CN104211741B - A kind of synthetic method of deuterated nucleoside phosphoramidites monomer - Google Patents
A kind of synthetic method of deuterated nucleoside phosphoramidites monomer Download PDFInfo
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- CN104211741B CN104211741B CN201410450722.7A CN201410450722A CN104211741B CN 104211741 B CN104211741 B CN 104211741B CN 201410450722 A CN201410450722 A CN 201410450722A CN 104211741 B CN104211741 B CN 104211741B
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Abstract
The invention discloses a kind of synthetic method of deuterated nucleoside phosphoramidites monomer.Technical scheme main points are：A kind of synthetic method of deuterated nucleoside phosphoramidites monomer, comprises the following steps：The oxidation of the hydroxyl of thymidine 3 ', the deuterated reduction of the carbonyl thymidines of 5 ' DMT 3 ' and the deuterated thymidines of 5 ' DMT 3 ' it is phosphorus acylated.The present invention uses 5 ' the deuterated nucleoside phosphoramidites monomers of DMT thymidine Material synthesis, raw material is easy to get, simplify synthesis step, suitable for large-scale production, 5 ' DMT thymidines are aoxidized using phase oxidative agent, obtain the carbonyl thymidine product purities of the DMT of key intermediate 5 ' 3 ' high, avoid column chromatography for separation process, improve product purity, reduce production cost, using boron deuterate sodium reduce at low temperature the carbonyl thymidines of 5 ' DMT 3 ' can Stereoselective generate the deuterated thymidines of 5 ' DMT 3 ', reaction has deuterated yield high, can be directly used for next step reaction.
Description
Technical field
The invention belongs to the chemosynthesis technical field of nucleotides, and in particular to a kind of deuterated nucleoside phosphoramidites monomer
Synthetic method.
Background technology
Deuterated nucleosides is the important construction unit for preparing preformed oligonucleotide acid of a class.The nucleotides and nucleosides list of deuterate
Unit be used to synthesize the RNA and DNA of chain.In nuclear magnetic resonance experiment research, one of non-deuterate of unit, adjacent cells deuterium
Generation, the sufficiently high nonoverlapping non-deuterate unit of resonance for making deuterate unit of deuterate resonates, therefore can be by mixing deuterated core
The technique study of glycosides has the higher structure of the DNA and RNA of bioactivity（Manalo MN, Pérez 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, the stretching vibration of C-D keys exists
2200cm is in infrared spectrum-1, the INFRARED ABSORPTION displacement of other functional groups in biomolecule is different from, therefore successfully used
In the dynamics research probe of the biomolecule such as protein, nucleic acid（L.B. Sagle, 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 nucleotides has intimate identical space structure with natural nucleotide, therefore deuterated nucleotides is mixed into tool
There is the nucleic acid of physiological function, the space structure of nucleic acid will not be destroyed, and the special infrared absorption wave number of C-D keys can be utilized to grind
Study carefully the dynamic process of functional nucleic acid.
The content of the invention
Present invention solves the technical problem that there is provided a kind of simple to operate and with low cost with natural thymidine as raw material
Deuterated nucleoside phosphoramidites monomer synthetic method.
The technical scheme is that：A kind of synthetic method of deuterated nucleoside phosphoramidites monomer, it is characterised in that including
Following steps：
（1）The oxidation of thymidine 3 '-hydroxyl
Input methylene chloride, 5 '-DMT- thymidines and phase oxidative agent in a kettle., is stirred at room temperature reaction 12h, mistake
Phase oxidative agent is filtered, solvent evaporated obtains crude product, and crude product is dissolved in into ethyl acetate, using saturated common salt water washing, had
Mutually using after anhydrous sodium sulfate drying, solvent evaporated obtains the-DMT-3 ' of product 5 '-carbonyl-thymidine to machine, and wherein phase oxidative agent is
It is loaded with the column chromatography silica gel of PCC drone salt PCC；
（2）The deuterated reduction of 5 '-DMT-3 '-carbonyl-thymidine
Solvent deuterated methanol, 5 '-DMT-3 '-carbonyl-thymidine and boron deuterate sodium are put into a kettle., in -10 DEG C of temperature
Stirring reaction 10h, is filtered to remove insoluble matter, and solvent evaporated is obtained crude product, and crude product uses petroleum ether-ethyl acetate column chromatography
Isolated-the DMT-3 ' of product 5 '-deuterated-thymidine；
（3）5 '-DMT-3 '-deuterated-thymidine it is phosphorus acylated
Under nitrogen protection to addition methylene chloride, 5 '-DMT-3 '-deuterated-thymidine, N, N- diisopropyls in reactor
Base ethylamine and 2- cyanoethyls N, N- diisopropyl chloro phosphoramidite, room temperature reaction 2h, plus solvent evaporated, make under nitrogen protection
The deuterated nucleoside phosphoramidites monomer of product is obtained with n-hexane-ethyl acetate column chromatography.
Step of the present invention（1）Described in the preparation method of phase oxidative agent be：Solvent dichloromethane is added in a kettle.
Alkane, PCC drone salt PCC and 200-300 mesh column chromatography silica gel, stir 3h at room temperature, and vacuum distillation removes solvent dichloromethane
Alkane, obtains solid oxidizer.
Key reaction equation in the synthetic method of deuterated nucleoside phosphoramidites monomer of the present invention is：
。
The invention has the advantages that：
（1）Using 5 ' the deuterated nucleoside phosphoramidites monomers of-DMT- thymidine Material synthesis, raw material is easy to get, and simplifies synthesis step,
Suitable for large-scale production；
（2）- the DMT-3 ' of column chromatography for separation 5 '-carbonyl-thymidine can cause Disassembling Products, and product purity reduction, the present invention makes
5 '-DMT- thymidines are aoxidized with phase oxidative agent ,-the DMT-3 ' of key intermediate 5 '-carbonyl-thymidine product purity is obtained high, it is to avoid
Column chromatography for separation process, improves product purity, reduces production cost；
（3）Using boron deuterate sodium reduce at low temperature 5 '-DMT-3 '-carbonyl-thymidine can Stereoselective generate 5 '-
DMT-3 '-deuterated-thymidine, reaction has deuterated yield high, can be directly used for next step reaction；
（4）The deuterated yield of reaction is detected using the method at 1H nuclear magnetic resonance characteristics peak, detection method simple and fast is easy to
The monitor in real time of course of reaction.
Specific embodiment
The above of the invention is described in further details by the following examples, but this should not be interpreted as this
The scope for inventing above-mentioned theme is only limitted to following embodiment, and all technologies realized based on the above of the present invention belong to this hair
Bright scope.
Embodiment 1
The synthesis of 5 '-DMT-3 '-carbonyl-thymidine
100mL methylene chlorides are put into 200mL reactors, the agent of 10g the phase oxidatives ,-DMT of 5.5g 5 ' is added
Thymidine, 12h is reacted under conditions of being stirred at room temperature.Solid insoluble is filtered to remove, solvent evaporated obtains crude product.Crude product is molten
In 100mL ethyl acetate, using 50mL saturated common salts water washing 3 times.Organic phase uses anhydrous sodium sulfate drying, is filtered to remove
Drier, is evaporated organic phase, obtains 5 '-DMT-3 '-carbonyl-thymidine 5g, and yield is 91%, and product purity is more than 96%, directly uses
In next step reaction, the preparation method of solid oxidizer is in building-up process：300mL solvents two are put into 500mL reactors
Chloromethanes, 2.6g PCCs drone salt PCC and 7.4g 200-300 mesh column chromatography silica gels, are stirred at room temperature 3h, and vacuum distillation is removed
Methylene chloride is gone, 10g solid oxidizers are obtained.
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
- the DMT-3 ' of 1.5g 5 '-carbonyl-thymidine, 1g boron deuterate sodium and deuterated first of 10mL solvents is put into 50mL reactors
Alcohol, in -10 DEG C of thermotonus 10h.It is filtered to remove insoluble matter, Distillation recovery deuterated methanol.Crude product uses petroleum ether-acetic acid second
Ester column chromatography for separation, obtains 5 '-DMT-3 '-deuterated-thymidine 0.8g, deuterated yield by 1 '-hydrogen in proton nmr spectra and 3 '-
The ratio calculation of position remaining hydrogen, 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 oligonucleotides
In 100mL reactors put into 60mL methylene chlorides, add-the DMT-3 ' of 3.2g 5 '-deuterated-thymidine,
2.6mL N, N- diisopropyl ethyl amines and 3mL 2- cyanoethyls N, N- diisopropyl chloro phosphoramidites, room temperature reaction 2h, plus
Solvent evaporated.Under nitrogen protection, using n-hexane-ethyl acetate column chromatography, it is white blister solid 6.5g to obtain product.Obtained
White-the DMT-3 ' of the blister solid 5 '-deuterated-thymidine phosphoramidite for obtaining is directly used in DNA synthesis in solid state oligonucleotides.
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 general principle of the invention, principal character and advantage, the technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, simply original of the invention is illustrated described in above-described embodiment and specification
Reason, under the scope for not departing from the principle of the invention, various changes and modifications of the present invention are possible, and these changes and improvements each fall within
The scope of protection of the invention.
Claims (1)
1. a kind of synthetic method of deuterated thymidine phosphoramidite monomer, it is characterised in that comprise the following steps：
（1）The oxidation of thymidine 3 '-hydroxyl
Input methylene chloride, 5 '-DMT- thymidines and phase oxidative agent in a kettle., is stirred at room temperature reaction 12h, crosses and filters
Phase oxidative agent is gone, solvent evaporated obtains crude product, and crude product is dissolved in into ethyl acetate, use saturated common salt water washing, organic phase
After anhydrous sodium sulfate drying, solvent evaporated obtains the-DMT-3 ' of product 5 '-carbonyl-thymidine, and wherein phase oxidative agent is load
There is the column chromatography silica gel of PCC drone salt PCC, the phase oxidative agent by adding methylene chloride, chlorine in a kettle.
Chromic acid pyridinium salt PCC and 200-300 mesh column chromatography silica gel stirs 3h at room temperature, and then vacuum distillation removes solvent dichloromethane
Alkane is obtained；
（2）The deuterated reduction of 5 '-DMT-3 '-carbonyl-thymidine
Input solvent deuterated methanol, 5 '-DMT-3 '-carbonyl-thymidine and boron deuterate sodium, stir in -10 DEG C of temperature in a kettle.
Reaction 10h, is filtered to remove insoluble matter, and solvent evaporated is obtained crude product, and crude product uses petroleum ether-ethyl acetate column chromatography for separation
Obtain-the DMT-3 ' of product 5 '-deuterated-thymidine；
（3）5 '-DMT-3 '-deuterated-thymidine it is phosphorus acylated
Under nitrogen protection to addition methylene chloride, 5 '-DMT-3 '-deuterated-thymidine, N, N- diisopropyl second in reactor
Base amine and 2- cyanoethyls N, N- diisopropyl chloro phosphoramidite, room temperature reaction 2h, plus solvent evaporated, nitrogen protection are lower using just
Hexane-ethylacetate column chromatography obtains the deuterated thymidine phosphoramidite monomer of product.
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| CN101048423A (en) * | 2004-08-26 | 2007-10-03 | 日本新药株式会社 | Phosphoramidite compound and method for producing oligo-rna |
| CN101050225A (en) * | 2007-05-11 | 2007-10-10 | 南开大学 | New method for synthesizing 2 - L - ribose from 2 - D - ribose |
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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 |
| CN1646452A (en) * | 2002-02-22 | 2005-07-27 | 霍尼韦尔国际公司 | Methods of producing phosphitylated compounds |
| CN101048423A (en) * | 2004-08-26 | 2007-10-03 | 日本新药株式会社 | Phosphoramidite compound and method for producing oligo-rna |
| CN101050225A (en) * | 2007-05-11 | 2007-10-10 | 南开大学 | New method for synthesizing 2 - L - ribose from 2 - D - ribose |
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Inventor after: Li Lingjun Inventor after: Zhu Anlian Inventor after: Zhang Chi Inventor before: Li Lingjun Inventor before: Zhu Anlian |
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Effective date of registration: 20210728 Address after: 201400 room 1020, floor 5, building 9, No. 1, Yangming Road, Xinghuo Development Zone, Fengxian District, Shanghai Patentee after: SHANGHAI ZHAOWEI BIOENGINEERING Co.,Ltd. Address before: 453007 No. 46 East Road, Makino District, Henan, Xinxiang Patentee before: HENAN NORMAL University |
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