Penicillin Acylase-catalyzed Synthesis Of N-bromoacetyl-7-aminocephalosporanic Acid

Cefathiamidine is a semi-synthetic ?-lactam antibiotic,which was synthesized successfully by the Chinese researchers for the first time.Currently,it has been widely used for the treatment and prevention of various bacterial infections in China.N-Bromoacetyl-7-aminocephalosporanic acid(N-Bromoacetyl-7-ACA)is the key intermediate for the production of cefathiamidine,which is industrially produced by a chemical method.It required use of highly active bromoacetyl bromide,harsh conditions;the chemical process was complex.In addition,a lot of waste was produced,which doesn't meet the requirements of green chemistry.Compared to chemical methods,the enzymatic routes appear to be preferable for the synthesis of semi-synthetic ?-lactam antibiotics because of many advantages such as mild reaction conditions,avoiding use of toxic chemicals,producing less waste,and being simple and environmentally friendly.With the increasing concern in environmental issues,enzymatic synthesis of semi-synthetic ?-lactam antibiotics has received more attention.In the thesis,the feasibility of the enzymatic synthesis of N-bromoacetyl-7-ACA from 7-ACA and bromoacetates using penicillin acylases(PAs)was demonstrated for the first time.The catalytic performance of the immobilized enzyme PGA-750 was explored.Then,to significantly improve the substrate(7-ACA)concentration in buffer,various alkalis were added.The enzymatic process at a high substrate concentration was optimized and the various side reactions were investigated.The results showed that various PAs were able to accept the esters without the aryl group(methyl/ethyl bromoacetate)as the substrates.Of the immobilized PAs tested,PA from Bacillus megaterium(PGA-750)was the best biocatalyst.The effect of key conditions such as acyl donors and substrate molar ratios on the enzymatic reaction was explored.The optimal acyl donor,molar ratio of acyl donor to 7-ACA,pH,temperature,7-ACA concentration,and enzyme dosage were methyl bromoacetate,3,7.5,20?,50 mM and 4 U/mL,respectively.Under the optimal conditions,the enzymatic N-acylation of 7-ACA with methyl bromoacetate afforded the desired product with the yield of 85% in 2 h,where the synthesis/hydrolysis(S/H)ratio was approximately 1.5.The immobilized enzyme PGA-750 exhibited good operational stability,and the relative yield of approximately 90% was achieved when it was reused in the7 th run.The effects of different alkalis on the soulibility of 7-ACA and the enzymatic synthesis of N-bromoacetyl-7-ACA were investigated.Sodium bicarbonate proved to be the optimal.Then,the effects of some key conditions such as the sodium bicarbonate concentrations and organic solvents on the enzymatic reaction at a high substrate concentration were studied.The optimal solvent,sodium bicarbonate concentration,molar ratio of acyl donor to 7-ACA,7-ACA concentration and enzyme dosage were deionic water,400 m M,2,200 mM and 5 U/mL,respectively.Under the optimal conditions,the enzymatic N-acylation of 7-ACA with ethyl bromoacetate afforded the desired product with the yield of 77% in 6 h.It was found that no significant improvements in the yields of N-bromoacetyl-7-ACA were achieved by optimizing the process.So,the side reactions in enzymatic synthesis of N-bromoacetyl-7-ACA were exploited,which may provide a new opportunity for improving the yield of the desired product.It was found that the following side reactions happened:(1)7-ACA could be hydrolyzed to 3-deacetyl-7-ACA and acetic acid in the presence of the immobilized enzyme;then,3-deacetyl-7-ACA was acylated by acetic acid,thus affording N-acetyl-3-deacetyl-7-ACA;also,the lactone derivative of N-acetyl-3-deacetyl-7-ACA was formed because of the occurrence of the lactonization between 3-hydroxymethyl and 4-carboxylic acid;and this lactone could be hydrolyzed to N-acetyl-3-deacetyl-7-ACA by the enzyme.(2)N-Bromoacetyl-3-deacetyl-7-ACA was synthesized from 3-deacetyl-7-ACA and ethyl bromoacetate in the presence of enzyme;additionally,a none-enzymatic reaction between3-deacetyl-7-ACA and ethyl bromoacetate gave the corresponding quaternary ammonium,which simultaneously reacted with NaHCO3,furnishing N-(2-ethoxy-2-oxoethyl)-3-deacetyl-7-ACA.Based on the SDS-PAGE analysis of the enzyme solution used for preparing the immobilized enzyme PGA-750,it was found that,in addition to ? and ? subunits of PAs,other impure proteins were present,which may lead to the above side reactions.An enzymatic approach to the key intermediate for preparing cefathiamidine(N-bromoacetyl-7-ACA)was successfully developed in an aqueous system.And the catalytic performance and behavior of this immobilized PA were uncovered in the synthesis ofN-bromoacetyl-7-ACA.The present study may provide the therotical guidance for the green large-scale production of cefathiamidine.