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

The traceless Staudinger ligation is an effective means to synthesize an amide bond between two groups of otherwise orthogonal reactivity: a phosphinothioester and an azide. An important application of the Staudinger ligation is in the ligation of peptides at a variety of residues. Here, we demonstrate that the traceless Staudinger ligation can be achieved in water with a water-soluble reagent. Those reagents that provide a high yield of amide product discourage protonation of the nitrogen in the key iminophosphorane intermediate. The most efficacious reagent, bis(pdimethylaminoethylphenyl)phosphinomethanethiol, mediates the rapid ligation of equimolar substrates in water. This reagent is also able to perform a transthioesterification reaction with the thioester intermediate formed during intein-mediated protein splicing. Hence, the traceless Staudinger ligation can be integrated with expressed protein ligation, extending the reach of modern protein chemistry.

The discovery of activation state dependent kinase inhibitors, which bind specifically to the inactive conformation of the protein, is considered to be a promising pathway to improved cancer treatments. Identifying such inhibitors is challenging, however, because they can have Kd values similar to molecules known to inhibit kinase function by interacting with the active form. Further, while inhibitor induced changes within the kinase tertiary structure are significant, few technologies are able to correctly assign inhibitor binding modes in a high-throughput fashion based exclusively on protein-inhibitor complex formation and changes in local protein structure. We have developed a new assay, using ion mobility-mass spectrometry, capable of both rapidly detecting inhibitor binding and classifying the resultant kinase binding modes. Here, we demonstrate the ability of our approach to classify a broad set of kinase inhibitors, using micrograms of protein, without the need for protein modification or tagging.

Chemical synthesis harbors the potential to provide ready access to natural proteins as well as to create nonnatural ones. The Staudinger ligation of a peptide containing a C-terminal phosphinothioester with a peptide containing an N-terminal azide gives an amide with no residual atoms. This method for amide bond formation is orthogonal and complementary to other ligation methods. Herein, we describe the first use of the Staudinger ligation to couple peptides on a solid support. The fragment thus produced is used to assemble functional ribonuclease A via native chemical ligation. The synthesis of a protein by this route expands the versatility of chemical approaches to protein production.