Synthesis of a docosapeptide comprising the hydrophobic membrane spanning region of glycophorin A.

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.