Fridkin Gil, Rahimipour Shai, Ben-Aroya Nurit, Kapitkovsky Aviva, Di-Segni Susanna, Rosenberg Masha, Kustanovich Irina, Koch Yitzhak, Gilon Chaim, Fridkin Mati
Department of Organic Chemistry, Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
J Pept Sci. 2006 Feb;12(2):106-15. doi: 10.1002/psc.696.
Five linear analogs of GnRH containing a p-aminophenylalanine (Pap) residue in their sequence and their six corresponding azo-bridged cyclic derivatives were synthesized. The precyclic peptides were prepared on solid-support, while azo-cyclization was performed in solution by diazotization of the p-aminophenylalanine residue followed by intramolecular coupling of the formed diazo salt with either tyrosine or histidine side chains present in the sequence. All peptides were examined for their binding ability to the GnRH receptor expressed on rat pituitary membranes and for their LH-release activity from dispersed rat pituitary cells. Linear analogs 1 i.e [Pap(5)] GnRH and 3, i.e. [Tyr(3), Pap(5)] GnRH, were found to bind to the GnRH receptors only slightly less avidly than native GnRH. Their cyclization, however, led to a marked reduction in the binding capacity, i.e. from IC(50) of 10(-9) M to the 10(-7) M range, and in biopotency, i.e. LH-release. All other linear and cyclic peptides were found to bind selectively to the GnRH receptor only in the low microM range. Only peptide 1 was found comparable to native GnRH in respect to LH-release activity and thus may potentially be a good agonist of the parent peptide. Peptides 1-4, the most potent GnRH receptor binders, were examined for their conformational properties using CD. Cyclic-azo peptides 2 and 4 were further evaluated by NMR spectroscopy in solution combined with molecular modeling. The structural information obtained explains in part the GnRH-like biological activity observed.
合成了5种在其序列中含有对氨基苯丙氨酸（Pap）残基的GnRH线性类似物及其6种相应的偶氮桥连环状衍生物。前环肽在固相载体上制备，而偶氮环化是通过对氨基苯丙氨酸残基进行重氮化，然后使形成的重氮盐与序列中存在的酪氨酸或组氨酸侧链进行分子内偶联在溶液中进行的。检测了所有肽与大鼠垂体膜上表达的GnRH受体的结合能力以及它们从分散的大鼠垂体细胞中释放LH的活性。发现线性类似物1即[Pap(5)]GnRH和3即[Tyr(3), Pap(5)]GnRH与GnRH受体的结合亲和力仅略低于天然GnRH。然而，它们的环化导致结合能力显著降低，即从IC(50)为10(-9)M降至10(-7)M范围，并且生物活性即LH释放也降低。发现所有其他线性和环状肽仅在低微摩尔范围内选择性地结合GnRH受体。仅发现肽1在LH释放活性方面与天然GnRH相当，因此可能潜在地是母体肽的良好激动剂。使用圆二色光谱法检测了肽1-4（最有效的GnRH受体结合剂）的构象性质。通过溶液中的核磁共振光谱结合分子建模进一步评估了环状偶氮肽2和4。获得的结构信息部分解释了观察到的GnRH样生物活性。
