Chemoenzymatic Production of Enantiocomplementary 2-Substituted 3-Hydroxycarboxylic Acids from L-α-Amino Acids

A two-enzyme cascade reaction plus in situ oxidative decarboxylation for the transformation of readily available canonical and non-canonical L-α-amino acids into 2-substituted 3-hydroxy-carboxylic acid derivatives is described. The biocatalytic cascade consisted of an oxidative deamination of L-α-amino acids by an L-α-amino acid deaminase from Cosenzaea myxofaciens, rendering 2-oxoacid intermediates, with an ensuing aldol addition reaction to formaldehyde, catalyzed by metal-dependent (R)- or (S)-selective carboligases namely 2-oxo-3-deoxy-l-rhamnonate aldolase (YfaU) and ketopantoate hydroxymethyltransferase (KPHMT), respectively, furnishing 3-substituted 4-hydroxy-2-oxoacids. The overall substrate conversion was optimized by balancing biocatalyst loading and amino acid and formaldehyde concentrations, yielding 36–98% aldol adduct formation and 91– 98% ee for each enantiomer. Subsequent in situ follow-up chemistry via hydrogen peroxide-driven oxidative decarboxylation afforded the corresponding 2-substituted 3-hydroxycarboxylic acid derivatives.


Endpoint study of PmaLAAD catalyzed oxidative deamination of amino acids
( Figure S1).
The amino acid (50 mM) was added to a suspension of PmaLAAD (1.1 U, 20 mg lyophilized cells), adjusted to 1 mL total volume with phosphate buffer (100 mM, pH 7) in a 6 mL glass vial with plastic screw caps. The vial was placed in a horizontal shaker for 24 h at 1000 rpm and room temperature. An aliquot was withdrawn and derivatized before HPLC measurements. Figure S1. Endpoint study of selected L-amino acid substrates 1 for oxidative deamination catalyzed by PmaLAAD. Conditions: 50 mM substrate, phosphate buffer (100 mM, pH 7), 24 h, room temperature, horizontal shaking.  Figure  S2). Figure S2. One-pot simultaneous multi-enzyme system for the synthesis of enantiomerically pure (R)-or (S)-3-(hydroxymethyl)-4-methyl-2-oxopentanoic acid starting from 1c and formaldehyde.

Initial conditions of one-pot cascade synthesis of (S)-and (R)-3-(hydroxymethyl)-4methyl-2-oxopentanoic acid ((S)-3c and (R)-3c) for optimization studies (
L-Leucine (1c, 50 mM) was added to a suspension of PmaLAAD (1.1 U, 20 mg lyophilized whole cells), containing NiCl 2 or CoCl 2 (0.6 mM) depending on the enzyme, carboligase (1 mg in case of KPHMT from a glycerol stock, or 2 mg in case of MBP-YfaU as a lyophilized powder), and formaldehyde (50 mM) adjusted to 1 mL total volume with MilliQ water in a 6 mL glass vial with plastic screw caps. The vial was placed in a horizontal shaker and shaken for 24 h at 1000 rpm and room temperature. An aliquot was withdrawn and derivatized before HPLC measurements. For optimization studies, the screened parameter was altered.
Conditions as described above with varying catalyst loading. In case of KPHMT-wt 1a was used as model substrate. and 4-hydroxy-3,3-dimethyl-2-oxobutanoic acid (3a). Endpoint study with varying catalyst load under equimolar conditions using MBP-YfaU-W23V and 1a in the case of KPHMT-wt.

Optimized cascade conditions for the synthesis of (S)-and (R)-3-(hydroxymethyl)-4-methyl-2-oxopentanoic acid ((S)-3c and (R)-3c).
L-Leucine (1c, 10 mM) was added to a suspension of PmaLAAD (1.1 U, 20 mg lyophilized whole cells), containing NiCl 2 or CoCl 2 (0.6 mM) depending on the enzyme, carboligase (1 mg of KPHMT from a glycerol stock, 2 mg of MBP-YfaU as a lyophilized powder, respectively), and formaldehyde (150 mM) adjusted to 1 mL total volume with MilliQ water in a 6 mL glass vial with plastic screw caps. The vial was placed in a horizontal shaker and shaken for 24 h at 1000 rpm and room temperature. An aliquot was withdrawn and derivatized before HPLC measurements.
L-Leucine (1c, 50 mM) was added to a suspension of PmaLAAD (1.1 U, 20 mg lyophilized whole cells), containing NiCl 2 or CoCl 2 (0.6 mM) depending on the enzyme, and formaldehyde (50 mM) adjusted to 1 mL total volume with MilliQ water in a 6 mL glass vial with plastic screw caps. The vial was placed in a horizontal shaker at 1000 rpm and room temperature. After 7 h the amino acid was completely consumed, the whole cells were removed, variant MBP-YfaU-W23V was added, and the mixture continued shaken for 14 h.
Then, an aliquot was withdrawn and derivatized before HPLC measurements.     Figure S6).