Deuterium exchange reactions as a probe of biomolecule structure. Traceless ligation of cysteine peptides using selective deselenization. Identification of pyridine synthase recognition sequences allows a modular solid-phase route to thiopeptide variants. Chemical generation and modification of peptides containing multiple dehydroalanines. Methods for converting cysteine to dehydroalanine on peptides and proteins. A biosynthetic route to dehydroalanine-containing proteins. Biliary excretion of a glutathione conjugate of busulfan and 1,4-diiodobutane in the rat. New insights into the biosynthetic logic of ribosomally synthesized and post-translationally modified peptide natural products. Reinvestigation of the histidine peak assignments. Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. Electron-nuclear interactions in two prototypical proteins: selective (chiral) deuteration and analysis of 1H and 2H NMR signals from the alpha and beta hydrogens of cysteinyl residues that ligate the iron in the active sites of human ferredoxin and Anabaena 7120 vegetative ferredoxin. Direct observation of protein vibrations by selective incorporation of spectroscopically observable carbon-deuterium bonds in cytochrome c. Application to assignment of 1H resonances of valine residues in dihydrofolate reductase. A novel method of preparing totally alpha-deuterated amino acids for selective incorporation into proteins. 1H nuclear magnetic resonance studies of the tyrosine residues of selectively deuterated Lactobacillus casei dihydrofolate reductase. High-resolution nuclear magnetic resonance spectra of selectively deuterated staphylococcal nuclease. Direct observation of structural heterogeneity in a beta-sheet. Segmental isotopic labeling of proteins for nuclear magnetic resonance. IR probes of protein microenvironments: utility and potential for perturbation. Efforts toward the direct experimental characterization of enzyme microenvironments: tyrosine100 in dihydrofolate reductase. Uniform and selective deuteration in two-dimensional NMR of proteins. Mechanistic deductions from isotope effects in multireactant enzyme mechanisms. The magnitude of the primary kinetic isotope effect for compounds of hydrogen and deuterium. The ready application on existing, intact protein constructs (without specialized culture or genetic methods) suggests this C–D labeling strategy as a possible tool in protein mechanism, structure, biotechnology and medicine. This analysis suggests, together with quantum mechanical calculations, stepwise deprotonations via on-protein carbanions and unexpected sulfonium ylides in the conversion of Cys to Dha, consistent with a ‘carba-Swern’ mechanism. It is used here to probe mechanisms of reactions used in protein bioconjugation. Here we describe a chemical method for selective protein α-carbon deuteration (proceeding from Cys to dehydroalanine (Dha) to deutero-Cys) allowing overall 1H→ 2H/D exchange at a nonexchangeable backbone site. The development of postbiosynthetic, C– 1H → C– 2H/D replacement in proteins could enable probing of mechanisms, among other uses. However, applications in proteins are largely limited to biosynthetic strategies or exchangeable (for example, N–H/D) labile sites only. Isotopic replacement has long-proven applications in small molecules.
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