Chiral-phase HPLC separation of (divinyl-)protochlorophyllide-a enantiomers as key precursors in chlorophyll biosynthesis from their 13 2 -stereoisomeric prime forms.

Protochlorophyllide(PChlide)-a and its 8-vinylated analog, divinyl(DV)-PChlide-a, are common and essential intermediates in the biosynthesis of all naturally occurring chlorophyll (Chl) pigments. These porphyrinoid-type pigments have a single optically active (asymmetric) carbon atom at the 13-position, so their stereoisomers are (13R)- and (13S)-enantiomers. The former and latter are called (DV-)PChlide-a and (DV-)PChlide-a', respectively. In this study, chiral-phase HPLC separation of enantiomeric (DV-)PChlides-a/a' was demonstrated. The (13R)-enantiomeric PChlide-a was eluted more slowly than the corresponding (13S)-enantiomeric PChlide-a' under the present HPLC conditions. On the other hand, the elution order of (13R)-DV-PChlide-a and (13S)-DV-PChlide-a' was reverse to that of PChlides-a/a'. After the separation of each enantiomer by the chiral-phase HPLC, the stereoisomeric configuration at the 13-position was characterized by means of circular dichroism spectroscopy. The present chiral-phase HPLC method enables us to evaluate optical purities of (DV-)PChlide-a species. For example, PChlide-a and/or DV-PChlide-a extracted from the spent medium and harvested cells of cultured purple photosynthetic bacterial mutants, the former of which has been often used as the source of (DV-)PChlide-a substrates for enzymatic reactions, were revealed to be mostly racemized, giving enantiomeric mixtures of (DV-)PChlides-a/a'.