Structure elucidation and conformational analysis of gonadotropin releasing hormone and its novel synthetic analogue [Tyr(OMe) 5, d-Lys 6, Aze 9NHEtGnRH: The importance of aromatic clustering in the receptor binding activity

Abstract

The conformational properties of gonadotropin releasing hormone (GnRH) in dimethylsulfoxide- d 6 were investigated by nuclear Overhauser effect (nOe) enhancement studies and were compared with the conformational properties of its analogue [Tyr(OMe) 5 GnRH resulting after methylation of the tyrosine hydroxyl. Assignment of all backbone and side-chain protons was possible by combining information from intraresidue nOe studies with two-dimensional correlated spectroscopy (COSY/TOCSY) studies. Saturation of distinct proton resonances of the three aromatic residues Tyr, His, Trp, in clear areas of the NMR spectrum of GnRH resulted in interresidue enhancements of aromatic resonances indicating the 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 × 10 −6 M) compared with that exerted by GnRH ( K d = 3.69 ± 0.89 × 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 rigid Aze residue [Tyr(OMe) 5 , d -Lys 6 , Aze 9 NHEt]GnRH significantly improved the binding affinity ( K d = 0.689 ± 10.15 × 10 −9 ) and this may be due to the restoration of the ring cluster. Overall, the clustering of the aromatic rings observed in GnRH was not seen in [Tyr(OMe) 5 ]GnRH and this conformational difference may be responsible for receptor recognition and higher binding of the parent peptide.