Protoporphyrin preparation

The second methodology involves direct introduction of glycosylated moieties onto a suitably functionalized meso-arylporphyrin scaffold, accessible from a natural source (protoporphyrin-IX) or by total synthesis. Several O-,133,145,146 S-,147 and N-glycoporphyrins148 (125-129) have thus been prepared (Fig. 12). Moreover, in order to explore the influence of the clustered peripheral saccharides around the porphyrin scaffolds, and to evaluate their photophysical properties, the synthesis of dodecavalent porphyrins bearing four trivalent glycodendrons via amide ligation (129) has been achieved.149... 

This type of double-seal gave very good protection against O2. Fe(III) protoporphyrin IX was prepared after the method of Falk (10). The Fe(III) complex was converted to Fe(II) using the method oF Brault and Rougee (11) in the N2-H2 atmosphere glove bag. 

Hb and Ni-reconstituted Hb were prepared according to reported methods (13). Mb was made by the method of Alston and Storm (14) Solutions of the proteins in 0.05 M phosphate buffer at pH 7.5 were used for obtaining spectra. The proteins are stable in air and do not photodecompose. Ni protoporphyrin IX (Ni(ProtoP)), Ni uroporphyrin I (Ni(UroP)), and Ni octaethylporphyrin (Ni(OEP)) were obtained from Porphyrin Products and used without further purification. All solvents were of highest purity obtainable from commercial sources. All materials showed the literature uv-visible absorption spectra. Absorption spectra were obtained on a Perkin-Elmer Model 330 spectrophotometer. 

Using a typical poly (vinyl chloride) (PVC)-based membrane with different ionophores - Zn-bis(2,4,4-trimethylpen-tyl) dithiophosphinic acid complex [450], protoporphyrin IX dimethyl ester [451], porphyrin derivative [452] and hemato-porphyrin IX [453], tetra(2-aminophenyl) porphyrin [454], cryptands [455, 456], 12-crown-4 [457], benzo-substituted macro-cyclic diamide [458], 5,6,14,15-dibenzo-l, 4-dioxa-8,l 2, diazacyclopentadecane-5,14-diene [459], and (A-[(ethyl-l-pyrrolidinyl-2 -methyl) ] methoxy-2-sulfamoyl-5 -benza-mide [460] - the sensors for zinc ions were prepared and investigated. The armed macrocycle, 5,7,7,12,14,14-hexamethyl-1,4,8,11 -tetraazacyclo tetradeca-4,11 -diene dihydrogen perchlorate was used for the preparation of polystyrene-based Zn(II)-sensitive electrode [461]. 

Killday etal. (1988) also provided evidence for internal autoreduction of ferric nitrosyl heme complexes, as previously proposed by Giddings (1977). Heating of chlorohemin( iron-III) dimethyl ester in dimethyl sulfoxide solution with imidazole and NO produced a product with an infrared spectra identical to that of nitrosyl iron(ll) protoporphyrin dimethyl ester prepared by dithionite reduction. Both spectra clearly showed the characteristic nitrosyl stretch at 1663 and 1665 cm. They thus proposed a mechanism for formation of cured meat pigment which includes internal autoreduction of NOMMb via globin imidazole residues. A second mole of nitrite is proposed to bind to the heat-denatured protein, possibly at a charged histidine residue generated in the previous autoreduction step. 

Carboxylic groups of the protoporphyrin IX ligand (Scheme 10) are phenomenal candidates for the targeted active-site modification of HRP and other heme-containing proteins. There is a clear parallel with the active site modification of GO (Section IV.A.2). The modification of hemin chloride by H2NCH2Fc in the presence of EDC and V-hydroxysuccinimide in DMF affording mono- and bis-amidated propionic acid residues is shown in Scheme 10 (144). The Fc Heme is actually a mixture of two diastereomers. -HRP has been prepared according to an acidic methyl ethyl ketone procedure of Teale (145)... 

The construction of an artificial protein-protein complex is an attractive subject to elucidate the electron-transfer process in biological systems. To convert Mb into an electron-transfer protein such as cytochromes, Hayashi and Ogoshi (101) prepared a new zinc Mb having a unique interface on the protein surface by the reconstitutional method as shown in Fig. 27. The modified zinc protoporphyrin has multiple functional groups, carboxylates, or ammonium groups, at the terminal of the two propionates. Thus, the incorporation of the... 

Protoporphyrin and its derivatives, because of their labile vinyl substituents, are susceptible to acid-catalyzed hydration. For this reason aqueous solutions should be avoided in the preparation of protoporphyrin dimethyl ester from protohemin, and subsequent reinsertion of the iron. Thus the direct esterification of protohemin under basic conditions appears to be the method of choice. More stable porphyrins such as H2oep are most conveniently prepared by the direct insertion of iron(lll) ion using the iron(III) chloride method5 the resultant Fe(IIl) (oep)Cl can be separated directly from the reaction medium as shining purple crystals. However, this method fails to give successful results with protoporphyrin derivatives. In general, the iron (II) sulfate method is the mildest and should be used for all prophyrins unless one is certain that the more rapid alternatives [boiling DMF with iron(II) chloride, or preferably the more stable iron(II) perchlorate] will have no ill effect on the peripheral substituents. 

Protoporphyrin IX dimethyl ester may be purchased (e.g., Porphyrin Products, Sigma, Strem) or prepared according to literature procedures.1 A convenient... 

One of the variables in the structures of the porphyrins present in heme proteins is the presence or absence of vinyl substituents on the periphery of the macrocycle. For example, b hemes have vinyl substituents whereas c hemes do not. Because of the sensitivity of such vinyl substituents during synthetic transformations, it has often been desirable to use octa-alkyl porphyrins in model studies of the spectroscopic properties of heme systems. The development of improved methods for the preparation of octa-alkyl porphyrins has likewise increased the availability of such porphyrins for model studies (20, 21). To assess the effect that replacement of the two vinyl substituents in protoporphyrin IX with alkyl (ethyl) groups has on the MCD properties of the heme system, an extensive and systematic study of the MCD properties of mesoheme IX-reconstituted myoglobin and horseradish peroxidase in comparison with the spectra of the native protoheme-bound proteins has been carried out (22). The structures of these two porphyrins are shown in Figure 3. 

The standard incubation mixture contained 100 fiL of 0.25 M Tris-HCl buffer (pH 7.4) containing 1.75 mM palmitic add and 1% (w/v) Tween-20 50 /x.L of enzyme preparation (about 0.5 mg protein), and 50 tL of 80 or 100 nM zinc acetate solution. After incubating 5 minutes at 37°C, the reaction was started by adding 50 / L of 100 /iM mesoporphyrin or protoporphyrin. The incubation was continued for 30 minutes at 37°C in the dark. The reaction was stopped by adding 1 mL of ice-cold methanol-DMSO (8 2, v/v) containing 13 nM Zn-deuteroporphyrin as internal standard. The mixture was cooled on ice and centrifuged before analysis of the supernate by HPLC. The... 

The molecular weight of cytochrome c peroxidase has been determined to be 34,100 on the basis of a sedimentation constant of 3.55 S, a diffusion constant of 9.44 F, and a partial specific volume of 0.733 ml/g (4 )-The enzyme exists as a monodisperse monomer containing one ferric protoporphyrin IX, which is noncovalently bound (/, , 14). No other transition metal is detected in crystalline preparations of the enzyme (22). The apoenzyme moiety is an acidic protein with an isoelectric point at pH... 

Modification of protoporphyrin IX at the propionate positions has been used by Traylor and coworkers and Casella eta/. to prepare porphyrin complexes with a covalently attached axial ligand or ligands. The latter workers... 

It should be noted here that there is a very wide latitude in the quality of the commercially available materials. Recently reported experiences with ferric hematoporphyrin derivatives are quite typical. In general, esters are better than the unesterified bases. For the blood-derived materials the usual order of quality goes deutero, hemato, and meso, with protoporphyrin the worst. For a well-defined preparation of natural porphyrins, it is always best to purify the starting material and to characterize it ° satisfactorily before carrying out any chemical modifications. 

Porphyrins are often employed in sensors on account of their ability to act as cation hosts and, with a suitable metal ion coordinated, as redox catalysts. Electropolymerised poly(metalloporphyrin)s have been used as potentiometric anion-selective electrodes [131] and as amperometric electrocatalytic sensors for many species including phenols [132], nitrous oxide [133] and oxygen [134]. Panasyuk et al. [135] have electropolymerised [nickel-(protoporphyrin IX)dimethylester] (Fig. 18.10) on glassy carbon in the presence of nitrobenzene in an attempt to prepare a nitrobenzene-selective amperometric sensor. Following extraction of the nitrobenzene the electrode was exposed to different species and cyclic voltammetric measurements made. A response was observed at the reduction potential of nitrobenzene (the polyporphyrin film acts only to accumulate the analyte and not in a catalytic fashion). Selectivity for nitrobenzene compared with w-nitroaniline and o-nitroto-luene was enhanced compared with an untreated electrode, while a glassy carbon-... 

Fig. 18.10. [Nickel-(protoporphyrin IX)dimethyester], which was electropolymerised on glassy carbon with nitrobenzene as template to prepare a nitrobenzene-selective sensor [135].

Antibodies that recognize transition states should function as catalysts, if our understanding of the importance of the transition state to catalysis is correct. The preparation of an antibody that catalyzes the insertion of a metal ion into a porphyrin nicely illustrates the validity of this approach. Ferrochelatase, the final enzyme in the biosynthetic pathway for the production of heme, catalyzes the insertion of Fe2+ into protoporphyrin IX. The nearly planar porphyrin must be bent for iron to enter. The recently determined crystal structure of the ferrochelatase bound to a substrate analog confirms that the enzyme does indeed bend one of the pyrole rings, distorting it 36 degrees to insert the iron. 

Figure 42. The preparation of a photoenzyme by the reconstitution of a heme protein with Co(II)-protoporphyrin IX and the chemical modification of the protein backbone with a tethered chro-mophore. Hydrogen evolution and hydrogenation of acetylene derivatives photobiocatalyzed by the assembly.

An alternative approach to the preparation of photoenzymes by the reconstitution of apo-proteins includes the application of metalloprotoporphyrins such as Zn(II)-protoporphyrin IX (Zn-P), which exhibits photophysical electron-transfer properties for the reconstitution of apo-proteins derived from hemoproteins. Func-... 

In this chapter we introduce compounds which have been successfully applied in the construction of supramolecular assemblies. Only the amphiphiles which have been prepared in sufficient quantities have been admitted milligram quantities being considered unacceptable as starting materials for the preparation, analysis and application of assemblies. Experience proves that complicated dyes, pore builders, receptors etc. never reappear in the literature after their syntheses and spectroscopic properties have been reported. On the other hand, such easily attainable synkinons and surfactants around the ten gram scale need not, of course, be too simple. On the contrary, they may contain all the components of the chiral pool, i.e. amino acids, carbohydrates, steroids etc., as well as all commercial dyes of interest such as protoporphyrin, phthalo-cyanines, carotenes, viologen and quinones. 

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