Pseudomonas aeruginosa ZJPH1504 and application thereof in preparation of sitagliptin chiral intermediate

A technology of Pseudomonas aeruginosa and sitagliptin, applied in the direction of bacteria, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of poor solubility of substrates and low yields, and achieve shortened reaction time, Improved reaction yield and high optical purity

Inactive Publication Date: 2016-09-07
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And by constructing an organic medium reaction system to effectively solve the problems of insoluble substrates and low yields

Method used

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  • Pseudomonas aeruginosa ZJPH1504 and application thereof in preparation of sitagliptin chiral intermediate
  • Pseudomonas aeruginosa ZJPH1504 and application thereof in preparation of sitagliptin chiral intermediate
  • Pseudomonas aeruginosa ZJPH1504 and application thereof in preparation of sitagliptin chiral intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: For catalytic reduction of 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine Screening of microbial strains of -7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one

[0034] Enrichment culture: Add 1g of fresh soil samples (collected from the campus of Zhejiang University of Technology (Hangzhou, Zhejiang)) into a 250mL shaker flask filled with 50mL of enrichment medium, culture at 30°C and 200rpm for 5 days, and let stand. Take 1 mL of the culture medium and transfer it to a fresh enrichment medium, continue to cultivate for 5 days, and repeat the enrichment process 3 times. The enrichment medium consists of: (NH 4 ) 2 SO 4 2g / L, KH 2 PO 4 1g / L, NaCl 0.5g / L, MgSO 4 ·7H 2 O 0.5g / L, the compound of formula (II) (2g / L) is the sole carbon source, the solvent is water, pH 6.5, and sterilized at 120°C for 20min.

[0035] Plate primary screening: the enriched culture solution was diluted 10 with normal saline 4 -10 6 After doubling, it w...

Embodiment 2

[0050] Example 2: Determination of the chiral configuration of the reduced product of strain ZJPH1504

[0051] With reference to the separation method of related substances described in the patent 201510289729.X (publication number CN 104893989 A), the biotransformation solution of the bacterial strain ZJPH1504 obtained in Example 1 was extracted twice with an equal volume of ethyl acetate, the solvent was removed by rotary evaporation, and then passed Preparative thin-layer chromatography (developing agent is ethyl acetate: sherwood oil: glacial acetic acid=80:20:0.1 (v / v / v); TLC plate is GF254; Color develops under the ultraviolet of wavelength 254nm) to separate, collect R f A product with a value of 0.44 gave the reduced product compound of formula (I). This substance corresponds to one of the peaks in the HPLC chromatogram of the compound of racemic formula (I), see image 3 . According to the standard method, the specific optical rotation (SOR) of the reduced product ...

Embodiment 3

[0052] Example 3: Obtaining of Resting Cell Enzyme Sources under Shake Flask Culture Conditions

[0053] (1) Slant culture: Pseudomonas aeruginosa ZJPH1504 was inoculated into the slant medium, and cultured at 30° C. for 36 hours to obtain mature cultured slant strains. The final concentration of the slant medium consists of: glucose 15g / L, peptone 7.5g / L, yeast extract 6g / L, (NH 4 ) 2 SO 4 3g / L, KH 2 PO 4 1.5g / L, NaCl 0.75g / L, MgSO 4 ·7H 2 O 0.75g / L, agar powder 20g / L, solvent is water, pH 6.5.

[0054] (2) Seed culture: inoculate the slant bacteria into the seed culture medium, cultivate at 30° C. and 200 rpm for 12 hours, and obtain the seed liquid. The final concentration of the seed medium consists of: glucose 15g / L, peptone 7.5g / L, yeast extract 6g / L, (NH 4 ) 2 SO 4 3g / L, KH 2 PO 4 1.5g / L, NaCl 0.75g / L, MgSO 4 ·7H 2 O 0.75g / L, solvent is water, pH 6.5.

[0055] (3) Fermentation culture: inoculate the seed liquid into the fermentation medium with an ino...

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Abstract

The invention discloses a pseudomonas aeruginosa ZJPH1504 and an application of pseudomonas aeruginosa ZJPH1504 in preparation of a sitagliptin chiral intermediate. The pseudomonas aeruginosa ZJPH1504 can be used for asymmetric reduction of a prochiral ketone compound (II) with high stereoselectivity to prepare a sitagliptin chiral intermediate (I-a) compound, the product prepared by using the pseudomonas aeruginosa ZJPH1504 is high in optical purity, and the e.e. (Errors Excepted) value is greater than 99.9%, 9g / L of a substrate is added in a phosphate buffer solution system of which the pH is 7.5, and reacted for 30 hours, and the yield of an S-reduction product is 60.2%. When an organic solvent is added in a reaction system, the catalytic efficiency can be effectively improved, the reaction yield can be increased, and the reaction time can be shortened, especially when di-n-butyl phthalate is added in the reaction system, the reaction yield can be increased to 75.6%, the reaction time can be shortened to 24 hours and the e.e. value is greater than 99.9%.

Description

(1) Technical field [0001] The invention relates to the preparation of a chiral intermediate of sitagliptin, in particular to a method for asymmetrically synthesizing the chiral intermediate of sitagliptin by biocatalysis of a new bacterial strain-Pseudomonas aeruginosa ZJPH1504. (2) Background technology [0002] 3-Hydroxy-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7-(8H)-yl ]-4-(2,4,5-trifluorophenyl) butan-1-one (I) (its molecular weight: 408.102) is the preparation formula (Ⅲ) compound (Sitagliptin, Sitagliptin, trade name ), wherein compound (I) S configuration, ie formula (I-a) is the key chiral intermediate for the preparation of sitagliptin mentioned in the present invention. The chemical name of formula (Ⅲ) is (3R)-3-amino-1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo[4 ,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one. [0003] [0004] Formula (I-a) is the (S)-isomer of formula (I), and formula (I-b) is the (R)-isomer ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P17/18C12R1/385
CPCC12P17/182C12N1/205C12R2001/385
Inventor 王普孙佳黄金沈琦
Owner ZHEJIANG UNIV OF TECH
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