Dehydroalanine has emerged in recent years as a non-proteinogenic residue with strong chemical utility in proteins for the study of biology. In this review we cover the several methods now available for its flexible and site-selective incorporation via a variety of complementary chemical and biological techniques and examine its reactivity, allowing both creation of modified protein side-chains through a variety of bond-forming methods (C-S, C-N, C-Se, C-C) and as an activity-based probe in its own right. We illustrate its utility with selected examples of biological and technological discovery and application.

Bioorthogonal covalent cross-linking of DNA-binding proteins (p53) to DNA was achieved through novel DNA probes bearing a reactive vinylsulfonamide (VS) group. The VS-modified dCTP served as building block for polymerase synthesis of modified DNA, which was readily conjugated with cysteine-containing peptides and proteins by Michael addition.

Biomimicry valuably allows the understanding of the essential chemical components required to recapitulate biological function, yet direct strategies for evaluating the roles of amino acids in proteins can be limited by access to suitable, subtly-altered unnatural variants. Here we describe a strategy for dissecting the role of histidine residues in enzyme active sites using unprecedented, chemical, post-translational side-chain-β,γ C–N bond formation. Installation of dehydroalanine (as a “tag”) allowed the testing of nitrogen conjugate nucleophiles in “aza-Michael”-1,4-additions (to “modify”). This allowed the creation of a regioisomer of His (iso-His, Hisiso) linked instead through its pros-Nπ atom rather than naturally linked via C4, as well as an aza-altered variant aza-Hisiso. The site-selective generation of these unnatural amino acids was successfully applied to probe the contributing roles (e.g., size, H-bonding) of His residues toward activity in the model enzymes subtilisin protease from Bacillus lentus and Mycobacterium tuberculosis pantothenate synthetase.

Aqueous-phase Heck coupling methodology was developed for direct attachment of butyl acrylate to 5-iodoracil, 5-iodocytosine, 7-iodo-7-deazaadenine, and 7-iodo-7-deazaguanine 2'-deoxyribonucleoside 5'-O-monophosphates (dNMPs) and 5'-O-triphosphates (dNTPs) and compared with the classical approach of phosphorylation of the corresponding modified nucleosides. The 7-substituted 7-deazapurine nucleotides (dA(BA)MP, dA(BA)TP, dG(BA)MP, and dG(BA)TP) were prepared by the direct Heck coupling of nucleotides in good yields (35-55%), whereas the pyrimidine nucleotides reacted poorly and the corresponding BA-modified dNTPs were prepared by triphosphorylation of the modified nucleosides. The acrylate-modified dN(BA)TPs (N = A, C, and U) were good substrates for DNA polymerases and were used for enzymatic synthesis of acrylate-modified DNA by primer extension, whereas dG(BA)TP was an inhibitor of polymerases. The butyl acrylate group was found to be a useful redox label giving a strong reduction peak at -1.3 to -1.4 V in cyclic voltammetry.

Isotopic replacement has long-proven applications in small molecules. However, applications in proteins are largely limited to biosynthetic strategies or exchangeable (for example, N-H/D) labile sites only. The development of postbiosynthetic, C-H → C-H/D replacement in proteins could enable probing of mechanisms, among other uses. Here we describe a chemical method for selective protein α-carbon deuteration (proceeding from Cys to dehydroalanine (Dha) to deutero-Cys) allowing overall H→H/D exchange at a nonexchangeable backbone site. It is used here to probe mechanisms of reactions used in protein bioconjugation. 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. 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.