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Sialyl Lewis x and derivatives have been synthesized using £-1,4-galactosyltransferase and recombinant a-2,3sialyltransferase and a-l,3-fucosyltransferase. The enzymatic glycosylations have been achieved on preparative scales with in situ regeneration of UDP-galactose, CMP-7V-acetylneuraminic acid, and GDP-fucose. Additionally, galactosyltransferase and fucosyltransferases have been studied with respect to their substrate specificity and inhibition. The enzymatic procedures have also been used in the synthesis of 2'-deoxy-LacNAc, 2'-amino-2'-deoxy-LacNAc, 2-azido-Lac, Lewis x, the Lewis x analog with GlcNAc replaced with 5-thioglucose, [Gal-l-13C]-LacNAc, [Gal-1 -13C]-sialyl Lewis x, and the corresponding terminal glycal. The synthesized 13C-labeled sialyl Lewis x and intermediates (including Lewis x and sialyl LacNAc) were used for conformational study using NMR techniques combined with calculations based on GESA and MM2 programs. GESA calculation of sialyl Lewis x gave four minimum-energy conformers, and the two (A and B) consistent with NMR results were further refined with MM2 calculation. The one (A') with lower energy was picked as the preferred conformer which had all intemuclear distances and glycosidic torsional angles consistent with the NMR analysis. The glycosidic torsional angle \p of Gal-GlcNAc, for example, was determined to be 18°on the basis of the coupling between Gal-l-13C and GlcNAc, while the predicted value was 15°. The tetrasaccharide appears to form a well-defined hydrophilic surface along NeuAc-Gal-Fuc, and a hydrophobic face underneath NeuAc-Gal-GlcNAc. Comparing the conformation of sialyl Lewis x to sialyl Lewis a indicates that the recognition domain of sialyl Lewis x mainly comes from the sialic acid-galactose-fucose residues.

The mechanism and substrate specificity of the phosphotriesterase from Pseudomonas diminuta have been examined. The enzyme hydrolyzes a large number of phosphotriester substrates in addition to paraoxon (diethyl p-nitrophenyl phosphate) and its thiophosphate analogue, parathion. The two ethyl groups in paraoxon can be changed to propyl and butyl groups, but the maximal velocity and Km values decrease substantially. The enzyme will not hydrolyze phosphomonoesters or -diesters. There is a linear correlation between enzymatic activity and the pKa of the phenolic leaving group for 16 paraoxon analogues. The beta value in the corresponding Brønsted plot is -0.8. No effect on either Vmax or Vmax/Km is observed when sucrose is used to increase the relative solvent viscosity by 3-fold. These results are consistent with rate-limiting phosphorus-oxygen bond cleavage. A plot of log V versus pH for the hydrolysis of paraoxon shows one enzymatic group that must be unprotonated for activity with a pKa of 6.1. The deuterium isotope effect by D2O on Vmax and Vmax/Km is 2.4 and 1.2, respectively, and the proton inventory is linear, which indicates that only one proton is "in flight" during the transition state. The inhibition patterns by the products are consistent with a random kinetic mechanism.

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