Precise Installation of Diazo-Tagged Side-Chains on Proteins to Enable In Vitro and In-Cell Site-Specific Labeling.

The chemistry of diazo compounds has generated a huge breadth of applications in the field of organic synthesis. Their versatility combined with their tunable reactivity, stability, and chemoselectivity makes diazo compounds desirable reagents for chemical biologists. Here, we describe a method for the precise installation of diazo handles on proteins and antibodies in a mild and specific approach. Subsequent 1,3-cycloaddition reactions with strained alkynes enable both bioimaging through an in-cell "click" reaction and probing of the cysteine proteome in cell lysates. The selectivity and efficiency of these processes makes these suitable reagents for chemical biology studies

Cyclic Sulfoxonium Ylides: Synthesis and Chemospecific Reactivity in the Catalytic Alkylation of Indoles

AbstractThe study of the reactivity of cyclic sulfoxonium ylides has been so far neglected, in particular for reactions that forms carbon‐carbon bond at the ylide carbon atom. Herein, we describe the synthesis of cyclic sulfoxonium ylides by palladium‐catalyzed intramolecular arylation and the reactivity of these ylides in the C3‐alkylation of indoles in the presence of either an acid catalyst or an iridium catalyst. This study revealed that acid catalysis is only efficient for cyclic sulfoxonium ylides in which the tether is a six‐membered lactone, whereas iridium catalysis was better suited to the reaction of cyclic sulfoxonium ylides in which the tether is a five‐membered ring ketone or lactone. The observed chemospecificity might be due to the relative basicity of the ylides under acid catalysis and to the steric hindrance around an iridium carbene intermediate when the reaction is conducted with the iridium catalyst.</jats:p

Efficient and irreversible antibody-cysteine bioconjugation using carbonylacrylic reagents.

There is considerable interest in the development of chemical methods for the precise, site-selective modification of antibodies for therapeutic applications. In this protocol, we describe a strategy for the irreversible and selective modification of cysteine residues on antibodies, using functionalized carbonylacrylic reagents. This protocol is based on a thiol-Michael-type addition of native or engineered cysteine residues to carbonylacrylic reagents equipped with functional compounds such as cytotoxic drugs. This approach is a robust alternative to the conventional maleimide technique; the reaction is irreversible and uses synthetically accessible reagents. Complete conversion to the conjugates, with improved quality and homogeneity, is often achieved using a minimal excess (typically between 5 and 10 equiv.) of the carbonylacrylic reagent. Potential applications of this method cover a broad scope of cysteine-tagged antibodies in various formats (full-length IgGs, nanobodies) for the site-selective incorporation of cytotoxic drugs without loss of antigen-binding affinity. Both the synthesis of the carbonylacrylic reagent armed with a synthetic molecule of interest and the subsequent preparation of the chemically defined, homogeneous antibody conjugate can be achieved within 48 h and can be easily performed by nonspecialists. Importantly, the conjugates formed are stable in human plasma. The use of liquid chromatography-mass spectrometry (LC-MS) analysis is recommended for monitoring the progression of the bioconjugation reactions on protein and antibody substrates with accurate resolution.We thank FAPESP (BEPE 2015/07509-1 and 2017/13168-8 to B.B., and 2013/25504-1 to A.C.B.B.), Xunta de Galicia (M.J.M.), FCT Portugal (FCT Investigator to G.J.L.B., IF/00624/2015), the EU (Marie Sklodowska-Curie ITN Protein Conjugates, GA 675007, including a PhD Studentship to X.F.), the Ministerio de Economía y Competitividad (projects CTQ2015-67727-R and UNLR13-4E-1931 to F.C. and CTQ2015-70524-R and RYC-2013-14706 to G.J.O.) and the Universidad de La Rioja (FPI Studentship to I.C.). We also thank S. Massa and N. Devoogdt (Vrije Universiteit Brussel (VUM), Brussels) for the generous gift of the Her2-targeting nanobody 2Rb17c, Genentech for providing Thiomab LC-V205C and trastuzumab antibodies, and D. Neri’s laboratory (Swiss Federal Institute of Technology (ETH Zürich), Zurich) for the generous gift of the F16 antibody. G.J.L.B. is a Royal Society University Research Fellow (UF110046 and URF/R/180019) and the recipient of a European Research Council Starting Grant (TagIt, GA 676832)