TY - JOUR
T1 - Fluorogenic kinetic assay for high-throughput discovery of stereoselective ketoreductases relevant to pharmaceutical synthesis
AU - Thai, Yen-Chi
AU - Szekrenyi, Anna
AU - Qi, Yuyin
AU - Black, Gary
AU - Charnock, Simon
AU - Fessner, Wolf-Dieter
PY - 2018/4/1
Y1 - 2018/4/1
N2 - Enantiomerically pure 1-(6-methoxynaphth-2-yl) and 1-(6-(dimethylamino)naphth-2-yl) carbinols are fluorogenic substrates for aldo/keto reductase (KRED) enzymes, which allow the highly sensitive and reliable determination of activity and kinetic constants of known and unknown enzymes, as well as an immediate enantioselectivity typing. Because of its simplicity in microtiter plate format, the assay qualifies for the discovery of novel KREDs of yet unknown specificity among this vast enzyme superfamily. The suitability of this approach for enzyme typing is illustrated by an exemplary screening of a large collection of short-chain dehydrogenase/reductase (SDR) enzymes arrayed from a metagenomic approach. We believe that this assay format should match well the pharmaceutical industry’s demand for acetophenone-type substrates and the continuing interest in new enzymes with broad substrate promiscuity for the synthesis of chiral, non-racemic carbinols.
AB - Enantiomerically pure 1-(6-methoxynaphth-2-yl) and 1-(6-(dimethylamino)naphth-2-yl) carbinols are fluorogenic substrates for aldo/keto reductase (KRED) enzymes, which allow the highly sensitive and reliable determination of activity and kinetic constants of known and unknown enzymes, as well as an immediate enantioselectivity typing. Because of its simplicity in microtiter plate format, the assay qualifies for the discovery of novel KREDs of yet unknown specificity among this vast enzyme superfamily. The suitability of this approach for enzyme typing is illustrated by an exemplary screening of a large collection of short-chain dehydrogenase/reductase (SDR) enzymes arrayed from a metagenomic approach. We believe that this assay format should match well the pharmaceutical industry’s demand for acetophenone-type substrates and the continuing interest in new enzymes with broad substrate promiscuity for the synthesis of chiral, non-racemic carbinols.
KW - Alcohol dehydrogenase
KW - Asymmetric synthesis
KW - Biocatalysis
KW - Chiral alcohols
KW - Enantioselectivity
UR - https://www.scopus.com/pages/publications/85019602758
U2 - 10.1016/j.bmc.2017.05.024
DO - 10.1016/j.bmc.2017.05.024
M3 - Article
SN - 0968-0896
VL - 26
SP - 1320
EP - 1326
JO - Bioorganic & Medicinal Chemistry
JF - Bioorganic & Medicinal Chemistry
IS - 7
ER -