Targeted covalent inhibitors (TCIs) are irreversible enzyme inhibitors that are designed to first bind to a targeted enzyme’s active site reversibly using non-covalent interactions between the molecular scaffold of the inhibitor and the surrounding amino acid residues of the enzyme’s binding site. They then form a covalent bond between the inhibitor’s electrophilic warhead and a nucleophilic amino acid residue located inside of the binding pocket. Cysteine (Cys), a redox-sensitive thiol, is found in many enzyme active sites and is used as the target for many current TCIs in clinical application. Electrophilic warheads such as acrylamides and chloroacetamides are known to readily undergo thiol-addition, and although they are commonly used in the development of enzyme inhibitors, few previous studies have explored the mechanism of thiol-addition and the intrinsic reactivities of these moieties. In this work, a robust kinetic assay was developed to perform mechanistic studies of thiol-addition to the electrophilic warhead derivatives N-phenylacrylamide (NPA), N-acryloylpiperidine (AcrPip), and N-phenylchloroacetamide (NPC). By reacting these warhead derivatives with thiol nucleophiles having various pKa values, we were able to construct Brønsted-type plots, resulting in shallow positive βNucRS- values for NPA, AcrPip and NPC (βNucRS- = 0.07 ± 0.04, 0.11 ± 0.03, and 0.21 ± 0.07, respectively), meaning that these electrophiles are relatively insensitive to thiolate nucleophilicity. However, while the trend in their reactivity across thiolate nucleophilicity is similar, their intrinsic reactivity was found to be vastly different. In conjunction with the Brønsted-type plot, temperature, ionic strength, and kinetic isotope effects were studied to afford information about the rate-limiting transition state and elucidate the mechanism of thiol-addition. NPA and AcrPip were found to undergo very similar thiol-additions, consistent with the microscopic reverse of the E1cbrev elimination, whereas NPC follows an SN2 type addition, consistent with the intuitive mechanism of addition to a haloacetamide.

Distinguishing 2,4-D and dicamba herbicide formulations in cotton and soybean tissue is challenging in regulation of crop injury from these herbicides. Additionally, stewardship of 2,4-D and dicamba technologies is important to maximize their longevity and efficacy. Research was conducted to (1) characterize cotton and soybean response to various formulations of 2,4-D or dicamba with or without glyphosate, (2) develop a method for classifying these formulations in crop tissue, and (3) optimize use of chloroacetamide herbicides in dicamba systems for mitigation of selection pressure on dicamba. Formulations evaluated include dicamba diglycolamine (DGA), dimethylamine (DMA), N,N-Bis-(3-aminopropyl) methylamine (BAPMA), and DGA plus potassium acetate (KAc); and 2,4-D DMA, acid, isooctyl ester (ESTER), and choline. Weed management by the chloroacetamides s-metolachlor and acetochlor was evaluated with applications preemergence (PRE), early postemergence (EP), late postemergence (LP), PRE followed by (fb) EP, PRE fb LP, and EP fb LP. Cotton and soybean response differed by 2,4-D and dicamba formulation, and glyphosate presence. Cotton yield was reduced by 200 to 500 kg ha-1 following exposure to 2,4-D choline or DMA relative to acid or ESTER. Glyphosate presence led to a reduction in cotton and soybean yield of 377 and 572 kg ha-1, respectively. Exposure to dicamba DMA resulted in a 263 kg ha-1 reduction in soybean yield relative to dicamba DGA, and glyphosate presence reduced yield by 439 and 246 kg ha-1 in cotton and soybeans, respectively. Chemometric analyses generated models capable of up to 85% accuracy in identifying dicamba formulation in cotton and soybean tissue, and up to 80% accuracy in identifying 2,4-D formulation. Split chloroacetamide applications improved cotton yield up to 60%, reduced weed densities up to 90%, and improved control up to 56% relative to single applications. Cotton height was reduced up to 23% if a single chloroacetamide application was made. Soybean yield was maximized following any chloroacetamide application timing except PRE alone, and weed control was reduced up to 31% following single chloroacetamide application relative to split applications. These results will aid regulatory bodies in managing use of new weed control technologies and will assist producers in stewarding these new technologies.