An Unexpected Cis Peptide Bond in the Minor Conformation of a Cyclic Hexapeptide Containing Only Secondary Amide Bonds

The conformation of the cyclic hexapeptide cyclo(-D-Ala¹-Phe²-Val³-Lys(Z)⁴-Trp⁵-Phe⁶-) [VDA008], a very potent inhibitor of the bile-acid transport system in hepatocytes, was investigated in hexadeuteriodimethyl sulfoxide solution by NMR spectroscopy and restraint MD calculations. Surprisingly, this peptide exhibits two conformations (94:6) in slow exchange on the NMR time scale at room temperature. Chemical exchange between both isomers was proven by 2D-ROESY and 2D-NOESY spectra. The dominant conformation has all-trans peptide bonds forming a βII,βII′ backbone conformation and preferred side chain orientations similar to that of the analogue cyclo(-D-Pro-Phe-Thr-Lys(Z)-Trp-Phe-), 008, whereas the minor conformation has a cis peptide bond between Lys(Z)⁴ and Trp⁵ forming a βVI turn about these residues. The conformational analyses of the backbones of both conformers are based on the temperature dependences of H_N chemical shifts, on scalar coupling constants, and on quantitative evaluation of ROEs and NOEs for restraint MD calculations. The populations of the side chain rotamers are derived from quantitative evaluation of homonuclear coupling constants and qualitative evaluation of heteronuclear coupling constants. For the MD calculations in vacuo, the side chains are fixed in the dominant rotamers.