Facile preparation of optically pure [α-2H]-α-amino acids

Summary Racemic [-²H]--amino acids were prepared by heating the corresponding amino acids (Phe, nor-Leu and Dopa) with 0.05 equivalents of benzaldehyde in deuterated-acetic acid. Based on¹H-nmr measurement, the isotopic purities of these racemized [-²H]--amino acids were found to be higher than 99.5%. Methylation of these isotope-labelled amino acids was achieved in methanol/thionyl chloride without affecting isotopic purity. Optically pure [-²H]--amino acids were obtained in high yield with high enantiomeric excess via alcalase catalysed resolution.     

Nuclear metabolism of benzo(a)pyrene and of (±)-trans-7,8-dihydroxy-7,8,-dihydrobenzo(a)pyrene: Comparative chromatographic analysis of alkylated DNA

Rat liver nuclei were incubated with [¹⁴C]benzo(a)pyrene (BP) or [³H](±)-trans-7,8-dihydrodiol of BP (³H-BP-7,8-diol) in the presence of a NADPH-generating system. The nuclei were able to form from BP the 9,10-, 4,5- and 7,8-dihydrodiols, the 3,6- and 1,6-quinones as well as the 3- and 9-phenols. The total nuclear metabolism was stimulated 11-fold by prior administration to the rats of 3-methylcholanthrene (3MC). BP-7,8-dihydrodiol formation, under these circumstances, was enhanced 29-fold. The rat liver nuclei were also able to form from [³H]BP-7,8-diol, (±)-7β,8α-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 1), (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 2), as well as three unknown metabolites. Diol epoxides 1 and 2 represented 23 and 65% of the total metabolites produced during the control nuclear incubation. Pretreatment of the rats with 3MC resulted in 4-fold increase in nuclear metabolic activity. Under the latter circumstances, the diol epoxides 1 and 2 represented 43 and 38%, respectively, of the total nuclear metabolites. Incubation of liver nuclei with labeled BP or BP-7,8-diol in the presence of NADPH resulted in alkylation of DNA. The alkylated deoxyribonucleosides were separated by Sephadex LH-20 chromatography. Two peaks of radioactivity were noted after incubation with the parent polycyclic hydrocarbon while only one peak was seen after incubation with the diol derivative. These results emphasize the importance of nuclei in the metabolism of BP and in the subsequent alkylation of DNA, reactions which may be related to mutagenesis or carcinogenesis.     

Lack of contribution of covalent benzo[a]pyrene-7,8-quinone–DNA adducts in benzo[a]pyrene-induced mouse lung tumorigenesis

Benzo[a]pyrene (B[a]P) is a potent human and rodent lung carcinogen. This activity has been ascribed in part to the formation of anti-trans-7,8-dihydroxy-7,8-dihydroB[a]P-9,10-epoxide (BPDE)-DNA adducts. Other carcinogenic mechanisms have been proposed: (1) the induction of apurinic sites from radical cation processes, and (2) the metabolic formation of B[a]P-7,8-quinone (BPQ) that can form covalent DNA adducts or reactive oxygen species which can damage DNA. The studies presented here sought to examine the role of stable BPQ-DNA adducts in B[a]P-induced mouse lung tumorigenesis. Male strain A/J mice were injected intraperitoneally once with BPQ or trans-7,8-dihydroxy-7,8-dihydroB[a]P (BP-7,8-diol) at 30, 10, 3, or 0 mg/kg. Lungs and livers were harvested after 24 h, the DNA extracted and subjected to ³²P-postlabeling analysis. Additional groups of mice were dosed once with BPQ or BP-7,8-diol each at 30 mg/kg and tissues harvested 48 and 72 h later, or with B[a]P (50 mg/kg, a tumorigenic dose) and tissues harvested 72 h later. No BPQ or any other DNA adducts were observed in lung or liver tissues 24, 48, or 72 h after the treatment with 30 mg/kg BPQ. BP-7,8-diol gave BPDE-DNA adducts at all time points in both tissues and B[a]P treatment gave BPDE-DNA adducts in the lung. In each case, no BPQ-DNA adducts were detected. Mouse body weights significantly decreased over time after BPQ or BP-7,8-diol treatments suggesting that systemic toxicity was induced by both agents. Model studies with BPQ and N-acetylcysteine suggested that BPQ is rapidly inactivated by sulfhydryl-containing compounds and not available for DNA adduction. We conclude that under these treatment conditions BPQ does not form stable covalent DNA adducts in the lungs or livers of strain A/J mice, suggesting that stable BPQ-covalent adducts are not a part of the complex of mechanisms involved in B[a]P-induced mouse lung tumorigenesis.     

Enzymatic preparation of optically pure (+)-2-azabicyclo[2.2.1]hept-5-en-3-one by (−)-γ-lactamase from Bradyrhizobium japonicum USDA 6

Whole cells of Bradyrhizobium japonicum USDA 6 showed both (+)-γ-lactamase activity and (−)-γ-lactamase activity. Insight into the genome of B. japonicum USDA 6 revealed two potential γ-lactamases: a type I (+)-γ-lactamase and a (−)-γ-lactamase, making it the first strain to contain two totally different enantioselective lactamases. Both recombinant enzymes could easily be used to prepare either optically pure (+)-γ-lactam ((+)-2-azabicyclo[2.2.1]hept-5-en-3-one) or optically pure (−)-γ-lactam ((−)-2-azabicyclo[2.2.1]hept-5-en-3-one), which are versatile synthetic building blocks for the synthesis of various carbocyclic nucleosides and carbocyclic sugar analogues. Bioinformatic analysis showed that the type I (+)-γ-lactamase belongs to the amidase signature family, with 504 amino acids; the (−)-γ-lactamase, which consists of 274 amino acids, belongs to the hydrolase family. Here, we report that B. japonicum USDA contains a (−)-γ-lactamase in addition to a (+)-γ-lactamase, and it is the (−)-γ-lactamase from this strain that is examined in detail in this Letter. Enzymatic synthesis of optically pure (+)-γ-lactam with nearly 50% isolated yield and >99% ee was achieved.     

On the kinetics of the enzyme-catalyzed hydrolysis of axial chiral alkyl allenecarboxylates: preparation of optically active R-(−)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid and its optically pure S-(+)-methylester

Pig liver esterase (PLE) was used for the preparation of optically active alkyl allenecarboxylates with axial chirality. Free and immobilized enzymes were used as biocatalysts for the kinetic resolution of racemic ester substrates. Whereas the biotransformations using the free biocatalyst resulted in moderately to high enantiomeric ratios, the immobilization significantly decreased the E-value. The reaction conditions were optimized with respect to the enantiomeric ratio and scaled up. The enantiomeric ratio (E-value) was thereby enhanced by a factor of four to E=60. Under optimized conditions (free enzyme, addition of acetone as a cosolvent and Triton X-100 as an emulgator) in a preparative scale biotransformation, 282 mg of optically pure S-(+)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid methylester (96% ee, 82% yield) and 257 mg of R-(−)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid (83% ee, 80% yield) could be synthesized from the racemic substrate.     

Structure and mechanism of error-free replication past the major benzo[a]pyrene adduct by human DNA polymerase κ

Benzo[a]pyrene (BP) is a well-known and frequently encountered carcinogen which generates a bulky DNA adduct (+)-trans-10S-BP-N²-dG (BP-dG) in cells. DNA polymerase kappa (polκ) is the only known Y-family polymerase that bypasses BP-dG accurately and thus protects cells from genotoxic BP. Here, we report the structures of human polκ in complex with DNA containing either a normal guanine (G) base or a BP-dG adduct at the active site and a correct deoxycytidine. The structures and supporting biochemical data reveal a unique mechanism for accurate replication by translesion synthesis past the major bulky adduct. The active site of polκ opens at the minor groove side of the DNA substrate to accommodate the bulky BP-dG that is attached there. More importantly, polκ stabilizes the lesion DNA substrate in the same active conformation as for regular B-form DNA substrates and the bulky BPDE ring in a 5′ end pointing conformation. The BP-dG adducted DNA substrate maintains a Watson–Crick (BP-dG:dC) base pair within the active site, governing correct nucleotide insertion opposite the bulky adduct. In addition, polκ''s unique N-clasp domain supports the open conformation of the enzyme and the extended conformation of the single-stranded template to allow bypass of the bulky lesion. This work illustrates the first molecular mechanism for how a bulky major adduct is replicated accurately without strand misalignment and mis-insertion.     

Mutagenicity and metabolism of dimethylnitrosamine and benzo[α]pyrene in tissue homogenates from inbred syrian hamsters treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls

There are significant differences between mice and hamsters in polycyclic hydrocarbon and nitrosamine metabolism. Homogenates of liver, lung and intestinal mucosa from 6 strains of Syrian golden hamster were compared for their ability to metabolize benzo[α]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains MHA/SsLak, LSH/SsLak, CB/SsLak, PD4/Lak LHC/Lak and Lak:LVG (SYR) were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagens. Dimethyl-nitrosamine demethylase (DMND) was assayed using 1 mM DMN as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured using benzo[α]pyrene as substrate. MC does not induce AHH activity in hamster liver, but is an excellent inducer of enzymes converting BP to mutagens. This lack of correlation between increased AHH activity and increased metabolism of BP to mutagen in liver is in marked contrast to correlations seen in mice. MC induces AHH in hamster lung and intestinal mucosa. AR induces AHH in liver, lung and intestinal mucosa. Activity of DMND in liver is not affected by treatment of hamsters with BP or AR, but is repressed approx. 30% by treatment with MC.     

Synthesis of indolizidines from optically pure α-amino acids via stereocontrolled rhodium-catalyzed hydroformylation of N-allylpyrroles

Indolizidine alkaloids have attracted considerable attention because of their vast array of structural diversity and varied biological activity. This article relates the results that we obtained in the field of the total synthesis of indolizidines from α-amino acids, based on the rhodium-catalyzed hydroformylation of N-allylpyrroles intermediates. The formed pyrrolylbutanals undergo an intramolecular cyclodehydration to 5,6-dihydroindolizines, which are fully hydrogenated to indolizidines. All reaction sequences are stereocontrolled: indeed, the chiral center in the starting amino acid is transferred into 5,6-dihydroindolizine moiety with complete stereochemical integrity and the new stereogenic center at C9 carbon atom in the final indolizidines is generated in only one configuration.     

Relationship between excision repair and the cytotoxic and mutagenic effect of the ‘anti’ 7,8-diol-9,10-epoxide of benzo[a]pyrene in human cells

The cytotoxic and mutagenic effect of (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE) in normally excision diploid human cells treated just prior to onset of S was compared with that of cells allowed ～ 16 h for excision repair before onset of S and with that observed in excision-deficient serodema pigmentosum (SP12BE) cells. The cells were synchronized by release from density inhibition of cell replication. DNA synthesis began ～ 22 h after the cells were plated at lower density (i.e., 1.4 × 10⁴ cells/cm²). The frequency of thioguanine-resistant mutants induced in normal cells treated just prior to onset of S was ～ 12- to 16-fold higher than that observed in cells treated in early G₁ or treated in G₀ (confluence) and then plated at lower density. The frequency approximated that expected for XP12BE cells from extrapolation of data obtained at lower doses. The frequency of mutants measured in normal cells treated in exponential growth was also much higher than that in the cells treated in early G₁ or in G₀, No such difference could be seen in XP12BE cells treated in exponential growth or in G₀. In contrast to the mutagenicity data in the normal cells, there was no significant difference in the slope of the survival curve of normal cells treated at various times prior to S phase at low densities. However, normal cells treated even at the onset of S exhibited survival equal to XP12BE cells give a 4- to 5-fold lower dose. The data support the hypothesis that DNA synthesis is the cellular event which converts unexcised DNA lesions into mutations. However, they indicate that S is not the event primarily responsible for translating DNA damage into cell death. Accompanying studies on the rate of excision of anti BPDE adducts from the normal cells during the period priot to S support the conclusions.     

Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis: application of engineered enzymes in a 2-propanol–water medium

We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)–water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The K _m and V _max values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA–water medium.     

Irreversible binding of [3H] (−) N-chloroethylnorapomorphine to mammalian brain tissue

A proposed dopamine (DA) receptor labeling agent, [³H] (?) N-chloroethylnorapomorphine (³H-NCA) underwent relatively little chemical change at 25°C and pH 6.4 up to an hour of incubation. At low (nM) concentrations it bound rapidly and avidly to a membrane preparation of calf caudate nucleus, but the binding did not saturate over two hours of incubation or at ligand concentrations between 0.2 nM and 10 μM. While similarly bound [³H]-(?) apomorphine was rapidly displaced by DA and other agents that interact with DA receptors, ³H-NCA could not be displaced by unlabeled DA, apomorphine and (+)butaclamol, nor by denaturation of the tissue with trichloracetic acid (TCA). There was no evidence of selectivity of binding of ³H-NCA in regions of rat brain, and binding occurred even to TCA-denatured caudate tissue. Catechol-aporphines prevented binding of ³H-NCA to calf caudate membranes by up to 30%, but this effect was not stereoselective and was lost at concentrations of ³H-NCA above 100 nM. In contrast, DA and ADTN as well as neuroleptics and adrenergic agonists had no such effect. The results suggest that while ³H-NCA may bind irreversibly, and possibly covalently, it does not have high selectivity for labeling dopamine D-3 or D-2 receptor sites, but may be partially selective for an aporphine binding site.     

Combinatorial screening of polymer precursors for preparation of benzo[α] pyrene imprinted polymer: an ab initio computational approach

A combinatorial screening procedure was used for the selection of polymer precursors in the preparation of molecularly imprinted polymer (MIP), which is useful in the detection of the air pollution marker molecule benzo[a]pyrene (BAP). Molecular imprinting is a technique for the preparation of polymer materials with specific molecular recognition receptors. The preparation of imprinted polymers requires polymer precursors such as functional monomer, cross-linking monomer, solvent, an initiator of polymerization and thermal or UV radiation. A virtual library of functional monomers was prepared based on interaction binding scores computed using HyperChem Release 8.0 software. Initially, the possible minimum energy conformation of the monomers and BAP were optimized using the semi-empirical (PM3) quantum method. The binding energy between the functional monomer and the template (BAP) was computed using the Hartree-Fock (HF) method with 6-31 G basis set, which is an ab initio approach based on Moller-Plesset second order perturbation theory (MP2). From the computations, methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) were selected for preparation of BAP imprinted polymer. The larger interaction energy (ΔE) represents possibility of more affinity binding sites formation in the polymer, which provides high binding capacity. The theoretical predictions were complimented through adsorption experiments. There is a good agreement between experimental binding results and theoretical computations, which provides further evidence of the validity of the usefulness of computational screening procedures in the selection of appropriate MIP precursors in an experiment-free way.     

Metabolism of benzo[a]pyrene: Effect of 3-methylcholanthrene pretreatment on metabolism by microsomes from lungs of genetically “responsive” and ”nonresponsive” mice

The metabolism of [¹⁴C]benzo[a]pyrene by microsomes from the lungs of normal and 3-methylcholanthrene-treated DBA/2J, C57BL/6J, and A/HeJ mouse strains was quantitatively analyzed by high-pressure liquid chromatography. The ratio of dihydrodiols of benzo[a]pyrene to total metabolites formed was greater with lung microsomes than with liver microsomes in all three strains. The ratio of epoxide hydrase to monooxygenase activity in mouse lung was shown to be considerably higher than in mouse liver. Benzo[a]pyrene metabolism by control lung microsomes showed some strain differences. C57BL/6J and A/HeJ mice formed twice as much dihydrodiols as a percentage of total metabolism compared to DBA/2J mice. DBA/2J mice produced somewhat less phenol 2 fraction and considerably more quinone 1 and 2 fractions than the other two mouse strains as a percentage of total metabolism. Treatment of C57BL/6J and DBA/2J mice with 3-methylcholanthrene resulted in a 20-fold increase in the metabolism of benzo[a]pyrene, while A/HeJ mice were induced more than 50-fold. The profiles of metabolites from the 3-methylcholanthrene-induced animals were nearly identical in all three mouse strains.     

α-Amino-oximes based on optically pure limonene: a new ligands family for ruthenium-catalyzed asymmetric transfer hydrogenation

A new family of bifunctional, optically pure α-amino-oxime ligands based on (R)-limonene has been synthesized and used as chiral inducers for enantioselective hydrogen transfer reactions on various ketones in the presence of ruthenium catalysts. The X-ray structures of Ru-amino-oxime complexes are also described.     

Cloning,characterization and tissue distribution of a pi-class glutathione S-transferase from clam (Venerupis philippinarum): Response to benzo[α]pyrene exposure

Glutathione S-transferases (GSTs) are phase II enzymes involved in major detoxification reactions of xenobiotics in many organisms. In this study, a full-length cDNA of GST-pi was cloned from the gill of Venerupis philippinarum by rapid amplification of cDNA ends (RACE) method for the first time. The full-length cDNA of V. philippinarum GST-pi (denoted as VpGSTp) was 1142 bp, with a 5′ untranslated region (UTR) of 87 bp, a 3′ UTR of 438 bp, and an open reading frame (ORF) of 618 bp encoding a protein of 205 amino acid residues with an estimated molecular mass of 23.9 kDa and an predicted isoelectric point (pI) of 7.9. The comparison of the deduced amino acid sequences with GSTs from other species showed that the enzyme belongs to the pi-class, and the amino acids defining the binding sites of glutathione (G-site) and for xenobiotic substrates (H-site) were highly conserved. Tissue distribution analysis of the VpGSTp mRNA revealed that the GST-pi expression level was observed higher in gill, adductor muscle, mantle and foot while lower in digestive gland. Using quantitative real-time PCR, the dose- and time-related effects of benzo[α]pyrene (B[α]P) on VpGSTp mRNA expression were investigated in gills and digestive gland. The results showed that a time-dependant increase in the expression of VpGSTp was induced by B[α]P and appeared a good linear relationship with B[α]P concentrations. All these results suggested that GST-pi in bivalve had an antioxidant role and VpGSTp expression may be a useful biomarker candidate for monitoring environmental contaminants such as PAHs.     

Induction of cytochrome P-450 and P-448 in the outer membrane of mouse liver nuclei by phenobarbital and benzo [α-]pyrene

This investigation confirms the presence of the inducible mixed function hydroxylase enzyme system in nuclear membranes. The cytochrome P-450 spectrum and demethylase activity, markers of the enzyme system, were used to define its localization to the outer membrane envelope. Intact BALB/c mouse liver nuclei isolated and purified in Mg⁺⁺ sucrose media of low ionic strength gave CO-dithionite reduced difference spectra of cytochrome P-450 and P-448. Phenobarbital induced P-450 by 40% while the carcinogenic hydrocarbon, benzo [α] pyrene, induced P-448 twofold. A corresponding increase was also observed in the microsomes of the same tissue preparations. No microsomal contamination of nuclear preparations was found. Intact nuclei stripped of their outer membrane by 0.5% Triton X-100 treatment resulted in a striking absence of the P-450 which, however, was found to be present in isolated outer nuclear membranes.     

Benzo[a]pyrene, aflatoxine B₁ and acetaldehyde mutational patterns in TP53 gene using a functional assay: relevance to human cancer aetiology

Mutations in the TP53 gene are the most common alterations in human tumours. TP53 mutational patterns have sometimes been linked to carcinogen exposure. In hepatocellular carcinoma, a specific G>T transversion on codon 249 is classically described as a fingerprint of aflatoxin B(1) exposure. Likewise G>T transversions in codons 157 and 158 have been related to tobacco exposure in human lung cancers. However, controversies remain about the interpretation of TP53 mutational pattern in tumours as the fingerprint of genotoxin exposure. By using a functional assay, the Functional Analysis of Separated Alleles in Yeast (FASAY), the present study depicts the mutational pattern of TP53 in normal human fibroblasts after in vitro exposure to well-known carcinogens: benzo[a]pyrene, aflatoxin B(1) and acetaldehyde. These in vitro patterns of mutations were then compared to those found in human tumours by using the IARC database of TP53 mutations. The results show that the TP53 mutational patterns found in human tumours can be only partly ascribed to genotoxin exposure. A complex interplay between the functional impact of the mutations on p53 phenotype and the cancer natural history may affect these patterns. However, our results strongly support that genotoxins exposure plays a major role in the aetiology of the considered cancers.