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Rhizopus microsporus root-shaped variant ZJPH1308 and application thereof in preparation of sitagliptin intermediate

A kind of technology of Rhizopus microspora and varieties, applied in the field of application of Rhizopus microspora whisker variety ZJPH1308 and in the preparation of sitagliptin intermediates

Active Publication Date: 2015-09-09
JIANGSU TIANHE PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently, the biocatalytic synthesis of (S)-3-hydroxy-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3- a] The method of pyrazin-7-(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one can only be found in Sanjeev Kumar Mendirata et al. (Mendirata S K , Pandey B, Joshi R, et al.Process for preparing an intermediate of Sitagliptin via enzymatic conversion: U.S.Patent Application 13 / 823,300[P].2011-10-10.) reported enzymatic asymmetric reduction of 4-oxo-4 -[3-(Trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7-(8H)-yl]-1-( Preparation of (S)-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

Method used

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  • Rhizopus microsporus root-shaped variant ZJPH1308 and application thereof in preparation of sitagliptin intermediate
  • Rhizopus microsporus root-shaped variant ZJPH1308 and application thereof in preparation of sitagliptin intermediate
  • Rhizopus microsporus root-shaped variant ZJPH1308 and application thereof in preparation of sitagliptin intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: the acquisition of wet thalline

[0055] The composition of PDA medium is: potato 200g / L, glucose 20g / L, agar 20g / L, solvent is distilled water, pH is natural;

[0056] The formula of the seed medium is as follows: glucose 25.0g / L, peptone 27.5g / L, ammonium sulfate 3.0g / L, potassium dihydrogen phosphate 1.0g / L, sodium chloride 0.3g / L, distilled water, pH 6.0.

[0057] The formula of the fermentation medium is as follows: dextrin 25.0g / L, beef extract 30.0g / L, ammonium sulfate 5.0g / L, calcium chloride 0.4g / L, sodium chloride 0.3g / L, cobalt chloride 0.05g / L , prepared in distilled water, pH 6.0.

[0058] Inoculate Rhizopus microsporin varietus ZJPH1308 into PDA medium, and culture at 30°C for 2-3 days; then transfer the mature slant strains into a 250mL shake flask containing 100mL seed medium, culture at 30°C, 200rpm After 22 hours, the seed liquid was transferred to a 250 mL shake flask containing 100 mL of fermentation liquid with an inoculum volume con...

Embodiment 2

[0060] The wet bacterium obtained in Example 1 was resuspended in 100mmol / L, pH 6.0 phosphate buffer, the wet bacterium was 350g / L in terms of buffer volume, and 4-oxo-4-[ 3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7-(8H)-yl]-1-(2, 4,5-trifluorophenyl)butan-2-one substrate, and adding 6% (v / v) of glycerol in the volume of the reaction medium as an auxiliary substrate, 30°C, 200rpm, shaking the reaction for 24h. Detected by liquid chromatography, the product (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3- a] The concentration of pyrazin-7-(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one is 0.233mmol / L, the e.e. value is >99.9%, and the yield is 23.3%. The liquid chromatograms of substrate and product standards are shown in figure 1 See figure 2 ;product 1 H-NMR nuclear magnetic resonance spectrum result is as follows: 1 H-NMR (400MHz.DMSO-d 6 )δ / ppm: 7.46-7.41(q,2H), 5.02-4.83(m,3H), 4.22(s,1H), 4.09(s,2H), 4.02-3.90(m,2H), 2.78-2.68...

Embodiment 3

[0063] The phosphate buffer of 100mmol / L, pH 6.0 among the embodiment 2 is replaced with distilled water of the same volume, other operation is the same as embodiment 2, product (S)-3-hydroxyl-1-[3-(trifluoromethyl)- 5,6-Dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7-(8H)-yl]-4-(2,4,5-trifluorophenyl) The concentration of butan-1-one was 0.801 mmol / L, the e.e. value was >99.9%, and the yield was 80.1%.

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Abstract

The invention discloses a rhizopus microsporus root-shaped variant ZJPH1308 and application thereof in preparation of a sitagliptin intermediate. A strain of the rhizopus microsporus root-shaped variant ZJPH1308 can be used for biologically and asymmetrically reducing 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazol[4,3-a]pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butanone so as to obtain (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazol[4,3-a]pyrazine-7-(8H)-yl]-4-(2,4,5-trifluorophenyl)butanone. The strain has the advantages of good three-dimensional selectivity, high product optical purity and the like. The rhizopus microsporus root-shaped variant ZJPH1308 is fermented and cultured to obtain a wet thallus which serves as chiral biological catalyst; when the substrate concentration is 10mmol / L, the reduction product yield is as high as 95%, and the e.e. value of the product is greater than 99.9%.

Description

(1) Technical field [0001] The present invention relates to the preparation of a sitagliptin intermediate, in particular to a new strain--Rhizopus microsporus var.rhizopodiformis ZJPH1308, and its asymmetric synthesis of sitagliptin by microorganism catalysis Chiral Intermediate (S)-3-Hydroxy-1-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine -7-(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one. (2) Background technology [0002] Sitagliptin (Sitagliptin, trade name ), the chemical name 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, the first dipeptidyl peptidase-4 (DPP- 4) Inhibitors for the treatment of type II diabetes. The drug has the functions of protecting pancreatic beta cells, slowing down the activity inhibition of hypoglycemic hormones, and regulating blood sugar in patients with type II diabetes. It was approved for marketing by the US Food and Drug Administratio...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12P17/18C12R1/845
Inventor 王普黄金丁徐中
Owner JIANGSU TIANHE PHARMA CO LTD
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