Journal
ACS CATALYSIS
Volume 1, Issue 9, Pages 1022-1034Publisher
AMER CHEMICAL SOC
DOI: 10.1021/cs2002884
Keywords
oligo(lysine); protease catalysis; peptide synthesis; bromelain; HPLC
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Funding
- Center for Biocatalysis and Bioprocessing of Macromolecules at NYU-POLY
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This paper describes the protease-catalyzed synthesis of oligo(L-Lys) from L-Lys ethyl ester (L-Lys-Et) in an aqueous reaction medium at controlled pH using a pH-stat. Four proteases (papain, bromelain, cc-chymotrypsin, and trypsin) were studied to determine their activity for L-Lys ethyl ester oligomerization at pH values ranging from 6 to 11. Bromelain was found to be preferred relative to the other protease catalysts because it gave the highest values of oligo(L-Lys) yield and optimal average chain length (DPavg). To evaluate reaction progress and product structure, H-1 NMR and HPLC-UV-MS methods were developed. A series of model oligo(L-Lys) compounds with chain lengths 2, 3, and 4 was obtained and analyzed to assign H-1 NMR signals. All components of the mixture were successfully resolved and analyzed by HPLC-UV-MS by ion-pairing reversed-phase chromatography using heptafluorobutyric acid as the ion-pairing agent. The effects of medium pH, L-Lys-Et concentration, bromelain concentration, reaction time, and reaction temperature on oligo(L-Lys) synthesis was evaluated. Oligomers with chain lengths more than 10 are formed within 5 min. By 30 min, the DPavg and longest oligomer chain lengths reached maximum values of similar to 3.6 and 12.0, respectively. Residual L-Lys-Et was only 2% by 40 min. Products formed by 30 min remained relatively unchanged as the reaction was continued for 2.5 h. The competitive reaction pathways during oligomer initiation, propagation and transamidation/hydrolysis pathways are presented and discussed relative to the results obtained herein.
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