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Table 1. Synthesis of Fmoc-aza-β 3 -Hse(R)-OH, step yields (%)Protecting group R CH 2 OEt Bn All Z(i) OR - protection 89 70 90 53(ii) acetal hydrolysis 5 73 79 88(iii) condensation 85 60 80 82(iv) reduction 53 89 81 70(v) reductive amination 95 88 80 89Then, the O-deprotection was performed after the peptidyl cleavage from the resin andremoval of the guanido protecting group. The yields resulting from the Bn and Allhydrogenolysis with Pd(C) or silane hydride under standard conditions were very low. Thereaction carried out under microwave-assisted conditions (40W, 45min, 333K) led to ahigher deprotection level (86% yield before purification) (Figure 2). Reaction effectivenesswas evaluated by combining analytical HPLC and MALDI-TOF mass spectrometry.Subsequently, crude aza-β 3 -peptide was purified by reversed-phase HPLC in MeCNgradient elution. Collected fractions were characterized by MALDI-TOF MS to lead to theexpected [aza-β 3 -Hse] 23 26RFa (20-26) analog. Nevertheless, its binding affinity and its abilityto increase the [Ca 2+ ] i on GPR103-transfected cells were, at least, twice lower than for26RFa (20-26) . However, this microwave-assisted specific efficiency retains our focus andopens perspectives to increase the reaction yield.Phe-Arg-Phe-NH 2(1a) Pd/C, 10%, MeOH, rt ∼ 8 %.HNMicrowave Conditions40W, 45min, Pd/C 10%MeOH/HCHO, 86% yieldHPLC before purificationOH-Gly-Gly-PheNHNO(1b) Pd/C,10%, Et 3SiH, CH 2Cl 2, MeOH, rt ~ 12%.(1c) Pd/C, 10%, MeOH /AcOH, rt~ 15 %.Phe-Arg-Phe-NH 2HNPhe-Arg-Phe-NH 2H-Gly-Gly-PheNHNO(2a) PhSiH 3 , Pd(PPh 3) 4, rt ~Ź9%.(2b) PMHS - ZnCl 2/Pd(PPh 3) 4, rt ~Ź14%.(2c) Pd/C, MeOH /AcOH 10%, ~ 12 %.H-Gly-Gly-PheNHNOPhe-Arg-Phe-NH 2NHO(3a) PhSiH 3 , Pd(PPh 3) 4, ~Ź9%.(3b) Pd(OAc) 2 , DMF, (1,4)-C 6H 8 ~7 %.HPLC after purificationOHHNOOOH-Gly-Gly-PheNNHOH-Gly 20 -Gly 21 -Phe 22 -aza β 3 -Hse 23 -Phe 24 -Arg 25 -Phe 26 -NH 2MALDI-TOF Š MS / [M+H+ ] thˇo : 845.50 / [M+H + ] exp.: 845.19Fig. 2. Aza-β 3 Hse (OR)-protecting group cleavages by standard and microwavecondition.References1. Busnel, O., et al J. Org. Chem. 26, 10701-10708 (2005).2. Dali, H., et al. Mol. Immunol. 44, 3024-3036 (2007).3. Laurencin, M., Legrand, B., Duval, E., Zatylny-Gaudin, C., Bondon, A., Baudy-Floc'h, M.submitted.4. Chartrel, N., et al. Proc. Natl. Acad. Sci. U.S.A. 100, 15247- 15252 (2003).95