Synthesis of 4,8-anhydro-2,3-dideoxy-d-galacto- and -d-gluco-non-3-enose dimethyl acetal and their use as new probes for determining by 1H-n.m.r. spectroscopy the steric course of protonation by glycoside hydrolases

Abstract

The title nonosulose derivatives 2(d-galacto) and 4-(d-gluco) were prepared by multistep syntheses. Addition of water to the enolic double bonds of both compounds was catalyzed only by the corresponding enzymes β-d-galactosidase from Escherichia coli, α-d-galactosidase from green coffee beans, and β-d-glucosidase from sweet almonds, α-d-glucosidase from yeast. The enzymic hydration of 2, performed in D2O to analyze the steric course of the addition, gave 2,3-dideoxy-α-d-galacto-(3-2H)-nonos-4-ulose dimethyl acetal (5), which when hydrolyzed gave an equilibrium mixture of the spiranes 16 and 17 as the main products (85%) and the fused-ring systems 18 and 19 as minor components (15%). Borohydride reduction of the product of enzymic hydration gave a separable mixture of the two epimers 14 and 15, convertible in acidic methanol for 8 h at 62° into 20 and 22, respectively. The rigid, bicyclic ring-systems allow facile assignment of the configuration at the monodeuterated C-3 as (S), thereby allowing determination of the steric course of the initial, enzyme-catalyzed step, the deuteration of the enolic double bond in the substrates used. © 1991.