Galardy R E, Kortylewicz Z P
Int J Pept Protein Res. 1985 Jul;26(1):33-48. doi: 10.1111/j.1399-3011.1985.tb03175.x.
The docosapeptide which constitutes the membrane spanning region (amino acid residues 73-94) of the human red blood cell protein glycophorin A has been synthesized. This may be the first example of the synthesis of the entire membrane embedded domain of a membrane spanning protein. Three fully protected fragments were prepared by stepwise elongation using dicyclohexylcarbodiimide and p-nitrophenyl ester activation of N alpha-tert.-butyloxycarbonyl amino acids. The three fragments represent amino acid residues 73-79, 80-86, and 87-94 in the sequence of glycophorin A and contain a large proportion of valine, leucine, and isoleucine residues but contain no amino acids with ionizable side chain functional groups. The three fragments were condensed using both the azide method and the dicyclohexylcarbodiimide method to give fully protected docosapeptide. Benzyl groups protecting the side chains of the docosapeptide were removed by prolonged hydrogenolysis to give the desired product N alpha-tert.-butyloxycarbonyldocosapeptide ethyl ester. High resolution proton n.m.r. spectra of the protected fragments in 100% deuterochloroform showed all resonances to be broadened with the amide resonances broadened beyond recognition. In perdeuterodimethylsulfoxide all resonances were relatively sharp with all amide resonances visible and showing coupling constants of 7-8 Hz. Solvent titration of the proton spectra of two of the fragments from 100% perdeuterodimethylsulfoxide to 100% deuterochloroform demonstrated a transition to the broadened spectrum, accompanied by a decrease in the coupling constant of the amide protons (JNH-CH alpha) suggesting solvent dependent onset of intramolecular secondary structure, possibly accompanied by aggregation. A proton n.m.r. spectrum of the docosapeptide in perdeuterodimethylsulfoxide shows a few resolved amide resonances with coupling constants of 7-9 Hz. Solvent titration with perdeuterochloroform again suggests a transition to a rigid intramolecular secondary structure.
构成人红细胞膜糖蛋白A跨膜区（氨基酸残基73 - 94）的二十二肽已被合成。这可能是合成跨膜蛋白整个膜嵌入结构域的首个实例。通过使用二环己基碳二亚胺和对硝基苯酯活化Nα - 叔丁氧羰基氨基酸进行逐步延伸，制备了三个完全保护的片段。这三个片段分别代表膜糖蛋白A序列中的氨基酸残基73 - 79、80 - 86和87 - 94，含有大量的缬氨酸、亮氨酸和异亮氨酸残基，但不含具有可电离侧链官能团的氨基酸。使用叠氮法和二环己基碳二亚胺法将这三个片段缩合，得到完全保护的二十二肽。通过长时间的氢解反应去除保护二十二肽侧链的苄基，得到所需产物Nα - 叔丁氧羰基二十二肽乙酯。在100%氘代氯仿中受保护片段的高分辨率质子核磁共振谱显示，所有共振峰都变宽，酰胺共振峰变宽到无法识别。在全氘代二甲亚砜中，所有共振峰相对较尖锐，所有酰胺共振峰都可见，且耦合常数为7 - 8 Hz。将其中两个片段的质子谱从100%全氘代二甲亚砜滴定到100%氘代氯仿，显示出向变宽谱的转变，同时酰胺质子的耦合常数（JNH - CHα）降低，这表明分子内二级结构的形成可能与溶剂有关，可能伴随着聚集。在全氘代二甲亚砜中二十二肽的质子核磁共振谱显示有一些分辨出的酰胺共振峰，耦合常数为7 - 9 Hz。用氘代氯仿进行溶剂滴定再次表明向刚性分子内二级结构的转变。
