反应详情

Based on the general enzymatic conditions described above, 1.50 g of mixed esters, 5,6-dihydro-4H-pyrrolo(1,2-b)pyrazole-2-carboxylic acid ethyl ester and 5,6-dihydro-4H-pyrrolo(1,2-b)pyrazole-3-carboxylic acid ethyl ester is hydrolyzed with about 100 mg of Pseudomonas sp. Lipase (L-6) in 50 ml 0.1M potassium phosphate buffer pH 7.25 to which, 0.5 ml acetonitrile is added for even distribution of the substrate in the buffer. The reaction is mixed at 37° C. The acid released after enzyme hydrolysis lowers the reaction pH. Periodically pH of the reaction is checked and if it is 7.0 or below, 1N sodium hydroxide is added to bring the pH to about 7.30. Thus, the pH is maintained above 7.0. After 72 h incubation the desired ester hydrolysis reached about 70%, based on HPLC analysis. Enzyme reaction is filtered through a 0.2μ membrane to remove the insoluble material from the crude enzyme preparation. The clear enzyme reaction mix is extracted twice with ethyl acetate (1:1) at pH 7.0, which removes all the remaining esters, leaving only 5,6-dihydro-4H-pyrrolo(1,2-b)pyrazole-2-carboxylic acid in the aqueous phase. The aqueous phase containing the acid product is lyophilized and the recovered solid is dissolved in methanol. The salts that were not soluble in methanol are separated and discarded. Methanol containing 5,6-dihydro-4H-pyrrolo(1,2-b)pyrazole-2-carboxylic acid (pale yellow) is evaporated and about 0.5 g of 5,6-dihydro-4H-pyrrolo(1,2-b)pyrazole-2-carboxylic acid is recovered. The LC-MS and NMR data are consistent and indicated that only the desired acid is present with isomeric purity at 100%. The acid purity by HPLC is over 97%. HPLC conditions are: Prodigy ODS3 column 4.6×150 mm. Solvent A: 0.02% Trifluoroacetic acid (TFA) in water. Solvent B: 0.02% TFA in acetonitrile. Gradient used is, 90% A and 10% B at zero time with gradient reaching at 20 minutes, 5% A and 95% B and continue until 25 minutes.

1. additionThe reaction is mixed at 37° C
2. temperatureThus, the pH is maintained above 7.0
3. filtrationEnzyme reaction is filtered through a 0.2μ membrane
4. customto remove the insoluble material from the crude
5. customenzyme preparation
6. customThe clear enzyme reaction
7. additionmix
8. extractionis extracted twice with ethyl acetate (1:1) at pH 7.0, which
9. customremoves all the remaining esters