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The Willstätter allomerization reaction of chlorophylls (Chl) has posed a difficult problem in Chl and photosynthesis research over the past 90 years. Here, we present strong additional evidence, based on (18)O-labeling and mass spectrometry, for the previously published free-radical allomerization (FRA) mechanism (Hynninen, Z. Naturforsch. 1981, 36b, 1010-1016). This mechanism is also complemented now by describing two alternative pathways for the formation of 13(2)(S/R)-hydroxy-Chl a. The results from the (18,18)O(2)-experiments suggest that the predominant route for the formation of the 13(2)(S/R)-hydroxy-Chl a under essentially anhydrous conditions (anhydrous Chl and thoroughly dried methanol) is the homolytic cleavage of the C-13(2)-hydroperoxide intermediate. However, if Chl dihydrate and undried methanol are used in the reaction mixture, the direct route from the Chl C-13(2) radical to 13(2)(S/R)-hydroxy-Chl a can be predicted to become significant. The results from the (18,18)O(2)-allomerization experiments described in this paper also verified that the 13(2)(S/R)-methoxy-lactone derivatives and the 15-glyoxylic acid derivative of Chl a incorporated each a single (18)O-atom, whereas 13(2)(R/S)-methoxy-Chl a remained unlabeled. Consequently, these allomers are formed via the pathways previously suggested in the original FRA mechanism. The possible factors contributing to the control of the allomerization reactions are considered. Finally, the relationship between the allomerization reactions of Chl a and those of Chl b and BChl a is briefly discussed.

The spontaneous autoxidation (allomerization) of 13'(R)-chlorophyll u in methanol produces seven main products containing three epimer pairs. The 'H and ' ' C NMR spectra of these products in acetone-d, were recorded on a 500 MHz spectrometer and fully assigned using two-dimensional HMQC and HMBC techniques. The absolute configurations of the oxidized carbons, originally C-13', were determined using the ROESY technique. In comparison with 13'(R)-chlorophyll u, the steric repulsion between the C-17 side-chain and the bulky substituents of the oxidized C-13' causes steric strain in the chiral part of an allomer, relieved by conformational changes in rings D and E and to a lesser extent also in the whole macrocycle. These changes were estimated for each allomer from the A6 values of the carbon and proton resonances and from protomproton coupling constants. Information about the orientation of the front part of the phytyl chain in 13z(R)-chlorophyll u and its methanolic allomers was obtained by analysing the variation in the form of the P1-CH, signal.

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Fast-atom bombardment (FAB) mass spectra have been measured for resolved 132-hydroxychlorophyll a epimers. The fragmentation of each epimer was studied by tandem mass spectrometry using linked scans at constant B/E. The FAB spectra showed a very intense radical cation (M+') and EI-type fragmentations. The most abundant fragment ions were formed by the losses of the phytyl, the 13'-hydroxy, and the [M -phytadiene -OH]' were found to be characteristic of both 132-hydroxychlorophyll u epimers. Nevertheless, it was shown that the loss of the 13*-hydroxy group accurs more easily from the 13'(S)-epimer than from the 132(R)-epimer. This fragmentation difference offers a new possibility for the determination of the stereochemistry at the 13'-chiral centre of purified 132-hydroxychlorophyll preparations.The 132(S)-and 132(H)-epimers of 132-hydroxychlorophyll a (Fig. 1) belong to the main autoxidation (allomerization) products of chlorophyll (Chl) These hydroxylated derivatives are easily formed from Chl a in methanolic solution via oxidation at C132 by triplet oxygen (Willstatter's allomerization reaction). Hydroxychlorophyll a was resolved for the first time into its 132-epimers by column chromatography on sucrose3 and, subsequently, by TLC on sucrose: as well as by reversed phase' and normal phase HPLC.' However, the assignment of the stereochemical configurations at C132 of the resolved hydroxychlorophyll epimers has posed a difficult task, which has been solved only recently after achieving the fully assigned 'H and 13C NMR spectra as well as ROESY spectra for the epimem6From the analytical point of view, a disadvantage in the use of NMR techniques is their relatively low sensitivity. Thus, for the characterization of Chl deriva-*