Photoacoustic Detection of Superoxide Using Oxoporphyrinogen and Porphyrin.

The superoxide (O2•-) ion is a highly reactive oxygen species involved in many diseases; hence, its noninvasive detection is desirable to identify the onset of pathological processes. Here, we employed photoacoustic (PA) spectroscopy, which enables imaging at ultrasound resolution with the sensitivity of optical modality, for the first time to detect O2•-, using stimuli-responsive contrast agents. meso-(3,5-Di-tert-butyl 4-hydroxyphenyl) porphyrins and oxoporphyrinogens were used as PA contrast agents, which trap the O2•- and enable its detection. The trapped O2•- increased the PA signal amplitude of chromophores up to 9.6-fold, and induced a red-shift in the PA signal maxima of up to 225 nm. Therefore, these trigger-responsive probes may be highly valuable as smart diagnostic PA probes to investigate pathological events stimulated by O2•- species.

NMR spectroscopic detection of chirality and enantiopurity in referenced systems without formation of diastereomers.

Enantiomeric excess of chiral compounds is a key parameter that determines their activity or therapeutic action. The current paradigm for rapid measurement of enantiomeric excess using NMR is based on the formation of diastereomeric complexes between the chiral analyte and a chiral resolving agent, leading to (at least) two species with no symmetry relationship. Here we report an effective method of enantiomeric excess determination using a symmetrical achiral molecule as the resolving agent, which is based on the complexation with analyte (in the fast exchange regime) without the formation of diastereomers. The use of N,N'-disubstituted oxoporphyrinogen as the resolving agent makes this novel method extremely versatile, and appropriate for various chiral analytes including carboxylic acids, esters, alcohols and protected amino acids using the same achiral molecule. The model of sensing mechanism exhibits a fundamental linear response between enantiomeric excess and the observed magnitude of induced chemical shift non-equivalence in the (1)H NMR spectra.

Liquid chromatography-tandem mass spectrometry of porphyrins and porphyrinogens in biological materials: separation and identification of interfering poly(ethylene) glycol by travelling wave ion mobility spectrometry/tandem mass spectrometry.

Biological and clinical samples for porphyrin and porphyrinogen analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS) are often contaminated with poly(ethylene)glycol (PEG), which complicates the interpretation of mass spectra and characterisation of new porphyrin metabolites. Two contaminating PEG molecules (m/z 833 and m/z 835) were completely separated from uroporphyrin I (m/z 831) by travelling wave ion mobility spectrometry and characterised by tandem mass spectrometry. One of the PEG species (m/z 835) also co-eluted with uroporphyrinogen I (m/z 837) and was unresolvable by travelling wave ion mobility spectrometry/MS, therefore contaminating the MS/MS mass spectra owing to isotope distribution. These PEG species, with the [M + H](+) ions at m/z at 833 and/or m/z 835, co-eluted with uroporphyrin I and uroporphyrinogen I by LC-MS/MS and could be wrongly identified as uroporphomethenes.

The syn and anti isomers of the porphyrinogen-like precursor of calix[4]phyrin: isolation, X-ray structure, anion binding and fluoride-ion-mediated proton-deuterium exchange studies.

Porphyrinogen-like precursors of calix[4]phyrins are presumed to be unstable owing to their auto-oxidation. In contrast to this, the syn and the anti isomers of a calix[4]pyrrole molecule containing pyridine moieties at the meso positions were isolated and their structures were determined by single crystal X-ray diffraction studies. Both the isomers gave the same calix[4]phyrin molecule upon oxidation. The anion binding properties of both the isomers were studied in DMSO-d6 by the EQNMR method, which showed that they have a preference of binding with the F(-) ion over the other large sized halide and oxo anions. In addition, the F(-) ion mediated H/D exchange process was monitored by the (19)F NMR method. The solution state structures of the 1 : 1 F(-) ion complexes containing deuterium atoms formed by a random but sequential substitution of NH protons by deuterium atoms were identified from their multiplicity patterns observed in the proton coupled (19)F NMR spectrum, which are supported by the proton decoupled (19)F NMR spectrum showing one singlet for each type of F(-) ion complex in solution for both the syn and anti isomers, correlating with their solid state structures.

Stepwise syntheses of tri- and tetraphosphaporphyrinogens.

A double ring-expansion methodology allows the stepwise synthesis of tri- and tetra-phosphaporhyrinogens. Structural and calculational methods reveal extended delocalisation about their phospholide centres.

Enhanced photocurrents via redox modulation by fluoride binding to oxoporphyrinogen in a zinc porphyrin-oxoporphyrinogen surface modified TiO2 supramolecular solar cell.

A novel approach for improving photocurrent in a supramolecular solar cell, composed of zinc porphyrin-oxoporphyrinogen (ZnP-OxP) surface-modified TiO(2), by redox tuning through fluoride anion binding to the redox active host, OxP is demonstrated.

Porphyrinogen fragmentation profiles by ultra-high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry.

An ultra-high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry system is described for the separation and characterisation of uroporphyrinogen, heptacarboxylic acid porphyrinogen, hexacarboxylic acid porphyrinogen, pentacarboxylic acid porphyrinogen and coproporphyrinogen. The separation was carried out on a 100 mm × 2.1 mm Thermo-Hypersil BDS column (2.4 µm average particle size) by gradient elution with a mixture of acetonitrile, methanol and 1 mol/L aqueous ammonium acetate buffer, pH 5.16, as eluent. The fragmentation pattern of each compound was established by collision-induced dissociation tandem mass spectrometry. The most characteristic fragmentation was ring opening at one of the four methylene bridges of the protonated porphyrinogen molecule followed by further cleavages of methylene bridges linking the four pyrrole rings at various points to give product ions with methylenepyrrolenine, methylene-dipyrrolenine and methylene-tripyrrolenine structures.

Effect of anion binding on charge stabilization in a bis-fullerene-oxoporphyrinogen conjugate.

Presence of strongly binding anion, F(-) stabilizes the photo-induced charge-separated states of a bis-fullerene-substituted oxoporphyrinogen due to the large shift in the oxidation potential of the oxoporphyrinogen moiety upon anion binding through hydrogen bonding at its core.

Normal and abnormal heme biosynthesis. 6. Synthesis and metabolism of a series of monovinylporphyrinogens related to harderoporphyrinogen. Further insights into the oxidative decarboxylation of porphyrinogen substrates by coproporphyrinogen oxidase.

A series of vinylporphyrinogens were prepared to probe the enzyme coproporphyrinogen oxidase (CPO). Six (2-chloroethyl)porphyrins were synthesized from a common dipyrrylmethane via a,c-biladiene intermediates in excellent yields. Subsequent dehydrohalogenation with DBU in refluxing DMF then gave the required vinylporphyrin methyl esters, including harderoporphyrin-I, harderoporphyrin-III, and isoharderoporphyrin. The corresponding porphyrinogen carboxylic acids were incubated with chicken red cell hemolysates, which contain the enzyme CPO, and the products analyzed. The 17-ethyl analogue of harderoporphyrinogen-III, but not its 13-ethyl isomer, was shown to be an excellent substrate for CPO in accord with a proposed model for the active site of this enzyme. In addition, harderoporphyrinogen-VII, the monovinyl intermediate in the metabolism of coproporphyrinogen-IV, was shown to be an equally good substrate for this enzyme. However, isoharderoporphyrinogen, which lacks the correct ordering of peripheral substituents, was also a substrate for CPO. Furthermore, a nonnatural type I isomer of harderoporphyrinogen was shown to be acted on by CPO, but in this case further metabolism was noted and this afforded an unprecedented trivinyl porphyrinogen product. The corresponding porphyrin methyl ester was isolated and characterized by FAB MS and proton NMR spectroscopy. The results from these studies allow the binding requirements of CPO to be further assessed and provide a series of substrates to investigate this poorly understood enzyme.

Nuclear magnetic resonance signaling of molecular chiral information using an achiral reagent.

Until now NMR spectroscopic detection of guest chirality using an achiral host has not been possible in the absence of a chiral medium or auxiliary since chiral discrimination is principally based on chiral discrimination by host and/or diastereomeric host-guest complex formation. In this paper, we demonstrate that an achiral oxoporphyrinogen works as a host capable of signaling chiral information of alpha-hydroxycarboxylic acids in (1)H NMR spectroscopy. In particular, enantiomeric excess (ee) can be determined by observing the splitting of (1)H NMR resonances of the achiral host. This differs from the case of chiral hosts (shift reagents) where % ee is generally determined from the ratio of peak areas due to diastereomeric host-guest complexes. UV/vis, CD, FT-IR, and NMR spectroscopic investigations suggest that the unusual phenomenon reported here is based on formation of a complex with 1:2 stoichiometry in concert with a protonation-driven tautomerization of the host.

Reduction of porphyrins to porphyrinogens with palladium on carbon.

Porphyrinogens serve as substrates for three heme biosynthetic enzymes. Porphyrinogens are highly unstable and must be generated as an integral part of enzyme assays. Methods commonly employed to generate porphyrinogens include chemical reduction using sodium amalgam or sodium borohydride and enzymatic generation from porphobilinogen. Chemical reduction yields porphyrinogens in highly alkaline solutions with high ionic strength, whereas enzymatic generation requires purified enzymes, deproteination, and complete buffer replacement. This article describes an improved method for reducing porphyrins to porphyrinogens using palladium on carbon as a catalyst under hydrogen at ambient temperature and pressure in the dark. The palladium catalyst is removed by filtration, the filtrate is blown dry with an inert gas, and the dried porphyrinogen can be dissolved in a buffer compatible with biological studies.

Binding and glutathione conjugation of porphyrinogens by plant glutathione transferases.

Overexpression in Escherichia coli of a tau (U) class glutathione transferase (GST) from maize (Zea mays L.), termed ZmGSTU1, caused a reduction in heme levels and an accumulation of porphyrin precursors. This disruption was highly specific, with the expression of the closely related ZmGSTU2 or other maize GSTs having little effect. Expression in E. coli of a series of chimeric ZmGSTU1/ZmGSTU2 proteins identified domains responsible for disrupting porphyrin metabolism. In addition to known heme precursors, expression of ZmGSTU1 led to the accumulation of a novel glutathione conjugate of harderoporphyrin(ogen) (2,7,12,18-tetramethyl-3-vinylporphyrin-8,13,17-tripropionic acid). Using the related protoporphyrinogen as a substrate, conjugation could be shown to occur on one vinyl group and was actively catalyzed by the ZmGSTU. In plant transgenesis studies, the ZmGSTUs did not perturb porphyrin metabolism when expressed in the cytosol of Arabidopsis or tobacco. However, expression of a ZmGSTU1-ZmGSTU2 chimera in the chloroplasts of tobacco resulted in the accumulation of the harderoporphyrin(ogen)-glutathione conjugate observed in the expression studies in bacteria. Our results show that the well known ability of GSTs to act as ligand binding (ligandin) proteins of porphyrins in vitro results in highly specific interactions with porphyrinogen intermediates, which can be demonstrated in both plants and bacteria in vivo.

Formation of superoxide anion on aerial oxidation of Cu(II)-porphyrinogen in the synthesis of tetrakis(cyclohexyl)porphyrinogenCu(III) anion.

Tetraethylammonium-tetrakis(cyclohexyl)porphyrinogenCu(II) (1) is spontaneously oxidized by aerial oxygen to the corresponding Cu(III) (2) species, producing 1 equiv of O2(-). Steric crowding of the peripheral hydrogens in 1 prevented any direct Cu-O2 bond formation in the oxidation process, which suggests an outer-sphere electron transfer reaction.

Dioxadiazuliporphyrin: a near-IR redox switchable chromophore.

The synthesis of dioxadiazuliporphyrinogen 7 and its oxidized forms: dioxadiazuliporphyrin 8 and dication 8(2+), is reported. These compounds were characterized in solution using UV-vis and 1H and 13C NMR spectroscopic means and in the solid state via single-crystal X-ray diffraction analysis. Dioxadiazuliporphyrin is a nonaromatic porphyrinoid, readily and reversibly oxidizable to its cation radical and to the aromatic carbaporphyrinoid dication, which can be viewed as a 21,23-dicarba-22,24-dioxaporphyrin with two fused tropylium rings. Further insight into the geometric and magnetic manifestations of aromaticity and antiaromaticity in the case of the redox couple 8, 8(2+) is obtained using density functional calculations and nucleus-independent chemical shifts.

Supramolecular triad and pentad composed of zinc-porphyrin(s), oxoporphyrinogen, and fullerene(s): design and electron-transfer studies.

By adopting a "covalent-coordinate" bonding approach, novel supramolecular pentad and triad molecules composed of zinc-porphyrin(s), fullerene(s), and oxoporphyrinogen redox-/photoactive entities have been constructed, and also characterized by means of spectral and electrochemical techniques. The geometry and electronic structures of the pentad and the triad were deduced by means of DFT calculations. Free-energy calculations suggested that the photoinduced electron/energy transfer from the zinc-porphyrin (ZnP) singlet-excited state to the imidazole modified fullerene (ImC(60)) acceptor and oxoporphyrinogen (OxP) entities is feasible for both the triad and the pentad. The charge-separation rates (k(CS)) determined from picosecond time-resolved emission studies were higher for pentad (C(60)Im:ZnP)(2)-OxP than for the corresponding triad, C(60)Im:ZnP-OxP. A comparison of the k(CS) values previously reported for the covalently linked bis(zinc-porphyrin)-oxoporphyrinogen triad suggests that employing a fullerene acceptor improves the electron-transfer rates. Nanosecond transient absorption studies provide evidence for the occurrence of electron-transfer processes. Lifetimes of the radical ion pairs (tau(RIP)) are in the range of hundreds of nanoseconds, which indicates that there is charge stabilization in the supramolecular systems.

Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase.

Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher K(m) for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.

Mechanical properties of calcium sulphate/hydroxyapatite cement.

Setting times, volume after setting, injectability and hardness (at 37 degrees C in contact with Ringer's solution) were determined for cements made of mixtures of calcium sulphate hemihydrate (CS) and hydroxyapatite (HA) with a range of compositions. The purpose of these experiments was to determine the behaviour of a mixture that could be used as an injectable cement for orthopaedic applications, including spinal fusion. A suitable mixture consisted of 60% CS and 40% HA by mass; a slurry was made by mixing solid (36 g) with water (15 cm(3)). The slurry had initial and final setting times of 5.7+/-1.3 min and 19.6+/-0.7 min (mean +/- standard deviation), respectively. The hardness of the cement did not systematically increase or decrease in the 72 h following the final setting time. The volume of the cement was 99.8+/-0.4% of the volume of the initial slurry, i.e. there was negligible shrinkage on setting. It was able to withstand a pressure of 7.3+/-1.2 MPa, applied by a hemispherical indenter before the onset of permanent damage, indicating adequate strength for spinal fusion.

Synthesis of substrate analogs of methyltransferases in the vitamin B(12) biosynthetic pathway and characterization of their enzymatic products.

The specificity toward substrate analogs of the first two methyltransferases in the vitamin B(12) biosynthetic pathway was probed with 15 synthetic porphyrinogens. Several novel methylated chlorins and isobacteriochlorins were isolated and characterized, suggesting the same methylation sequence C-2>C-7>C-20 as for the natural substrate, uro'gen III. The results allow us to narrow down possible structural requirements concerning substrate recognition by the methyltransferase enzymes.

Direct incorporation of a ferric ion in the porphyrinogen core: tetrakis(cyclohexyl)iron porphyrinogen anion with different conformers and its reaction with iodine.

Et(4)N[L' 'Fe(III)].3DCM (1) is directly synthesized by adding ferric chloride into a solution of a lithium salt of tetrakis(cyclohexyl)porphyrinogen (L' '). [L' '](4-) is a good chelating ligand for both Fe(III) and Fe(II) ions. It is an avid proton scavenger but not a reducing agent. 1 showed a magnetic moment (mu(eff)) of 4.3 micro(B) in the solid, which changed to 6.0 micro(B) in solution. This change in spin state is common for all iron porphrinogens. 1 showed polymorphism, and with pyridine in the lattice, it changed to Et(4)N[L' 'Fe(III)].DCM(0.5)Py(1.5) (2), possessing two different conformers. Calculation of these conformers at the density functional theory level showed the relative energies of all d orbital changes in three conformers, highlighting the influence of the disposition of a peripheral ligand. Iodine oxidation of 1 yielded [L' '(DeltaDelta)Fe(II)I][I(3).I(2)(+).I(3)(-)] (3) with the introduction of two C(alpha)-C(alpha) bonds with concomitant reduction of Fe(III) to Fe(II). Its mu(eff) (5.4 mu(B)) in the solid changed to 4.8 micro(B) in solution, suggesting a high spin state (S = 2) for Fe(II).

Metabolism of pentacarboxylate porphyrinogens by highly purified human coproporphyrinogen oxidase: further evidence for the existence of an abnormal pathway for heme biosynthesis.

An abnormal series of porphyrin tetracarboxylic acids known as the isocoproporphyrins, are commonly excreted by patients suffering from the disease porphyria cutanea tarda (PCT). These porphyrins appear to arise by bacterial degradation of dehydroisocoproporphyrinogen that is generated by the premature metabolism of the normal pentacarboxylate intermediate (5dab) by coproporphyrinogen oxidase (copro'gen oxidase). This porphyrinogen can be further metabolized by uroporphyrinogen decarboxylase to give harderoporphyrinogen, one of the usual intermediates in heme biosynthesis. Therefore, it is possible that some of the heme formed under abnormal conditions may originate from the 'isocopro-type' porphyrinogen intermediate. In order to investigate the feasibility of alternative pathways for heme biosynthesis, the four type III pentacarboxylate isomeric porphyrinogens were incubated with purified, cloned human copro'gen oxidase at 37 degrees C with various substrate concentrations under initial velocity conditions. Of the four isomers, only 5dab was a substrate for copro'gen oxidase and this gave dehydroisocoproporphyrin. The structure of the related porphyrin tetramethyl ester was confirmed by proton NMR spectroscopy and mass spectrometry. The K(m) value for proto'gen-IX formation from copro'gen, an indicator of molecular recognition, was similar to the K(m) value for monovinyl product formation with 5dab, although copro'gen-III has an approximately twofold higher K(cat) value. Although 5dab is a slightly poorer substrate than copro'gen-III, these results support the hypothesis that an abnormal route for heme biosynthesis is possible in humans suffering from PCT or related syndromes such as hexachlorobenzene poisoning.