Protonation-deprotonation equilibria in tetrapyrroles Part 2: Mono- and diprotonation of methyl pyropheophorbide a in methanolic hydrochloric acid as verified by the 1H, 13C HSQC and 1H, 15N HMBC NMR experiments

Abstract

The two-dimensional 1H, 13C and 1H, 15N heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) NMR techniques were applied to investigate the formation of N-protonated cationic species of methyl pyropheophorbide a in methanolic hydrochloric acid (CD3OH-HCl). The 1H, 13C HSQC and 1H, 15N HMBC NMR spectra, recorded at the temperature of 278 K, verified that the CD3OH-HCl solution with [H]+ = 0.021 M, contained the N22-protonated monocations of methyl pyropheophorbide a, whereas the CD3OH-HCl solution with [H]+ = 5.0 M contained the N22, N24-protonated dications of the phorbin. No further protonations to form the trication or tetracation were observed. Consequently, the two middle electronic spectra, which were previously tentatively interpreted as representing the dication and trication, were now attributed to conformational alterations originating from steric hindrance between the central hydrogens and changes in counter-ion and solvation interactions and/or conformational alterations intimately connected to NH-tautomerism. These possibilities to explain the previously observed middle electronic spectra are briefly discussed.