Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents

Maleimides remain the reagents of choice for the preparation of therapeutic and imaging protein conjugates despite the known instability of the resulting products that undergo thiol-exchange reactions $\textit{in vivo}$. Here we present the rational design of carbonylacrylic reagents for chemoselective cysteine bioconjugation. These reagents undergo rapid thiol Michael-addition under biocompatible conditions in stoichiometric amounts. When using carbonylacrylic reagents equipped with PEG or fluorophore moieties, this method enables access to protein and antibody conjugates precisely modified at pre-determined sites. Importantly, the conjugates formed are resistant to degradation in plasma and are biologically functional, as demonstrated by the selective imaging and detection of apoptotic and HER2+ cells, respectively. The straightforward preparation, stoichiometric use and exquisite cysteine selectivity of the carbonylacrylic reagents combined with the stability of the products and the availability of biologically relevant cysteine-tagged proteins make this method suitable for the routine preparation of chemically defined conjugates for $\textit{in vivo}$ applications.FAPESP (Grant IDs: 2012/22274-2; BEPE 2015/07509-1, 2013/25504-1), Xunta de Galicia, FCT Portugal (FCT Investigator, SFRH/BPD/103172/2014 Postdoctoral fellowship, SFRH/BD/111556/2015 PhD Studentship), European Union (Marie-Sklodowska Curie ITN Protein Conjugates), Engineering and Physical Sciences Research Council, MECD (‘Salvador Madariaga’ mobility grant PRX15/00638), MINECO (CTQ2015-70524-R, RYC-2013-14706 ), Royal Society, European Research Council Starting Grant (TagIt

A new entry to the synthesis of substituted azetidines: [2+2] cycloaddition reaction of four-membered endocyclic enamides to ketenes

The first example of a [2+2] cycloaddition reaction of a four-membered endocyclic enamide (2-azetine) to dichloroketene is described and constitutes a new entry to the synthesis of substituted azetidines. Preliminary studies concerning the Baeyer Villiger oxidation of the [2+2] cycloadduct revealed an unusual regioselectivity. The synthesis of a new azetidine-3-carboxylic acid derivative from the [2+2] cycloadduct is also presented. (c) 2006 Elsevier Ltd. All rights reserved.47366377638

Asymmetric synthesis of cis-2,4-disubstituted azetidin-3-ones from metal carbene chemistry

Several chiral cis-2,4-disubstituted azetidin-3-ones were prepared as single diastereoisomers from N-protected amino acids, employing a highly stereoselective copper carbenoid N-H insertion reaction of diazoketones. These azetidin-3-ones were then converted into fully substituted azetidines in a few steps in good to high yields. (c) 2005 Elsevier B.V. All rights reserved.69024-255636564

Stereoselective synthesis of the conformationally constrained glutamate analogue, (-)-(2R,3S)-cis-2-carboxyazetidine-3-acetic acid, from (S)-N-tosyl-2-phenylglycine

The stereoselective synthesis of a novel cis conformationally constrained glutamate analogue containing an azetidine framework was accomplished from (S)-N-tosyl-2-phenylglycine in moderate overall yield. The key steps in the synthesis involved a N-H carbenoid insertion promoted by Cu(acaC)(2), a very efficient Wittig olefination of an azetidin-3-one, followed by a highly stereoselective rhodium-catalyzed hydrogenation. Epimerization of the cis to the trans analogue was performed using DBU as base in toluene at reflux.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.101559156

Theoretical studies of the asymmetric alkylation reaction on chiral enamines

Semi-empirical calculations on the asymmetric alkylation of beta-ketoesters described by Koga and co-workers were carried out with the use of the MNDO semi-empirical method. This reaction is dependent on the kind of solvent used as additive for the generation of the asymmetric center with chirality control. Investigations were carried out to test how different types of additives, such as HMPA and THF, affect the establishment of this stereogenic center. (c) 2004 Elsevier B.V. All rights reserved.7164169910310

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

Precise installation of diazo-tagged side-chains on proteins to enable in vitro and in-cell site-specific labelling

The chemistry of diazocompounds has generated a huge breadth of applications in the field of organic synthesis. Their versatility combined with their tuneable 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