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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Imide-Click Ligation and Click-Unclick Peptide-BasedProdrug StrategyReda Mhidia 1 , Nicolas Bézière 1 , Nicole Pommery 2 , Annick Blanpain 1 ,and Oleg Melnyk 11 CNRS UMR 8161 Institut Pasteur de Lille, Univ. Lille Nord de France, IFR 142, Lille,59021, France; 2 Univ. Lille Nord de France, Lille, 59006, FranceIntroductionPolypeptide-drug conjugates are frequently used for targeting drugs to specific biomarkers.Release of the active substance at the delivery site can be triggered by a specificbiochemical event. In particular, the design of self-immolative linkers, whose fragmentationis triggered by a specific enzyme, is of utmost interest. However, preparation of suchcarrier-linked prodrugs requires multi-step procedures and complex protection schemes.We disclose here a novel strategy named click-unclick allowing the easy assembly and thecontrolled disassembly of peptide-based prodrugs (Figure 1) [1]. First, a click-typereaction, i.e. imide ligation between a peptide thioacid and an azidoformate derivative ofthe drug, allows the formation of an imide linker between a drug and a peptide carrier. Thesecond unclick step is defined as a process leading to the elimination of the linker byintramolecular cyclization and to the release of the drug. Essential to the unclick concept isthe participation of the bond formed by click chemistry in the disassembly mechanism.Results and DiscussionThe reaction of thioacids with electron-deficient azides such as sulfonyl azides gives accessto the corresponding amides in good yield [2,3]. Azidoformates have been scarcely studiedin this field. The strategy described in Figure 1 involves as a first step an imide ligationbetween peptide thioacid carrier 1 and a drug activated by an azidocarbonyl group 2.Ligation proceeds very cleanly at pH 2.5. At this pH, ε or α-amino groups are protected byprotonation. At higher pH, azidoformate 2 was able to react partially with amino groups.No side reactions with amino acids such as His, Tyr, Ser, Asn or Glu have been observed.The compatibility of imide ligation with Cys remains to be established since this aminoacid has been shown to be incompatible with sulfo-click ligation between thioacids andsulfonyl azides [3]. Imides such as 3 can be purified by RP-HPLC using water/acetonitrilelinear gradients containing TFA and lyophilized without detectable degradation. Imideligation features most of the characteristics displayed by click reactions: the reaction ischemoselective, racemization free, gives good yields using simple reaction conditions inwater, requires only readily available starting materials. It is illustrated in Figure 1 with thesynthesis of imide 3. The modest yield of 28% is due to the small scale of synthesis (fewmgs). Isolated yields are usually in the range 50-60%. Conjugate 3 features a maskedAc-GISSGYCarrierNHO1SHN 3CLICKImide ligationOON NOwater, pH 2.528%OS2PSAAc-GISSGYHNONHO3H 2 NdrugUNCLICKONH5OdrugAc-GISSGY-OH4ONHHONHON N6OOS7Fig. 1. Illustration of the click-unclick strategy. An imide-click reaction allows thechemoselective and racemization free assembly of peptide-drug conjugate. The drug isunclicked by the action of an enzyme.20