Investigation of the Substrate Range of CYP199A4: Modification of the Partition between Hydroxylation and Desaturation Activities by Substrate and Protein Engineering

The cytochrome P450 enzyme CYP199A4, from Rhodopseudomonas palustris HaA2, can efficiently demethylate 4‐methoxybenzoic acid. It is also capable of oxidising a range of other related substrates. By investigating substrates with different substituents and ring systems we have been able to show that the carboxylate group and the nature of the ring system and the substituent are all important for optimal substrate binding and activity. The structures of the veratric acid, 2‐naphthoic acid and indole‐6‐carboxylic acid substrate‐bound CYP199A4 complexes reveal the substrate binding modes and the side‐chain conformational changes of the active site residues to accommodate these larger substrates. They also provide a rationale for the selectivity of product oxidation. The oxidation of alkyl substituted benzoic acids by CYP199A4 is more complex, with desaturation reactions competing with hydroxylation activity. The structure of 4‐ethylbenzoic acid‐bound CYP199A4 revealed that the substrate is held in a similar position to 4‐methoxybenzoic acid, and that the C^β C? H bonds of the ethyl group are closer to the heme iron than those of the C^α (3.5 vs. 4.8 Å). This observation, when coupled to the relative energies of the reaction intermediates, indicates that the positioning of the alkyl group relative to the heme iron may be critical in determining the amount of desaturation that is observed. By mutating a single residue in the active site of CYP199A4 (Phe185) we were able to convert the enzyme into a 4‐ethylbenzoic acid desaturase.     

The Stereoselective Oxidation of para-Substituted Benzenes by a Cytochrome P450 Biocatalyst

The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates >60 nmol.(nmol-CYP)⁻¹.min⁻¹) and with total turnover numbers of up to 20,000. Ethyl α-hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to >98 %). The crystal structure of 4-methoxybenzoic acid-bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.     

The crystal structures of 4-methoxybenzoate bound CYP199A2 and CYP199A4: structural changes on substrate binding and the identification of an anion binding site

The crystal structures of the 4-methoxybenzoate bound forms of cytochrome P450 enzymes CYP199A2 and CYP199A4 from the Rhodopseudomonas palustris strains CGA009 and HaA2 have been solved. The structures of these two enzymes, which share 86% sequence identity, are very similar though some differences are found on the proximal surface. In these structures the enzymes have a closed conformation, in contrast to the substrate-free form of CYP199A2 where an obvious substrate access channel is observed. The switch from an open to a closed conformation arises from pronounced residue side-chain movements and alterations of ion pair and hydrogen bonding interactions at the entrance of the access channel. A chloride ion bound just inside the protein surface caps the entrance to the active site and protects the substrate and the heme from the external solvent. In both structures the substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of these enzymes for oxidative demethylation of the substrate. Mutagenesis studies on CYP199A4 highlight the involvement of hydrophobic (Phe185) and hydrophilic (Arg92, Ser95 and Arg243) amino acid residues in the binding of para-substituted benzoates by these enzymes.     

Tandem Acid/Pd-Catalyzed Reductive Rearrangement of Glycol Derivatives

Herein, we describe the acid/Pd-tandem-catalyzed transformation of glycol derivatives into terminal formic esters. Mechanistic investigations show that the substrate undergoes rearrangement to an aldehyde under [1,2] hydrogen migration and cleavage of an oxygen-based leaving group. The leaving group is trapped as its formic ester, and the aldehyde is reduced and subsequently esterified to a formate. Whereas the rearrangement to the aldehyde is catalyzed by sulfonic acids, the reduction step requires a unique catalyst system comprising a Pd^II or Pd⁰ precursor in loadings as low as 0.75 mol % and α,α′-bis(di-tert-butylphosphino)-o-xylene as ligand. The reduction step makes use of formic acid as an easy-to-handle transfer reductant. The substrate scope of the transformation encompasses both aromatic and aliphatic substrates and a variety of leaving groups.     

Pyrene.pyrene complexes at the active site of cytochrome P450 3A4: evidence for a multiple substrate binding site

Cytochrome P450 monooxygenases (CYPs) metabolize nearly all drugs and toxins. Recently, it has become clear that CYPs exhibit both homotropic and heterotropic allosteric kinetics for many substrates. However, the mechanism of cooperative kinetics has not been established for any specific human CYP/substrate combination. Suggested mechanisms include binding of multiple substrates within distinct, static, subsites of a single large active site or binding of multiple substrates within a single fluid active site. CYP3A4 hydroxylates pyrene with positive cooperativity. Therefore, experiments were designed to exploit the fluorescence properties of pyrene, which diagnostically distinguish between pyrene.pyrene complexes versus spatially separated pyrene substrates. Pyrene complexes (excimers) yield an emission spectrum clearly distinct from pyrene monomers. In lipid-free aqueous/glycerol solutions of CYP3A4, addition of pyrene affords a concentration-dependent low-spin to high-spin conversion of the CYP3A4 heme prosthetic group, indicating occupancy of the active site by pyrene. Under the same conditions, in the presence of CYP3A4 but not other heme proteins, the excimer/monomer ratio (E/M) of pyrene was decreased in emission spectra, compared to pyrene alone. However, excitation spectra indicate a CYP3A4-dependent increase in the wavelength shift for the excimer excitation spectrum versus the monomer excitation spectrum, as well as changes in the excimer excitation peak shape and vibronic structure. These changes are reversed by the CYP3A4 substrate testosterone. Together, the results demonstrate that pyrene.pyrene ground-state complexes occupy the CYP3A4 active site, and they provide the first spectroscopic evidence for substrate complexes within a single fluid active site. Functional implications include the possibility that turnover rate, regioselectivity, and stereoselectivity of the reaction are determined by the substrate.substrate complex rather than individual substrates.     

Catalytic oxidative reactions of organic compounds by nitrogen‐containing copper complexes

The dimeric copper(II) complex di‐µ‐chloro‐bis[chloro(di‐3,5‐dimethylpyrazole)copper(II)] (A) in the presence of co‐oxidant hydrogen peroxide acts as a catalyst for the oxidation of benzylic alcohols to give the corresponding aldehydes. In the presence of hydrogen peroxide it also catalyses the oxidation reaction of 2,6‐dimethylphenol to 4,4′‐dihydroxy‐3,5,3′,5′‐tetramethylbiphenyl. The oxidative reactions by bis‐pyridinium tetrachlorocopper(II) (B) in the presence of hydrogen peroxide were compared for similar catalytic reactions of A, and it is observed that B can catalyse the oxidation of aromatic diols, 2,6‐dimethylphenol and thiophenol, but is not suitable for oxidation of benzylic alcohols. Bis‐(N‐phenyl‐3,5‐dimethylpyrazole)copper(II) nitrate monohydrate (C) has a suitable redox potential for one‐electron oxidation. It can oxidize ferrocene to the ferricinium cation, and it can liberate bromine from tetra‐alkylammonium bromides. The complex is catalytically effective for the oxidation of different aromatic and aliphatic aldehydes to the corresponding carboxylic acids. The compound is also effective in transforming benzylic amine to benzylalcohol and benzaldehyde. It can also oxidize diphenylmethane to give benzophenone and diphenylmethanol. It is observed that in each of these complexes a quasi‐reversible Cu(I)–Cu(II) species is present and facilitates the single‐electron oxidation process. Copyright © 2004 John Wiley & Sons, Ltd.     

Study of the Synthesis of Dextran Derivatives

Abstract

The synthesis of biologically active high-molecular compounds offers an interesting subject for study in polymer chemistry. Dextran, one of the best blood plasma expanders, is a good product for use in synthesizing biologically active water-soluble polymers. This paper discusses the synthesis of water-soluble derivatives of dextran which can be used as drug carriers or as independent physiologically active compounds. The synthesis of these derivatives involved oxidation of dextran, O-alkylation of dextran, subsequent transformation of ethers of dextran containing reactive functional groups, and graft polymerization. Periodate oxidation of dextran resulted in the production of a derivative of dextran containing aldehyde groups: dialdehydedextran. Subsequent oxidation of dialdehyde-dextran with sodium chlorite permitted the introduction of carboxyl groups into the macromolecule of dextran. In order to introduce sulfonic acid, phosphonic, mercapto, chlorhydrinic, cyano, and aromatic amino groups into the macromolecule of dextran, dextran was alkylated with various alkylating reagents. The synthesized products were sulfopropyl, phosphonomethyl, mercaptoethyl, 3-chloro-2-hydroxypropyl, cyanoethyl, and 2-(3′-amino-4′-methoxyphenyl)-sulfonylethyl ethers of dextran. Treatment of the 3-chloro-2-hydroxypropyl ether of dextran with ammonia, amines, and aliphatic and aromatic amino acids produced ethers of dextran containing primary, secondary, and tertiary amino groups and quaternary ammonium groups, as well as residual aliphatic and aromatic amino acids.

Cyanethyl ether of dextran was used to synthesize derivatives of dextran containing thioamide and hydrazidine groups.

Graft copolymers of dextran and polyacrylic acid and of dextran and poly-2-methyl-5-vinylpyridine were synthesized. Redox systems were utilized to initiate graft copolymerizatLon with tetravalent cerium compounds used as oxidants and pentavalent vanadium with dextran or 2-(3′-amino-4′-methoxyphenyl)-sulfonylethyl ether of dextran used as reductant. This method produced graft copolymers with short graft chains.

The availability of the above derivatives of dextran has permitted the linking of drugs to dextran by different types of chemical bonds.     

Dioxomolybdenum(VI) complexes of hydrazones of two substituted salicylaldehydes: synthesis,structures, and catalytic properties

Dioxomolybdenum(VI) complexes [MoO₂L(CH₃OH)] (L?=?L¹?=?N′-(2-hydroxy-5nitrobenzylidene)isonicotinoylhydrazide for 1, L?=?L²?=?N′-(4-diethylamino-2-hydroxybenzylidene)-4-methylbenzohydrazide for 2) were prepared and structurally characterized by physicochemical and spectroscopic methods and single-crystal X-ray determination. Mo in the complexes is octahedrally coordinated. Both complexes show effective catalysis in oxidation of cyclohexene, vinylbenzene, 1-butylene, and 1-pentene, to their corresponding epoxides. In general, high epoxide yields (over 89%) and selectivity (100%) were observed for all aliphatic and aromatic substrates.     

A study of the acylation of 4-hydroxy-6-methyl-2-pyrone and 4 hy(lroxy-6-phenyl-2-pyrone

The acylation of 4-hydroxy-6-melhyl-2-pyrone (1) and 4-hydroxy-6-phenyl-2-pyrone (XXII) with aliphatic acid anhydrides, or with aliphatic acid chlorides in trifluoroacetic acid, has been found to product; first the corresponding ester, which then rearranges, partially or wholly, depending on the conditions and on the nature of the acyl moiety, to form the corresponding 3-acylpyrone. With aromatic acid chlorides in pyridine or in trichloroacetic acid only the corresponding ester was obtained. These esters, however, could be rearranged to the corresponding 3-acylpyrones with aluminum chloride. Acetoaeetylation of 1 and 4-hydroxycoumarin (XXIX) with diketene gave, in both cases, the C-acetoacetylated product.     

Asymmetric benzophenone-series aromatic diamines and related soluble polyimides

Asymmetric aromatic diamines of the benzophenone series (3,4′-diamino-4-methylbenzophenone and 3,4′-diamino-4-methoxybenzophenone) were synthesized via the Friedel-Crafts reaction of 4-nitrobenzoyl chloride with an equimolar amount of toluene or anisole; the subsequent nitration of the products (4′-nitro-4-methylbenzophenone and 4′-nitro-4-methoxybenzophenone), yielding 3,4′-dinitro-4-methylbenzophenone and 3,4′-dinitro-4-methoxybenzophenone; and the reduction of the latter compounds. The high-temperature polycyclocondensation of the above diamines with aromatic tetracarboxylic dianhydrides in phenol solvents gave methyl-and methoxy-substituted aromatic polyimides soluble in phenol and amide solvents.     

New approaches to reversible crosslinking in epoxy resins

The objective of the research is to investigate epoxy resins where crosslinks formed in curing reactions can be cleaved and reformed without significant sacrifice in resin properties. Experimental disulfide-containing crosslinking agents have been selected for the study, including dihydrazides of dithiodicarboxylic acids (aliphatic and aromatic) and dithioaromatic diamines. Conditions for curing reactions and for subsequent cleavage of crosslinks by reduction have been determined in model systems and confirmed for a prototype epoxy resin. A comparative evaluation of cure kinetics, crosslink density, and thermal and mechanical properties has been carried out for resin cured with aromatic curing agents (dithiodianiline and methylenedianiline) and with aliphatic compounds (3, 3′-dithiopropionic acid dihydrazide and hexamethylenediamine). It has been shown that in resin fully cured with disulfide-containing crosslinking agents, crosslinks are ruptured by reduction, and re-established by oxidation under mild conditions, or by reaction of thiol groups with bifunctional alkylating agents.     

The Syntheses and Reactions of 3‐Arylsydnone‐4‐carboxamide Phenylhydrazones

Reactions of aniline with 3‐arylsydnone‐4‐carbohydroximic acid chlorides ( 1 ) gave the de sired substitution products 5 . 3‐Arylsydnone‐4‐carboxamide phenylhydrazones ( 7 ) were obtained unexpectedly by the reaction of carbohydroximic acid chlorides 1 with phenylhydrazine in suitable conditions. Compounds 7 could react with both aromatic and aliphatic aldehydes in the presence of acid catalyst to give 3‐aryl‐4‐(1′‐phenyl‐5′‐substituted‐1′,2′,4′‐triazol‐3′‐yl)sydnones ( 11 ).     

A study of the conditions of the formation of the cis and trans isomers of N-substituted 4-aminothiolan-3-ol 1,1-dioxides

A mixture of cis- and trans-4-alkyl(heteryl, aryl)aminothiolan-3-ol 1,1-dioxides and (3-hydroxy-1,1-dioxothiolan-4-ylamino)acetic acids have been obtained by the reaction of 2,3-dihydrothiophene-3-ol 1,1-dioxide with aliphatic, heterocyclic, and aromatic amines and aminoacetic acid. The structures of the compounds isolated has been shown by chemical and spectral methods.Translated from Khimiya Geterotskiklicheskikh Soedinenii, No. 4, pp. 475–478, April, 1980.     

Albumin-directed stereoselective reduction of 1,3-diketones and β-hydroxyketones to anti diols

The reduction of 1,3-diketones and β-hydroxyketones with NaBH(4) in aqueous acetonitrile is highly stereoselective in the presence of stoichiometric amounts of bovine or human albumin, giving anti 1,3-diols with d.e. up to 96%. The same reaction, without albumin, gives syn and anti 1,3-diols in approximately 1:1 ratio. The presence of an aromatic carbonyl group is essential for diastereoselectivity in the NaBH(4)/albumin reduction of both 1,3-diketones and β-hydroxyketones. Thus, 3-hydroxy-1-(p-tolyl)-1-butanone is stereoselectively reduced in the presence of albumin, while reduction of its isomer 4-(p-tolyl)-4-hydroxy-2-butanone is not stereoselective. The albumin-controlled reduction is not stereospecific as both enantiomers of 1-aryl-3-hydroxy-1-butanones are reduced to diols with identical stereoselectivities. Circular dichroism of the bound substrates confirms that aromatic ketones are recognized by the protein's IIA binding site. Binding studies also suggest that 1,3-diketones are recognized in their enol form. From the effect of pH on binding of a diketone it is concluded that, in the complex with the substrate, ionizable residues His242 and Lys199 are in the neutral and protonated forms, respectively. A homology model of BSA was obtained and docking of model substrates confirms the preference of the protein for aromatic ketones. Modelling of the complexes with the substrates also allows us to propose a mechanism for the reduction of 1,3-diketones in which the chemoselective reduction of the first (aliphatic) carbonyl is followed by the diastereoselective reduction of the second (aromatic) carbonyl. The role of albumin is thus a combination of chemo- and stereocontrol.     

Preparation and properties of aromatic–aliphatic copolyamides from aromatic diisocyanates and dicarboxylic acids

Aromatic–aliphatic random copolyamides of high molecular weights were prepared by the high-temperature solution polycondensation from a combination of aromatic diisocyanates, 4,4′-methylenedi(phenyl isocyanate), and 2,4-tolylene diisocyanate, and a mixture of isophthalic acid and aliphatic dicarboxylic acids with 4–10 methylene groups. Reaction conditions, such as solvent, temperature, time, and catalyst were studied to determine the optimum conditions for the preparation of high molecular weight polymers. Glass transition temperatures of the copolyamides were in the range of 131–244°C and varied with combination and composition of the diisocyanates and dicarboxylic acids used. The copolyamides prepared from 2,4-tolylene diisocyanate had greater solubility and higher glass transition temperatures than those obtained from 4,4′-methylenedi(phenyl isocyanate).     

Regioselective Hydroxylation of C12–C15 Fatty Acids with Fluorinated Substituents by Cytochrome P450 BM3

We demonstrate herein that wild‐type cytochrome P450 BM3 can recognize non‐natural substrates, such as fluorinated C₁₂–C₁₅ chain‐length fatty acids, and show better catalysis for their efficient conversion. Although the binding affinities for fluorinated substrates in the P450 BM3 pocket are marginally lower than those for non‐fluorinated substrates, spin‐shift measurements suggest that fluoro substituents at the ω‐position can facilitate rearrangement of the dynamic structure of the bulk‐water network within the hydrophobic pocket through a micro desolvation process to expel the water ligand of the heme iron that is present in the resting state. A lowering of the Michaelis–Menten constant (K_m), however, indicates that fluorinated fatty acids are indeed better substrates compared with their non‐fluorinated counterparts. An enhancement of the turnover frequencies (k_cat) for electron transfer from NADPH to the heme iron and for C? H bond oxidation by compound I (Cpd I) to yield the product suggests that the activation energies associated with going from the enzyme–substrate (ES state) to the corresponding transition state (ES^≠ state) are significantly lowered for both steps in the case of the fluorinated substrates. Delicate control of the regioselectivity by the fluorinated terminal methyl groups of the C₁₂–C₁₅ fatty acids has been noted. Despite the fact that residues Arg47/Tyr51/Ser72 exert significant control over the hydroxylation of the subterminal carbon atoms toward the hydrocarbon tail, the fluorine substituent(s) at the ω‐position affects the regioselective hydroxylation. For substrate hydroxylation, we have found that fluorinated lauric acids probably give a better structural fit for the heme pocket than fluorinated pentadecanoic acid, even though pentadecanoic acid is by far the best substrate among the reported fatty acids. Interestingly, 12‐fluorododecanoic acid, with only one fluorine atom at the terminal methyl group, exhibits a comparable turnover frequency to that of pentadecanoic acid. Thus, fluorination of the terminal methyl group introduces additional interactions of the substrate within the hydrophobic pocket, which influence the electron transfers for both dioxygen activation and the controlled oxidation of aliphatics mediated by high‐valent oxoferryl species.     

Enantioselective hydrogenation of α,β-unsaturated carboxylic acids over cinchonidine-modified Pd catalysts: effect of substrate structure on the adsorption mode

Systematic variation of the substrate structure in the enantioselective hydrogenation of α,β-unsaturated carboxylic acids over cinchonidine-modified Pd catalysts indicated that aryl substituents in β position changed the adsorption mode of the substrates on the metal surface and eventually the configuration of the products formed in excess as compared to aliphatic substrates. The configuration of the products formed in excess by hydrogenation of eight α,β-unsaturated carboxylic acids, of which two have not been described yet, indicated that the substrates bearing aromatic ring in β position were adsorbed on the opposite face of the CC group compared to acids having aliphatic group in β position.     

Oxidation of carboxylic acids by horseradish peroxidase results in prosthetic heme modification and inactivation

Hemoproteins are powerful oxidative catalysts. However, despite the diversity of functions known to be susceptible to oxidation by these catalysts, it is not known whether they can oxidize carboxylic acids to carboxylic radicals. We report here that incubation of horseradish peroxidase (HRP) at acidic pH with H(2)O(2) in acetate buffer results in rapid modification of the heme group and loss of catalytic activity. Mass spectrometry and NMR indicate that an acetoxy group is covalently bound to the delta-meso-carbon in the modified heme. A heme with a hydroxyl group on the 8-methyl is also formed as a minor product. These reactions do not occur if protein-free heme and H(2)O(2) are co-incubated in acetate buffer, if the HRP reaction is carried out at pH 7, in the absence of H(2)O(2), or if citrate rather than acetate buffer is used. A similar heme modification is observed in incubations with n-caproic and phenylacetic acids. A mechanism involving oxidation of the carboxyl group to a carboxylic radical followed by addition to the delta-meso-position is proposed. This demonstration of the oxidation of a carboxylic acid solidifies the proposal that a carboxylic radical mediates the normal covalent attachment of the heme to the protein in the mammalian peroxidases and CYP4 family of P450 enzymes. The hemoprotein-mediated oxidation of carboxylic acids, ubiquitous natural constituents, may play other roles in biology.     

Tryptophan in horseradish peroxidase

Fluorescent derivatives of horseradish peroxidase C were prepared by replacing protoheme by protoporphyrin or mesoporphyrin. Calculations according to F?rster on energy transfer allowed the determination of the distances of greater than 2.2 nm between tryptophan and porphyrin (heme) and greater than 2 nm between tryptophan and substrate-binding site. The modification of the single tryptophan with 2-hydroxy-5-nitrobenzyl bromide (Koshland's reagent) did not affect the enzyme's activity towards hydrogen peroxide or ascorbate. Modified and unmodified peroxidase showed the same affinity for aromatic substrates.