Investigation of steric and functionality limits in the enzymatic dihydroxylation of benzoate esters. Versatile intermediates for the synthesis of pseudo-sugars, amino cyclitols, and bicyclic ring systems

A series of benzoate esters (methyl, ethyl, n-Pr, i-Pr, n-Bu, t-Bu, allyl, and propargyl) were subjected to enzymatic dihydroxylation by E. coli JM 109(pDTG 601) strain in a whole-cell fermentation. Thecis-cyclohexadienediols were obtained in yields of ~1g/L except for n-propyl- and i-propyl benzoate which were found to be poor substrates. n-Butyl and t-butyl benzoates were not oxidized at all. The absolute stereochemistry for all metabolites was determined by comparison with a standard prepared from (1S-cis)-3-bromo-3,5-cyclohexadiene-1,2-diol, whose absolute con.guration is well established. The free diols were found to be quite stable compared to other cis-dihydrodiols of this type, however, their acetonides underwent a dimerization via a regio- and stereoselective Diels–Alder cycloaddition. The diol derived from ethyl benzoate was subjected to a stereo- and regioselective inverse electron demand Diels–Alder cycloadditions with several dienophiles. The new adducts were completely characterized. The hetero-Diels–Alder reaction of this diol with an acyl nitroso dienophile yielded regio- and stereoselectively a bicyclic oxazine, which upon reduction provided a useful derivative of amino shikimate that can be exploited in an approach to oseltamivir (Tami.u) and other amino cyclitols. The diol was also converted to carba-a-L-galactopyranose to demonstrate its potential utility as a source of pseudo sugars. Experimental and spectral data are provided for all new compounds.