(S)‐Selectivity in Phenylacetyl Carbinol Synthesis Using the Wild‐Type Enzyme Acetoin:Dichlorophenolindophenol Oxidoreductase from Bacillus licheniformis

Abstract

Thiamine diphosphate (ThDP)-dependent enzymes are well known biocatalysts for the asymmetric synthesis of α-hydroxy ketones with preferential (R)selectivity. Pharmaceutically relevant phenylacetyl carbinol (PAC) is prepared with absolute (S)-configuration only in few occasions using enzyme variants suitably designed through rational site-directed mutagenesis approaches. Herein, we describe the synthesis of (S)-phenylacetyl carbinol products with extended reaction scope employing the readily available wild-type ThDP-dependent enzyme acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) from Bacillus licheniformis. On a semipreparative scale, cross-benzoin-like condensations of methylacetoin (donor) and differently substituted benzaldehydes proceed with almost complete chemoselectivity yielding the target (S)-1-hydroxy-1phenylpropan-2-one derivatives with high conversion efficiencies (up to 95%) and good enantioselectivities (up to 99%).Ao:DCPIP OR accepts hydroxy-and nitro-benzaldehydes and also sterically demanding substrates such as 1naphthaldehyde and 4-(tert-butyl)benzaldehyde, which are typically poor acceptors in enzymatic transformations. The explorative synthesis of (S)-phenylpropionyl carbinol mediated by Ao:DCPIP OR via carboligation of benzaldehyde with 3,4-hexanedione is also reported.

Thiamine‐Diphosphate‐Dependent Enzymes as Catalytic Tools for the Asymmetric Benzoin‐Type Reaction

“…[66] Several PDC variants with improved carboligation activity have been obtained by site-directed mutagenesis. The previously mentioned ZmPDC-Glu473Gln variant (Section 4) catalyzes PAC formation with a 20-fold increased rate, affording a three-times higher yield (from 30 to 98 %) than the wt enzyme ( [66] 2 ZmPDC 30; 98 (R) [34a] 3 ScPDC 75; 90 (R) [66] 4 ZmPDC-Glu473Gln 98; 98.4 (R) [34a] 5 ZmPDC-Glu473Phe 93; 99.6 (R) [41] 6 ZmPDC-Glu473Gly 95; 76 (S) [41] 7 ApPDC-Glu469Gly 95; 70 (S) [31] 8 EcAHAS-I 75; 90 (R) [69] 9 CDH 75; 90 (R) [71] 10 Ao:DCPIP OR 95; 94 (S) [72] This improved performance is the result of stabilization of the enamine/carbanion intermediate, which is protonated 2000 times more slowly in the enzyme variant because of the loss of GLu473 general acid catalysis. This residue is highly conserved in PDCs and thus also found in PDCs from S. cerevisiae [67] (position 477) and A. pasteurianus [31] (position 469).…”

“…Very recently, our group demonstrated that acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) can also be used to catalyze the asymmetric synthesis of PAC analogues. [72] This enzyme, the name of which comes from its ability to catalyze the 2,6-dichlorophenolindophenol-dependent cleavage of acetoin into acetate and acetaldehyde, is involved in the bacterial deg- Table 13. Monosubstituted PAC analogues synthesized by CDH [71] and acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR).…”

“…as donor. [72] radation of acetoin, together with the other components of the acetoin dehydrogenase enzyme system (ADH-ES). [73] As in the cases of most of the enzymes discussed up until this point, a side carboligation activity has also been reported for Ao:DCPIP OR; its exploitation for the asymmetric synthesis of tertiary alcohols is discussed in the next section.…”

“…[76] With regard to the production of (S)-1-hydroxy-1-phenylpropan-2-one, we have recently proposed a preliminary study in which we have demonstrated that, under non-optimized conditions, the combined use of Ao:DCPIP OR as catalyst with hexane-3,4-dione as propionyl anion donor affords (S)-1hydroxy-1-phenylpropan-2-one (ee 94 %) in 60 % isolated yield (Table 16, Entry 2). [72]…”

Structural and Mutagenesis Studies of the Thiamine‐Dependent, Ketone‐Accepting YerE from Pseudomonas protegens

“…[5] Recently,G iovannini et al reported the highly enantioselective synthesis of (S)-phenylacetylcarbinol [(S)-PAC] and its derivatives in good yields by the wild-type Ao:DCPIP OR using methylacetoin as ad onors ubstrate. [8] Beforet hat, the synthesis of (S)-PAC was achieved only by rationally designed variantsofR-selective enzymes. [9] The relatede nzymeA coAB from Bacillus subtilis plays ak ey role in the biodegradation of methylacetoin.…”