Post-translational site-selective protein backbone α-deuteration.

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-1H → C-2H/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 1H→2H/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.

Synthesis of modified proteins via functionalization of dehydroalanine.

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.

Precise Probing of Residue Roles by Post-Translational ß,γ-C,N Aza-Michael Mutagenesis in Enzyme Active Sites.

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-ß,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.

Cleavage of DNA containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases.

A systematic study of the cleavage of DNA sequences containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases (REs) was performed and the results compared with the same sequences containing natural pyrimidine bases, uracil or 5-methylcytosine. The results show that some REs recognize fluorine as a hydrogen on cytosine and cleave the corresponding sequences where the presence of m5dC leads to blocking of the cleavage. However, on uracil, the same REs recognize the F as a methyl surrogate and cleave the sequences which are not cleaved if uracil is incorporated instead of thymine. These results are interesting for understanding the recognition of DNA sequences by REs and for manipulation of the specific DNA cutting.

Azidopropylvinylsulfonamide as a New Bifunctional Click Reagent for Bioorthogonal Conjugations: Application for DNA-Protein Cross-Linking.

N-(3-Azidopropyl)vinylsulfonamide was developed as a new bifunctional bioconjugation reagent suitable for the cross-linking of biomolecules through copper(I)-catalyzed azide-alkyne cycloaddition and thiol Michael addition reactions under biorthogonal conditions. The reagent is easily clicked to an acetylene-containing DNA or protein and then reacts with cysteine-containing peptides or proteins to form covalent cross-links. Several examples of bioconjugations of ethynyl- or octadiynyl-modified DNA with peptides, p53 protein, or alkyne-modified human carbonic anhydrase with peptides are given.

Aqueous Heck cross-coupling preparation of acrylate-modified nucleotides and nucleoside triphosphates for polymerase synthesis of acrylate-labeled DNA.

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.

Vinylsulfonamide and acrylamide modification of DNA for cross-linking with proteins.

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.

Aggregation effects in visible-light flavin photocatalysts: synthesis, structure, and catalytic activity of 10-arylflavins.

A series of 10-arylflavins (10-phenyl-, 10-(2',6'-dimethylphenyl)-, 10-(2',6'-diethylphenyl)-, 10-(2',6'-diisopropylphenyl)-, 10-(2'-tert-butylphenyl)-, and 10-(2',6'-dimethylphenyl)-3-methylisoalloxazine (2 a-f)) was prepared as potentially nonaggregating flavin photocatalysts. The investigation of their structures in the crystalline phase combined with (1)H-DOSY NMR spectroscopic experiments in CD(3)CN, CD(3)CN/D(2)O (1:1), and D(2)O confirm the decreased ability of 10-arylflavins 2 to form aggregates relative to tetra-O-acetyl riboflavin (1). 10-Arylflavins 2 a-d do not interact by π-π interactions, which are restricted by the 10-phenyl ring oriented perpendicularly to the isoalloxazine skeleton. On the other hand, N3-H⋅⋅⋅O hydrogen bonds were detected in their crystal structures. In the structure of 10-aryl-3-methylflavin (2 f) with a substituted N3 position, weak C-H⋅⋅⋅O bonds and weak π-π interactions were found. 10-Arylflavins 2 were tested as photoredox catalysts for the aerial oxidation of 4-methoxybenzyl alcohol to the corresponding aldehyde (model reaction), thus showing higher efficiency relative to 1. The quantum yields of 4-methoxybenzyl alcohol oxidation reactions mediated by arylflavins 2 were higher by almost one order of magnitude relative to values in the presence of 1.