Porphyrin Esters Research Articles

Synthesis and DNA‐Recognition and ‐Cleavage Properties of Multiply Charged Porphyrin Esters

A series of systematically modified porphyrin esters, compounds 1-6, with multiple, permanent positive charges introduced at the meso-positions via N-methylated 4-, 3-, or 2-pyridyl moieties, were prepared and characterized. Their singlet-oxygen production, CT-DNA-binding properties, and plasmid-DNA photocleavage propensities were determined spectroscopically and by gel electrophoresis, and compared to those of the known, fourfold-charged parent porphyrin 4,4',4'',4'''-porphyrin-5,10,15,20-tetrayltetrakis(1-methylpyridinium) (TMPyP4). Some interesting structure-activity relationships could be established to rationalize effects affecting DNA binding mode and cleavage ability.

In vivo transport and pharmacokinetic behavior of tumour photosensitizers.

The mechanisms by which photodynamic sensitizers are transported in the bloodstream influence their distribution among normal and tumour tissues, as well as their partitioning among the various compartments of tumour tissues. Column chromatographic analysis and density gradient ultracentrifugation of sera obtained from both patients and experimental animals show that hydrophilic photosensitizers (e.g. haematoporphyrin, and tetrasulphonated porphyrins and phthalocyanines) are largely transported by albumin and globulins and mainly deposited in the vascular stroma of tumours. More hydrophobic photosensitizers (haematoporphyrin oligomers, porphyrin esters, monosulphonated or unsubstituted phthalocyanines) are preferentially incorporated in the lipid core of lipoproteins. Tightly aggregated dyes partly circulate as unbound pseudomicellar structures which can be entrapped in the interstitial regions of the tumour, localize in macrophages, or enter neoplastic cells via pinocytotic processes. Low density lipoproteins (LDL), which are endocytosed by neoplastic cells through a specific receptor-mediated pathway, display the most selective release of photosensitizers to tumours. The binding of the injected photosensitizer to LDL can be enhanced by preincorporation of the dye in liposomal vesicles which are in a quasi-solid state at the body temperature.

Two-photon absorption properties of substituted porphyrins

The two-photon absorption (TPA) properties of two newly synthesized substituted porphyrins: 5,10,15,20-tetra-(4-pyrrolidinylphenyl)porphyrin (1) and 5,10,15,20-tetra-(4-diphenylamino-benzoic acid phenyl) porphyrin ester (3) were measured by the femtosecond Z-scan technique at 800 nm Ti : sapphire laser and by direct nonlinear optical transmission (NLT) using nanosecond Nd : YAG laser. The substituent effects, pumped wavelength and the environment upon the molecular TPA cross-section were investigated. It is found that the TPA cross-section for 3 is enhanced under excitation of 800 nm, relative to its precursor, 5,10,15,20-tetra(4-hydroxyphenyl) porphyrin (2), due to the former possessing extension conjugation of "D-A-core-A-D" molecular architecture. Also sample 3 obtained an increased TPA cross-section in rigid film relative to that in solution, suggesting that the enhancement is facilitated by the quasi-planar structure which allows direct interbranch conjugation throughout the molecule.

On-line LC-MS analysis of urinary porphyrins.

A reversed-phase high-performance liquid chromatography-mass spectrometry (LC-MS) method is described for the separation and simultaneous analysis of porphyrins related to disorders of heme biosynthesis (uro-, heptacarboxylic, hexacarboxylic, pentacarboxylic, and coproporphyrins). The method involves initial porphyrin esterification and extraction from urine. Detection and quantification is performed from the extracts by separation with a Hypersil BDS column and on-line detection by MS through coupling with an atmospheric pressure chemical ionization interface. The porphyrin esters are detected as protonated molecules [M + H]+. Their mass spectra also exhibit an [M + Na]+ fragment of lower intensity. The analytical performance of this method is compared with those of LC with UV and fluorescence detection. LC-MS used in selective [M + H]+ ion monitoring provides the lowest detection and quantitation limits. In scan mode, this LC-MS method affords, without further isolation or concentration steps, the measurement of mass spectra of unknown compounds present in the urine of patients with altered porphyrin excretion.

A comparative study of normal and reverse phase high pressure liquid chromatography for analysis of porphyrins accumulated after 5-aminolaevulinic acid treatment of colon adenocarcinoma cells

Primary adenocarcinoma cells of the rectosigmoid colon (WiDr-cells) were treated with 5-aminolevulinic acid (5-ALA). Cellular porphyrins were separated and quantified by high performance liquid chromatography (HPLC), both as free porphyrin acids after an easy extraction method with a subsequent reverse phase technique, and then as porphyrin esters after a more laborious extraction method and subsequent normal phase technique. The porphyrins were detected by means of a fluorescence detector. Analysis by normal phase HPLC indicated that 81% (739 pmol/mg protein) of the total amounts of fluorescing porphyrins accumulated was protoporphyrin IX, while similar analysis by reverse phase HPLC indicated that PpIX constituted 91% (622 pmol/mg protein) of the accumulated porphyrins. In addition to protoporhyrin IX, copro-, hexa-, hepta- and uroporphyrins were observed in extracts from 5-ALA-treated cells by both methods. The discrepancy between the two methods increased with increasing hydrophilicity of the analysed porphyrins, with uroporphyrin estimated to be 6-fold higher (63 vs. 10 pmol/mg protein) by normal than by reverse phase HPLC.

Synthesis of Formyl(2,2,2-trifluoro-1-hydroxyethyl)deuteroporphyrins and Their Derivatization to Porphyrin Derivatives with Elongated Side-Chains.

We previously synthesized 3- and 8-mono, and 3, 8-bis(2, 2, 2-trifluoro-1-hydroxyethyl)deuteroporphyrin dimethyl esters (2-4) by Friedel-Crafts reaction of deuteroporphyrin dimethyl ester (1) with trifluoroacetaldehyde, and trifluorohematoporphyrin analogs (5 and 6) by acetylation of 2 and 3 followed by reduction. These fluorine analogs of natural porphyrins have interesting biological properties; some of them accumulate selectively in certain tumor cells. In order to introduce larger substituents at the 8- or 3-position of 2 and 3, 2 and 3 were converted in several steps to 8- and 3-formyl derivatives (13 and 14), respectively. These were treated with vinylmagnesium bromide to give 8- or 3-(1-hydroxy-2-propenyl) derivatives (15 or 16). Further, reaction of 7 or 8 with the enol ether of 2-octanone gave the 8- or 3-(3-oxo-1-nonenyl) compounds (17 or 18). The affinities of the porphyrin derivatives, obtained by hydrolysis of the above porphyrin esters, for tumor tissues were examined. Among them, the porphyrin obtained by hydrolysis of 16 was found to accumulate in liver cancer transplanted in nude mice to a greater extent than hematoporphyrin.

PHOTOPHYSICAL AND PHOTOBIOLOGICAL PROPERTIES OF DIPORPHYRIN ETHERS

Spectral properties of several diporphyrin ethers were assessed in different solvents and after accumulation by leukemia L1210 cells in vitro. To facilitate studies in a variety of solvents, both tetramethylesters of the diporphyrin ethers and free acids were employed. For comparison, studies on the corresponding porphyrin monomers were also carried out. The joining of two porphyrins by an ether linkage had several consequences. We observed a blue shift in the Soret band of the ethers, but not of the corresponding simple porphyrins, in protic solvents. This phenomenon is likely related to ether aggregation under conditions which promote H-bonding. The presence of an ether linkage was associated with enhanced fluorescence at 630-640 nm and decreased fluorescence lifetimes and yields, especially in protic solvents. The ether linkage was unaffected by intracellular enzymes, but porphyrin esters were readily hydrolyzed upon accumulation by L1210 cells. The joining of two hematoporphyrin molecules by an ether linkage promoted dye accumulation by L1210 cells. In contrast, accumulation of mesoporphyrin and protoporphyrin was thereby retarded.

POLYMER BOUND PYRROLE COMPOUNDS–VI. PHOTOPHYSICAL PROPERTIES OF MONOMERIC MODELS FOR POLYSTYRENE-BOUND PORPHYRINS

Abstract— Several porphyrin esters used as models for polystyrene-bound porphyrins have been prepared and their excited states have been studied by laser flash photolysis, IR phosphorescence of singlet molecular oxygen, O2(1Δg), and steady-state fluorescence. The photophysical properties of the porphyrin esters in solution are affected by the presence of nitro group(s) in the chain. In this case, an important decrease in φf, φT and φδ (to ca 0.7–0.4 of the value for the parent dimethyl ester) is observed. This is mainly due to intramolecular electron-transfer quenching [by the nitro group(s)] of the first excited singlet state of the porphyrin. The thermodynamic feasibility of this deactivation pathway has been confirmed polarographically. Quenching of the porphyrin triplet state and of O2(1Δg) by the nitro groups is negligible. The present conclusions explain also the results obtained previously for the photooxidation of bilirubin sensitized by the parent insoluble polystyrene-bound porphyrins. In that case the photooxidation rates were correlated directly with the quantum yield of O2(1Δg) production by the sensitizer. The consequences of these results for the use of polystyrene-bound porphyrins in sensitized photooxidation processes are discussed.

The structure of the active material in hematoporphyrin derivative.

Abstract The structure of the active material in hematoporphyrin derivative is shown to be a condensation polymer of hematoporphyrin linked by ether functional groups. When a mixture of the monoacetates and the diacetate of hematoporphyrin is treated with dilute sodium hydroxide solution a polymeric fraction is formed which constitutes the active material of hematoporphyrin derivative. This fraction is stable to basic hydrolysis using conditions which are shown to hydrolyse porphyrin esters, but is hydrolysed by acidic conditions which cleave porphyrin ethers as well as esters. When hematoporphyrin diacetate is similarly treated with base a polymeric fraction is formed which is hydrolysed by both acidic and basic conditions showing it to be ester linked. This ester linked polymer is unstable in aqueous solution at pH 7 and converts to the polyether within 2 days at room temperature.

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.

Controlled targeting of different subcellular sites by porphyrins in tumour-bearing mice.

Unilamellar liposomes of dipalmitoyl-phosphatidylcholine can incorporate various porphyrins in either the phospholipid bilayer or the internal aqueous compartment depending on the water-/lipo-solubility of the drug. Intraperitoneal injection of the liposome-bound porphyrins to mice bearing a MS-2 fibrosarcoma results in remarkably more efficient tumour targeting than that obtained by administration of the same porphyrins dissolved in homogeneous aqueous solution. Moreover, also water-insoluble porphyrins can be transported to the tumour via liposomes. Fractionation of liver and neoplastic cells indicates that the subcellular distribution of liposome-delivered porphyrins is also dependent on their solubility properties: thus, relatively polar porphyrins, such as tetra(4-sulfonatophenyl)porphine and uroporphyrin, are mainly recovered from the soluble fraction, whereas hydrophobic porphyrins, such as haematoporphyrin or porphyrin esters, preferentially partition in the cytoplasmic membrane. As a consequence, different subcellular sites can be targeted by porphyrins and possibly photodamaged through a suitable choice of the drug-carrier system.

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 isocratic high-performance liquid chromatography of porphyrin esters on aminopropyl-bonded silica

A simple isocratic procedure for the separation of porphyrin esters according to the number of carboxyl groups using an aminopropyl-bonded silica column and a simple binary eluent is suggested. In this way, problems with the choice of a suitable unmodified silica column for isocratic separation are circumvented; entirely different aminopropyl-bonded silicas of various origins exhibit almost identical behaviour. The influence of end-capping of residual silanols and the chemical substitution of the primary amino group on the separation was studied. To achieve an isocratic separation of porphyrin esters on the bare silica used, the concentration of surface silanol groups must be decreased; suitable concentrations are tailored by means of partial trimethylsilylation. The isocratic separation of porphyrin esters can also be achieved on cyanopropyl-bonded silica. The suggested optimum procedure, using primary amino-bonded silica, gives the highest resolution and allows the baseline separation of porphyrin esters with two to eight carboxyl groups together with their Cu 2+ complexes and synthetic tetraphenylporphyrin as the internal standard.

Effect of carbamazepine on haem biosynthesis in man.

The effects of the enzyme-inducing anticonvulsant carbamazepine on haem biosynthesis in healthy male subjects is reported. A dose-dependent increase in the activity of leucocyte delta-aminolaevulinic-acid synthase, the rate-limiting enzyme of haem biosynthesis, was noted following 400 and 600 mg carbamazepine daily in the same eight subjects. This rise was maximal after 1 week's treatment (400 mg: 509% +/- 285 of baseline; 600 mg: 1062% +/- 170 of baseline; P less than 0.01). Values fell from this peak during the second week despite continuing carbamazepine administration. This pattern was confirmed in a further six subjects taking carbamazepine 400 mg daily for 3 weeks in whom more frequent enzyme measurements were made. The activity of uroporphyrinogen-1-synthase in the erythrocyte fell by 10-15% during the treatment periods (P less than 0.01). Uroporphyrin and penta-, hexa- and hepta-carboxylic porphyrins appeared in the urine of all subjects during CBZ therapy. Changes in daily urinary porphyrin and precursor excretion were inconsistent. CBZ is a porphyrinogenic drug which mimics the changes in enzyme activities and urinary porphyrin ester profile found in patients with latent acute intermittent porphyria, who have a genetic deficiency in uroporphyrinogen-1-synthase activity. Leucocyte delta-aminolaevulinic-acid synthase may provide a suitable in vivo system for testing the porphyrinogenicity of drugs in man.

Reverse-phase purification and silica gel thin-layer chromatography of porphyrin carboxylic acids

Thin-layer chromatography on silica gel 60 plates in the solvent N,N-dimethylformamide/methanol/ethylene glycol/glacial acetic acid/1-chlorobutane/chloroform (4/35/6/0.4/18/20 by volume) separates porphyrin carboxylic acids by the number of free carboxyl groups. Coproporphyrins I and III and isocoproporphyrin are separated in 30 min, other porphyrins in 15 min. The N,N-dimethylformamide and acetic acid in the solvent strongly increase porphyrin fluorescence on the plates. Fading and diffusion of the fluorescent patterns is prevented by storage of the plates in the cold and dark without oxygen and with desiccant. In a preliminary step, porphyrins are purified in high yields, concentrated, and deacidified rapidly (2 min) by reversephase chromatography on cartridges containing a C 18 spacer or on Amberlite XAD-2 columns. The methods are applied to urines of porphyria patients and for following porphyrin ester hydrolysis.

Rapid semiquantitative measurement of total porphyrins in urine and feces by magnetic circular dichroism.

Magnetic circular dichroism is a suitable technique for semiquantitative determination of total porphyrins in urine and feces, being rapid, reliable, and involving little sample preparation. The sensitivity of the assay suffices to distinguish normal from above-normal concentrations of total porphyrins in urine and feces. Results for urine compare well with those obtained by thin-layer chromatography of porphyrin esters (n = 42, r = 0.92) or ion-exchange column chromatography of porphyrin acids (n = 19, r = 0.80).

Synthesis of [ 14C]coproporphyrin III by yeast cell-free extracts

The procedure described utilizes a crude cell-free extract from the yeast Saccharomyces cerevisiae as enzymatic source for the synthesis of coproporphyrin III from [ 14C]δ-aminolevulinic acid with a high yield of conversion (⋍60%). Both specific radioactivity and total radioactivity of coproporphyrin III can be adjusted fairly well. This procedure is not time consuming for yeast acellular extracts or porphyrin ester preparations. The acellular extracts can be stored frozen (−30°C) for at least 1 year without loss of enzymatic activity. The same procedure can be used for [ 14C]protoporphyrin preparation.

Uroporphyrinogen Decarboxylase

A method for measuring the activity of uroporphyrinogen decarboxylase from a variety of sources is described. Porphyrinogen substrates are generated by enzymic synthesis or chemical reduction. Substrates and enzyme are incubated under standardized conditions. The reactions are then stopped and the reaction products, as well as unmetabolized substrate, are absorbed on talc, eluted and esterified. Porphyrin esters are then separated and quantified using high performance liquid chromatography.

Chloroplast biogenesis: biosynthesis of protochlorophyll(ide) via acidic and fully esterified biosynthetic branches in higher plants.

The operation of two parallel biosynthetic pathways during the biosynthesis of the protochlorophyll(ide) pool of cucumber cotyledons was probed with the use of several [14C]tetrapyrroles in vitro. A comparison of the ratio (R) of delta-amino[14C]levulinic acid incorporation into protochlorophyllide/14C incorporation into protochlorophyllide ester with other incorporation ratios, RX, where X represents any of several [14C]tetrapyrrole substrates, allowed us to determine which exogenous 14C-labeled substrate was common precursor of protochlorophyllide and protochlorophyllide ester and which one was not. From such comparisons, a biosynthetic pathway of protochlorophyll(ide) biosynthesis is inferred. According to this pathway, protochlorophyllide is formed via an acidic (mono/dicarboxylic) biosynthetic branch, while protochlorophyllide ester is formed via a fully esterified (neutral) biosynthetic branch. The two biosynthetic branches are linked together by porphyrin ester synthetases at the level of protochlorophyllides and possibly at the level of the photoporphyrin IX and the magnesium protoporphyrin monoester pools.

Biosynthesis of porphyrins and heme from gamma, delta-dioxovalerate by intact hepatocytes.

The purpose of this study was to assess the ability of hepatocytes to synthesize porphyrins and heme from delta-aminolevulinic acid derived from gamma, delta-dioxovalerate and alanine. An alternate pathway for delta-aminolevulinic acid synthesis, in contrast to the condensation of succinate and glycine by delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] has been suggested. This has been supported by the isolation of gamma, delta-dioxovalerate transaminase from liver mitochondria (Varticovski, L., Kushner, J. P. & Burnham, B. F. (1980) J. Biol. Chem. 255, 3742-3747). Gamma, delta-dioxovalerate transaminase catalyzes the formation of delta-aminolevulinic acid by a transamination reaction involving gamma, delta-dioxovalerate and alanine. To assess the significance of this alternate route, we prepared suspensions of respiring rat hepatocytes, which were incubated with optimal concentrations of various additives and then analyzed spectrophotometrically for synthesized porphyrins. No porphyrin synthesis was detected in cell suspensions incubated with succinate(1 mM) and glycine (20 mM). Cell suspensions incubated with gamma, delta-dioxovalerate (0.5-1.0 mM) and alanine (20 mM) synthesized 0.19 nmol of porphyrin per 10(7) cells per 2 hr (SD, 0.057). Cell suspensions incubated with delta-aminolevulinic acid (0.1 mM) synthesized 1.26 nmol of porphyrin per 10(7) cells per 2 hr (range, 1.18-1.32). Incubations with chemically synthesized gamma, delta-dioxo[5-14C]valerate were followed by extraction and purification of porphyrin esters and heme. Liquid scintillation counting revealed radiolabel incorporation into both porphyrins and heme. These studies demonstrate significant tetrapyrrole synthesis by the gamma, delta-dioxovalerate transaminase reaction. That gamma, delta-dioxovalerate is an important precursor of heme in vivo must be considered.