Chiral dimethyl 2-methylsuccinate (
1) is a very important building block for the manufacturing of many active pharmaceutical ingredients and fine chemicals. The asymmetric reduction of C=C double bond of dimethyl citraconate (
2), dimethyl mesaconate (
3) or dimethyl itaconate (
4) by ene-reductases (ERs) represents an attractive straightforward approach, but lack of high-performance ERs, especially (
S)-selective ones, has limited implementing this method to prepare the optically pure dimethyl 2-methylsuccinate. Herein, three ERs (Bac-OYE1 from
Bacillus sp.,
SeER from
Saccharomyces eubayanus and
AfER from
Aspergillus flavus) with high substrate tolerance and stereoselectivity towards
2,
3 and
4 have been identified. Up to 500 mM of
3 was converted to (
S)-dimethyl 2-methylsuccinate ((
S)-
1) by
SeER in high yields (80%) and enantioselectivity (98%
ee), and 700 mM of
2 and 400 mM of
4 were converted to (
R)-
1 by Bac-OYE1 and
AfER, respectively, in high yields (86% and 77%) with excellent enantioselectivity (99%
ee). The reductions of diethyl citraconate (
5), diethyl mesaconate (
6) and diethyl itaconate (
7) were also tested with the three ERs. Although up to 500 mM of
5 was completely converted to (
R)-diethyl 2-methylsuccinate ((
R)-
8) by Bac-OYE1 with excellent enantioselectivity (99%
ee), the alcohol moiety of the esters had a great effect on the activity and enantioselectivity of ERs. This work provides an efficient methodology for the enantiocomplementary production of optically pure dimethyl 2-methylsuccinate from dimethyl itaconate and its isomers at high titer.
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