| Summary: | Chiral dimethyl 2-methylsuccinate (<b>1</b>) 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 (<b>2</b>), dimethyl mesaconate (<b>3</b>) or dimethyl itaconate (<b>4</b>) by ene-reductases (ERs) represents an attractive straightforward approach, but lack of high-performance ERs, especially (<i>S</i>)-selective ones, has limited implementing this method to prepare the optically pure dimethyl 2-methylsuccinate. Herein, three ERs (Bac-OYE1 from <i>Bacillus</i> sp., <i>Se</i>ER from <i>Saccharomyces eubayanus</i> and <i>Af</i>ER from <i>Aspergillus flavus</i>) with high substrate tolerance and stereoselectivity towards <b>2</b>, <b>3</b> and <b>4</b> have been identified. Up to 500 mM of <b>3</b> was converted to (<i>S</i>)-dimethyl 2-methylsuccinate ((<i>S</i>)-<b>1</b>) by <i>Se</i>ER in high yields (80%) and enantioselectivity (98% <i>ee</i>), and 700 mM of <b>2</b> and 400 mM of <b>4</b> were converted to (<i>R</i>)-<b>1</b> by Bac-OYE1 and <i>Af</i>ER, respectively, in high yields (86% and 77%) with excellent enantioselectivity (99% <i>ee</i>). The reductions of diethyl citraconate (<b>5</b>), diethyl mesaconate (<b>6</b>) and diethyl itaconate (<b>7</b>) were also tested with the three ERs. Although up to 500 mM of <b>5</b> was completely converted to (<i>R</i>)-diethyl 2-methylsuccinate ((<i>R</i>)-<b>8</b>) by Bac-OYE1 with excellent enantioselectivity (99% <i>ee</i>), 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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