Creation of a Tailored Aldolase for the Parallel Synthesis of Sialic Acid Mimetics

Abstract

Broadening substrate specificity: A modified form of sialic acid aldolase (E192N) exhibits a 640‐fold switch in substrate specificity relative to the wild‐type enzyme. Ozonolysis of the unsaturated amides 1, followed by an E192N‐mediated step, was exploited in the parallel synthesis of 14 different sialic acid mimetics of general structure 2.

Enzyme‐Catalyzed Aldol Additions

“…NeuA-catalyzed aldol additions of pyruvate to four carbon sugar such as l-and d-erythrose and threose reacting at between 0.3 and 5% of the rate of N-acetylmannosamine, whereas two-and three-carbon aldehydes are not substrates [37]. To overcome this limitation, NeuA was engineered to accept four-carbon aldehydes of general structure 40 (Scheme 8.8) into the corresponding sialic acid mimetics 41 and 42 [38]. Analysis of the X-ray crystallographic structure of the homologous sialic acid aldolase from Haemophilus influenzae, which has 35% identity and 59% similarity to the corresponding E. coli protein, revealed three residues in the E. coli protein, Asp191, Glu192, and Ser208, which were targeted separately by using saturation mutagenesis and screened.…”

“…The single point mutant, NeuA Glu192Asn, showed rather broad substrate spectrum, and with a stereofacial selectivity of ∼80 : 20 being the most promiscuous mutant among those screened in terms of substrate selectivity (Scheme 8.8). Moreover, in the retroaldol direction, this mutant catalyzes the cleavage of (5R,6R)-6-dipropylcarbamoyl-2-oxo-4,5,6-trihydroxyhexanoic acid (DPAH) (41c/42c) into pyruvate and (2R,3S)-2,3-dihydroxy-4-oxo-N,N-dipropylbutanamide (40c) five times more effectively that the wild-type enzyme catalyzes the cleavage of N-acetylneuraminic acid, its natural substrate [38].…”

Computational Tools for Rational Protein Engineering of Aldolases

“…It is used for the synthesis of sialic acid derivatives. To extend the substrate scope of NANA of E. coli a semirational approach was used [95, 96]. As there was no structure available for the E. coli enzyme in complex with a substrate analog the structure of a complex of a related enzyme (35% identity) was used to identify residues that interact with the acceptor substrate.…”

“…The new aldolase (E192N) shows a 49-fold increase in catalytic efficiency towards the screening substrate compared to wt [96] and an almost 6-fold higher catalytic efficiency towards the new substrate than NANAwt towards its natural substrate. NANA E192N was successfully applied for the synthesis of sialic acid mimetics from substrates with differently substituted tertiary amides [95]. The products were obtained in a ≈ 80:20 mixture of the epimers.…”

Fluorescence Assays for Biotransformations

“…[14] and Woodhall et al . [15] for evolving sialic acid aldolases to accept non -natural aldehyde acceptors.…”