Proceedings book download - 5Z.com

Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010OOPhoto-Uncaging of Neurotransmitter Amino Acids FromFluorescent 5,6-Benzocoumarinyl PrecursorsMaria José G. Fernandes, M. Sameiro T. Gonçalves, andSusana P.G. CostaCentre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, PortugalIntroductionPhotoactivable precursors of bioactive molecules (caged compounds) are importantresearch tools for tracking molecular dynamics with high spatiotemporal resolution inbiological systems [1]. A diversity of structures, such as 2-nitrobenzyl [2], 1-(4,5-dimethoxy-2-nitrophenyl)-ethyl [3], 4,5-dimethoxy-2-nitrobenzyl [4], 6-bromo-7-hydroxycoumarin-4-yl-methyl[5], 7-[(diethyl-amino)-coumarin-4-yl]-methyl [6] and 7-dinitroindolinyl[7] have been evaluated as caging groups for the photo-regulation of calcium ions,neurotransmitters, carboxylic acids, proteins, nucleotides, peptides, RNAs and DNAs [8].Having in mind these facts and as a continuation of our work related with theinvestigation of alternative photolabile protecting groups [9], we now report the evaluationof the release under irradiation of β-alanine, tyrosine, 3,4-dihydroxyphenylalanine (DOPA)and glutamic acid neurotransmitters from the corresponding 5,6-benzocoumarinylfluorescent conjugates. Photo-uncaging studies were carried out at 254, 300 and 350 nmand cleavage kinetic data obtained by HPLC/UV monitoring.Results and Discussion5,6-Benzocoumarinyl-neurotransmitter amino acids conjugates 1a-e were obtained bycleavage of N-benzyloxycarbonyl or N-tert-butyloxycarbonyl groups under usual chemicaldeprotection conditions from the corresponding precursors with β-alanine, tyrosine, 3,4-OorOOOOORnNH 21a-dO O 1ea n = 2, R = Hb n = 1, R =NH 2OOHOhνHOc n = 1, R =d n = 1, R =HOOORornNH 22a-dONH 2OOOHOOH+photoby-productsFig. 1. Photo-uncaging of neurotransmitter amino acids 2a-efrom the corresponding 5,6-benzocoumarinyl conjugates 1a-e.2edihydroxyphenylalanineand glutamic acid [9c](Figure 1). The benzocoumarinylmoiety willbe designated in thisreport by a three-lettercode (Bba) for simplicityof naming the variousfluorescent conjugates,as indicated in Table 1.The sensitivity ofconjugates 1a-e towardsUV-visible irradiationwas evaluated byexposing solutions of thementioned compounds inACN/HEPES buffer(80:20) solution in aRayonet RPR-100reactor at 254, 300 and350 nm. The course ofthe photocleavage reaction was followed by reverse phase HPLC with UV detection. Theplots of peak area (A) of the starting material versus irradiation time were obtained for eachcompound at the considered wavelengths. Peak areas were determined by HPLC, whichrevealed a gradual decrease with time, and were the average of three runs. The determinedirradiation time represents the time necessary for the consumption of the starting materialsuntil less than 5% of the initial area was detected (Table 1).82