STRUCTURE ELUCIDATION AND CONFORMATIONAL-ANALYSIS OF GONADOTROPIN-RELEASING-HORMONE AND ITS NOVEL SYNTHETIC ANALOG [TYR(OME)(5), D-LYS(6), AZE(9)NHET]GNRH - THE IMPORTANCE OF AROMATIC CLUSTERING IN THE RECEPTOR-BINDING ACTIVITY

The conformational properties of gonadotropin releasing hormone (GnRH) in dimethylsulfoxide-d(6) were investigated by nuclear Overhauser eff ect (nOe) enhancement studies and were compared with the conformationa l properties of its analogue [Tyr(OMe)(5)]GnRH resulting after methyla tion of the tyrosine hydroxyl. Assignment of all backbone and side-cha in protons was possible by combining information from intraresidue nOe studies with two-dimensional correlated spectroscopy (COSY/TOCSY) stu dies. Saturation of distinct proton resonances of the three aromatic r esidues Tyr, His, Trp, in clear areas of the NMR spectrum of GnRH resu lted in interresidue enhancements of aromatic resonances indicating th e proximity of the three aromatic rings. This spatial proximity is not observed in [Tyr(OMe)(5)]GnRH and is correlated with a lower receptor binding affinity in the rat pituitary (K-d = 1.53 +/- 0.35 x 10(-6) M ) compared with that exerted by GnRH (K-d = 3.69 +/- 0.89 x 10(-9) M). However, substitution of Gly at position 6 of [Tyr(OMe)(5)]GnRH with D-Lys(6) and further replacement of Pro at position 9 with the more ri gid Ate residue [Tyr(OMe)(5), D-Lys(6), Aze(9)NHEt]GnRH significantly improved the binding affinity (K-d = 0.689 +/- 10.15 x 10(-9)) and thi s may be due to the restoration of the ring cluster. Overall, the clus tering of the aromatic rings observed in GnRH was not seen in [Sr(OMe) (5)]GnRH and this conformational difference may be responsible for rec eptor recognition and higher binding of the parent peptide.