Chlorophyll Pigments and Their Synthetic Analogs.

Oxygenic phototrophs use chlorophylls (Chls) as photosynthetically active pigments. A variety of Chl molecules have been found in photosynthetic eukaryotes including green plants, algae, and cyanobacteria. Here we review their molecular structures with stereochemistry, occurrence in light-harvesting antennas and reaction centers, biosyntheses in the late stage, chemical stabilities, and visible absorption maxima in diethyl ether. The observed maxima are comparable to those of semisynthetic Chl analogs, methyl pyropheophorbides, in dichloromethane. The effects of their peripheral substituents and core π-conjugation on the maxima of the monomeric states are discussed. Notably, the oxidation along the molecular x-axis in Chl-a produces its accessory pigments, Chls-b/c, and introduction of an electron-withdrawing formyl group along the y-axis perpendicular to the x-axis affords far-red light absorbing Chls-d/f.

Extra-Annulated Chlorophyll Derivatives by Scholl Reaction.

The ferric chloride assisted Scholl reaction of methyl (3,5-dimethoxyphenyl)carbonyl-pyropheophorbide-a produced an extra-annulated, didehydrogenated chlorophyll derivative with a six-membered ring fused at the pyrrole A-ring. The steric interaction of the substituents on the additional tetralone and B-rings induced molecular helicity. The helically cyclized stereoisomers were separated by recrystallization, and their (P/M)-stereochemistry was confirmed by circular dichroism spectra. The distortion of their chlorin π-systems broadened electronic absorption bands to cover all visible regions and reach the near-infrared region.

Genes for the type-I reaction center and galactolipid synthesis are required for chlorophyll a accumulation in a purple photosynthetic bacterium.

Anoxygenic photosynthesis is diversified into two classes: chlorophototrophy based on a bacterial type-I or type-II reaction center (RC). Whereas the type-I RC contains both bacteriochlorophyll and chlorophyll, type-II RC-based phototrophy relies only on bacteriochlorophyll. However, type-II phototrophic bacteria theoretically have the potential to produce chlorophyll a by the addition of an enzyme, chlorophyll synthase, because the direct precursor for the enzyme, chlorophyllide a, is produced as an intermediate of BChl a biosynthesis. In this study, we attempted to modify the type-II proteobacterial phototroph Rhodovulum sulfidophilum to produce chlorophyll a by introducing chlorophyll synthase, which catalyzes the esterification of a diterpenoid group to chlorophyllide a thereby producing chlorophyll a. However, the resulting strain did not accumulate chlorophyll a, perhaps due to absence of endogenous chlorophyll a-binding proteins. We further heterologously incorporated genes encoding the type-I RC complex to provide a target for chlorophyll a. Heterologous expression of type-I RC subunits, chlorophyll synthase, and galactolipid synthase successfully afforded detectable accumulation of chlorophyll a in Rdv. sulfidophilum. This suggests that the type-I RC can work to accumulate chlorophyll a and that galactolipids are likely necessary for the type-I RC assembly. The evolutionary acquisition of type-I RCs could be related to prior or concomitant acquisition of galactolipids and chlorophylls.

Pyrobacteriopheophorbide-a derivatives possessing various hydrophilic esterifying groups at the C17-propionate residues for photodynamic therapy.

Aiming at the application to photodynamic therapy, natural bacteriochlorophyll-a was converted to chemically stable free-base derivatives possessing different kinds of hydrophilic C17-propionate residues. These semi-synthetic bacteriochlorins were found to have self-assembling ability in an aqueous environment and formed stable J-type aggregates in a cell culture medium containing 0.2% DMSO. The electronic absorption spectra of all the sensitizers showed Qy absorption maxima at 754 nm in DMSO as their monomeric states, while a drastic shift of the red-most bands to ca. 880 nm was observed in the aqueous medium. The circular dichroism spectra in the medium showed much intense signals compared to those measured in DMSO, supporting the formation of well-ordered supramolecular structures. By introducing hydrophilic side chains, the bacteriochlorin sensitizers could be dispersed in the aqueous medium as their J-aggregates without the use of any surfactants. Cellular uptake efficiencies as well as photodynamic activities were evaluated using human cervical adenocarcinoma HeLa cells. Among the 11 photosensitizers investigated, the best result was obtained for a charged derivative possessing trimethylammonium terminal (17-CH2CH2COOCH2CH2N+(CH3)3I-) and photocytotoxicity of EC50 = 0.09 µM was achieved by far-red light illumination of 35 J/cm2 from an LED panel (730 nm).

Solitary and Synergistic Effects of Different Hydrophilic and Hydrophobic Phospholipid Moieties on Rat Behaviors.

Glycerophospholipids have hydrophobic and hydrophilic moieties. Previous studies suggest that phospholipids with different moieties have different effects on rodent behavior; however, the relationship between chemical structures and behavioral effects remains unclear. To clarify the functions of phospholipid moieties, we injected male rats with phospholipids with different moieties and conducted behavioral tests. Exploratory activity was reduced by phosphatidylethanolamine (PE)(18:0/22:6) but not PE(18:0/18:0) or PE(18:0/20:4). Conversely, exploratory activity was increased by plasmanyl PE(16:0/22:6), which harbors an alkyl-ether linkage, but not by phosphatidylcholine (PC)(16:0/22:6) or plasmanyl PC(16:0/22:6). Docosahexaenoic acid (DHA)(22:6) and an alkyl-ether linkage in PE were thus postulated to be involved in exploratory activity. Anxiety-like behavior was reduced by plasmenyl PC(18:0/20:4), which harbors a vinyl-ether linkage, but not by PC(18:0/20:4) or plasmanyl PC(18:0/20:4), suggesting the anxiolytic effects of vinyl-ether linkage. The activation of social interaction was suppressed by PE(18:0/18:0), PE(18:0/22:6), PC(16:0/22:6), plasmanyl PE(16:0/22:6), and plasmanyl PC(16:0/22:6) but not by PE(18:0/20:4), plasmenyl PE(18:0/20:4), or plasmanyl PC(18:0/22:6). DHA may suppress social interaction, whereas arachidonic acid(20:4) or a combination of alkyl-ether linkage and stearic acid(18:0) may restore social deficits. Our findings indicate the characteristic effects of different phospholipid moieties on rat behavior, and may help to elucidate patterns between chemical structures and their effects.

Determination of N-centered stereochemistry in N22-methylated chlorophyll-a derivatives and their epimer-dependent optical spectra.

An N-centered epimeric mixture of chlorophyll-a derivatives methylated at the inner nitrogen atom was separated by reverse-phase high-performance liquid chromatography. Circular dichroism (CD) spectroscopic analyses of the epimerically pure N22-methyl-chlorins revealed that the minor first-eluted and major second-eluted stereoisomers were (22S)- and (22R)-configurations, respectively. Their visible absorption and CD spectra in solution were dependent on the N22-stereochemistry. The epimer-dependent spectral changes were independent of the substituents at the peripheral 3-position of the core chlorin chromophore.

Self-aggregation of zinc bacteriochlorophyll-d analogs with an acylhydrazone moiety as the 13-keto-carbonyl alternative.

Zinc methyl 3-hydroxymethyl-pyropheophorbides-a possessing an acylhydrazinylidene group at the 131-position were prepared by chemically modifying chlorophyll-a, which were models of bacteriochlorophyll-d as one of the light-harvesting pigments in photosynthetic green bacteria. Similar to the self-aggregation of natural bacteriochlorophyll-d in the antenna systems called chlorosomes, some of the synthetic models self-aggregated in an aqueous Triton X-100 solution to give red-shifted and broadened visible absorption bands. The newly appeared oligomeric bands were ascribable to the exciton coupling of the chlorin π-systems along the molecular y-axis, leading to intense circular dichroism bands in the red-shifted Qy and Soret regions. The self-aggregation in the aqueous micelle was dependent on the steric size of the terminal substituent at the 13-acylhydrazone moiety. An increase in the length of the oligomethylene chain as the terminal moved the red-shifted Qy maxima to shorter wavelengths, and branched alkyl and benzyl substitutes afforded no more self-aggregates to leave monomeric species in the hydrophobic environment inside the micelle. These results indicated that the acyl groups on the 13-hydrazone as the alternative of the natural 13-ketone regulated the chlorosome-like self-aggregation.

In vitro demetalation of central magnesium in various chlorophyll derivatives using Mg-dechelatase homolog from the chloroflexi Anaerolineae.

In the metabolic pathway of chlorophylls (Chls), an enzyme called STAY-GREEN or SGR catalyzes the removal of the central magnesium ion of Chls and their derivatives to their corresponding free bases, including pheophytins. The substrate specificity of SGR has been investigated through in vitro reactions using Chl-related molecules. However, information about the biochemical properties and reaction mechanisms of SGR and its substrate specificity remains elusive. In this study, we synthesized various Chl derivatives and investigated their in vitro dechelations using an SGR enzyme. Chl-a derivatives with the C3-vinyl group on the A-ring, which is commonly found as a substituent in natural substrates, and their analogs with ethyl, hydroxymethyl, formyl, and styryl groups at the C3-position were prepared as substrates. In vitro dechelatase reactions of these substrates were performed using an SGR enzyme derived from an Anaerolineae bacterium, allowing us to investigate their specificity. Reactivity was reduced for substrates with an electron-withdrawing formyl or sterically demanding styryl group at the C3-position. Furthermore, the Chl derivative with the C8-styryl group on the B-ring was less reactive for SGR dechelation than the C3-styryl substrate. These results indicate that the SGR enzyme recognizes substituents on the B-ring of substrates more than those on the A-ring.

Supramolecular chirality in self-assembly of zinc protobacteriochlorophyll-d analogs possessing enantiomeric esterifying groups.

Zinc 3-hydroxymethyl-pyroprotopheophorbides-a esterified with a chiral secondary alcohol at the 17-propionate residue were prepared as bacteriochlorophyll-d analogs. The synthetic zinc 31-hydroxy-131-oxo-porphyrins self-aggregated in an aqueous Triton X-100 micellar solution to give red-shifted and broadened Soret and Qy absorption bands in comparison with their monomeric bands. The intense, exciton-coupled circular dichroism spectra of their self-aggregates were dependent on the chirality of the esterifying groups. The observation indicated that the self-aggregates based on the J-type stacking of the porphyrin cores were sensitive to the peripheral 17-propionate residues. The supramolecular structures of the present J-aggregates as models of bacteriochlorophyll aggregates in natural chlorosomes were remotely regulated by the esterifying groups.

Photomultiplication Behavior of Chlorophyll-Based Photodetector under Biased Voltage.

In this study, we fabricated a photodetector (PD) with two types of chlorophyll derivatives, namely, zinc methyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (ZnChl) and methyl 131-deoxo-131-dicyanomethylene-pyropheophorbide-a (H2Chl'), via a two-step drop-coating process. In the absorption range of ZnChl/H2Chl' films, the maximum external quantum efficiency of ZnChl/H2Chl'-based devices reached 1363% at -8 V and 1345% at 2.5 V, exhibiting the photomultiplication (PM) phenomenon. The PM phenomenon of ZnChl/H2Chl'-based devices is attributed to hole tunneling injection from the external circuit assisted by electron accumulation in the ZnChl and H2Chl' under light illumination. Through the investigation of the responsivity (R) of ZnChl/H2Chl'-based devices, it has been found that achieving a high R is easier under forward bias compared with reverse bias (7706 mA/W at -8 V and 7629 mA/W at 2.5 V). The organic PDs based on ZnChl/H2Chl' exhibit PM behavior, offering a promising approach to improve the device's responsivity.

Self-aggregation behavior of dimeric chlorophyll-a derivatives linked with ethynylene and m-phenylene moieties.

Chlorophyll(Chl)-a derivatives inserting an ethynylene-m-phenylene group between a zinc chlorin ring and a hydroxymethyl group, in which various substituents were introduced on the benzene spacer, were prepared as model compounds for the light-harvesting antennae (chlorosomes) of photosynthetic green bacteria. These compounds were synthesized from a C3-ethynylated Chl-a derivative via sequential Sonogashira cross-coupling reaction, and the effects of the substituents on the phenylene linker on their self-aggregation behaviors were investigated by electronic absorption, circular dichroism, and infrared absorption spectroscopic measurements. These studies exhibited that some compounds gave the disordered self-assemblies including several species; however, the zinc complex of the dimeric Chl-a derivative primarily allowed a single J-aggregate species in an aqueous Triton X-100 micellar solution. Additional control experiments revealed that its self-assembly was constructed through the hydrogen and coordination bonding involving the hydroxymethyl group on benzene ring, keto-carbonyl group at C13-position, and central zinc atom, and this is consistent with a conventional chlorosomal manner.

Remodeling Zinc Deposition via Multisite Zincophilic Chlorophyll for Powerful Aprotic Zinc Batteries.

The organic electrolyte can resolve the hurdle of hydrogen evolution in aqueous electrolytes but suffers from sluggish electrochemical reaction kinetics due to a compromised mass transfer process. Herein, we introduce a chlorophyll, zinc methyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (Chl), as a multifunctional electrolyte additive for aprotic zinc batteries to address the related dynamic problems in organic electrolyte systems. The Chl exhibits multisite zincophilicity, which significantly reduces the nucleation potential, increases the nucleation sites, and induces uniform nucleation of Zn metal with a nucleation overpotential close to zero. Furthermore, the lower LUMO of Chl contributes to a Zn-N-bond-containing SEI layer and inhibits the decomposition of the electrolyte. Therefore, the electrolyte enables repeated zinc stripping/plating up to 2000 h (2 Ah cm-2 cumulative capacity) with an overpotential of only 32 mV and a high Coulomb efficiency of 99.4%. This work is expected to enlighten the practical application of organic electrolyte systems.

Predicted Structure of the BciC Enzyme Catalyzing the Removal of the C132-Methoxycarbonyl Group for Biosynthesis of Chlorosomal Chlorophylls: A Mechanism for Dual Catalytic Functions of Hydrolysis and Decarboxylation inside Its Active Site.

Green photosynthetic bacteria, one of the phototrophs, have the largest and most efficient light-harvesting antenna systems, called chlorosomes. The core part of chlorosomes consists of unique bacteriochlorophyll c/d/e molecules. In the biosynthetic pathway of these molecules, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. Two sequential reactions have been proposed for the BciC enzymatic demethoxycarbonylation: the BciC enzyme would catalyze the hydrolysis of the C132-methoxycarbonyl group, and the resulting carboxylic acid would be rapidly decarboxylated to generate pyrochlorophyllide a. In this study, we computationally predicted the three-dimensional structure of the BciC protein. Its active site was proposed based on structural analysis using docking simulation. In vitro enzymatic reaction assays of mutated BciC supported the prediction. The BciC enzymatic hydrolysis would be an aspartic/glutamic acid hydrolase, which involves the amino residues E85 and D180. Furthermore, Y58 and H126 might depend on stabilization and/or recognition with the substrate. Most importantly, H137 would protonate 13-C═O or deprotonate C132-COOH in the hydrolyzed product to promote decarboxylation. In conclusion, the BciC enzyme has the dual functions of hydrolysis and decarboxylation.

Ring-shaped self-assembly of a naphthalene-linked chlorophyll dimer.

Light-harvesting antennas, for example the LH2 complex in purple bacteria, sophisticatedly align chlorophyll molecules in a cyclic fashion by using protein scaffolds. However, artificial preparation of the circular LH antenna model without any templates has not been reported. We demonstrated the construction of ring-shaped supramolecules by self-assembly of a semisynthetic chlorophyll dimer through a transformation from wavy fiber-like aggregates.

Chiral-phase HPLC separation of (divinyl-)protochlorophyllide-a enantiomers as key precursors in chlorophyll biosynthesis from their 132-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 132-position, so their stereoisomers are (132R)- and (132S)-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 (132R)-enantiomeric PChlide-a was eluted more slowly than the corresponding (132S)-enantiomeric PChlide-a' under the present HPLC conditions. On the other hand, the elution order of (132R)-DV-PChlide-a and (132S)-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 132-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'.

In vitro reversible dehydration in C3-substituents of zinc chlorophyll analogs by BchF and BchV enzymes: Stereoselectivity and substrate specificity in the dehydration.

In the biosynthetic pathway of bacteriochlorophyll(BChl)-a/b/c/d/e molecules, BchF and BchV enzymes catalyze the hydration of a C3-vinyl to C3-1-hydroxyethyl group. In this study, the in vitro reactions catalyzed by BchF and BchV partially afforded a C31-epimeric mixture of the hydrated products (secondary alcohols), with the primary recovery of the C3-vinylated substrate. The stereoselectivity and substrate specificity for the in vitro reverse enzymatic dehydration were examined using zinc chlorophyll analogs as model substrates by BchF and BchV, which were obtained from extracts of Escherichia coli overexpressing the respective genes from Chlorobaculum tepidum and used without further purification. Both BchF and BchV preferred dehydration of the (31R)-epimers over the (31S)-epimers. The (31R)-epimer was directly dehydrated by BchF and BchV to give the C3-vinylated product. By contrast, two reaction pathways for BchF and BchV dehydrations of the (31S)-epimer were proposed: (1) the (31S)-epimer would be directly dehydrated to C3-vinyl group. (2) the (31S)-epimer would be epimerized to the (31R)-epimer, and the resulting epimer was dehydrated. The results indicated that both BchF and BchV did function as a hydratase/dehydratase and could play a role in the C31-epimerization. An increase in the alkyl size at the C8-position gradually suppressed the BchF and BchV-catalyzed dehydration in vitro, while the C121- and C20-methylation only slightly affected the reaction. Using the BchF dehydration, a large amount of 3-vinyl-bacteriochlorophyllide-a was successfully prepared, with the retention of the chemically labile, central magnesium atom.

Photoinduced Catalytic Organic-Hydride Transfer to CO2 Mediated with Ruthenium Complexes as NAD+ /NADH Redox Couple Models.

The catalytic organic-hydride transfer to CO2 was first achieved through the photoinduced two-electron reduction of the [Ru(bpy)2 (pbn)]2+ /[Ru(bpy)2 (pbnHH)]2+ (bpy=2,2'-bipyridine, pbn=2-(pyridin-2-yl)benzo[b]-1,5-naphthyridine, and pbnHH=2-(pyridin-2-yl)-5,10-dihydrobenzo[b]-1,5-naphthyridine) redox couple in the presence of 1,3-dimethyl-2-phenyl-2,3-dihydro-1H-benzo[d]imidazole (BIH). The active species for the catalytic hydride transfer to carbon dioxide giving formate is [Ru(bpy)(bpy⋅- )(pbnHH)]+ formed by one-electron reduction of [Ru(bpy)2 (pbnHH)]2+ with BI⋅.

Synthesis and Self-Aggregation of Chlorophyll-a Derivatives Possessing a Hydroxymethyl Group in the C20-Substituent with Ethynylene and/or Phenylene Linkers.

Chlorophyll(Chl)-a derivatives containing some rigid linkers in the C20 substituents, which were inserted between a hydroxymethyl group and the chlorin π-skeleton, were synthesized and their self-aggregation abilities were investigated. Measurements of electronic absorption, circular dichroism and infrared absorption spectra revealed that the zinc complex of a Chl-a derivative bearing a (p-ethynyl)benzyl alcoholic moiety at the C20-position gave its J-aggregates in an aqueous micellar solution through the hydrogen and coordination bonding. These results exhibited the potential of Chl-a derivatives bearing a hydroxyl group in the C20 substituents as the model compounds for chlorosomal light-harvesting antennas.

Characterization of regioisomeric diterpenoid tails in bacteriochlorophylls produced by geranylgeranyl reductase from Halorhodospira halochloris and Blastochloris viridis.

Geranylgeranyl reductase (GGR) encoded by the bchP gene catalyzes the reductions of three unsaturated C = C double bonds (C6 = C7, C10 = C11, and C14 = C15) in a geranylgeranyl (GG) group of the esterifying moiety in 17-propionate residue of bacteriochlorophyll (BChl) molecules. It was recently reported that GGR in Halorhodospira halochloris potentially catalyzes two hydrogenations, yielding BChl with a tetrahydrogeranylgeranyl (THGG) tail. Furthermore, its engineered GGR, in which N-terminal insertion peptides characteristic for H. halochloris were deleted, performed single hydrogenation, producing BChl with a dihydrogeranylgeranyl (DHGG) tail. In some of these enzymatic reactions, it remained unclear in which order the C = C double bond in a GG group was first reduced. In this study, we demonstrated that the (variant) GGR from H. halochloris catalyzed an initial reduction of the C6 = C7 double bond to yield a 6,7-DHGG tail. The intact GGR of H. halochloris catalyzed the further hydrogenation of the C14 = C15 double bonds to give a 6,7,14,15-THGG group, whereas deleting the characteristic peptide region from the GGR suppressed the C14 = C15 reduction. We also verified that in a model bacterium, Blastochloris viridis producing standard BChl-b, the reduction of a GG to phytyl group occurred via 10,11-DHGG and 6,7,10,11-THGG. The high-performance liquid chromatographic elution profiles of BChls-a/b employed in this study are essential for identifying the regioisomeric diterpenoid tails in the BChls of phototrophic bacteria distributed in nature and elucidating GGR enzymatic reactions.

Intramolecular axial α/ß-coordination of the 132-terminal pyridyl group to the central zinc atom in chlorophyll-a derivatives.

Chlorophyll(Chl)-a derivatives possessing a zinc center and a C132-alkanoate residue with a terminal pyridyl group were synthesized. Their C132-epimerically pure products were isolated by preparative reverse phase HPLC. The C132-stereochemistry resulted in two directed terminal pyridinyl groups that coordinate with the central zinc atom in each stereoisomeric molecule: α/ß-intramolecular axial coordination. The asymmetric axial coordinations mimic the immobilization manner of Chl-a in photosynthetically active proteins. The diastereomerically dependent conformers in solution were characterized by 1D/2D NMR, UV-visible absorption, and fluorescence emission as well as circular dichroism (CD) spectroscopy. The 1H NMR and DOSY spectra revealed that the stereoselectively intramolecular coordination occurred in less coordinative deuterated chloroform, whereas a highly coordinative deuterated pyridine molecule replaced the terminal pyridine moiety as the axial ligand to form a mixture of α/ß-coordinated species. Their optical spectra in pyridine were nearly independent of the C132-stereochemistry and the linkers in the C132-substituents. The CD bands of ß-coordinated species in chloroform were more intense than those of the corresponding α-coordinated stereoisomers, indicating that the former had a larger distortion of the chlorin π-plane than the latter. Therefore, the α-coordinated Chl complex is more conformationally stable than the ß-complex.