Fast‐atom bombardment mass spectra of the 13²‐epimers of 13²‐hydroxychlorophyll a

Abstract

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-*

Development of an accurate and high-throughput methodology for structural comprehension of chlorophylls derivatives. (I) Phytylated derivatives

“…It is possible that the modifications introduced in the original protocol, to increase the amount of hydroxyl-derivatives, may have modified the ratio among epimers raising the trend to S configuration what results in a minimal presence of product ions containing hydroxyl group at C13 2 in MS 2 analyses. In fact, there are MS 2 analysis in which the product ions retaining hydroxyl groups are the predominating ones [20,21] and vice versa [22,24]. The application of the post-processing software to MS 2 spectra for determining the structural configuration of the product ions has allowed the identification of some new ones for hydroxyl chlorophyll derivatives.…”

“…With the exception of the CO fragmentation, there is no other difference in the fragmentation profile of a and b series of native chlorophylls and pheophytins ( Table 3). The 13 2 -hydroxyl derivatives have been only extensively analyzed independently, by MS 2 , in the case of 13 2 -hydroxy chlorophyll-a [20,22,23,31], 13 2 -hydroxy chlorophyll-b [21] and 13 2 -hydroxy pheophytin-a [24]. In this work, we performed the analysis of all these hydroxyl derivatives as well as the 13 2 -hydroxy pheophytin-b (not described so far, Fig.…”

“…Among the allomerization products identified and studied in depth are 15 1 -methoxy-lactone-, 13 2 -methoxy-, and 13 2 -hydroxy-chlorophyll. Although outstanding contributions have dealt with the MS/MS characterization of allomerized chlorophylls [20][21][22], it is important to highlight that 15 1 -methoxy-lactone-and 13 2 -methoxy-chlorophyll derivatives are not produced in vivo, as naturally chlorophyll compounds are not in contact with alcoholic solutions, so the natural substituent in chlorophyll derivatives is the hydroxyl group, forming 13 2 -hydroxy-or 15 1 -hydroxy-lactone chlorophyll derivatives. In fact, the common technique to produce the 13 2 -hydroxy chlorophyll-a and -b used for MS analyses is the methanolic allomerization [21][22][23][24], which leads to non-specific oxidation and calls for large volumes of initial substrates, because the hydroxyl derivatives are produced in minor amounts.…”

Identification of diastereomeric chlorophyll allomers by atmospheric pressure chemical ionisation liquid chromatography/tandem mass spectrometry

“…Structural elucidation by NMR is rigorous but time‐consuming and often requires milligram quantities of highly purified sample. By contrast, mass spectrometry has proved to be a very efficient technique for the analysis of allomerisation products of chlorophylls and has superior limits of detection 7–9. To this end, high‐performance liquid chromatography (HPLC) coupled with mass spectrometry has provided a powerful tool for the identification and separation of the allomers of chlorophylls 10–13.…”

Principal components scores and loadings plots for visualisation of the electrospray ionisation liquid chromatography mass spectra of a mixture of chlorophyll degradation products at different cone voltages

“…Chlorophyll allomers have been characterised in previous papers. 11,15,23,24 The next phase is to scale the data as described above, and then perform PCA, retaining the three most significant principal components. Three-dimensional (3-D) plots of scores and loadings were generated, and rotated to emphasise the most important features.…”