Omar Boutureira

Site-selective protein-modification chemistry for basic biology and drug development

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.

Advances in Chemical Protein Modification

O.B. thanks the European Commission (Marie Curie CIG) and Ministerio de Ciencia e Innovacion, Spain (Juan de la Cierva Fellowship). G.J.L.B. thanks his generous sources of funding: Royal Society, FCT Portugal (FCT Investigator), European Commission (Marie Curie CIG), and the EPSRC. G.J.L.B. is a Royal Society University Research Fellow. The authors thank Paula Boutureira Regla and Francisco Pinteus da Cruz Lopes Bernardes for inspiration.

Methods for converting cysteine to dehydroalanine on peptides and proteins

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 site-selective synthesis of a glycosylated antibody.

Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis

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.

QuaNCAT: quantitating proteome dynamics in primary cells

Here we demonstrate quantitation of stimuli-induced proteome dynamics in primary cells by combining the power of bio-orthogonal noncanonical amino acid tagging (BONCAT) and stable-isotope labeling of amino acids in cell culture (SILAC). In conjunction with nanoscale liquid chromatography–tandem mass spectrometry (nanoLC-MS/MS), quantitative noncanonical amino acid tagging (QuaNCAT) allowed us to monitor the early expression changes of >600 proteins in primary resting T cells subjected to activation stimuli.

Synthetically defined glycoprotein vaccines: current status and future directions

We highlight current glycovaccines in the clinic and derive principles for the construction of the next generation of synthetically defined glycoconjugate vaccines.

Rapid Cross-Metathesis for Reversible Protein Modifications via Chemical Access to Se-Allyl-selenocysteine in Proteins

Cross-metathesis (CM) has recently emerged as a viable strategy for protein modification. Here, efficient protein CM has been demonstrated through biomimetic chemical access to Se-allyl-selenocysteine (Seac), a metathesis-reactive amino acid substrate, via dehydroalanine. On-protein reaction kinetics reveal a rapid reaction with rate constants of Seac-mediated-CM comparable or superior to off-protein rates of many current bioconjugations. This use of Se-relayed Seac CM on proteins has now enabled reactions with substrates (allyl GlcNAc, N-allyl acetamide) that were previously not possible for the corresponding sulfur analogue. This CM strategy was applied to histone proteins to install a mimic of acetylated lysine (KAc, an epigenetic marker). The resulting synthetic H3 was successfully recognized by antibody that binds natural H3-K9Ac. Moreover, Cope-type selenoxide elimination allowed this putative marker (and function) to be chemically expunged, regenerating an H3 that can be rewritten to complete a chemically enabled “write (CM)–erase (ox)–rewrite (CM)” cycle.

A Coordinated Synthesis and Conjugation Strategy for the Preparation of Homogeneous Glycoconjugate Vaccine Candidates

Glycoconjugates are the center of many therapeutic strategies and carbohydrate-based vaccines in particular hold great promise. The development of glycovaccines, however, can be hindered by the limited access offered by natural sources to homogeneous antigenic carbohydrates; efficient chemical synthesis offers an attractive route to pure samples of these carbohydrates. Furthermore, for an optimal immune response, the carbohydrate antigen should be conjugated to an immunogenic carrier, usually a protein. 10] The synthesis and use of well-defined glycoprotein therapeutics and glycovaccines—uniform in sugar, site, and level of protein attachment—is rare and most constructs are prepared and administered as complex mixtures. 10] Even strategies that utilize pure synthetic glycan may employ non-selective methods for subsequent conjugation to a protein carrier. 7, 11, 12] Given the unknown influence of conjugation site on immunogenic response, it is remarkable that, to our knowledge, no homogeneous glycovaccine has been studied. To fully evaluate the structure–activity relationships (SARs) between glycoprotein and immunogenicity, we have initiated a program for the construction of such “pure” or uniform glycoconjugate vaccines. We report a coherent strategy for homogenous glycoprotein construction that coordinates both carbohydrate synthesis and conjugation methodology. This approach features glycosyl disulfides as versatile donors in complex carbohydrate synthesis, providing strategic access to glycosyl thiols that can be site-specifically attached to a protein carrier through a well-defined thioether linkage (Scheme 1).

Site-selective chemoenzymatic construction of synthetic glycoproteins using endoglycosidases

Combined chemical tagging followed by Endo-A catalysed elongation allows access to homogeneous, elaborated glycoproteins. A survey of different linkages and sugars demonstrated not only that unnatural linkages can be tolerated but they can provide insight into the scope of Endo-A transglycosylation activity. S-linked GlcNAc-glycoproteins are useful substrates for Endo-A extensions and display enhanced stability to hydrolysis at exposed sites. O-CH2-triazole-linked GlcNAc-glycoproteins derived from azidohomoalanine-tagged protein precursors were found to be optimal at sterically demanding sites.

Selenenylsulfide‐Linked Homogeneous Glycopeptides and Glycoproteins: Synthesis of Human “Hepatic Se Metabolite A”

Introducing selenium: The synthesis and full characterization of human hepatic Se metaboliteA has been accomplished by using a robust, efficient, and Cys-specific selenenylation protocol (see scheme; NaPi=sodium phosphate). The selenenylsulfide linkage is sufficiently stable to allow quantification of Se-containing glycoconjugates in biological fluids by using atomic detection methods. Copyright