INHIBITION OF EPSP SYNTHASE BY ANALOGS OF THE TETRAHEDRAL INTERMEDIATE AND OF EPSP

A number of analogues of the intermediate (1) and product of the enzyme EPSP synthase are described. Inert analogues that show high affinity for the enzyme from Petunia hybrida are produced by replacing the labile ketal phosphate moiety of this intermediate with a phosphonate (4) or by stabilizing the ketal phosphate against ionization through introduction of fluorine substituents (5 and 6). The most potent inhibitor is the R-stereoisomer of the difluoromethyl derivative, (R)-6, with a K(i) value of 4 nM. The uncertain stereochemical preference exhibited by the enzyme for the side-chain stereoisomers of these tetrahedral analogues and for the reduced derivatives of EPSP itself (8) suggests that the methyl binding site is indistinct and that the phosphate and carboxylate moieties may interchange binding sites without undue penalty. Fluorine or methyl substitution at the 9-Z position of EPSP reduces the affinity of these compounds by 1 order of magnitude only, but it abolishes their ability to act as substrates. The analogues were synthesized by a general strategy (Chart I) that involves introduction of the C-5 side chain onto a 3,4 protected shikimate, lactonization to facilitate separation and assignment of the diastereomers and to allow selective introduction of the allylic phosphate, and final deprotection. Novel synthetic methods developed in this connection include the multistep, single-flask generation of the ketal phosphates (viz. 13a --> 18 --> 19 --> 20 --> 14c) and an improvement in the procedure for double deprotection of bis(p-nitrophenethyl) phosphate triesters under basic conditions. A model study involving pyruvate ketal phosphates substituted with a single electron-withdrawing substituent demonstrated the stability of this functional group and helped to define methods for its construction.