This journal is © The Royal Society of Chemistry 2014 Chem. Commun. Cite this: DOI: 10.1039/c3cc48871c First enzymatic hydrolysis/thio-Michael addition cascade route to synthesis of AChE inhibitors† Emilia Oueis, a Florian Nachon, b Cyrille Sabot* a and Pierre-Yves Renard* a The irreversible Michael addition of thiols to acrylamides is reported as a new tool for the kinetic target-guided synthesis. In an unprecedented enzymatic hydrolysis/thio-Michael addition procedure, potent and selective acetylcholinesterase inhibitors are assembled by the enzyme using both its esterasic and templating ability. Target-guided synthesis (TGS) is an unconventional fragment-based lead discovery methodology that relies on the multi-binding biological target (BT) itself to build its own inhibitors. 1 Indeed, the reaction that occurs between ligands bearing complementary reactive functions is dramatically accelerated in the presence of the BT, leading to the formation of highly selective inhibitors. Different TGS approaches have already been developed and proved to be efficient for the discovery of potent inhibitors of various enzymes. Worth noting among them, is the dynamic combinatorial chemistry (DCC) 2 which involves reversible bond formations, and the kinetically controlled target-guided synthesis (KTGS) which is based on irreversible reactions. 1,3 As an eminent example of KTGS, the in situ click chemistry tool developed by Sharpless et al. uses the 1,3-dipolar cycloaddition between the bioorthogonal azides and alkynes (Scheme 1A) as the irreversible templated reaction to create new C–N bonds and obtain triazole- based heterodimeric inhibitors. 4 Aside from the Huisgen reaction, which was used on a dozen occasions for the discovery of enzymatic triazole-based inhibitors, only a handful of other irreversible reactions have been engaged in KTGS approaches. 3,5 Notably, the synthesis of amide bonds via the sulfo-click reaction between thio acids and sulfonyl azides was achieved by Manetsch et al. for the discovery of inhibitors of a Bcl-2 protein family member (Scheme 1B). 5b–d Huc et al. explored the utility of the nucleophilic substitution of alkyl chlorides by mercaptoarylsulfonamide that generates a new C–S bond (Scheme 1C) to identify inhibitors of the zinc-containing metalloenzyme bovine carbonic anhydrase (CAII). 5a However, due to the high reactivity of alkyl chlorides generating a background activity, only binary competition assays were conducted, in which the enzyme favoured the assembly of the best inhibitors. Even though the efficiency of the KTGS approach in drug discovery has been proven, its common use is still hampered by the low availability of adaptable reactions. In this context, we considered a new amenable reaction for KTGS: the Michael addition of thiols to acrylamide derivatives. Maleimides are widely used for thio-mediated conjugation of biomolecules due to their high reactivity, specificity and strong C–S bond formation. We questioned whether less reactive acrylamide derivatives would limit the formation of 1,4-adducts in the absence of the target enzyme, and remain suitable Michael acceptors for KTGS. Interestingly, this reaction proved to be irreversible (see also ESI†), 6 in sharp contrast to the reaction carried out with enones whose utility had already been established for the discovery of the dynamic inhibitors. 7 However, in the latter strategy cautious reaction conditions are required: sequential additions, and freezing of the final equilibrium before analysis. Scheme 1 KTGS reactions: (A) Huisgen reaction between azides and alkynes. (B) Amidation reaction between sulfonyl azides and thio acids. (C) S N 2 reaction between mercaptoarylsulfonamides and alkyl chlorides. (D) Thio-Michael addition of thiols to acrylamides (this study). a Normandie Univ, COBRA, UMR 6014 & FR 3038; Univ Rouen; INSA Rouen; CNRS, 1 rue Tesnie `re 76821 Mont-Saint-Aignan, Cedex, France. E-mail: pierre-yves.renard@univ-rouen.fr; Fax: +33 2 35 52 29 71 b Institut de Recherche Biome ´dicale des Arme ´es, BP73, F-91993 Bre ´tigny-sur-Orge, France † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3cc48871c Received 20th November 2013, Accepted 19th December 2013 DOI: 10.1039/c3cc48871c www.rsc.org/chemcomm ChemComm COMMUNICATION Published on 20 December 2013. Downloaded by University of St Andrews Library on 13/01/2014 14:21:23. View Article Online View Journal