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Table 1. Proline cis/trans ratios of RGD-related analogues (m,n=1) calculated from NMRspectra a and preferred guanidine orientations according to a molecular modeling studyX NH 2 NH-Me PyrrolidinePro cis/trans 65:35 90:10 20:80Preferredtc (Cis) tc (Cis) cc (Trans)guanidinect (Trans) ct (Trans) ct (Trans)orientation(s)b,c tt (Trans)tc (Cis)a Water, 700 MHz, 320 K; b From the most to the least preferred; c The prolineconformation preferred for each guanidine orientation is given in bracketsABAcHNH TyrOmGly-Asp-ProNHm, n = 1, 3XNHX = = -NH 2 , -NH-Me,HNOHNGly-PheXNHHNHNX = =S, -S-Me, -NH 2 , -NH-Me,Fig. 2. A, RGD-related cyclicanalogues. B, Enkephalin cyclicanalogues.NOnONNH 2NH 2(Figure 2). The Asp-Pro peptide bond showed both cis and trans conformations. Theproline cis/trans ratio was measured and found to vary significantly as a function of thedegree of bridge substitution, at least for the shortest analogues (m = n = 1) (Table 1). Inparticular, an inversion of this ratio was observed between the mono- and the di-substitutedanalogues. To explain these results, a molecular modeling study (Vconf, Verachem LLC)helped by quantum chemical computations was performed. As it could be expected, thisstudy indicated that the guanidine orientation could be driven by potential steric clashesbetween substituents. As a result, the cc orientation is disfavored for non- and monosubstitutedanalogues while the tc and ct isomeries are the only ones favored for the monosubstitutedanalogue (Table 1). The di-substitutedanalogue is a special case as steric clashes shouldoccur for all orientations but the cc orientationappeared as the most favored. The study alsoindicated that, for unknown reasons, eachguanidine orientation would favor either cis ortrans proline. For instance, the tt and ccorientations were preferentially associated with atrans proline, while a cis proline was preferred forthe tc isomer. Taken together, the modeling resultswere in accordance with NMR data.As we showed that the degree of guanidinebridge substitution could modulate theconformation of a given peptide, we explored itspotential influence on the biological activity ofanother model peptide. A series of enkephalincyclic analogues was prepared (Figure 2). Theywere submitted to various biological assays,including binding to mu and delta opioid receptorsand functional assays (G-protein activation, GPIand MVD bioassays). All compounds were foundmore or less selective for the mu receptor and fullagonists. A significant variation in mu affinity andselectivity for this receptor was evidenced betweennon- (Ki = 19 nM; delta/mu = 39), mono- (Ki = 1.5nM; 41) and di-substituted (Ki = 107 nM; 10)analogues. NMR study is currently performed to try to explain these variations. Then, it isinteresting to note that the analogue with a thiourea bridge possessed a very good affinityfor the mu receptor with a Ki of 0.4 nM but with a mu selectivity of only 13 fold.In conclusion, guanidine bridges appeared as interesting tools to build cyclic peptideswith substitution-dependent cycle conformations. At least for short cyclic peptides, weshowed that, for a same sequence, a diversely substituted bridge could modulate theconformation and the biological activity of a peptide. Then, the ability to prepare suchanalogues together with the thiourea and S-methyl-isothiourea intermediates from a uniqueprecursor makes our solid phase method a powerful tool to rapidly attain large diversity.AcknowledgmentsWe thank Pierre Sanchez for mass spectrometry analyses.7