2.20 (m, 2H), 1.46 (m, 3H), 1.22-1.17 (m, 12H). 31P NMR δ 149.1 (s), 148.7 (s).
Embodiment above describes ultimate principle of the present invention, principal character and advantage; the technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; under the scope not departing from the principle of the invention; the present invention also has various changes and modifications, and these changes and improvements all fall into the scope of protection of the invention.
Claims (3)
1. a synthetic method for deuterated nucleoside phosphoramidites monomer, is characterized in that comprising the following steps:
(1) oxidation of thymidine 3 '-hydroxyl
Drop into methylene chloride, 5 '-DMT-thymidine and phase oxidative agent in a kettle., stirring at room temperature reaction 12h, cross and filter phase oxidative agent, solvent evaporated obtains crude product, crude product is dissolved in ethyl acetate, uses saturated common salt water washing, after organic phase uses anhydrous sodium sulfate drying, solvent evaporated obtains product 5 '-DMT-3 '-carbonyl-thymidine, and wherein phase oxidative agent is the column chromatography silica gel that load has pyridinium chlorochromate drone salt PCC;
The deuterated reduction of (2) 5 '-DMT-3 '-carbonyl-thymidine
Drop into solvent deuterated methanol, 5 '-DMT-3 '-carbonyl-thymidine and boron deuterate sodium in a kettle., in-10 DEG C of temperature stirring reaction 10h, cross and filter insolubles, solvent evaporated obtains crude product, and crude product uses petroleum ether-ethyl acetate column chromatography for separation to obtain product 5 '-DMT-3 '-deuterated-thymidine;
(3) 5 '-DMT-3 '-deuterated-thymidine phosphorus acylated
Methylene chloride, 5 '-DMT-3 '-deuterated-thymidine, N is added under nitrogen protection in reactor; N-diisopropyl ethyl amine and 2-cyanoethyl N; N-di-isopropyl chloro phosphoramidite; room temperature reaction 2h; add solvent evaporated, under nitrogen protection, use n-hexane-ethyl acetate column chromatography to obtain the deuterated nucleoside phosphoramidites monomer of product.
2. the synthetic method of deuterated nucleoside phosphoramidites monomer according to claim 1, it is characterized in that the preparation method of the phase oxidative agent described in step (1) is: add methylene chloride, pyridinium chlorochromate drone salt PCC and 200-300 order column chromatography silica gel in a kettle., stirred at ambient temperature 3h, underpressure distillation removing methylene chloride, obtains solid oxidizing agent.
3. the synthetic method of deuterated nucleoside phosphoramidites monomer according to claim 1, is characterized in that the principal reaction equation in the synthetic method of described deuterated nucleoside phosphoramidites monomer is:
。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105348343A (en) * | 2015-11-25 | 2016-02-24 | 北京大学 | Chromophore-modified deoxynucleoside phosphoramidite monomer compound, preparation method therefor and application thereof |
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2014
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| CN1072414A (en) * | 1992-11-17 | 1993-05-26 | 蒋飞龙 | Protection deoxynucleoside-3 '-cyanoethyl-N, the preparation method of N-diisopropylphosphoramidite |
| CN1122040C (en) * | 1994-11-02 | 2003-09-24 | Icn药品公司 | Suger modified nucleosides and their use for synthesis of oligonucleotides |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105348343A (en) * | 2015-11-25 | 2016-02-24 | 北京大学 | Chromophore-modified deoxynucleoside phosphoramidite monomer compound, preparation method therefor and application thereof |
| CN105348343B (en) * | 2015-11-25 | 2019-01-04 | 北京大学 | The deoxynucleoside phosphoramidite monomeric compound and its preparation method and application of chromophore modification |
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