Synthesis of screening substrates for the directed evolution of sialic acid aldolase: towards tailored enzymes for the preparation of influenza A sialidase inhibitor analogues

Abstract

The stereoselective synthesis of two epimeric screening substrates, (4R, 5R, 6R)- and (4S, 5R, 6R)-6-dipropylcarbamoyl-2-oxo-4,5,6-trihydroxy-hexanoic acid, for the directed evolution of sialic acid aldolase is described. The complementary methods relied on stereoselective indium-mediated additions of ethyl alpha-bromomethyl acrylate to functionalised aldehydes. With an alpha-hydroxy aldehyde, (2R, 3R)-2,3-dihydroxy-4-oxo butanoic acid dipropylamide, the addition was chelation controlled, and the syn product, (6R, 5R, 4S)-6-dipropylcarbamoyl-2-methylidene-4,5,6-trihydroxy-hexanoic acid ethyl ester, was obtained. In contrast, the stereochemical outcome of the addition to (2R, 3R)-N,N-dipropyl-2,3-O-isopropylidene-4-oxobutyramide was consistent with Felkin-Anh control, and the anti adduct, (4R, 5R, 6R)-6-dipropylcarbamoyl-2-methylidene-4-hydroxy-5,6-O-isopropylidene-hexanoic acid ethyl ester, was the major product. Ozonolysis and deprotection gave the screening substrates as mixtures of furanose and pyranose forms, in good yields.

Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution

“…1), mimics the substrate DHOB, but the reactive aldehyde has been reduced to an alcohol, preventing nucleophilic attack of pyruvate enamine. THB was synthesized, 20 and steady-state enzyme kinetics showed that it is a competitive inhibitor of the E192N NAL reaction (Fig. 6).…”

“…One unit of aldolase activity is defined as the amount of enzyme that catalyzes the oxidation of 1 μmol min − 1 NADH in this system, using the molar extinction coefficient of NADH (6220 M − 1 cm − 1 ). The triol, THB, was synthesized as previously described, 20 and inhibition of the NAL reaction was measured by including THB (0.25–2 mM) in the standard enzyme assay. Steady-state kinetic parameters k cat , K m , and K i were all estimated by nonlinear regression to the appropriate rate equations.…”

Development of an organo- and enzyme-catalysed one-pot, sequential three-component reaction

“…5c, 6 We envisaged, however, that it might be possible to develop a three-component synthesis of similar products (such as 7) in a single pot (Scheme 2). Thus, it was hoped that organocatalysed condensation 7 between a glyoxylamide 4 and acetaldehyde would yield an aldol product 6.…”

Biocatalytic Methods for CC Bond Formation

“…The substrate specificity of NeuA is limited to C5 and C6 aldehydes, whereas short aldehydes are poor acceptors. To optimize NeuA aldolases for other substrates directed evolution approaches were applied and the novel designed enzymes were used for the synthesis of potent influenza inhibitors like zanamivir,51d as well as tertiary amides52 and different sialic acid mimetics 52 with high stereoselectivity, which can be switched by using a proper mutant (Scheme ) 53…”