John L. Huppatz

Expression of a bacterial gene in transgenic tobacco plants confers resistance to the herbicide 2,4-dichlorophenoxyacetic acid

Plants resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were produced through the genetic engineering of a novel detoxification pathway into the cells of a species normally sensitive to 2,4-D. We cloned the gene for 2,4-D monooxygenase, the first enzyme in the plasmid-encoded 2,4-D degradative pathway of the bacterium Alcaligenes eutrophus, into a cauliflower mosaic virus 35S promoter expression vector and introduced it into tobacco plants by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing the highest levels of the monooxygenase enzyme exhibited increased tolerance to 2,4-D in leaf disc and seed germination assays, and young plants survived spraying with levels of herbicide up to eight times the usual field application rate. The introduction of the gene for 2,4-D monooxygenase into broad-leaved crop plants, such as cotton, should eventually allow 2,4-D to be used as an inexpensive post-emergence herbicide on economically important dicot crops.

Stereospecific Inhibitor Probes of the PS II Herbicide Binding Site

The influence of steric factors on the activity of 2-cyanoacrylic esters as inhibitors of the Hill reaction was examined. The spatial arrangement of the different groups in the inhibitor molecule was found to be an important factor in determining potency. The positioning of the phenyl ring in aralkylamino derivatives and the steric properties of the β-substituent are particularly significant in the interaction of molecules with the) hydrophobic domain of the receptor site. The difference in activity observed with optically active α-methylbenzylamino derivatives confirmed the importance of the orientation of the phenyl ring and indicated an interaction with a specific hydrophobic region.

Cyanoacrylate inhibitors of photosynthetic electron transport. Nature of the interaction with the receptor site

The inhibitory activities (pI50 values) of a series of 3-alkylamino-2-cyanoacrylic acid ester derivatives in relation to photosypthetic electron transport in isolated pea chloroplast suspensions are reported. Assuming such pI50 values reflect the relative binding affinities for the inhibitor receptor site, the results suggest that several groups in these molecules interact specifically with the binding domain so that a multi-point attachment is formed. Stereochemical factors appear to play a significant role in the interaction.

Cyanoacrylate Inhibitors of the Hill Reaction V. The Effect of Chirality on Inhibitor Binding

Optically active α-methylbenzylamino 2-cyanoacrylic esters were synthesized and assayed as inhibitors of the Hill reaction in isolated pea chloroplast fragments. The 5-isomers were more potent inhibitors than the S-isomers with discriminations of from ten to greater than 100-fold being observed. A β-alkyl substituent in the cyanoacrylate molecule affected both the level of activity and the difference in activity between the isomers. An α,α-dimethylbenzylamino derivative was also active at about the same level as the corresponding α-methylbenzylamino racemate. This result could be explained in terms of the orientation of the phenyl ring in the receptor site. Replacement of the α-methylbenzylamino group by other α-alkyl and α-phenyl substituents had little effect on activity. However, an α-benzyl group was beneficial.

Cyanoacrylate Inhibitors of Photosynthetic Electron Transport in Atrazine Susceptible and Atrazine Resistant Brassica Chloroplasts

Comparison of the p/50 values for a series of cyanoacrylate derivatives in chloroplasts isolated from atrazinc susccptiblc (wild type) and atrazine resistant (mutant) Brassica napus biotvpes reveal that the degree and direction of discrimination can vary from being 200- fold more active against the wild type to 10-fold more active against the mutant. There appears to be a direct correlation between the level of inhibitory activity in thylakoids isolated from “susceptible” chloroplasts and the level of discrimination between “susceptible” and “resistant” chloroplasts - a correlation which can be improved by allowing for variations in molecular hydrophobicity. Studies with optically active ethoxyethyl-3-alkyl-2-cyano-3-α-methylbenzylamino acrylates suggest that there are specific receptor sites present in both “susceptible” and “resistant” chloroplasts for both the a-methylbenzyl chiral centre and the 3-alkyl moiety. There is a direct relationship between photosynthetic electron transport inhibitory activity and herbicidal activity of optical isomers.

Cyanoacrylate Inhibitors of the Hill Reaction IV. Binding Characteristics of the Hydrophobic Domain

Aryl- and aralkyl-amino derivatives of 2-cyanoacrylic esters were synthesized and assayed as inhibitors of the Hill reaction in isolated pea chloroplast fragments. Aryl- and aralkyl-amino 2-cyanoacrylates unsubstituted in the β-position were weak inhibitors but activity could be greatly enhanced by inclusion of a β-alkyl group with the increase in potency being dependent on the carbon chain length of the β-substituent. The magnitude of this effect appeared independent of the nature of the aryl- or aralkyl-amino function. Inclusion of a carbon chain between the phenyl and amino functions of phenylamino-2-cyano- acrylates produced a stepwise increase in activity with increasing chain length, indicating a prefer- red orientation for the phenyl ring within the hydrophobic binding domain.

Cyanoacrylate inhibitors as probes for the nature of the photosystem II herbicide binding site

Three series of phenyl-substituted 2-cyanoacrylates were evaluated using simple quantitative structure activity relationships (QSAR) in an attempt to elucidate the nature of the regions of the binding site occupied by different parts of the molecules. Inhibition of the Hill reaction by substituted 3-phenylamino-2-cyanoacrylates correlated well with the lipophilicity of the substituent. The hydrophobic effect was also dominant when the Hill activity of a series of 3-benzylamino-2-cyanoacrylates was analyzed, although potency was considerably higher in the latter series. Lipophilicity and the electronic nature of the substituents were not major determinants in the Hill inhibitory activity of a series of substituted phcnoxycthyl 2-cyanoacrylic esters. In this case, a significant correlation was found with the molar rcfractivity (MR) of meta substituents, a parameter reflecting substituent size. The results indicate that the phenyl moiety of substituted 3-phenylamino- and 3-bcnzyl- amino-2-cyanoacrylates interacts with an essentially lipophilic binding domain, though it is likely that the two series are oriented differently with the 3-bcnzylamino series able to bind with greater affinity. In the phcnoxycthyl ester series, the substituted phenyl group interacts with a different environment, wherein ortho- and we7tf-substitution is tolerated, dependent on the bulk of the substituent, but /wra-substitution is detrimental to affinity for this region of the site.

Acetohydroxyacid synthase inhibitors: N-phthalyl-L-valine anilide and related compounds.

Abstract The potency of ʟ-valine as an inhibitor of Zea mays acetohydroxyacid synthase (AHAS) is increased more than 80110-fold on conversion to its N-phthalyl anilide derivative which is active at 2 µᴍ. The ᴅ-valine, α-aminobutyric acid, isoleucine and phenylalanine analogs are 11- to 43-fold less potent, and similar N-phthalyl anilide derivatives of other branched-chain amino acids are essentially inactive. Full potency is retained on replacing the phthalimide moiety of the valine anilide with cyclohexane-1,2-dicarboximide or 1-cyclohexene-1.2-dicarboximide groups and partial activity with 4-cyclohexene-1,2-dicarboximide and methyl- or dimethylmaleimide groups. Inhibition of the enzyme and of root growth by the valine derivatives may result from binding at or near the site involved in feedback control of AHAS by ʟ-valine.

Cyanoacrylate Inhibitors of the Hill Reaction III. Stereochemical and Electronic Aspects of Inhibitor Binding

Ethoxyethyl 3-octylamino-2-cyanoacrylate and related compounds in which the amino group was replaced by N-CH3, S, O and CH2 were synthesized and their activity as inhibitors of the Hill reaction in isolated pea chloroplasts determined. All compounds showed moderate to high activity but there was no obvious correlation between activity and the electronic character of the ester carbonyl group. The stereochemistry of the various inhibitor molecules was deduced from the PMR spectra and the possible influence of stereochemistry on Hill inhibitory activity discussed. Replacement of the olefinic proton in the 2-cyanoacrylates with a β-alkyl substituent was examined and a specific relationship between the length of the alkyl chain and activity was observed.

Vinylogous Sulfonylureas: A New Class of Acetohydroxyacid Synthase Inhibitors Incorporating a Large Bridging Moiety

Several acrylic acid derivatives incorporating elements of sulfonylurea structure around a central vinylogous core were synthesized and found to be moderate inhibitors of the enzyme, acetohydroxyacid synthase (AHAS). Some com pounds showed post-emergence herbicidal activity against mustard. Biological activity was found to be markedly influenced by the nature of the 2-arylcarbamoyl group. When this group contained a phenyl ring the compounds were inactive. A 4,6-dim ethoxypyrimidin-2-ylcarbamoyl function resulted in the best activity but this activity was highly dependent on the nature of the 4,6-substituents on the pyrimidine ring, with 4,6-dim ethylpyrimidines showing very little activity. The structures of the most active compound, ethyl 3-(2-chlorophenyl)sulfonylamino-3-m ethylthio-2-[(4,6-dimethoxypyrimidin- 2-yl)carbam oyl]acrylate, and an inactive analogue, 4,6-dimethylpyrimidin-2-yl-3-(2-chlorophenyl) sulfonylamino-3-methylthio-2-cyanoacrylate, were determined by X -ray crystallography. The resulting structures showed several conformational differences which may play an important role in inhibitor binding to AHAS and the resultant herbicidal activity.