Cleavages from long aliphatic chains

According to the molecular composition predicted from the exact mass value, a series of fragments are proposed from cleavages of the two aliphatic side chains (side chains in sites C-17 and C-29 are named chain A and chain

B hereafter, which are highlighted in Scheme 6.1), and summarized in Table 6.1.

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Scheme 6.1 Labelling the proposed cleavages sites of chain A and B in ring IV&V

Apart from the cluster around m/z 614.2, the other two high intensity peaks at m/z 583.2190 and m/z 555.2241 generated by all three methods are assigned to the loss of the phytyl group and CH3O/CH3COO of the B chain respectively. Moreover, a range of peaks, which are also proposed from the cleavages from chain A and B, are identified. These peaks have a relatively higher intensity as well in EID and IRMPD spectra (Relative intensity can be found in Table E.1). The presence of this series suggests that the spatial positions of chain A and chain B may allow interaction, where cyclization could occur between the two cleavages sites due to their proximity.

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Table 6.1 Summary of fragments proposed cleavages from Chain A & B Proposed formula Theoretical mass EID ion (m/z) Internally calibrated Error (ppm) CAD ion (m/z) Internally calibrated Error (ppm) IRMPD ion (m/z) Internally calibrated Error (ppm) Proposed cleavage C35H35O5N4Mg 615.2452 615.2453 0.16 615.2454 0.32 615.2456 0.65 a C35H34O5N4Mg* 614.2374 614.2374 --- 614.2374 --- 614.2374 --- a- H• C34H31O4N4Mg* 583.2190 583.2190 --- 583.2193 --- 583.2191 --- ag C34H30O4N4Mg 582.2112 582.2112 0 582.2114 0.34 582.2113 0.17 ag - H• C33H31O3N4Mg 555.2241 555.2240 -0.18 555.2242 0.18 555.2243 0.36 af C33H31O2N4Mg 539.2292 539.2292 0 539.2290 -0.37 539.2294 0.37 bf/cg C33H30O2N4Mg 538.2214 538.2213 -0.19 538.2216 0.37 bf/cg - H• C32H29ON4Mg 509.2186 509.2184 -0.39 509.2189 0.59 cf - H2 C31H25O2N4Mg 509.1822 509.1823 0.20 eg - H2 C32H28ON4Mg 508.2108 508.2108 0 508.2110 0.39 cf - H2 - H• C31H27ON4Mg 495.2030 495.2030 0 495.2035 1.01 df - H2 C30H25ON4Mg* 481.1873 481.1873 --- 481.1873 --- 481.1878 --- ef - H2 Average 0.03 0.12 0.33 SD (σ) 0.17 0.29 0.47

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Cleavage preference among the different bonds of side chain A and B are summarized on the basis of the fragments detected and their relative intensity (Table 6.1 and Table E.1). Comparatively, cleavages from sites ‘a’, ‘f’, ‘e’, are preferred (Scheme 6.1). As described, site ‘a’ between the ester group and phytyl chain is most fragile. Sites ‘e’ and ‘f’ which are adjacent to the conjugated macrocyclic ring also contribute to several intense peaks. This order generally follows the bond strength information provided by crystal x-ray experiments of chlorophyllide-a dihydrate.310 Additionally, loss of H2 was detected almost universally. This loss is most likely from C-17 and C-18 of ring IV (Figure 6.1), leading to a larger conjugated structure which is thermodynamically favourable.

Though similar peaks were formed with EID, IRMPD, and CAD, more radical ions were generated by EID after detailed assignment of the fragments described above (Figure 6.3 and Figure 6.4). As expected, cleavages leading to radical ions are more common during EID than CAD and IRMPD, due to the combination of vibrational excitation and electronic excitation.70 However, odd-electron fragments are also observed in IRMPD, and at very low abundance in CAD as well. Figure 6.5 shows the abundance change of several odd-electron species with increasing the laser power in IRMPD. Although the relative abundance of these species is still quite low compared to their abundance in EID, it increases with increase in laser influence. The observation of these unusual odd-electron species in CAD and IRMPD spectra of chlorophyll-a may be related to a structural property of the macrocyclic ring, as a similar phenomenon was observed in octaethylporphyrin using low energy CAD in Rosario’s work.306

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considering the discovery of ECD was because of an UV photodissociation experiment, where secondary electrons were emitted by UV photons and lead to fragmentation.60 In the IRMPD experiment, it is unlikely that secondary electrons are formed by IR photons, but the intensity of EID-like odd-electron fragments suggests otherwise, and the overall intensity of all fragments is 10 times higher in the IRMPD spectrum than in the EID spectrum. Further experimental and computational investigations are necessary to understand the results.

Figure 6.4 From left to right are expansions of m/z regions 582-586, 538-541 and 508-511 (a) CAD; (b) IRMPD; (c) EID; d-f are the corresponding simulations of the elemental compositions in the highlight regions respectively

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Figure 6.5 Plotting of intensity changes of several radical ions according to the laser power in IRMPD

Two low intensity peaks at m/z 833.5215 (C53H69O3N4Mg) and m/z 541.2085 (C32H29O3N4Mg) (Table E.2), repeated in the EID and IRMPD spectra, are interesting. The peak at m/z 833.5215 seems to be the only product ion detected between m/z 614 and m/z 893 from chlorophyll-a. As proposed, this peak can be assigned to the facile losses of side chain B and one H atom from C-29 from the protonated molecular ion.311 In addition, losses of chain A and the H atom from C-18, resulting in a double bond between C-17 and C-18, is speculated to generate the peak at m/z 541.2085. Otherwise, no extensive cleavage within the phytyl chain was detected in these experiments. As an unidentified doubly-charged impurity, which has a similar m/z (893.7459) with chlorophyll-a, was isolated together with our target ion; several fragments were induced from it as well. However, based

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on the charge state and accurate mass value, most of the peaks from the impurity can be distinguished.

In addition to fragments from the cleavages of two long side chains, several peaks that are probably from cleavages of ring V or rearrangements are present (Table E.2). In particular, the high intensity peak at m/z 455.2081 (C29H27N4Mg) (Figure E.2), whose formula suggests all five oxygen atoms are lost from the molecular ion, along with its elemental composition further implying that ring V is probably cleaved.