The synthesis of new photolabeling analogues of luteinizing hormone releasing hormone is described. Peptides were prepared by the solid-phasc method, and the photolabeling moiety was introduced as the 4'-nitrophenylalanine precursor. This amino acid, either of the L or u configuration, was placed at different positions and was modified afterwards to 4'-azidophenylalanine. Structures with agonistic or antagonistic character were prepared, together with a peptide containing simultaneously the photolabeling moiety and a biotin substituent for future receptor isolations. Binding studics on placental and pituitary membranes indicatc thal these compounds represent promising candidates for receptor labeling studies.
Introduction. -The photoaffinity labeling method has gained wide acceptance in most areas of biochemistry, particularly in the area of peptide hormones and their receptors [l] [2]. Generally, two methods of photolabel introduction are utilized: either by modification of an already existing peptide with e.g. an active ester containing a photosensitive moiety or, second, by synthetic introduction of a photolabeling amino acid into the chosen sequence during peptide synthesis [3], an approach we propose to name 'intrinsical labeling'. Many peptide hormones have been treated with either one or even both approaches, thus helping to characterize their respective receptors. Both methods, the intrinsical and the modification pathway, have permitted to identify the receptor of angiotensin I1 [4] [5]. Only the intrinsical photolabel, however, achieved a receptor incorporation rate over 40 %, sufficiently high to observe very significant irreversible biological effects [5] [6], and biochemical isolation of the receptor protein.
The standard procedure for the introduction of an intrinsic photolabel is the use of "-protected 4'-nitrophenylalanine in solid-phase or classical peptide synthesis and the conversion of this residue in the completed peptide into 4'-azidophenylalanine, the photolabeling moiety. A further advantage of the intrinsic photolabels is the relatively small structural disturbance caused by this moiety, compared to a prosthetic photolabeling group (modification pathway) which can disturb strongly the biological action and significance of a peptide. I ) This work has been financcd by grants from the Medical Research Council of Canada, by the Cunudiun Kidney Foundation, and the Canadian Heart Foundutiurz. S. B., N . G.-P., J.-G. L., and E.E. are scholars of the Fonds de la Recherche en SantP du Quihcc. Abbreviations of amino-acid residues according to IUPAC/IUB [la]; moreover, Glp = pyroglutamyl, CI2Z = 2,4-dichlorobenzyloxycarbonyl, Tos = 4-toluolsulfonyl, HOBt = Nhydroxybenrotriazole, DCC = dicyclohexylcarbodiiniid, BNp = 2-naphthy1, Biot = biotinyl.