Paavo H. Hynninen

High-performance liquid chromatographic separation and isolation of the methanolic allomerization products of chlorophyll a

Abstract Isocratic normal-phase HPLC on a silica column with diode-array detection provided a powerful means for the analytical separation and preparative isolation of seven methanolic allomerization products of chlorophyll a. Diastereomeric selectivity was achieved for 132(R,S)-hydroxychlorophyll a, 132(R,S)-methoxychlorophyll a and the Mg complex of 31,32-didehydro-151-hydroxy-151(R,S)-methoxyrhodochlorin-15-acetic acid δ-lactone 152-methyl 173-phytyl ester. The Mg complex of 31,32-didehydrorhodochlorin-15-glyoxylic acid 131,152-dimethyl 173-phytyl ester was also isolated in high purity. Reversed-phase HPLC did not result in an acceptable separation in spite of using several different brands of reversed-phase C18 columns and mobile phase compositions. The identification of the allomerization products was based on UV-Vis and 1H NMR spectra, retention times and co-elution with authentic samples. The observed formation of small amounts of numerous side-products is interpreted as further evidence for the free-radical mechanism of the allomerization.

Ultrafast charge transfer in phytochlorin-[60]fullerene dyads: influence of the attachment position

Abstract A femtochemistry study has been performed on two phytochlorin–[60]fullerene donor–acceptor dyads, in which the fullerene moiety is linked via a pyrrolidine bridge either to the position 3 or 7 of the phytochlorin ring. Both dyads form an emissive intramolecular exciplex in less than 1 ps, but only the former dyad relaxes to the ground state via a charge transfer (CT) state.

Liquid chromatographic separation and mass spectrometric identification of chlorophyll b allomers

Abstract Seven methanolic allomers of Chl b were separated and isolated by using sucrose column chromatography and normal-phase HPLC. Diastereomeric selectivity was achieved for 13 2 ( R,S )-methoxychlorophyll b and the magnesium complex of 3 1 ,3 2 -didehydro-15 1 -hydroxy-15 1 ( R,S )-methoxy-7 1 -oxo-rhodochlorin-15-acetic acid δ-lactone 15 2 -methyl-17 3 -phytyl ester. 13 2 ( S )-Hydroxy-10-methoxychlorophyll b , 13 2 -hydroxychlorophyll b and the magnesium complex of 3 1 ,3 2 -didehydro-7 1 -oxo-rhodochlorin-15-glyoxylic acid 13 1 ,15 2 -dimethyl-17 3 -phytyl ester were also isolated in high purity. The allomers were mainly identified by using UV–Vis spectrophotometry and FAB–MS. The allomerization of Chl b clearly differed from that of Chl a. The most striking difference was the formation of 13 2 ( S )-hydroxy-10-methoxychlorophyll b , as the corresponding derivative has never been observed to form in the allomerization of Chl a .

Vectorial photoinduced electron transfer in alternating Langmuir–Blodgett films of phytochlorin–[60]fullerene dyad and regioregular poly(3-hexylthiophene)

Abstract Alternating Langmuir–Blodgett (LB) bilayer structures, consisting of a donor–acceptor (DA) layer of a phytochlorin–fullerene (PF) dyad and a layer of a regioregular poly(3-hexylthiophene) (PHT) polymer were used to study interlayer vectorial photoinduced electron transfer (VPET). As the dyad PF undergoes, under light illumination, an intramolecular ET from the phytochlorin to the fullerene moiety an intralayer VPET takes place in the LB monolayer. When PF was deposited on the PHT layer and excited the second ET took place from the PHT layer to the phytochlorin cation. Thus the PHT layer can act as a secondary electron donor and accompany the primary photoinduced electron transfer in the PF layer by a spontaneous interlayer electron transfer. Important characteristic properties of the VPET bilayer are the longer distance of charge separation (CS) and the longer lifetime of the charge separated state (lifetime from microsecond to second) as compared to VPET of the PF monolayer alone (where the lifetime of CS state was ≈30 ns). The CT measurements were carried out for different molecular orientations and film structures. Models for the multistep photochemical reactions are discussed.

Electrophilic substitution at the δ-methine bridge of pheophorbide a and a′

Abstract A chloro-substituted derivative was prepared from chlorophyll a ′. The derivative was shown to be δ-chloro-methyl pheophorbide a by UV/VIS, MS and ′H NMR.

Application of elution analysis to the study of chlorophyll transformations by column chromatography on sucrose

Abstract The transformations of chlorophyll (Chl) a b were studied by applying elution analysis to chromatographic separations on a sucrose column. When the Chl a —water adduct was heated in 1-propanol at 50° for 15 min, subsequent separation on sucrose revealed three principal products which were identical with Chl a according to their visible absorption spectra and yielded positive reactions to the Molisch phase test. Only minor amounts of oxidation products were observed in this instance. When water-free Chl a was healed under similar conditions, separation on sucrose yielded, in addition to Chl a , considerable amounts of Chl a′ and oxidation products. On standing in 1-propanol at 25° for 4 days, Chl a was completely allomerized. Separation on sucrose revealed three pairs of oxidation products, each pair consisting of the 10( S ) and ( R ) isomers. When a light petroleum solution of chloroplast pigments was extracted with 70% aqueous methanol, the separation of the precipitated chlorophylls on sucrose yielded three chlorophyll components in both series but virtually no oxidation products. As the most plausible interpretation, it is suggested that the most slowly migrating chlorphylls arose from the presence of considerable amounts of the enol of chlorophyll in the samples. When these samples were introduced on to the sucrose column, the hydrogen-bonded water molecules were removed from the enol. This rendered the enol susceptible to transformation during the separation on sucrose. As very little oxygen was present, principally Chl a and a′ were produced. Although no enol could be detected in the effluent, the lifetime of the enol was considered to be long enough to cause the appearance of shoulders corresponding to the slowly migrating chlorophylls. These results confirm the enol forms of chlorophyll play a key role in chlorophyll oxidation and epimerization. They also show that the use of methanol—water mixtures for the purification of chlorophyll preparations requires special precautions in order to prevent the enolization and epimerization reactions.

Regioselective oxidation of the phytyl chain of pyrochlorophyll a

Abstract A regioselective oxidation of pyrochlorophyll a is described. Selenium dioxide is introduced for the hydroxylation or carbonylation of the P-4 allylic position in the phytyl chain ofthe phorbin.

PHOTOELECTRIC SIGNALS OF CHLOROPHYLL A LANGMUIR-BLODGETT FILMS

Abstract Photoelectric cells of ITO/insulator/chlorophyll a /insulator/InGa liquid metal, containing 1, 2, 4 or 10 LB monolayers of chlorophyll a as photoactive layers and 9–10 stearyl amine films as insulator layers have been studied by measuring the transient photovoltage and transient photocurrent responses in the micro- to millisecond and second time domains, respectively. The signals were created either by a laser pulse or by step-wise excitation. The cells exhibited light-induced charge transport inside the chlorophyll layers in the direction from the InGa to the ITO electrode. The charge movement is explained by an internal electric field due to the metal surface potentials, and was confirmed by measurements for when an external voltage was applied. A model based on a single-step light-induced charge shift is discussed, and the quantum yield for the light-induced charge generation has been estimated to be close to unity.

Conformations of Chlorophylls a and a′ and their Magnesium-Free Derivatives as Revealed by Circular Dichroism and Proton Magnetic Resonance

Abstract The electronic absorption (UV/VIS), circular dichroism (CD) and proton magnetic resonance (1H NMR) spectra have been recorded for C-10 epimeric chlorophylls a and a′, pheophytins a and a′ as well as pheophorbides a and a′. Although the epimers in each pair showed virtually identical UV/VIS spectra, their CD spectra were profoundly different and exhibited opposite signs at most wavelengths in the UV region. The differences were interpreted as arising, in part, from different C-10 configurations, and, in part, from conformational alterations induced by the steric strain in the crowded periphery of the macrocycle. The conformational alterations were also clearly indicated by the 1H NMR Δδ-values observed for the α,β and δ methine protons, the C-10 protons and most methyl group protons of the epimers in each pair. This was considered to imply changes in the geometry of the whole macrocycle. The Δδ-values were larger for the Mg-free epimers than for the chlorophyll epimers, which shows that the central Mg-atom makes the macrocycle more rigid. Correlations between the signs of the CD bands and configurations are discussed.

Reduction of chlorophyll a, a′ and b by sodium borohydride: separation of diastereoisomeric desoxo-chlorophyll alcohols on a sucrose column

Abstract Chlorophyll a , a′ and b were reduced with sodium borohydride in pyridine and the desoxo-chlorophyll alchohols (DOCA) produced were separated on a sucrose column. Reduction of chlorophyll a (Chl a ) for 15 min resulted in one principal product, the 9( R ) 10( R ) DOCA a . Similar treatment of Chl a′ yielded as the principal reduction product a different diastereomer, presumably the 9( S ) 10( S ) DOCA a . More unreduced Chl remained also in the latter instance. This was interpreted as an indication of the steric hindrance of the 9-oxo group by the methoxycarbonyl group in Chl a′ . Reduction of Chl a for 30 min resulted in three diastereomeric DOCA as evidenced by the separtion on a sucrose column. These were presumably the 9( S ) 10( S ), 9( R ) 10( R ) and 9( S ) 10( R ) forms of DOCA a . The 9( R ) 10( R ) form was, however, the principal reduction product also in this instance. When Chl a′ was reduced for 30 min, the separation on sucrose yielded possibly four diastereomeric DOCA which were thought to possess the 9( S ) 10( S ), 9( R ) 10( S ), 9( R ) 10( R ) and 9( S ) 10( R ) configurations. The 9( S ) 10( S ) form was still the principal reduction product. Chl a′ also yielded appreciable amounts of magnesium-free pigments, which was interpreted as further evidence for the higher pheophytinization tendency of Chl a′ in comparison with that of Chl a′ . The separation of the products from the reduction of Chl b for 30 min yielded five components. Two of them were 10-epimers of Chl b -3-methanol and the three others, different diastereomers of DOCA b -3-methanol.