John N. Phillips

The indirect role of 2,4-D in the maintenance of apical dominance in decapitated sunflower seedlings (Helianthus annuus L.)

When sufficient 2,4-D to maintain apical dominance for at least 21d was applied to the cut stem interface of sunflower seedlings which had been decapitated in the epicotyl, it could not be detected in the vicinity of the inhibited axillary buds 7d after application. Rather the 2,4-D concentrated at the stump apex where it was associated with formation of meristematic tissue. The results indicate an indirect role for 2,4-D in the maintenance of apical dominance in this system, possibly involving the induced meristematic activity.

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 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.

Hydrogen Bonding of Cyanoacrylates with the D 1 Peptide

Abstract Representatives of two structural types of cyanoacrylate PS II inhibitors have been studied in respect to their pI50 values and qualitative rate of binding with wild type and S 264 G mutant thylakoids isolated from Brassica napus. Both are potent inhibitors of photosynthetic electron transport and both show a large discrimination between wild type and mutant thylakoids under equilibrium conditions. However one, an N-methylanilino cyanoacrylate, has an initial rapid reaction with both wild type and mutant thylakoids, but continues to react slowly with the wild type species until equilibrium is reached, while the other, a benzylamino cyanoacry late, equilibrates rapidly with both species as does the classical PS II inhibitor, atrazine. These differences in kinetic behaviour have been interpreted in terms of different H-bond interactions with the serine-264 hydroxyl group. It is suggested that the slow binding reaction is due to the N -methylanilino compound interacting as an H-bond acceptor with the serine-264 hydroxyl hydrogen thus disrupting an intramolecular ser-264-his-252 H-bond within the D 1 peptide. Rapid equilibration on the other hand, has been attributed to the benzylamino derivative acting as an H-bond donor to the serine-264 hydroxyl oxygen and strengthening the 264 -252 H -bond by conjugation. It is proposed that atrazine and other classical PS II inhibitors act in this way and that this may explain their ability to inhibit trypsin degradation of the D 1 peptide, if the 264-252 intramolecular H-bond plays an important role in stabilizing the peptide conform ation. It is also speculated that photodegradation may be related to the ability of QB- to act as an H-bond acceptor and disrupt the 264-252 H-bond.

X-RAY STRUCTURE ANALYSIS OF A CYANOACRYLATE INHIBITOR OF PHOTOSYSTEM II ELECTRON TRANSPORT

Abstract X-ray crystallographic data for the highly potent cyanoacrylate photosynthetic electron transport inhibitor, (Z)-ethoxyethyl 3-(4-chlorobenzylamino)-2-cyano-4-methylpent-2-enoate, are presented. This compound has a particularly high affinity for the photosystem II (PS II) herbicide receptor with a p I50 value of 9.5 (in the Hill reaction under uncoupled conditions with a chlorophyll concentration of 0.1 μg/ml). Data regarding the structure of small ligands, such as this potent cyanoacrylate, which bind to the site with high affinity may be used to provide the basis for modelling studies of PS II/herbicide complexes. The X-ray data presented confirm the Z-stereochemistry of active cyanoacrylates and demonstrate the presence of a planar core stabilized by an intramolecular hydrogen bond between the ester carbonyl oxygen and a benzylamino hydrogen atom. In order to assess the importance of the benzylamino -NH -group in this type of cyanoacrylate, analogues containing a methylene group in its place were synthesized and found to be 100-and 1000-fold less active as Hill inhibitors.

The Binding Behaviour of Cyanoacrylate PS11 Inhibitors

Many compounds, including a number of commercial herbicides, are known to inhibit photosynthetic electron transport (PET) close to the photosystem 11 (PS11) reaction centre (RC) in plant chloroplasts. Such PS11 PET inhibitors are believed to act by displacing a plastoquinone molecule from its binding niche on the D1, (32 Kd) peptide associated with the PS11RC. In spite of their common mode of action and their ability to competitively displace each other from the site of action, their structural diversity is such as to suggest that, within the one plastoquinone binding domain, many different but overlapping inhibitor interaction sites may be involved. Evidence for this has come from studies where such competitive inhibitors have been found to show different levels of discrimination between electron transport in thylakoids・isolated from wild type and mutant plant species. The D1 peptide in these biotypes differs in only one amino acid residue with the serine264 moiety having been replaced by a glycine. [1,2]