Analogues of N-acetylneuraminic acid (sialic acid, NANA, Neu5Ac), including 6-dipropylcarboxamides, have been found to be selective and potent inhibitors of influenza sialidases. Sialic acid analogues are, however, difficult to synthesize by traditional chemical methods and the enzyme N-acetylneuraminic acid lyase (NAL) has previously been used for the synthesis of a number of analogues. The activity of this enzyme towards 6-dipropylcarboxamides is, however, low. Here, we used structure-guided saturation mutagenesis to produce variants of NAL with improved activity and specificity towards 6-dipropylcarboxamides. Three residues were targeted for mutagenesis, Asp191, Glu192 and Ser208. Only substitution at position 192 produced significant improvements in activity towards the dipropylamide. One variant, E192N, showed a 49-fold improvement in catalytic efficiency towards the target analogue and a 690-fold shift in specificity from sialic acid towards the analogue. These engineering efforts provide a scaffold for the further tailoring of NAL for the synthesis of sialic acid mimetics.

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.

The stereochemical course of aldolase-catalysed reaction between fluoropyruvate and aldehydes is described.

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.

Diisopropylsilyl ethers were activated with N-bromosuccinimide, and reacted with a fluorous-tagged alcohol, to yield tethered substrates for ring-closing metathesis reactions.

No 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.

The bisindolylmaleimides are selective protein kinase inhibitors that can adopt two limiting diastereomeric (syn and anti) conformations. The configurational stability of a range of substituted and macrocyclic bisindolylmaleimides was investigated by using appropriate techniques. With unconstrained bisindolylmaleimides, the size of the 2-indolyl substituents was found to affect configurational stability, though not sufficiently to allow atropisomeric bisindolylmaleimides to be obtained. However, with a tether between the two indole nitrogen atoms in place, the steric effect of 2-indolyl substituents was greatly exaggerated, leading to large differences in configurational stability. The rate of interconversion of the syn and anti conformers varied by over twenty orders of magnitude through substitution of a bisindolylmaleimide ring system, which was constrained within a macrocyclic ring. Indeed, the first examples of configurationally stable atropisomeric bisindolylmaleimides are reported; the half-life for epimerisation of these compounds at room temperature was estimated to be >10(7) years.