No abstract

Dehydroalanine is a synthetic precursor to a wide array of protein modifications. We describe multiple methods for the chemical conversion of cysteine to dehydroalanine on peptides and proteins. The scope and limitations of these methods were investigated with attention paid to side reactions, scale, and aqueous-and bio-compatibility. The most general method investigated-a bis-alkylation-elimination of cysteine to dehydroalanine-was applied successfully to multiple proteins and enabled the siteselective synthesis of a glycosylated antibody.Scheme 1 Dehydroalanine is a precursor to modified peptides and proteins.

Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.

The detection of tuberculosis currently relies upon insensitive and unspecific techniques; newer diagnostics would ideally co-opt specific bacterial processes to provide real-time readouts. The trehalose mycolyltransesterase enzymes (antigens 85A, 85B and 85C (Ag85A, Ag85B, Ag85C)) serve as essential mediators of cell envelope function and biogenesis in Mycobacterium tuberculosis. Through the construction of a systematically varied sugar library, we show here that Ag85 enzymes have exceptionally broad substrate specificity. This allowed exogenously added synthetic probes to be specifically incorporated into M. tuberculosis growing in vitro and within macrophages. Even bulky substituents, such as a fluorescein-containing trehalose probe (FITC-trehalose), were incorporated by growing bacilli, thereby producing fluorescent bacteria; microscopy revealed selective labeling of poles and membrane. Addition of FITC-trehalose to M. tuberculosis–infected macrophages allowed selective, sensitive detection of M. tuberculosis within infected mammalian macrophages. These studies suggest that analogs of trehalose may prove useful as probes of function and for other imaging modalities.

Here we demonstrate that quantitation of stimuli-induced proteome dynamics in primary cells is feasible by combining the power of Bio-Orthogonal Non Canonical Amino acid Tagging (BONCAT) and Stable Isotope Labelling of Amino acids in Cell culture (SILAC). In conjunction with nanoLC-MS/MS QuaNCAT allowed us to monitor the early expression changes of > 600 proteins in primary resting T cells subjected to activation stimuli.