Comparison of two assay methods for activities of uroporphyrinogen decarboxylase and coproporphyrinogen oxidase.

Uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen oxidase (copro'gen oxidase) are two of the least well understood enzymes in the heme biosynthetic pathway. In the fifth step of the pathway, UROD converts uroporphyrinogen III to coproporphyrinogen III by the decarboxylation of the four acetic acid side chains. Copro'gen oxidase then converts coproporphyrinogen III to protoporphyrinogen IX via two sequential oxidative decarboxylations. Studies of these two enzymes are important to increase our understanding of their mechanisms. Assay comparisons of UROD and copro'gen oxidase from chicken blood hemolysates (CBH), using a newly developed micro-assay, showed that the specific activity of both enzymes is increased in the micro-assay relative to the large-scale assay. The micro-assay has distinct advantages in terms of cost, labor intensity, amount of enzyme required, and sensitivity.

Kinetic studies of novel di- and tri-propionate substrates for the chicken red blood cell enzyme coproporphyrinogen oxidase.

Coproporphyrinogen oxidase is an important enzyme in heme biosynthesis and catalyses the sequential oxidative decarboxylation of propionates on the A and B rings of the porphyrinogen ring. The effects of substituents on the C and D rings have not been systematically evaluated for their effects on the kinetic constants, K(m) and V(max). A series of synthetic porphyrinogens have been tested for their ability to affect these kinetic constants for the chicken enzyme. The enzyme exhibited the largest V(max) when incubated with the authentic substrate and was clearly able to distinguish between various substituents on the C and D rings of the macrocycle. When co-incubated with substrate, the authentic product, protoporphyrinogen-IX, appears to inhibit coproporphyrinogen oxidase and this may have an important role in the regulation of this enzyme. Thus the model for the active site of this enzyme should be modified to take these factors into account.

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.

Porphyrins with exocyclic rings. 16. Synthesis and spectroscopic characterization of fluoranthoporphyrins, a new class of highly conjugated porphyrin chromophores.

Porphyrins with fused aromatic rings are under detailed investigation due to their unique spectroscopic properties. To gain more insights into the effects due to ring annealation on the porphyrin chromophore, a series of fluoranthoporphyrins have been synthesized. Reaction of 3-nitrofluoranthene with isocyanoacetate esters in the presence of a phosphazene base afforded good yields of the fluorantho[2,3-c]pyrrole esters 8. Cleavage of the ester moiety with KOH in ethylene glycol afforded the parent heterocycle 9, and this condensed with 2 equiv of acetoxymethylpyrroles 10 in refluxing acetic acid-2-propanol to afford tripyrranes 11. Following cleavage of the tert-butyl ester protective groups with TFA, "3 + 1" condensation with pyrrole dialdehyde 12 gave the fluoranthoporphyrins 13 in good overall yields. In addition, reaction of tripyrrane 11 with acenaphthopyrrole dialdehyde 16 gave the mixed acenaphthofluoranthoporphyrin 17 in excellent yields. A difluoranthoporphyrin 18 was also prepared via a "2 + 2" MacDonald condensation. Reaction of fluoranthopyrrole 8a with dimethoxymethane in the presence of p-toluenesulfonic acid gave the symmetrical dipyrrylmethane 19, and following ester saponification, this was condensed with a dipyrrylmethane dialdehyde to afford the adj-difluoranthoporphyrin 18. The UV--vis spectra for these fluoranthoporphyrins gave a series of three broadened absorptions in the Soret band region, although the Q-bands were little effected by ring fusion. The nickel(II), copper(II), and zinc chelates were more unusual, showing strong absorptions near 600 nm. Difluoranthoporphyrin 18 showed many of the same spectroscopic features, although the presence of two ring fusions gave rise to an increase in the spectroscopic shifts. The mixed system 17 gave spectra that showed larger red shifts due to the acenaphthylene unit combined with the features due to the fluoranthene rings. This work further demonstrates the utility of aromatic ring fusion in altering the properties of porphyrinoid systems.

Palladium (II) complexes of oxybenziporphyrin.

8,19-Dimethyl-9,13,14,18-tetraethyloxybenziporphyrin coordinates palladium(II) to form the four-coordinate anionic complex [(OBP)Pd(II)](-). The NMR data provide evidence for the retention of macrocyclic aromaticity and coordination via a carbon sigma-donor. Protonation of the external oxygen atom to give [(HOBP)Pd(II)] switches the molecule to a less aromatic phenol-like state, which is manifested by a significant reduction of the macrocyclic ring current. [(AcOBP)Pd(II)] and [(TsOBP)Pd(II)], two ester derivatives of [(OBP)Pd(II)](-), are similar to the protonated species, and their benzenoid character is more pronounced. However, reaction of [(OBP)Pd(II)](-) with methyl iodide leads to selective methylation of the coordinating C(22) atom to form a novel organopalladium complex (OBPMe)Pd(II). The strong shielding of the inner Me(22) (delta((1)H) -2.00 ppm in CDCl(3)) indicates that the aromaticity of the macrocycle has been retained. At the same time the (13)C chemical shift of C(22) (44 ppm) shows that the palladium-bound carbon has undergone a drastic hybridization change. Alkylation with n-BuI yields a mixture of the O-substituted [(n-BuOBP)Pd(II)] and the C-substituted [(OBP-n-Bu)Pd(II)], which confirms the ambident nucleophilicity of [(OBP)Pd(II)](-). DFT calculations carried out for six tautomers of oxybenziporphyrin and the 22-methylated palladium species provide further insight into the electronic structure of the ligand and its complexes. Relative energies of the tautomers, increasing in the order [CH,NH,N,NH,O] < [CH,N,NH,N,OH] < [CH(2),N,NH,N,O] < [CH(2),N,N,N,OH], have been used to estimate the accessibility of four limiting delocalization modes postulated for oxybenziporphyrin and its derivatives. The state of macrocyclic aromaticity observed experimentally in the free base and the phenolic aromaticity of the O-protonated tautomer are the most favorable, and the latter has its energy higher by only 13 kcal/mol. The peculiar bonding situation in (OBPMe)Pd(II), which can be inferred from the NMR data, is also predicted by the DFT methods, which show a strongly distorted tetrahedral environment of C(22).

Porphyrins with exocyclic rings. 15.(1) synthesis of quino- and isoquinoporphyrins, aza analogues of the naphthoporphyrins.

Porphyrins with fused isoquinoline and quinoline units have been prepared by the "3 + 1" methodology. 5-Nitroisoquinoline and 6-nitroquinoline condensed with ethyl isocyanoacetate in the presence of a phosphazene base to give isoquino- and quinopyrroles, respectively. Ester saponification and decarboxylation with KOH in ethylene glycol at 190 degrees C gave the parent azatricycles, and these were further condensed with 2 equiv of an acetoxymethylpyrrole to give the corresponding tripyrranes protected at the terminal positions as their tert-butyl esters. In a one-pot procedure, the ester protective groups were cleaved with TFA, and following dilution with dichloromethane, "3 + 1" condensation with a pyrrole dialdehyde and dehydrogenation of the phlorin intermediate with DDQ gave the targeted azanaphthoporphyrins in excellent yields. Although the UV-vis spectra of these new porphyrin systems are unexceptional, they show promise for further functionalization and applications in the development of porphyrin arrays. In addition, a zinc chelate of the isoquinoporphyrin system shows a high degree of regioselective intermolecular interaction/aggregation in chloroform solution that may lead to selectivity in molecular recognition studies.

Porphyrins with exocyclic rings. 14. Synthesis of tetraacenaphthoporphyrins, a new family of highly conjugated porphyrins with record-breaking long-wavelength electronic absorptions.

The Soret band for porphyrins is usually observed in the near-ultraviolet at approximately 400 nm, and few examples of "nonexpanded" porphyrins with this major absorption band at values above 500 nm have previously been reported in the literature. Ring fusion with aromatic ring systems such as naphthalene, anthracene, or phenanthrene generally only produces minor bathochromic shifts to this diagnostic absorption band. In this paper, the synthesis of a series of tetraacenaphthoporphyrins and their metal chelates is reported. The compact nature of the acenaphthylene ring system allows the introduction of meso substituents using the Lindsey methodology. meso-Tetraphenylporphyrin 10a shows the presence of a Soret band at 556 nm, while p-methoxy and p-nitro substituents in 10f and 10g, respectively, further shift this band to 560 and 570 nm. Addition of TFA produces the corresponding dications with slightly higher wavelength Soret bands at 565, 573, and 588 nm. These values compare to 525 nm for the dication of tetraacenaphthylene 8, which lacks the meso-aryl substituents, indicating that steric crowding and its resulting distortion of the macrocyclic conformation is responsible for a significant albeit minor portion of these shifts. The nickel(II), copper(II), and zinc chelates of 10a produce Soret bands at 528, 545, and 558 nm, respectively, demonstrating that the trend for increasing red shifts in metalloporphyrins across the periodic table is retained for this series. The lead(II) chelate 19d gave an additional "hyper" shift that brought the Soret band to 604 nm. A similar red shift could be achieved by introducing four phenylethynyl substituents at the meso positions, and this highly conjugated porphyrin (20) also showed a Soret band at 604 nm, while the corresponding dication afforded this absorption band at 629 nm. The essentially additive "hyper" shift due to lead chelation brought the Soret band for the related lead(II) complex 22d to 642 nm. These effects are by far the largest ever observed for true porphyrins and demonstrate that the Soret band can be fined tuned to virtually any part of the visible spectrum.

Effect of covalent modification on coproporphyrinogen oxidase from chicken red blood cells.

The biosynthesis of heme is a complex multi-step pathway requiring the efforts of eight enzymes. The initial enzymes in the heme biosynthetic pathway have been well characterized in relation to their mechanisms. Coproporphyrinogen oxidase (Copro'gen oxidase) is one of the last three enzymes in the pathway and is one of the least well understood. Copro'gen oxidase converts coproporphyrinogen III to protoporphyrinogen IX via oxidative decarboxylation of the 3- and 8-propionic side chain moieties. To further our understanding of the recognition and binding of substrate, Copro'gen oxidase was partially purified from chicken red blood cell hemolysates then incubated with covalent modifiers of specific amino acids. Incubation with tetranitromethane, p-hydroxyphenylglyoxal, N-acetylimidazole, or trinitrobenzenesulfonic acid resulted in substantial reduction of Copro'gen oxidase activity implying the presence of critical tyrosine, arginine and lysine residues. We conclude that these amino acids play important roles in the enzymic mechanism (for both binding and catalysis) of Copro'gen oxidase.

Proline betaine is a highly effective osmoprotectant for Staphylococcus aureus.

Proline betaine is an osmoprotectant that is at least as effective as glycine betaine, and more effective than L-proline, for various strains of Staphylococcus aureus, and Staphylococcus epidermidis and Staphylococcus saprophyticus. 13C NMR studies revealed that proline betaine accumulated to high levels in osmotically stressed S. aureus, but was also detected in organisms grown in its presence in the absence of osmotic stress. Competition experiments indicated that proline betaine was taken up by the proline transport systems of S. aureus, but not by the high affinity glycine betaine transport system.

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.

Transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine by Salmonella typhimurium.

The transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine (MAHMP) by Salmonella typhimurium was studied using synthetic [methyl-3H3]MAHMP. It was found that an active transport system existed for MAHMP, having Km of 0.07 microM and Vmax 45 nmol.min-1.(g dry wt. cells)-1, that required glucose as a source of energy and was pH and temperature dependent. Uptake was inhibited by cyanide, azide, N-ethylmaleimide, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Uptake was also weakly inhibited by oxythiamine, but not by thiamine, 2-methyl-4-amino-5-aminomethylpyrimidine, or 4-amino-5-hydroxymethylpyrimidine, indicating that the transport system is specific for MAHMP.

Transport of thiamine and 4-methyl-5-hydroxyethylthiazole by Salmonella typhimurium.

The transport of thiamine and 4-methyl-5-hydroxyethylthiazole (MHET), its thiazole moiety, was studied using whole cells of Salmonella typhimurium. It was found that the bacteria possessed an active transport system for thiamine that had Km 0.21 microM and Vmax 33 nmol.min-1.(mg dry wt. cells)-1. Transport of thiamine was glucose dependent, whereas MHET uptake was dependent on both glucose and 2-methyl-4-amino-5-hydroxymethylpyrimidine (MAHMP), the pyrimidine moiety of thiamine. Uptake of both thiamine and MHET was severely curtailed by cyanide, azide, N-ethylmaleimide and carbonyl cyanide m-chlorophenylhydrazone. Oxythiamine inhibited thiamine, but not MHET, uptake and thiamine slightly inhibited MHET uptake. 2-Methyl-4-amino-5-methoxymethylpyrimidine and 4-amino-5-hydroxymethylpyrimidine were unable to replace MAHMP as stimulators of MHET uptake, but 2-methyl-4-amino-5-aminomethylpyrimidine was marginally effective in this regard. Similar results were obtained with attempts to replace MAHMP as a growth requirement for a purD mutant of Salmonella typhimurium. MHET uptake showed saturation kinetics only in the presence of MAHMP, and is not otherwise actively transported.