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A) PSA, few seconds73B) PSA, 45 minC) PSA, 80 min7 3D) PSA, 3.5 hAc-GISSGY-OH7E) Without PSA, 3 h3Fig. 2. Unmasking of the drug using an endopeptidase (PBS pH 7.4, 37°C). Conjugate 3(40 µM) was incubated with (A-D, 10 µg/mL) or without (E) PSA. RP-HPLC analysis ofthe reaction mixtures on a C18 Nucleosil column (215 nm).COX-2 inhibitor and a peptide substrate for prostate specific antigen (PSA). COXs areinvolved in a number of diseases. In particular, COX-2 is over-expressed in prostate cancer,and is considered as a molecular target in this disease. PSA is an chymotrypsin-likeendopeptidase which is relatively specific to prostate tissue and overexpressed in prostatecancer cells. This enzyme has been used by several groups for vectorization of anticancerdrugs because PSA is active only in the prostate cells microenvironment.Next, conjugate 3 was incubated with PSA, which cleaves after Tyr residue present inthe penultimate position (Figure 2A, D). Figure 2A-D shows that unmasking of 7 wasalmost complete after 3h of PSA digestion (t 1/2 ~ 40 min). Unmasking of 7 occurred in themixture because rearrangement of intermediate 5 (Figure 1) did not occur in the eluent usedfor RP-HPLC analysis (pH 2) due to protonation of the amino group. Stability of 5 in acidicmedia was confirmed with a reference compound obtained by reacting Boc-Ala-SH withazidoformate 2, followed by Boc removal in TFA. We have also demonstrated theformation of hydantoin 6 in stoichiometric amounts. The early eluting peak in Figure 2A,Dcorresponded, by LC-MS, to peptide Ac-GISSGY-OH, showing the Tyr-Ala peptide bondcleavage by PSA. The buffer alone led to the release of a small proportion of 7 due to slowhydrolysis or degradation of the imide bond in water (Figure 2E, t 1/2 ~ 15 h).In conclusion, imide ligation is a useful ligation method in peptide chemistry whichallowed the facilitated assembly of peptide-drug conjugates and the design of a novelself-immolative linker.AcknowledgmentsWe thank financial support from Région Nord Pas-de-Calais and from Cancéropôle Nord-Ouest. Wethank Emmanuelle Boll (CNRS) for NMR experiments.References1. Mhidia, R., Beziere, N., Blanpain, A., Pommery, N., Melnyk, O. Org. Lett. 12, 3982-3985 (2010).2. Shangguan, N., Katukojvala, S., Greenberg, R., Williams, L.J. J. Am. Chem. Soc. 125, 7754-7755(2003).3. Rijkers, D.T., Merkx, R., Yim, C.B., Brouwer, A.J., Liskamp, R.M. J. Pept. Sci. 16, 1-5 (2010).21