Enhanced Synthetic Access to Tris-CF3-Substituted Corroles.

Separate focus on the oligomerization and oxidative cyclization steps required for the synthesis of 5,10,15-tris(trifluoromethyl)corrole revealed [bis(trifluoroacetoxy)iodo]benzene (PIFA) as a superior alternative oxidant. Under optimized conditions, the pure free-base corrole was obtained with a 6-fold increase in chemical yield and an 11-fold rise in isolated material per synthesis. The corresponding gallium(III) and manganese(III) complexes were isolated by adding the appropriate metal salt prior to corrole purification.

Tolyporphins L-R: Unusual Tetrapyrroles from a Brasilonema sp. of Cyanobacterium.

Tolyporphins L-R (2-8) have been isolated from a mixed cyanobacterium-microbial culture. The structures of tolyporphins L and M have been revised to four constitutional isomers, isolated as two mixtures of dioxobacteriochlorins (2/3 and 4/5). In contrast, tolyporphin P (6) is a fully oxidized tetrapyrrole, while tolyporphins Q and R (7 and 8) are oxochlorins. X-ray structures are reported for the first time for tolyporphins A (1), R (8), and E (9), revealing unexpected stereochemical variation within the series.

A Cytotoxic Porphyrin from North Pacific Brittle Star Ophiura sarsii.

Triple-negative breast cancer (TNBC) represents the deadliest form of gynecological tumors currently lacking targeted therapies. The ethanol extract of the North Pacific brittle star Ophiura sarsii presented promising anti-TNBC activities. After elimination of the inert material, the active extract was submitted to a bioguided isolation approach using high-resolution semipreparative HPLC-UV, resulting in one-step isolation of an unusual porphyrin derivative possessing strong cytotoxic activity. HRMS and 2D NMR resulted in the structure elucidation of the compound as (3S,4S)-14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid. Never identified before in Ophiuroidea, porphyrins have found broad applications as photosensitizers in the anticancer photodynamic therapy. The simple isolation of a cytotoxic porphyrin from an abundant brittle star species we describe here may pave the way for novel natural-based developments of targeted anti-cancer therapies.

Anti-biofilm, nitric oxide inhibition and wound healing potential of purpurin-18 phytyl ester isolated from Clinacanthus nutans leaves.

AIMS: Clinacanthus nutans (C. nutans) has demonstrated anti-inflammatory activity, however, the active compound generating this activity remains unknown. The aim of this study was to identify the bioactive compound in C. nutans responsible for its anti-inflammatory, in-vitro wound healing, and anti-biofilm activities. MAIN METHODS: A pure compound was isolated from the chloroform extract (CE) of C. nutans leaves by chromatographic techniques and bioassay-guided fractionation. This compound's structure was determined by spectroscopic analyses (FTIR/NMR/HRES-MS). Biological activities were evaluated using cytotoxicity, nitric oxide (NO), wound scratch, anti-microbial activity, and anti-biofilm assays; and the compound's bactericidal depth into the biofilm was visualized by confocal laser scanning microscopy. KEY FINDINGS: CE and its pure isolated compound, purpurin-18 phytyl ester (P18PE), significantly inhibited lipopolysaccharide (LPS)-induced NO production in RAW 264.7 cells at concentrations of 100 µg/ml and 10-100 µg/ml, respectively. These concentrations significantly induced wound closure by human gingival fibroblasts. CE (100-1000µg/ml) and P18PE (1-500 µg/ml) did not inhibit Streptococcus (S.) mutans growth. However, these concentrations significantly reduced S. mutans biofilm formation below 50% at 250 µg/ml for CE, and 25 µg/ml for P18PE (p<0.05). SIGNIFICANCE: C. nutans contains a bioactive compound, P18PE, which exhibits anti-inflammatory, in-vitro wound healing, and anti-biofilm activities.

Photo-induced antibacterial activity of a porphyrin derivative isolated from the harmful dinoflagellate Heterocapsa circularisquama.

The dinoflagellate Heterocapsa circularisquama is highly toxic to bivalves. However, significant toxicity to finfish species has not been reported. We previously found that H. circularisquama has light-dependent haemolytic agents. Purification and chemical structural analyses revealed that the haemolytic agent H2-a is a porphyrin derivative, which exhibits light-dependent cytotoxicity toward tumour cells. To clarify the biological activity of H2-a further, its antibacterial activities against Gram-positive and Gram-negative bacteria were investigated in this study. A fraction (F5) equivalent to H2-a purified from the methanol extract of H. circularisquama showed potent light-dependent bactericidal activity toward Staphylococcus aureus, and the activity was concentration- and light illumination time-dependent; however, Escherichia coli was highly resistant to F5. Electron microscopic observation suggested that F5 induces morphological changes in S. aureus in a light-dependent manner. Further analysis using other bacterial species showed that the Gram-positive bacterium Bacillus subtilis was more sensitive than the Gram-negative bacteria Pseudomonas aeruginosa and Vibrio alginolyticus. These results indicate that F5 is a photo-induced antibacterial agent with relatively higher specificity to Gram-positive bacteria. Iodometric assay suggested that singlet oxygen was generated from light-illuminated F5. Histidine, a specific singlet oxygen scavenger, markedly inhibited the photosensitising antibacterial activity of F5 against S. aureus, suggesting the involvement of singlet oxygen in antibacterial activity. The antibacterial spectrum of F5 was evidently different from that of 5,10,15,20-tetra (N,N,N-trimethylanilinium) porphyrin tetratosylate, a commercially available porphyrin compound with antibacterial activity. Our results demonstrate that H. circularisquama has a novel antibacterial photosensitiser, a porphyrin derivative, with relatively higher specificity to Gram-positive bacteria. To the best of our knowledge, this is the first study to discover a porphyrin derivative with antibacterial activity in marine microalga.

Single-molecule porphyrin-metal ion interaction and sensing application.

It remains a significant challenge to study the interactions between metal ions and porphyrin molecules at single ion level. Here, we constructed a nanopore-based sensing for label-free and real-time analysis of the interaction between Cu2+ and 5,10,15,20-tetrakis(4-sulfonatophenyl)-porphyrin (TPPS). The results demonstrate that emerging electronic signatures of the Cu2+-TPPS complex that is completely different form the original free TPPS were observed in the α-hemolysin (α-HL) nanopore. Based on the distinctive electronic signal patterns between TPPS and Cu2+-TPPS complex, the unique nanopore sensor can achieve a highly sensitive detection of Cu2+ in aqueous media. The frequency of signature events showed a linear response toward the concentration of Cu2+ in the range of 0.03 µM - 1.0 µM, with a detection limit of 16 nM (S/N = 3). The sensing system also exhibited high selectivity against other metal ions, and the feasibility of this approach for practical applications was demonstrated with the determination of Cu2+ in running water.

Compositional analysis of endogenous porphyrins from Helicobacter pylori.

Bacteria able to accumulate porphyrins can be inactivated by visible light irradiation thanks to the photosensitizing properties of this class of aromatic pigments (photodynamic therapy, PDT). Since the bacterial resistance to antibiotic is growing, PDT is becoming a valid alternative. In this context, the pathogen Helicobacter pylori (Hp) is a suitable target for PDT since it spontaneously produces and accumulates porphyrins. It is then important to understand the spectroscopic behavior of these endogenous species to exploit them as photosensitizers, thus improving the results given by the application of PDT in the treatment of Hp infections. In this work we extracted porphyrins from both a laboratory-adapted and a virulent strain of Hp, and we performed spectroscopic and chromatographic experiments to collect information about the composition and the spectrophotometric features of the extracts. The main components of the porphyrin mixtures were identified and their relative contribution to the global red fluorescence was examined.

Tapping the biotechnological potential of insect microbial symbionts: new insecticidal porphyrins.

BACKGROUND: The demand for sustainable agricultural practices and the limited progress toward newer and safer chemicals for use in pest control maintain the impetus for research and identification of new natural molecules. Natural molecules are preferable to synthetic organic molecules because they are biodegradable, have low toxicity, are often selective and can be applied at low concentrations. Microbes are one source of natural insecticides, and microbial insect symbionts have attracted attention as a source of new bioactive molecules because these microbes are exposed to various selection pressures in their association with insects. Analytical techniques must be used to isolate and characterize new compounds, and sensitive analytical tools such as mass spectrometry and high-resolution chromatography are required to identify the least-abundant molecules. RESULTS: We used classical fermentation techniques combined with tandem mass spectrometry to prospect for insecticidal substances produced by the ant symbiont Streptomyces caniferus. Crude extracts from this bacterium showed low biological activity (less than 10% mortality) against the larval stage of the fall armyworm Spodoptera frugiperda. Because of the complexity of the crude extract, we used fractionation-guided bioassays to investigate if the low toxicity was related to the relative abundance of the active molecule, leading to the isolation of porphyrins as active molecules. Porphyrins are a class of photoactive molecules with a broad range of bioactivity, including insecticidal. The active fraction, containing a mixture of porphyrins, induced up to 100% larval mortality (LD50 = 37.7 µg.cm-2). Tandem mass-spectrometry analyses provided structural information for two new porphyrin structures. Data on the availability of porphyrins in 67 other crude extracts of ant ectosymbionts were also obtained with ion-monitoring experiments. CONCLUSIONS: Insect-associated bacterial symbionts are a rich source of bioactive compounds. Exploring microbial diversity through mass-spectrometry analyses is a useful approach for isolating and identifying new compounds. Our results showed high insecticidal activity of porphyrin compounds. Applications of different experiments in mass spectrometry allowed the characterization of two new porphyrins.

Isolation of Monoterpene Dihydrochalcones from Piper montealegreanum Yuncker (Piperaceae).

Four new compounds were isolated from the branches of Piper montealegreanum Yuncker, a shrub found in the Amazon rainforest, including two new dihydrochalcones named claricine (1) and maisine (2), a cinnamic acid derivative 3 and a phenylalkanoid 4, along with a porphyrin identified as the known compound phaeophytin a (5). The structures were established using spectroscopic experiments, including 1D and 2D NMR and HRESIMS experiments, performed on the two monoterpene dihydrochalcones and their monoacetyl derivatives. The structural diversity of these substances is very important for the Piper genus chemotaxonomy.

Quantification of a Novel Photosensitizer of Chlorin e6-C15-Monomethyl Ester in Beagle Dog Plasma Using HPLC: Application to Pharmacokinetic Studies.

Chlorin e6-C15-monomethyl ester (CMME) is a novel photosensitizer, which is synthetized from the degradation products of silkworm excrement. Preclinical studies on the promising photosensitizer CMME are necessary to determine its therapeutic efficacy and druglikeness. A high-performance liquid chromatography with UV detection (HPLC-UV) method was established for the determination of CMME in beagle dog plasma. The sample preparation involved a protein-precipitation method with acetonitrile after the addition of tanshinone IIA as an internal standard (IS). CMME and the IS were separated on a Diamonsil C18 (2) column (100 mm × 4.6 mm, 5 µm) with a isocratic system of methanol-water containing 20 mM ammonium acetate with 0.3% glacial acetic acid (85:15, v/v). The flow rate was 1.0 mL/min with UV detection using a wavelength of 400 nm. The method was sensitive enough with a lower limit of quantitation (LLOQ) of 0.05 µg/mL and had a good linearity (r² > 0.999) over the linear range of 0.05-5.00 µg/mL. The intra-day and inter-day accuracies ranged from 98.5% to 102.8% and precisions (RSD) were within 6.8%. The validated method was successfully applied to the pharmacokinetic study of CMME after intravenous administration of single and multiple doses in beagle dogs.

Community analysis of pigment patterns from 37 microalgae strains reveals new carotenoids and porphyrins characteristic of distinct strains and taxonomic groups.

Phytoplankton, with an estimated 30 000 to 1 000 000 species clustered in 12 phyla, presents a high taxonomic and ecophysiological diversity, reflected by the complex distribution of pigments among the different algal classes. High performance liquid chromatography is the gold standard method for qualitative and quantitative analysis of phytoplankton pigments in seawater and culture samples, but only a few pigments can be used as robust chemotaxonomic markers. A major challenge is thus to identify new ones, characteristic of a strain, species, class or taxon that cannot be currently identified on the basis of its pigment signature. Using an optimized extraction process coupled to a HPLC de-replication strategy, we examined the pigment composition of 37 microalgae strains, representative of the broad taxonomic diversity of marine and freshwater species (excluding cyanobacteria). For each species, the major pigments already described were unambiguously identified. We also observed the presence of several minor unidentified pigments in each chromatogram. The global analysis of pigment compositions revealed a total of 124 pigments, including 98 pigments or derivatives unidentified using the standards. Absorption spectra indicated that 35 corresponded to chlorophyll/porphyrin derivatives, 57 to carotenoids and six to derivatives having both spectral signatures. Sixty-one of these unidentified or new carotenoids and porphyrin derivatives were characteristic of particular strains or species, indicating their possible use as highly specific chemotaxonomic markers capable of identifying one strain out of the 37 selected. We developed a graphical analysis using Gephi software to give a clear representation of pigment communities among the various phytoplankton strains, and to reveal strain-characteristic and shared pigments. This made it possible to reconstruct the taxonomic evolution of microalgae classes, on the basis of the conservation, loss, and/or appearance of pigments.

Preclinical Study of Antineoplastic Sinoporphyrin Sodium-PDT via In Vitro and In Vivo Models.

Photodynamic therapy (PDT) investigations have seen stable increases and the development of new photosensitizers is a heated topic. Sinoporphyrin sodium is a new photosensitizer isolated from Photofrin. This article evaluated its anticancer effects by clonogenic assays, MTT assays and xenograft experiments in comparison to Photofrin. The clonogenicity inhibition rates of sinoporphyrin sodium-PDT towards four human cancer cell lines ranged from 85.5% to 94.2% at 0.5 µg/mL under 630 nm irradiation of 30 mW/cm² for 180 s. For MTT assays, the IC50 ranges of Photofrin-PDT and sinoporphyrin sodium-PDT towards human cancer cells were 0.3 µg/mL to 5.5 µg/mL and 0.1 µg/mL to 0.8 µg/mL under the same irradiation conditions, respectively. The IC50 values of Photofrin-PDT and sinoporphyrin sodium-PDT towards human skin cells, HaCaT, were 10 µg/mL and 1.0 µg/mL, respectively. Esophagus carcinoma and hepatoma xenograft models were established to evaluate the in vivo antineoplastic efficacy. A control group, Photofrin-PDT group (20 mg/kg) and sinoporphyrin sodium group at three doses, 0.5 mg/kg, 1 mg/kg and 2 mg/kg, were set. Mice were injected with photosensitizers 24 h before 60 J 630 nm laser irradiation. The tumor weight inhibition ratio of 2 mg/kg sinoporphyrin sodium-PDT reached approximately 90%. Besides, the tumor growths were significantly slowed down by 2 mg/kg sinoporphyrin sodium-PDT, which was equivalent to 20 mg/kg Photofrin-PDT. In sum, sinoporphyrin sodium-PDT showed great anticancer efficacy and with a smaller dose compared with Photofrin. Further investigations are warranted.

The main pigment of the dormant Mycobacterium smegmatis is porphyrin.

Upon transition of Mycobacterium smegmatis into the dormant state, accumulation of a dark brown fluorescent pigment was observed. This pigment gave bright red fluorescence in both cells and the culture medium. Based on 1H-NMR, MALDI and UV spectra, the fluorescent compounds, extracted from the culture medium as well as from the dormant cells, were concluded to be a mixture of free coproporphyrin III and uroporphyrin III and their corresponding methyl esters. A possible significance of porphyrin pigment accumulation in the dormant cells is discussed.

Mass-spectrometric profiling of porphyrins in complex biological samples with fundamental, toxicological, and pharmacological applications.

Rapid, high-throughput, and quantitative evaluations of biological metabolites in complex milieu are increasingly required for biochemical, toxicological, pharmacological, and environmental analyses. They are also essential for the development, testing, and improvement of new commercial chemical products. We demonstrate the application of ultra-high performance liquid chromatography-mass spectrometry (uHPLC-MS), employing an electrospray ionization source and a high accuracy quadrupole time-of-flight mass analyzer, for the identification and quantification of a series of porphyrin derivatives in liver: a matrix of particular relevance in toxicological or pharmacological testing. Exact mass is used to identify and quantify the metabolites. Chromatography enhances sensitivity and alleviates potential saturation issues by fanning out the contents of a complex sample before their injection into the spectrometer, but is not strictly necessary for the analysis. Extraction and sample treatment procedures are evaluated and matrix effects discussed. Using this method, the known mechanism of action of a well-characterized porphyrinogenic agent was verified in liver extracts from treated rats. The method was also validated for use with bacterial cells. This exact-mass method uses workhorse instruments available in many laboratories, providing a highly flexible alternative to existing HPLC- and MS/MS-based approaches for the simultaneous analysis of multiple compounds in biological media.

Chromatographic enrichment and subsequent separation of nickel and vanadyl porphyrins from natural seeps and molecular characterization by positive electrospray ionization FT-ICR mass spectrometry.

We report a novel chromatographic method to enrich and separate nickel and vanadyl porphyrins from a natural seep sample and combine molecular level characterization by positive-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Vanadyl and nickel porphyrin model compound elution from primary secondary amine (PSA) stationary phase combined with UV-vis spectroscopy confirms enrichment and subsequent fractionation of nickel and vanadyl porphyrins into polarity-based subfractions. A more than 100-fold increase in signal-to-noise ratio for nickel porphyrins enables unequivocal elemental composition assignment confirmed by isotopic fine structure for all isotopes >1% relative abundance, and the first mass spectral identification of (61)Ni porphyrin isotopologues derived from natural seeps. Oxygen-containing vanadyl porphyrins and sulfur-containing vanadyl porphyrins are isolated in the same fraction simultaneously from the same sample. We provide the first chromatographic evidence of carboxylic acid functionalities peripheral to the porphyrin core, in agreement with previous studies.

Pressure-assisted electrokinetic injection stacking for verteporfin drug to achieve highly sensitive enantioseparation and detection in artificial urine by capillary electrophoresis.

Pressure-assisted electrokinetic injection (PAEKI) was applied for negatively charged verteporfin (VER) overloading and inline stacking, which targeted highly sensitive enantioseparation by CE. The essential step of PAEKI is a constant pressure used to counterbalance the electroosmotic flow (EOF), consequently, the large amount of analyte could be permitted into capillary and concentrated at the motionless boundary of the sample zone and background electrolyte (BGE). Aiming to know the balance, the velocity of the whole BGE in capillary by the impetus of pressure (0.2-2.0psi), and the velocity of EOF depending on the length of sample plug and voltage (5.0-20kV) was investigated, respectively. The velocity of bulk flow in capillary has good linearity with the pressure or applied voltage. Through the pattern of EOF marked peak and analyte peaks (dissolved in pure water), the constant pressure (0.8psi) vs. the added voltage (-10.3kV) during PAEKI was confirmed to immobilize the bulk flow of BGE, thus the sample injection time could sustain 2.0min without compromising separation efficiency. The obtained LOD (S/N=3) of each isomer at UV detection (428nm) was around 10.3µg/L, which was improved to 116 and 39-fold in comparison with normal hydrodynamic injection (HDI) and electrokinetic injection (EKI). The LOD is far below the reported value with LIF detection of VER. The RSD (n=5) of migration time and peak area was, respectively, around 3.5% and 5.7% for the proposed PAEKI method. Finally, PAEKI was used for the detection of VER in artificial urine to investigate the matrix interference.

Hemoglobin-derived porphyrins preserved in a Middle Eocene blood-engorged mosquito.

Although hematophagy is found in ~14,000 species of extant insects, the fossil record of blood-feeding insects is extremely poor and largely confined to specimens identified as hematophagic based on their taxonomic affinities with extant hematophagic insects; direct evidence of hematophagy is limited to four insect fossils in which trypanosomes and the malarial protozoan Plasmodium have been found. Here, we describe a blood-engorged mosquito from the Middle Eocene Kishenehn Formation in Montana. This unique specimen provided the opportunity to ask whether or not hemoglobin, or biomolecules derived from hemoglobin, were preserved in the fossilized blood meal. The abdomen of the fossil mosquito was shown to contain very high levels of iron, and mass spectrometry data provided a convincing identification of porphyrin molecules derived from the oxygen-carrying heme moiety of hemoglobin. These data confirm the existence of taphonomic conditions conducive to the preservation of biomolecules through deep time and support previous reports of the existence of heme-derived porphyrins in terrestrial fossils.

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.

Indole-porphyrin hybrids produced by metagenomics.

New indole-porphyrin hybrid molecules were isolated from Escherichia coli harboring metagenomic DNA from the Japanese marine sponge Discodermia calyx. The indole was appended to the reactive vinyl substituent of the harderoporphyrin chromophore, encoded by the insert DNA. Thus, the chimeric pathway between the heterologously expressed porphyrins and the endogenous indole generated new indole-conjugated chiral porphyrins in E. coli.