NMR verification of helical conformations of phycocyanobilin in organic solvents

Selective NMR decoupling and nuclear Overhauser effect (NOE) experiments with phycocyanobilin (PCB) show proton-proton interactions between the terminal rings A and D, viz. the chiral C(2) methine center and the ethyl substituent at C(18), as a result of the helical conformation of this open-chain tetrapyrrole in solution. Quantitative NOE measurements and a combination of force-field and semiempirical calculations (FSC) afford inter-proton distances across the helical gap of 4.2-4.6 (NOE) and 3.2-4.2 A degrees (FSC). The NOE and FSC, in conjunction with a qualitative evaluation of the steric interactions in the two optimized helices, suggest furthermore that, in solution, the helix M is somewhat more stable than P. The coexistence of at least two diastereoisomers is corroborated also by the circular dichroism (CD) spectra of PCB in MeOH/EtOH which point to a temperature-dependent equilibrium in solution, and by a considerable increase of this CD upon changing the solvent from the achiral alcohols to ethyl (-)-(S)-lactate which reflects a selective solvent-induced CD differentiating between diastereoisomers.