Darryl Cleary

EPSP Synthase: The Design and Synthesis of Bisubstrate Inhibitors Incorporating Novel 3-Phosphate Mimics

Abstract Novel aromatic bisubstrate inhibitors of the enzyme EPSP (5-enolpyruvoylshikimate-3-phosphate) synthase (EC 2.5.1.19) have been designed and synthesized as structural analogs of the single, catalytic intermediate 1 utilized by the enzyme. These aromatic inhibitors incorporate novel α-hydroxyphosphonates, malonate ethers and α-hydroxymalonates as replacements for the hydrolytically labile 3-phosphate group. These 3-phosphate mimics were much preferred to the corresponding methylene and vinylic phosphonates, malonates and phosphonomethyl ethers.

EPSP synthase inhibitor design II. The importance of the 3-phosphate group for ligand binding at the shikimate-3-phosphate site & the identification of 3-malonate ethers as novel 3-phosphate mimics.

Abstract Studies using alternate substrates, inhibitor product mimics and new derivatives of 4,5-dideoxy-shikimate-3-phosphate (ddS3P) are reported which indicate that the 3-phosphate group contributes significantly to substrate and inhibitor recognition at the shikimate 3-phosphate (S3P) site and that 3-malonate ethers will function as suitable 3-phosphate replacements for substrate and inhibitor binding to the S3P site of this enzyme.

New EPSP synthase inhibitors: synthesis and evaluation of an aromatic tetrahedral intermediate mimic containing a 3-malonate ether as a 3-phosphate surrogate.

A new analog of the EPSP synthase enzyme reaction intermediate 1, containing a 3-malonate ether moiety in place of the usual 3-phosphate group, was synthesized from 3,5-dihydroxybenzoic acid. This simple, synthetically accessible aromatic compound (5) is an effective competitive inhibitor versus S3P with an apparent Ki of 1.3 +/- 0.22 microM. This result demonstrates that a simple benzene ring can be a suitable achiral substitute for the more complex shikimate ring in the design of EPSP synthase inhibitors. Furthermore, the greater potency of 5 versus the phenol 6, glycolate 7 and the gallic acid analog 8 demonstrates the requirement for multiple anionic charges at the dihydroxybenzoate 5-position in order to attain effective inhibition of this enzyme. However, this 3-malonate ether substituted compound was at least 10-fold less effective as a bisubstrate inhibitor than the corresponding 3-phosphate. This suggests that tetrahedral intermediate mimics possessing a 3-malonate ether moiety are less effective than their corresponding 3-phosphates in accessing the optimal enzyme conformation stabilizing 1.

An Enzyme-Targeted Herbicide Design Program Based on EPSP Synthase: Chemical Mechanism and Glyphosate Inhibition Studies

The herbicide markets of the late 1990’s and beyond will demand high performance products with stringent environmental acceptability requirements. We have initiated a multi-disciplinary herbicide discovery program directed toward the inhibition of key plant enzymes as one approach to meet these challenges. Plants contain a variety of biosynthetic pathways which are essential for their growth. Effective, plant-specific enzyme inhibitors offer the opportunity to satisfy the herbicide performance needs of the marketplace while exhibiting favorably low mammalian toxicity properties.1 Our enzyme-targeted research effort systematically integrates mechanistic biochemistry, molecular biology, modeling, inhibitor recognition, and structural biology information with organic synthesis through a series of collaborations within and outside Monsanto. A thorough understanding of the chemical mechanism for a particular enzyme target is an essential first step for the design of potent inhibitors.