Porphyrin Methyl Esters Research Articles

MTHPP monoacetic ester: unexpected formation, zinc metalation, thermal and photophysical properties

In the current investigation we report an unexpected methyl esterification occurred during the coupling reaction of mTHPP monoacetic acid 2 with 3-amino-1,2,4-triazole in the presence of HBTU/DIPEA. The mechanism of this unexpected methyl esterification was studied, and the structure of the formed methyl ester 5 was confirmed by the means of 1H, 13C NMR in addition to (MALDI-TOF and ESI-HRMS) spectrometry. The formation of 5 during the coupling reaction was also chemically supported by an alternative synthetic method involving a direct monosubstitution reaction of mTHPP 1 with methyl bromoacetate. We also investigated the metalation of 5 with zinc and studied the thermal properties along with differential scanning calorimetry (DSC) of the zinc porphyrin 6. The photophysical properties of porphyrin methyl ester 5 and its zinc complex 6 were also investigated.Graphical

Normal and abnormal heme biosynthesis. Part 7. Synthesis and metabolism of coproporphyrinogen-III analogues with acetate or butyrate side chains on rings C and D. Development of a modified model for the active site of coproporphyrinogen oxidase

Analogues of coproporphyrinogen-III have been prepared with acetate or butyrate groups attached to the C and D pyrrolic subunits. The corresponding porphyrin methyl esters were synthesized by first generating a,c-biladienes by reacting a dipyrrylmethane with pyrrole aldehydes in the presence of HBr. Cyclization with copper(II) chloride in DMF, followed by demetalation with 15% H2SO4–TFA and reesterification, gave the required porphyrins in excellent yields. Hydrolysis with 25% hydrochloric acid and reduction with sodium-amalgam gave novel diacetate and dibutyrate porphyrinogens 9. Diacetate 9a was incubated with chicken red cell hemolysates (CRH), but gave complex results due to the combined action of two of the enzymes present in these preparations. Separation of uroporphyrinogen decarboxylase (URO-D) from coproporphyrinogen oxidase (CPO) allowed the effects of both enzymes on the diacetate substrate to be assessed. Porphyrinogen 9a proved to be a relatively poor substrate for CPO compared to the natural substrate coproporphyrinogen-III, and only the A ring propionate moiety was processed to a significant extent. Similar results were obtained for incubations of 9a with purified human recombinant CPO. Diacetate 9a was also a substrate for URO-D and a porphyrinogen monoacetate was the major product in this case; however, some conversion of a second acetate unit was also evident. The dibutyrate porphyrinogen 9b was only recognized by the enzyme CPO, but proved to be a modest substrate for incubations with CRH. However, 9b was an excellent substrate for purified human recombinant CPO. The major product for these incubations was a monovinylporphyrinogen, but some divinyl product was also generated in incubations using purified recombinant human CPO. The incubation products were converted into the corresponding porphyrin methyl esters, and these were characterized by proton NMR spectroscopy and mass spectrometry. The results extend our understanding of substrate recognition and catalysis for this intriguing enzyme and have allowed us to extend the active site model for CPO. In addition, the competitive action of both URO-D and CPO on the same diacetate porphyrinogen substrate provides additional perspectives on the potential existence of abnormal pathways for heme biosynthesis.

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.

Investigation of solvent effects in capillary electrophoresis for the separation of biological porphyrin methyl esters

The effects of organic solvents on the capillary electrophoresis (CE) separation of a number of important biological porphyrin methyl esters - six weakly basic, hydrophobic cyclic tetrapyrroles possessing two and four to eight methyl ester groups around the periphery of the porphyrin ring - were investigated in the mode of micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic chromatography (MEEKC), and nonaqueous CE. In aqueous MEKC, partial separation of the six neutral porphyrin methyl esters was obtained with an organic modifier (acetonitrile) in the concentration range between 20 and 40%, in which sodium dodecyl sulfate (SDS) molecules might be present in the form of SDS micelles and/or SDS micelle-like aggregates. Relatively stable SDS micelles can be formed in nonaqueous MEKC using formamide as the separation medium, but the separation of the target analytes remained unsatisfactory. Improved resolution of all six porphyrin methyl esters was obtained using MEEKC with the running buffer consisting of 0.8% w/w n-heptane (oil phase), 2.25% w/w SDS and 1.0% w/w Brij 35 (mixed surfactant), 6.6% w/w 1-butanol (cosurfactant), and 30% v/v 2-propanol (second cosurfactant), but reproducibility in terms of peak areas for certain porphyrins (especially uroporphyrin I octamethyl ester) was found to be very poor. Best separation performances were achieved with nonaqueous CE separations in which the weakly basic porphyrin methyl esters were protonated under strongly acidic conditions (e.g., using 10 mM perchloric acid) in mixed organic solvents. For example, using a 50:50 mixture of methanol and acetonitrile as the separation medium, baseline separation of all six (positively charged) porphyrin methyl esters can be obtained within 3 min and the average precision (RSD, N = 13) in terms of migration time and peak area were 0.55 and 2.16%, respectively.

Normal and Abnormal Heme Biosynthesis. 3.1 Synthesis and Metabolism of Tripropionate Analogues of Coproporphyrinogen-III: Novel Probes for the Active Site of Coproporphyrinogen Oxidase

Coproporphyrinogen oxidase (copro'gen oxidase) catalyses the oxidative decarboxylation of two propionate side chains on coproporphyrinogen-III to produce protoporphyrinogen-IX. This process is very poorly understood at a molecular level, and copro'gen oxidase remains one of the least well-characterized enzymes in the heme biosynthetic pathway. To provide a rigorous test for a proposed model for substrate recognition and binding by this enzyme, two tripropionate analogues of copro'gen-III were prepared where an ethyl group replaced one of the usual propionate residues on positions 13 or 17. Although the required substrate probes are porphyrinogens (hexahydroporphyrins), the corresponding porphyrin methyl esters were initially synthesized via tripyrrene and a,c-biladiene intermediates. These were hydrolyzed and reduced with 3% sodium-amalgam to give the unstable porphyrinogens needed for the biochemical investigations. The modified structure with a 13-ethyl moiety was metabolized by avian preparations of copro'gen oxidase to give a monovinylic product, but the isomeric 17-ethylporphyrinogen afforded a divinylic product, albeit with poorer overall conversion. These results strongly support the proposed model for substrate binding at the active site of copro'gen oxidase.

Normal and Abnormal Heme Biosynthesis. 1. Synthesis and Metabolism of Di- and Monocarboxylic Porphyrinogens Related to Coproporphyrinogen-III and Harderoporphyrinogen: A Model for the Active Site of Coproporphyrinogen Oxidase

Coproporphyrinogen oxidase (copro'gen oxidase), which catalyses the conversion of coproporphyrinogen-III via a monovinylic intermediate to protoporphyrinogen-IX, is one of the least well understood enzymes in the heme biosynthetic pathway. To develop a model for the substrate recognition and binding recognition for this enzyme, a series of substrate analogues were prepared with two alkyl substituents on positions 13 and 17 in place of the usual propionate residues. Although the required substrate probes are porphyrinogens (hexahydroporphyrins), the corresponding porphyrin methyl esters were initialy synthesized via a,c-biladiene intermediates. These were hydrolyzed and reduced with 3% sodium amalgam to give the unstable porphyrinogens needed for the biochemical investigations. These modified structures were metabolized by avian preparations of copro'gen oxidase to give monovinylic products, but the second propionate residue was not further metabolized. In three cases, the metabolites were isolated and further characterized by proton NMR spectroscopy and mass spectrometry. When methyl or ethyl groups were placed at the 13 and 17 positions, the resulting porphyrinogens were very good substrates (although the ethyl version, mesoporphyrinogen-VI, gave slightly better results), but when propyl units were introduced metabolism was significantly inhibited and the butyl-substituted structure was only slightly transformed after long incubation periods. These results suggest the presence of an active-site lipophobic region near the catalytic site for copro'gen oxidase. The observation that the related 3-vinyl- and 3-ethylporphyrinogens with 13,17-diethyl substituents were not substrates for this enzyme confirmed the need for a second propionate residue to hold the substrate in place at the catalytic site.

Preparation and separation of hydroxy derivatives of uroporphyrinogen I by high-performance liquid chromatography with electrochemical detection

The preparation and high-performance liquid chromatography (HPLC) separation of meso-hydroxyuroporphyrinogen I, hydroxyacetic acid uroporphyrinogen I and β-hydroxypropionic acid uroporphyrinogen I is described. meso-Hydroxyuroporphyrin I, hydroxyacetic acid uroporphyrin I and β-hydroxypropionic acid uroporphyrin I were isolated from the urine of a patient with congenital erythropoietic porphyria. The porphyrins were reduced to the corresponding porphyrinogens with 3% (w/w) Na/Hg amalgam. The hydroxy porphyrinogens were separated on a Hypersil ODS column with 4% (v/v) acetonitrile in 1 M ammonium acetate buffer, pH 5.16, containing EDTA (0.27 m M) as the mobile phase, and detected electrochemically. Reduction of meso-hydroxyuroporphyrin I and hydroxyacetic acid uroporphyrin I, followed by HPLC analysis, showed that, in addition to the expected formation of meso-hydroxyuroporphyrinogen I and hydroxyacetic uroporphyrinogen I, respectively, uroporphyrinogen I was also produced. Reduction of β-hydroxypropionic acid uroporphyrin I, however, gave β-hydroxypropionic acid uroporphyrinogen I, acrylic acid uroporphyrinogen I and uroporphyrinogen I as the products. The peaks were identified by conversion into the porphyrin methyl esters and analysed by liquid secondary-ion mass spectrometry.

Urinary porphyrin profiles by laser desorption/ionization time‐of‐flight mass spectrometry without the use of classical matrices

A method is described for the rapid analysis of mixtures of porphyrin methyl esters using laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS). Ions corresponding to [M+H]+ for each component have been observed without the need for added matrix. LDI-TOF MS has been applied to the analysis and characterization of porphyrin methyl esters derived from urine samples of patients suffering from various porphyrias. This technique has been shown to provide "fingerprint" profiles of the porphyrins present in each urine sample, characteristic of the porphyria, which compare favourably with those obtained with the established method of high-performance liquid chromatography. LDI-TOF MS is a rapid and reliable method for the screening of urine from patients suffering from porphyria.

Laser fluorometric detection of porphyrin methyl esters for high-performance thin-layer chromatography

A new detection method is presented for the determination of porphyrins present in biological materials. Separation is accomplished by high-performance thin-layer chromatography after esterification of individual carboxylic acid porphyrins. Detection is achieved by utilizing one of the visible lines of an argon-ion laser for fluorometric excitation. Good selectivity and detectability are demonstrated for the determination of porphyrin profiles in human urine. The detection limits for uro-, heptacarboxy-, hexacarboxy-, pentacarboxy-, copro-, and mesoporphyrin methyl esters are in the 18-35 pg range.

Structural Characterization of Nitrimyoglobin

Nitrimyoglobin was formed in greater than 94% yield by a simple reaction between excess nitrite and horse heart metmyoglobin at pH 5.5. This dark green pigment was shown by 1H NMR spectroscopy to be a single, pure product with a well defined tertiary structure that is highly similar to the starting myoglobin. Electronic spin states parallel those of myoglobin, although the relaxation times differ. Ligand binding reactions of nitrimyoglobin parallel those of normal myoglobin, but lead to a unique series of UV-visible spectra. In the ferrous state, nitrimyoglobin reversibly binds O2 with half-saturation of sites at an O2 partial pressure of 10.4 +/- 1.4 mm Hg. 1H NMR data indicate that the altered heme of nitrimyoglobin has not undergone reaction at any meso proton position, nor has it been partially saturated to the level of a chlorin. 15N NMR spectra indicate that only a single nitrogen was added to the protein as a nitro group. Extraction of the modified heme from nitrimyoglobin and spectroscopic characterization of the nitriheme by infrared spectroscopy and of the free base porphyrin methyl ester derived from nitriheme by 1H NMR indicate that the modification is regiospecific. The heme in nitrimyoglobin is 3-(trans-2-nitrovinyl)-2,7,12,18-tetramethyl-8-vinylporphyrin-13,1 7-dipropionic acid. In the Fisher nomenclature scheme, the 2-vinyl substituent is the site of modification and has been converted to a nitrovinyl group by substitution of a proton by -NO2.

Solid phase extraction and isocratic separation of urinary porphyrins by HPLC.

A method for determining urine porphyrins by HPLC is described. In the preliminary step, porphyrins are purified in high yields and concentrated by low pressure reverse-phase chromatography on C18 (octadecylsilane bonded silica) cartridge. Porphyrins are stable for 10 days after adsorption on C18 cartridge. The separation of porphyrin esters is performed on an aminopropyl-bonded silica column with an eluting system containing n-heptane and ethyl acetate. The system enables rapid isocratic separation of porphyrin methyl esters with high selectivity. The simplicity and reproducibility of the whole procedure allows its application to the routine analysis of urinary porphyrins in the clinical laboratory.

40] High-performance liquid chromatography of porphyrin methyl esters

Publisher Summary This chapter discusses the high-performance liquid chromatography (HPLC) of porphyrin methyl esters. Separation of porphyrin esters by HPLC is performed on silica columns using mixtures of organic solvents. The porphyrins elute from the column with increasing polarity. Chromatographic systems are therefore designed and can be modified predictably for highly specific applications. Samples of dried porphyrin esters are dissolved completely in a known volume of CHCl 3 . A measured portion is injected onto the column and chromatographed. The volume of CHCl 3 used for dissolving the sample must be empirically determined and depends on porphyrin concentrations, the type of injector used, the column dimensions and resolution capabilities, and the type of detector used. The amount of porphyrin ester represented by a chromatographic peak is directly proportional to both the area under the peak and the peak height over a broad range, using either fluorescence or absorbance detection. Reliable separation and quantification of porphyrin esters require rigorous control of chromatographic conditions. For this reason, the following should always be observed: (1) a standard mixture of porphyrin esters should be chromatographed at least once a day to monitor instrument and column performance and to adjust chromatographic conditions if necessary, (2) all solvents should be HPLC grade or better. Solvent mixtures should be thoroughly mixed and then filtered and degassed by vacuum prior to use, (3) all samples should be rendered free of particulate material which could block the column by filtration through 0.45- μ m filters. Regenerated cellulose or PTFE filters are most effective. After filtration, the samples should be blown dry, then redissolved in a known volume of filtered CHCl 3 (stabilized with 1-hexene) prior to chromatography, and (4) system backpressure should be routinely monitored.

Rapid procedure for fecal porphyrin assay.

Hydrochloric acid extraction of feces in the presence of ether yields an extract suitable for spectrophotometric estimation of total porphyrin and for further separation by "high-performance" liquid chromatography (HPLC) or thin-layer chromatography. A total porphyrin reference interval of less than 200 nmol/g dry weight of feces was established from data on 106 normal subjects on an unrestricted diet. Total fecal porphyrin values in 11 porphyria cutanea tarda patients were considerably higher than given by the widely used Rimington method (respective means, 652 and 239 nmol/g dry weight). Our HPLC method for separation of porphyrin methyl esters on a silica column, with quantification by fluorescence, is described. HPLC separations performed on 23 porphyria cutanea tarda patients gave the following mean proportions of total fecal porphyrins: dicarboxylics 21%, coproporphyrin 9%, isocoproporphyrins 28%, pentacarboxylporphyrin 9%, hexacarboxylporphyrin 11%, heptacarboxylporphyrin 18%, and uroporphyrin 4%.

H.p.l.c. analysis of di- and tri-carboxylic porphyrins in porphyric patients.

New h.p.l.c. methods have been developed for the quantitative determination of di- and tri-carboxylic porphyrin methyl esters, and applied to the analysis of faecal extracts from patients with four different types of porphyria.

High-performance liquid chromatographic analysis of porphyrins and their isomers with radial compression columns

Porphyrin methyl esters and the isomers of uroporphyrin and heptacarboxylic porphyrin were separated by high-performance liquid chromatography. Isocoproporphyrin was also separated from coproporphyrin. By slight modifications to the solvent mixture, the separation of all biological polycarboxylic porphyrins was achieved. These separations were made possible through the high efficiency of 10- or 5-μm particle-size Radial-PAK cartridges, which have been used in the separation of porphyrins in various excreta and tissues in a number of porphyrias.

Dealkylation during electron impact mass spectrometric analysis ofN- monoalkylated porphyrin methyl esters

The N-monoethylated, N-monoethylated and N-mono-1-propylated homologues of mesoporphyrin IX dimethyl ester and the corresponding methylated and 1-propylated homologues of coproporphyrin III tetramethyl ester have been prepared. Substituted porphyrins in a series could be distinguished from each other and from the parent porphyrin by their dication absorption spectra. All compounds gave molecular ions of varying intensities during electron impact mass spectrometry but thermal N-dealkylation to mesoporphyrin IX or coproporphyrin III methyl esters occurred readily and it was difficult to obtain spectra not showing fragmentation ions derived from this process. The compounds serve as models for the ‘green pigments’ produced in vivo by the action of some drugs and illustrate the problem of dealkylation during their analysis by electron impact mass spectrometry.

High-performance liquid chromatography of porphyrin esters: Identification of mixed esters generated in sample preparation

Uroporphyrin and its naturally occurring decarboxylated derivatives can be conveniently separated, identified, and quantified chromatographically after formation of their methyl esters. Use of high-performance liquid chromatography has revealed the presence of previously unobserved components in such mixtures. One of these components was isolated and identified as uroporphyrin heptamethyl monoethyl ester. The family of uroporphyrin methylethyl esters was synthesized and its chromatographic behavior investigated. The quantity of ethanol present in solvents, e.g., chloroform, which are used during the standard preparation of porphyrin methyl esters is sufficient for the synthesis of significant amounts of methyl-ethyl esters. The presence of methyl-ethyl esters can lead to errors in chromatographic purification, identification, and/or quantification of components in mixtures of porphyrins.

Second derivative-high-performance liquid chromatographic-fluorometric detection of porphyrins in chick embryo liver cell culture medium

A high-performance liquid chromatographic system is described which is suitable for the separation and quantitative determination of a mixture of porphyrin esters in nanogram quantities from the culture medium used for maintenance of a monolayer culture of chick embryo liver cells. The desired sensitivity was obtained by coupling a high-performance liquid chromatograph with a second derivative-fluorometric detection system. The most effective method for preparation of the porphyrin methyl esters prior to chromatography was found to be lyophilization of the culture medium prior to esterification with 5% sulphuric acid in methanol.

Preparation of porphyrin methyl esters for high pressure liquid chromatography

High pressure liquid chromatography has distinct advantages over thin-layer chromatography for the rapid separation and quantitative determination of porphyrin methyl esters [1–3]. Our initial experience with the talc method of. extracting porphyrins from urine [3] was disappointing, poor recoveries being the rule rather than the exception. We therefore have developed new techniques for extracting and methylating porphyrins from biological samples, prior to HPLC.

Quantitation of red cell porphyrins by fluorescence scanning after thin-layer chromatography

This paper reports a new method for the quantitative determination of erythrocyte and plasma porphyrins by fluoroscanning of the methyl esters separated by thin-layer chromatography (TLC). After esterification overnight in the dark the polycarboxylated porphyrin methyl esters were extracted into chloroform and aliquots applied to the TLC plates within the range of quantities shown in a preliminary study to be directly proportional to fluorescence intensity. The accuracy and reliability of the technique was tested by comparison of RBC protoporphyrin values obtained by TLC with an established quantitative porphyrin solvent extraction method and with a rapid method presently in common use. Good correlation was demonstrated between the solvent extraction and the TLC techniques. The high sensitivity and adaptability of the TLC technique and its ability to clearly separate all the porphyrins present in the samples are discussed, along with the fluorescence mechanisms involved and the effects of instrumentation.