Creation of a Pair of Stereochemically Complementary Biocatalysts

Abstract

N-Acetylneuraminic acid lyase (NAL) exhibits poor facial selectivity during carbon-carbon formation, and as such, its utility as a catalyst for use in synthetic chemistry is limited. For example, the NAL-catalyzed condensation between pyruvate and (2R,3S)-2,3-dihydroxy-4-oxo-N,N-dipropylbutyramide yields ca. 3:1 mixtures of diastereomeric products under either kinetic or thermodynamic control. Engineering the stereochemical course of NAL-catalyzed reactions could remove this limitation. We used directed evolution to create a pair of stereochemically complementary variant NALs for the synthesis of sialic acid mimetics. The E192N variant, a highly efficient catalyst for aldol reactions of (2R,3S)-2,3-dihydroxy-4-oxo-N,N-dialkylbutyramides, was chosen as a starting point. Initially, error-prone PCR identified residues in the active site of NAL that contributed to the stereochemical control of an aldolase-catalyzed reaction. Subsequently, an intense structure-guided program of saturation and site-directed mutagenesis was used to identify a complementary pair of variants, E192N/T167G and E192N/T167V/S208V, which were approximately 50-fold selective toward the cleavage of the alternative 4S- and 4R-configured condensation products, respectively. It was shown that wild-type NAL could not be used for the highly stereoselective synthesis of a 6-dipropylamide sialic acid mimetic because the 4S-configured product was only approximately 3-fold kinetically favored and only approximately 3-fold thermodynamically favored over the alternative 4R-configured product. However, the complementary 4R- and 4S-selective variants allowed the highly (>98:<2) diastereoselective synthesis of both 4S- and 4R-configured products under kinetic control from the same starting materials. Conversion of an essentially nonselective aldolase into a pair of complementary biocatalysts will be of enormous interest to synthetic chemists. Furthermore, since residues identified as critical for stereoselectivity are conserved among members of the NAL superfamily, the approach might be extended to the evolution of other useful biocatalysts for the stereoselective synthesis of biologically active molecules.

Extending enzyme molecular recognition with an expanded amino acid alphabet

“…1A). The substrate specificity and stereochemistry of this enzyme is highly malleable through protein engineering and directed evolution (34,35), and we have shown that an enzyme bearing the Nca γ-thialysine (2-aminoethyl cysteine) in place of the catalytic Lys-165 retains activity (36). Here we have explored the effect of introducing Ncas throughout the active site on the reaction catalyzed.…”

“…Aldolases are an important class of biocatalysts that are finding increasing uses in the synthesis of complex compounds (33); they are particularly useful in that they can create two stereochemical centers during their reaction (34,(44)(45)(46) and the reactions can be carried out in aqueous solvent without the use of protecting groups. However, the substrate specificity of natural aldolases often has to be engineered to produce enzymes with the required substrate specificity (33,47).…”

Enzyme‐Catalyzed Aldol Additions

“…Then, a structure-guided program of saturation and site-directed mutagenesis was conducted. As a result two variants, E192N/T167G and E192N/T167V/S208V, allowed the highly (>98 : <2) diastereoselective synthesis of both (4S,5R,6R)-and (4R,5R,6R)-6-dipropylcarbamoyl-2-oxo-4,5,6-trihydroxy-hexanoic acid (41c/42c) (Scheme 8.9) under kinetic control from the same starting materials [40].…”

Aldolase-catalysed stereoselective synthesis of fluorinated small molecules

“…[7] Furthermore, protein engineering has been shown to be valuable for increasing further the synthetic value of aldolase enzymes, for example by broadening the range of substrates accepted or by modifying the stereochemistry of carboncarbon bond formation. [8][9][10][11][12][13][14] Recently, aldolases have emerged as a useful class of catalysts for controlling the formation of fluorine-bearing stereocentres. [15 16 Approaches for the synthesis of fluorinated small molecules are important because fluorination can tune exquisitely small molecule conformation and biological activity.…”