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Chemically Modified “Polar Patch” Mutants of Subtilisin in Peptide Synthesis with Remarkably Broad Substrate Acceptance: Designing Combinatorial Biocatalysts
Author(s) -
Matsumoto Kazutsugu,
Davis Benjamin G.,
Jones J. Bryan
Publication year - 2002
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/1521-3765(20020916)8:18<4129::aid-chem4129>3.0.co;2-v
Subject(s) - subtilisin , chemistry , yield (engineering) , stereochemistry , substrate (aquarium) , peptide synthesis , amino acid , mutagenesis , serine protease , peptide , protein engineering , site directed mutagenesis , enzyme , combinatorial chemistry , protease , mutant , organic chemistry , biochemistry , materials science , oceanography , metallurgy , gene , geology
A significant enhancement of the applicability of the serine protease subtilisin Bacillus lentus (SBL) in peptide synthesis was achieved by using the strategy of combined site‐directed mutagenesis and chemical modification to create chemically modified mutant (CMM) enzymes. The introduction of polar and/or homochiral auxiliary substituents, such as X=oxazolidinones, alkylammonium groups, and carbohydrates at position 166 at the base of the primary specificity S 1 pocket created SBL CMMs S166C‐S‐X with strikingly broad structural substrate specificities. These CMMs are capable of catalyzing the coupling reactions of not only L ‐amino acid esters but also D ‐amino acid esters as acyl donors with glycinamide to give the corresponding dipeptides in good yields. These powerful enzymes are also applicable to the coupling of L ‐amino acid acyl donors with α ‐branched acyl acceptor, L ‐alaninamide. Typical increases in isolated yields of dipeptides of 60–80 % over SBL‐WT (e.g. 0 % yield of Z‐ D ‐Glu‐GlyNH 2 using SBL‐WT→74 % using S166C‐S‐(CH 2 ) 2 NMe 3 + ) demonstrate the remarkable synthetic utility of this “polar patch” strategy. Such wide‐ranging systems displaying broadened and therefore similarly high, balanced yields of products (e.g. 91 % Z‐ L ‐Ala‐GlyNH 2 and 86 % yield of Z‐ D ‐Ala‐GlyNH 2 using S166C‐S‐(3 R ,4 S )‐indenooxazolidinone) may now allow the use of biocatalysts in parallel library synthesis.