Synthesis of screening substrates for the directed evolution of sialic acid aldolase: towards tailored enzymes for the preparation of influenza A sialidase inhibitor analogues.
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.
Woodhall
Thomas
T
School of Chemistry, University of Leeds, Leeds, UK LS2 9JT .
Williams
Gavin
G
Berry
Alan
A
Nelson
Adam
A
D019020:Directed Molecular Evolution; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D009980:Influenza A virus / Q000201:enzymology*; D008958:Models, Molecular; D009439:Neuraminidase / Q000037:antagonists & inhibitors*; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D009439:Neuraminidase; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase