Joel E. Ream

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.

Synthesis and evaluation of two new inhibitors of EPSP synthase

Abstract The enzyme EPSP synthase, EPSPS, (EC 2.5.1.19) catalyzes an unusual transfer reaction of the enolpyruvoyl moiety from phosphoenol pyruvate ( 2 , PEP) regiospecifically to the 5-OH of shikimate 3-phosphate ( 1 , S3P) to form 5-enol-pyruvoylshikimate 3-phosphate ( 3 , EPSP). Two new inhibitors, ( 4 , and 5 ) were prepared to probe the S3P binding site.

EPSP synthase inhibitor design IV. New aromatic substrate analogs and symmetrical inhibitors containing novel 3-phosphate mimics.

Abstract New 4-deoxy aromatic analogs of S3P and EPSP have been synthesized as potential substrate-based inhibitors of EPSPS to evaluate various 3-phosphate replacements in combination with the aromatic ring system. These studies identified 3-malonate ethers and α-hydroxymethylphosphonates as suitable 3-phosphate mimics in this series and led to the discovery of two unexpectedly potent symmetrical aromatic inhibitors 6 and 7 .

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.

Functionalized 3,5-dihydroxybenzoates as potent novel inhibitors of EPSP synthase

Aromatic analogues of the EPSP synthase enzyme substrate (S3P), reaction intermediate (1), and product (EPSP) were synthesized from 3,5-dihydroxybenzoic acid and were evaluated as inhibitors of E. coli EPSP synthase. These simple, synthetically accessible aromatic analogues are highly effective competitive inhibitors versus S3P with an apparent Ki for the tetrahedral intermediate analogue 4 of 160 +/- 40 nM. This demonstrates that a simple benzene ring is a quite suitable substitute for the complex shikimate ring in the design of EPSP synthase inhibitors.

Synthesis & characterization of N-amino-glyphosate as a potent analog inhibitor of E. coli EPSP synthase

Abstract All previous attempts to identify glyphosate analogs which retain their potency against the known biological target, EPSP synthase, have been unsuccessful. Consequently, the glyphosate binding site was thought to be extremely specific in this system. Here we report the novel N-amino glyphosate analog 3 as the first successful modification of the glyphosate skeleton which exhibits inhibitor properties comparable to glyphosate.

New aromatic inhibitors of EPSP synthase incorporating hydroxymalonates as novel 3-phosphate replacements

A new, aromatic analogue of the EPSP synthase enzyme reaction intermediate 1 has been identified, which contains a 3-hydroxymalonate moiety in place of the usual 3-phosphate group. This simplified inhibitor was readily prepared in five steps from ethyl 3,4-dihydroxybenzoate. The resulting tetrahedral intermediate mimic 9 is an effective, competitive inhibitor versus S3P with an apparent Ki of 0.57 +/- 0.06 microM. This result demonstrates that 3-hydroxymalonates exhibit potencies comparable to aromatic inhibitors containing the previously identified 3-malonate ether replacements and can thus function as suitable 3-phosphate mimics in this system. These new compounds provide another example in which a simple benzene ring can be used effectively in place of the more complex shikimate ring in the design of EPSP synthase inhibitors. Furthermore, the greater potency of 9 versus the glycolate derivative 10 and the 5-deoxy-analog 11, again confirms the requirement for multiple anionic charges at the dihydroxybenzoate 5-position in order to attain effective inhibition of this enzyme.

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.

High Fermentable Corn Hybrids for the Dry-Grind Corn Ethanol Industry

The biofuel corn ethanol helps provide a sustainable and secure non-petroleum source of energy. The dry-grind ethanol industry is the customer for about one-third of US-produced corn grain. Getting the most ethanol from sourced corn grain is important to the economics of a commercial ethanol plant. Near infrared transmittance spectroscopy (NIT), backed by calibrations built with robust reference chemistry, is used to predict the fermentability of whole corn grain. Ethanol yield predicted by NIT has been shown to be highly correlated with commercial ethanol yield. High fermentable corn hybrids identified using NIT have been designated by commercial seed producers and made available to corn growers. The combination of a robust, commercially validated NIT calibration and a rigorous corn hybrid designation process has been used to identify high fermentable corn hybrids to enable higher ethanol yields for the dry-grind ethanol industry.