Proteins have become accessible targets for chemical synthesis. The basic strategy is to use native chemical ligation, Staudinger ligation, or other orthogonal chemical reactions to couple synthetic peptides. The ligation reactions are compatible with a variety of solvents and proceed in solution or on a solid support. Chemical synthesis enables a level of control on protein composition that greatly exceeds that attainable with ribosome-mediated biosynthesis. Accordingly, the chemical synthesis of proteins is providing previously unattainable insight into the structure and function of proteins.

The traceless Staudinger ligation enables the formation of an amide bond between a phosphinothioester (or phosphinoester) and an azide without the incorporation of residual atoms. Here, the coupling of peptides by this reaction was characterized in detail. Experiments with [(18)O]H(2)O indicated that the reaction mediated by (diphenylphosphino)methanethiol proceeded by S-->N acyl transfer of the iminophosphorane intermediate to form an amidophosphonium salt, rather than by an aza-Wittig reaction and subsequent hydrolysis of the resulting thioimidate. A continuous (13)C NMR-based assay revealed that the rate-determining step in the Staudinger ligation of glycyl residues mediated by (diphenylphosphino)methanethiol was the formation of the initial phosphazide intermediate. Less efficacious coupling reagents and reaction conditions led to the accumulation of an amine byproduct (which resulted from a Staudinger reduction) or phosphonamide byproduct (which resulted from an aza-Wittig reaction). The Staudinger ligation mediated by (diphenylphosphino)methanethiol proceeded with a second-order rate constant (7.7 x 10(-3) M(-1) s(-1)) and yield (95%) that was unchanged by the addition of exogenous nucleophiles. Ligations mediated by phosphinoalcohols had lower rate constants or less chemoselectivity. Accordingly, (diphenylphosphino)methanethiol was judged to be the most efficacious known reagent for effecting the traceless Staudinger ligation.

The Staudinger ligation between an azido-protein and a phosphinothioester-derivatized surface is demonstrated to be an effective means for the site-specific, covalent immobilization of a protein. Immobilization yields of >50% are obtained in <1 min, and immobilized proteins have >80% of their expected activity. No other method enables more rapid immobilization or a higher yield of active protein. Because azido-peptides and azido-proteins are readily attainable by synthesis, biosynthesis, or semisynthesis, the Staudinger ligation could be of unsurpassed utility in creating microarrays of functional peptides and proteins.

Generating highly selective probes to interrogate protein kinase function in biological studies remains a challenge and new strategies are required. Herein, we describe the development of the first highly selective and cell permeable inhibitor of c-Src, a key signaling kinase in cancer. Our strategy involves extension of traditional inhibitor design by appending functionality proposed to interact with the phosphate-binding loop of c-Src. Using our selective inhibitor, we demonstrate that selective inhibition is significantly more efficacious than pan-kinase inhibition in slowing the growth of cancer cells. We also show that inhibition of c-Abl kinase, an off-target of most c-Src inhibitors, promotes oncogenic cell growth.

Proline is unique among the natural amino acids in the similar propensity of its peptide bond to be in the cis or trans conformation. This attribute affects many processes, including the rate at which proteins fold, their structures, and their activities. Other aliphatic amino acids can serve as mimics for proline residues with trans peptide bonds. In contrast, chemical synthesis is needed to create surrogates for cis prolyl peptide bonds. Here, 1,5-disubstituted-[1,2,3]-triazoles were assessed as cispeptide bond surrogates. Huisgen's 1,3-dipolar cycloaddition reaction of amino alkynes and azido acids and a Ru(II) catalyst were used to synthesize a variety of Xaa-1,5-triazole-Ala modules in moderate-to-high yields. Two of these modules, along with their 1,4-triazole regioisomers, were installed in a turn region of bovine pancreatic ribonuclease by using expressed protein ligation. The resulting semisynthetic enzymes displayed full enzymatic activity, indicating the maintenance of native structure. The 1,5-triazole surrogates instilled conformational stability that was comparable to that of Xaa-cis-Pro segments, whereas the 1,4-triazoles conferred markedly less stability. The stability conferred by both surrogates was independent of the Xaa residue, eliminating an uncertainty in protein design. We conclude that Xaa-1,5-triazole-Ala modules can serve as viable mimics of Xaa-cis-Pro segments. The possibility of synthesizing this surrogate by the ligation of fragments in situ and the emergence of biocompatible catalysts for that process portends its widespread use.Proline is unique among the natural amino acids in the similar propensity of its peptide bond to be in the cis or trans conformation. 1 This attribute affects many processes, including the rate at which proteins fold, 2 their structures, 3 and their activities. 4 Other aliphatic amino acids can serve as mimics for proline residues with trans peptide bonds. In contrast, chemical synthesis is needed to create surrogates for cis prolyl peptide bonds. 1,5-7