protoporphyrin-IX Dimethyl Ester Research Articles

Investigation of protoporphyrin IX standard materials used in acid-extraction methods, and a proposed correction for the millimolar absorptivity of protoporphyrin IX.

Erythrocyte protoporphyrin (EP) has been used for more than 30 years as an indicator of lead intoxication, iron deficiency, and porphyrias. Recently, numerous analytical problems associated with various EP methods have been reported, including a lack of consensus among investigators regarding the best calibration material or analytical procedure. We investigated commercially available protoporphyrin IX (PPIX) standard materials and measured the millimolar absorptivity (m epsilon) of these materials, focusing on variables affecting the determination of their absorptivities. Among the five forms of PPIX available, PPIX dimethyl ester, when hydrolyzed to PPIX free acid, gave the most consistent and reproducible results. This work confirmed our earlier observations, made on more than 600 separate occasions during 12 years, that the m epsilon of PPIX free acid in 1.5 mol/L HCl at the Soret maximum is 297 +/- 1.3 L.mmol-1.cm-1, 19% higher than the arbitrary value of 241 L.mmol-1.cm-1 generally accepted by most investigators but based on unpublished data. We propose that the m epsilon of 297 L.mmol-1.cm-1 for PPIX be adopted and that PPIX dimethyl ester be used for the calibration of acid-extraction methods. A detailed protocol for the preparation and verification of PPIX from the dimethyl ester is available upon request.

Syntheses of methyl-devinylporphyrins related to protoporphyrin-IX. Initial studies on the mechanism of the copper (II) catalysed cyclization of 1′,8′-dimethyl-a,c-biladiene salts

Using copper (II) catalysed cyclization of a,c-biladiene dihydrobromide salts, porphyrins [(3)–(5)] related to protoporphyrin-IX dimethyl ester (8), but in which both vinyls are replaced with methyls, or where either the 2- or 4-vinyls are individually replaced with methyls, are synthesized. These compounds are required for reconstitution of the corresponding hemes into hemoproteins, to enable the study of the rotational disorder of prosthetic heme groups in reconstituted hemoproteins. By way of 13C n.m.r. spectroscopy of enriched a,c-biladienes and porphyrins, the copper(II) catalysed cyclization of 1′,8′-dimethyl-a,c-biladiene dihydrobromides is shown to afford porphyrins in which one of the 1′- and 8′-methyl groups becomes the new linking meso carbon atom.

Chromatographic Behavior of Metalloporphyrins by Cellulose Thin-layer Chromatography

Abstract Cellulose thin-layer chromatographic separation of chlorophyll a, b, and c, pheophytin a, b, and c, Zn(II) and Cu(II)-complexes of protoporphyrin-IX dimethyl ester (PPDE) was studied by using a light petroleum–acetone developing solvent system. The hRf values of metal–PPDE chelates were found to be virtually linearly related with the electronegativities-to-radius ratio (En/ri) of central-metal ions. The hRf values of metalloporphyrins become progressively greater with increase in En/ri values of the central metal. This suggests that a dominant factor influencing the hRf values of metal–porphyrins is the extent of electron transfer from the four nitrogens and/or the π-electron system of porphyrin to central metal.

New syntheses of deuterated protoporphyrin-IX derivatives for heme protein nmr studies

An efficient total synthesis of 1,5-di(trideuteromethyl)protoporphyrin-IX ( 3) dimethyl ester from monopyrrole precursors is described, the synthesis proceeding through crystalline tripyrrene and a,c-biladiene salt intermediates. The 2- and 4-vinyl groups in ( 3) are formed from the corresponding (2-chloroethyl) substituents by way of base-promoted dehydrochlorination. In protio solvents, this synthetic step is shown to exchange out preferentially deuterons in the 1-methyl group, and this observation is exploited in an efficient synthesis of the 1,3-di(trideuteromethyl)protoporphyrin-IX ( 22) dimethyl ester from 2,4-diacetyldeuteroporphyrin-IX ( 20) dimethyl ester (which is in turn accessible from commercially available protoporphyrin-IX ( 5)). Thus, basic exchange in deuterated solvent of ( 20) gives the deuterated analog, which after reduction and dehydration gives the 1,3-di(trideuteromethyl)protoporphyrin-IX analog ( 22), in which the vinyl H 2 and propionic C H 2·CO functions have also become deuterated.

The nmr spectra of porphyrins—15 : Self-aggregation in zinc(II) protoporphyrin-IX dimethyl ester

Complex formation in Zn(II) protoporphyrin-IX dimethyl ester has been studied by proton and 13C NMR spectroscopy. The large concentration dependence of the spectra has been studied by the technique of porphyrin/axial ligand titration, which together with selective decoupling and regiospecific deuterium labelling allows the assignment of all the peripheral proton and 13C nuclei in both the monomeric and aggregated species. Titration of the metalloporphyrin with various basic ligands (pyrrolidine, pyridine, lutidine) showed that dissociation of the aggregate was complete for a 1:1 porphyrin/added base ratio. The concentration dependence of the spectra was then analyzed to give the monomer and monomer-dimer shifts for all the assigned nuclei. Analysis of the monomer-dimer shifts in terms of the ring current model gives good agreement with a dimer geometry in which the inter-ring separation is ca. 4.5 Å and there is a smaller lateral displacement of the porphyrin rings. The dimer geometry is such that rings A and B of one porphyrin molecule are situated over rings C and D of the other. These results confirm our earlier suggestions of intermolecular metal-to-porphyrin binding in these metalloporphyrins, and further suggest that charge-transfer interactions may also be present in appropriate cases. The discrepancy between the absolute values of the observed and calculated monomer-dimer shifts, which was formerly attributed to multiple aggregation, is now suggested to be due to ensemble-averaging in the dimer structure.

New syntheses of chlorocruoro- and isochlorocruoro-porphyrins from protoporphyrin-IX

Treatment of protoporphyrin-IX dimethyl ester with picryl azide affords an equimolar mixture of chlorocruoro- and isochlorocruoro-porphyrin dimethyl esters.

Excited states of protoporphyrin IX dimethyl ester: reaction on the triplet with carotenoids

The triplet and singlet excited states of protoporphyrin IX dimethyl ester (PPIXDME) have been identified by means of pulse radiolysis experiments in benzene solution. The absorption spectrum, extinction coefficients, energy level and rate of oxygen quenching of the triplet state have been measured. Triplet energy transfer from PPIXDME to a group of polyenes of differing conjugated double-bond chain length has been studied. Pariser–Parr–Pople m.o. calculations have been performed to find the variation in the energy of the lowest triplet excited state with the number of polyene conjugated double bonds. The quenching data are compared with similar previous results for chlorophyll triplet; they are discussed in connection with the role of protoporphyrin IX in the light-sensitive human disease erythropoietic porphyria and the treatment of this disease by induced carotenemia.

Pyrroles and related compounds—XXXV: A stepwise, general synthesis of unsymmetrically substituted porphyrins

Using a new, general approach involving the stepwise progression through pyrrole, dipyrrole, tripyrrole, to tetrapyrrole, followed by cyclisation of the resulting a, c-biladiene by the copper salt method, syntheses of isocoproporphyrin tetramethyl ester ( 1b), coproporphyrin-III tetramethyl ester ( 24), protoporphyrin-IX dimethyl ester ( 26), 2,4,6,7-tetrakis (2-methoxycarbonylethyl)-5-methoxycarbonylmethyl-1,3,8-trimethylporphin ( 29) (the ester of the pentacarboxylic porphyrin recently associated with haem metabolism), rhodoporphyrin-XV dimethyl ester ( 27) and 2,4,7-triethyl-6-methoxycarbonyl-1,3,5,8-tetramethylporphin ( 28), are described. The route employs condensation of unsymmetrically substituted pyrromethanes with 2-formyl-5-methylpyrroles to give crystalline and fully characterised tripyrrene salts in high yield. These are then condensed with a second mole of a different 2-formyl-5-methylpyrrole to give very high yields of a, c-biladiene dihydrobromides; cyclisation with copper(II) chloride indimethylformamide gives copper(II) porphyrins which are demetallated in trifluoroacetic acid containing sulphuric acid to give high overall yields of the corresponding metal free porphyrins.

Pyrroles and related compounds—XXXVI : Transformations of protoporphyrin-IX into harderoporphyrin, pemptoporphyrin, chlorocruoroporphyrin and their isomers

Treatment of protoporphyrin-IX dimethyl ester ( 8b) with 2 equiv of thallium(III) nitrate in methanol followed by chromatography affords good yields of a mixture of mono-acetal-mono-vinyl isomers ( 11b and 12b) which can be converted into the corresponding (2-hydroxyethyl)-vinyldeuteroporphyrins ( 13b and 14b) by hydrolysis and reduction with sodium borohydride. After separation by preparative TLC these compounds are transformed into isoharderoporphyrin trimethyl ester ( 3b) and harderoporphyrin trimethyl ester ( 2b) by treatment with CBr 4/Ph 3P (to afford the 2-bromoethyl derivatives), then sodium cyanide in 1-methylpyrrolidone (giving the 2-cyanoethyl compounds), followed by methanolysis. Formal syntheses of pemptoporphyrin dimethyl ester ( 4b) and its isomer ( 5b) are achieved by devinylation (resorcinol melt) of the haemins from the pure (2-hydroxyethyl)-vinyl isomers ( 14b and 13b) respectively, followed by demetallation. Controlled oxidation of protoporphydrin-IX dimethyl ester ( 8b) with osmium tetroxide gives starting material, mono-glycol-mono-vinyl isomers ( 23b and 24b), and bis-glycol ( 25b); this mixture can be separated by column chromatography. Further separation of the mono-glycols through preparative TLC followed by treatment with sodium periodate gives pure samples of chlorocruoroporphyrin dimethyl ester ( 6b) and its isomer ( 7b). In the TLC separations of unsymmetrically 2,4-substituted deuteroporphyrins studied, the 2-vinyl substituted isomers proved always to be the more polar (less mobile) componnets of the mixtures of isomers.

Polarographic studies of some porphyrins and metalloporphyrins in N, N′-dimethylformamide

Electrochemical behavior of protoporphyrin IX dimethylester (PPDM), mesoporphyrin IX dimethylester, Fe(III)Cl-protoporphyrin, Ni(II)-, Cu(II)-, Zn(II)-, Co(II)- and Mn(III)Cl-PPDM was investigated in dimethylformamide by use of d.c. polarography, a.c. polarography, cyclic voltammetry and radio-frequency polarography. These porphyrins and divalent metal complexes of prophyrin gave, for the most part, three reduction waves. The first and second waves were reversible one-electron steps in each case but the third one was irreversible. Kinetic parameters of the first two steps were determined by radio-frequency polarographic method.

ChemInform Abstract: POLAROGRAPHIC STUDIES OF SOME PORPHYRINS AND METALLOPORPHYRINS IN N,N‐DIMETHYLFORMAMIDE

Electrochemical behavior of protoporphyrin IX dimethylester (PPDM), mesoporphyrin IX dimethylester(MPDM), Fe(III)Cl-protoporphyrin, Ni(II)-, Cu(II)-, Co(II)-, Zn(II)- and Mn(III)Cl·H2O–PPDM was investigated in dimethylformamide by use of AC polarography, cyclic voltammetry and DC polarography. These porphyrins and divalent metal complexes of PPDM gave, for the most part, three reduction waves. The first and second waves were reversible one-electron steps in each case, but the third step was an irreversible one. Mn(III)-complex gave four reduction waves and one oxidation wave. The first three reduction waves were one-electron steps and the oxidation wave was identified as that due to chloride anion. Fe(III)-complex gave five reduction waves and one oxidation wave. The first two reduction waves were one-electron steps. All these compounds showed specific adsorption at the potential more positive than −1.1 V vs. SCE. Assignment of the reduction steps of the metalloporphyrins is described. The observed order ...

Pyrroles and related compounds. Part XXXIII. Total synthesis of deuteriated derivatives of protoporphyrin-IX for nuclear magnetic resonance studies of haemoproteins

In order to settle the precise identity of certain resonances in the paramagnetic n.m.r. spectra of low-spin haemoproteins, a variation of the MacDonald approach to porphyrins from pyrromethanes has been developed and applied to the synthesis of protoporphyrin-IX dimethyl ester (1a) and its 5,8-bis(trideuteriomethyl)-, 1,3-bis(trideuteriomethyl)-, and αγ-dideuterio-derivatives [(1b), (1d), and (14b), respectively]. Synthesis of 1,8-bis(trideuteriomethyl)protoporphyrin-IX dimethyl ester (1c) by the b-oxobilane route completed the series to be used for unambiguous assignment of the four methyl resonances in the contact shift n.m.r. spectra of the corresponding iron(III) cyanides.

Polarographic Studies of Some Porphyrins and Metalloporphyrins in N,N-Dimethylformamide

Abstract Electrochemical behavior of protoporphyrin IX dimethylester (PPDM), mesoporphyrin IX dimethylester(MPDM), Fe(III)Cl-protoporphyrin, Ni(II)-, Cu(II)-, Co(II)-, Zn(II)- and Mn(III)Cl·H2O–PPDM was investigated in dimethylformamide by use of AC polarography, cyclic voltammetry and DC polarography. These porphyrins and divalent metal complexes of PPDM gave, for the most part, three reduction waves. The first and second waves were reversible one-electron steps in each case, but the third step was an irreversible one. Mn(III)-complex gave four reduction waves and one oxidation wave. The first three reduction waves were one-electron steps and the oxidation wave was identified as that due to chloride anion. Fe(III)-complex gave five reduction waves and one oxidation wave. The first two reduction waves were one-electron steps. All these compounds showed specific adsorption at the potential more positive than −1.1 V vs. SCE. Assignment of the reduction steps of the metalloporphyrins is described. The observed order of increasing negative half-wave potentials is discussed in terms of Zerner and Gouterman’s molecular orbital calculation.

Biosynthesis of porphyrins and related macrocycles. Part III. Rational syntheses of [β-13C]-, [γ-13C]-, and [δ-13C]-protoporphyrin-IX: assignment of the13C nuclear magnetic resonance signals from the meso-carbon atoms of protoporphyrin-IX dimethyl ester

The 13C signals arising from the meso-carbon atoms ot protoporphyrin-IX are of key importance for biosynthetic studies. Rigorous assignment is made of the signals from the β- and δ-carbon atoms by synthesis of [β-13C]- and [δ-13C]-protoporphyrin-IX dimethyl ester from unsymmetrical pyrromethenes in 42% yield. The formation of porphyrins from a,c-biladienes and formaldehyde is studied and leads to synthesis of [γ-13C]protoporphyrin-IX dimethyl ester. The spectrum of this product allows assignment of the two remaining signals, from the γ- and α-carbon atoms. The three 13C-labelled samples of protoporphyrin-IX dimethyl ester are converted into 2,4-diacetyldeuteroporphyrin-IX dimethyl ester; assignment is thereby made of the well-spread 13C signals from the meso-carbon atoms of this porphyrin. 13C Chemical shifts are reported for a variety of pyrrole derivatives and porphyrins.

Coproporphyrin-III from protoporphyrin-IX

Manipulation of the vinyl side-chains of the readily available protoporphyrin-IX dimethyl ester (2) allows the synthesis of coproporphyrin-III tetramethyl ester (1) in an overall yield of 37%.

Pyrroles and related compounds. Part XVI. Synthesis of protoporphyrin-IX by the a- and b-oxobilane routes

2-Ethyl-2-devinylprotoporphyrin-IX dimethyl ester has been synthesised by the a-oxobilane route, the 4-vinyl group being introduced by a new method, viz. transformation of a β-acetoxyethyl side-chain present in one of the intermediate pyrroles. Protoporphyrin-IX dimethyl ester has also been prepared by both the a- and b-oxobilane routes, again with β-acetoxyethyl groups as precursors of the two vinyl groups in the porphyrin. The use of a pentachlorophenyl pyrromethane ester in the a-oxobilane synthesis and of a t-butyl pyrromethane ester in the b-oxobilane route enables these syntheses to be carried out in an entirely rational fashion without involving symmetrical intermediates, because the nuclear pentachlorophenyl ester can be selectivety cleaved by mild alkaline hydrolysis, and the nuclear t-butyl ester by trifluoroacetic acid in the presence of nuclear benzyl esters. The intermediate oxophlorin formed in the b-oxobilane route undergoes exchange with tritium at the meso-position opposite the oxo-group, and thus the oxo-function facilitates the synthesis of δ-meso-tritiated protoporphyrin-IX required for biosynthetic experiments in the chlorophyll field. Attempts to prepare the 2,4-divinyl-β-oxophlorin were frustrated by preferential formation of a cyclic ether from the 4-substituent and the β-oxygen atom.