TEMPLATE FOR STABILIZATION OF A PEPTIDE ALPHA-HELIX - SYNTHESIS AND EVALUATION OF CONFORMATIONAL EFFECTS BY CIRCULAR-DICHROISM AND NMR

The bicyclic diacid 1 was designed as a semi-rigid template for the hy drogen-bonding pattern of a peptide alpha-helix. The protected precurs or 7 was synthesized in eight steps from tert-butyl 3,5-dimethoxybenzo ate and linked to L-alanine and L-lactic acid to provide derivatives a ppropriate for coupling to a peptide. Both the amide 8-N and the ester 12-O were obtained in each of the four diastereomeric forms. The stru cture of R,R-8-N was determined by X-ray crystallography, which facili tated assignment of the diastereomers and confirmed the intended confo rmational effects of the quaternary methyl groups. The bicyclic amide and ester derivatives were appended to the peptide EALAKA-NH2, and the ir influence on the conformation was evaluated in aqueous solution usi ng circular dichroism and NMR. The amide analogs have only a slight ef fect on the appended peptide, whereas the ester-linked template in S,S -9-O induces 32-50% helical character at 23 degrees C and 49-77% at 0 degrees C, depending on the method of determination; significant helic al character persists even at 70 degrees C. The ability of the templat e 1 to induce the helical conformation is related to its structural an d electronic complementarity to the N-terminus of the peptide; templat ing ability disappears when the carboxylate in S,S-9-O is protonated, and it is not observed in the dimethylamide S,S-9-N-a. The structural and dynamical properties of conjugate S,S-9-O were studied by NMR and compared with those of the acetylated heptapeptide 13. The dispersion and temperature dependence of amide hydrogen chemical shifts and the p attern observed for intra- and inter-residue nuclear Overhauser enhanc ements are all consistent with a significant population of helical con formers within the conformational ensemble of conjugate S,S-9-O, in co ntrast to the unstructured peptide 13. The generalized order parameter S-2 was derived for each residue from the N-15 T-1 and T-2 relaxation rate constants and H-1-N-15 heteronuclear NOEs determined for the N-1 5-labeled derivative of S,S-9-O; these parameters demonstrate a high d egree of conformational rigidity along the peptide chain at 4 degrees C, with relative motion increasing for the C-terminal residues. These data are consistent with the chiroptical studies and demonstrate that the template is exceptionally effective in inducing helical behavior i n an appended peptide.