Molecular dissection of the biosynthetic relationship between phthiocerol and phthiodiolone dimycocerosates and their critical role in the virulence and permeability of Mycobacterium tuberculosis

Phthiocerol dimycocerosates and related compounds are important molecules in the biology of Mycobacterium tuberculosis, playing a key role in the permeability barrier and in pathogenicity. Both phthiocerol dimycocerosates, the major compounds, and phthiodiolone dimycocerosates, the minor constituents, are found in the cell envelope of M. tuberculosis, but their specific roles in the biology of the tubercle bacillus have not been established yet. According to the current model of their biosynthesis, phthiocerol is produced from phthiodiolone through a two-step process in which the keto group is first reduced and then methylated. We have previously identified the methyltransferase enzyme that is involved in this process, encoded by the gene Rv2952 in M. tuberculosis. In this study, we report the construction and biochemical analyses of an M. tuberculosis strain mutated in gene Rv2951c. This mutation prevents the formation of phthiocerol and phenolphthiocerol derivatives, but leads to the accumulation of phthiodiolone dimycocerosates and glycosylated phenolphthiodiolone dimycocerosates. These results provide the formal evidence that Rv2951c encodes the ketoreductase catalyzing the reduction of phthiodiolone and phenolphthiodiolone to yield phthiotriol and phenolphthiotriol, which are the substrates of the methyltransferase encoded by gene Rv2952. We also compared the resistance to SDS and replication in mice of the Rv2951c mutant, deficient in synthesis of phthiocerol dimycocerosates but producing phthiodiolone dimycocerosates, with those of a wild-type strain and a mutant without phthiocerol and phthiodiolone dimycocerosates. The results established the functional redundancy between phthiocerol and phthiodiolone dimycocerosates in both the protection of the mycobacterial cell and the pathogenicity of M. tuberculosis in mice.     

Distribution of some mycobacterial waxes based on the phthiocerol family

Characteristic waxes, based on methoxy and keto long-chain diols, members of the phthiocerol family, have been isolated from representatives of Mycobacterium bovis, M. kansasii, M. marinum, M. microti and M. tuberculosis. M. kansasii produced essentially di-esters of the ketodiol phthiodiolone A, but the remaining species also had waxes based on the methoxy-diols phthiocerol A and phthiocerol B. Gas chromatography of derivatives of the components of the waxes showed that the phthiocerol A components from M. bovis, M. microti and M. microti and M. tuberculosis were qualitatively similar, being mainly C34 and C36, but potentially significant differences were seen in the proportions of the components from M. bovis. The phthiocerols A from M. marinum were C28 and C30 and the phthiodiolones A from M. kansasii were C25 and C27. The multimethyl-branched acids from the waxes of M. bovis were quantitatively different from those of M. microti and M. tuberculosis but all these mycocerosic acids ranged in size from C23 or C24 to C32, with C29 or C30 being the major component in most cases. M. marinum and M. kansasii strains had mainly C26 or C27 and C29 or C30 multimethyl-branched acids, respectively.     

Side Chain Hydroxylation of Aromatic Compounds by Fungi. Part 6. Biotransformation of Olefins by Mortierella Isabellina

The fungus Mortierella isabellina ATCC 42613 converts phenyl-substituted olefins to vicinal diols. The substrate selectivity and product stereochemistry of this biotransformation can be rationalized by application of the model developed for benzylic hydroxylation of aromatic hydrocarbons by M isabellina.     

Stereochemical aspects of the hydration of trans-anethole epoxide in the rat

Racemic trans-anethole epoxide [1-(4′-methoxyphenyl)-propane-1,2-oxide] was incubated with water, buffers, and rat liver microsomes and cytosol and the stereochemistry of the diols produced was determined by HPLC as their dicamphanyl esters. The diol metabolites were isolated by HPLC from the urine of rats administered [1′-¹⁴C] trans-anethole and their stereochemistry determined after derivatization to their camphanyl esters. The stereochemical course of the metabolism of trans-anethole by rat liver microsomes and cytosol is discussed. © 1995 Wiley-Liss, Inc.     

Side Chain Hydroxylation of Aromatic Compounds by Fungi. Part 6. Biotransformation of Olefins by Mortierella Isabellina

The fungus Mortierella isabellina ATCC 42613 converts phenyl-substituted olefins to vicinal diols. The substrate selectivity and product stereochemistry of this biotransformation can be rationalized by application of the model developed for benzylic hydroxylation of aromatic hydrocarbons by M isabellina.     

Configurational assignment of optically active hydroperoxy homoallylic alcohols and the corresponding diols by circular dichroism and multidimensional gas chromatography

Allylic hydroperoxides are a class of compounds of versatile synthetic utility. Optically active diastereomeric hydroperoxy homoallylic alcohols and their corresponding diols are easily available through horseradish peroxidase (HRP)-catalyzed kinetic resolution of racemic hydroperoxides. Here we describe the assignment of the absolute configuration of the optically active products and substrates obtained after HRP-catalysis by the circular dichroism exciton chirality method. Moreover, the analytical-scale separation of the enantiomers based on multidimensional gas chromatography on chiral columns is presented. Since the enantiomeric elution order on the ciral columns was constituted, the absolute stereochemistry of optically active allylic diols can easily be deduced by their retention times on β-cyclodextrins. Chirality 9:69–74, 1997. © 1997 Wiley-Liss, Inc.     

Stereochemistry of the porphyrin-protein bond of cytochrome c. Stereochemical comparison of Rhodospirillum rubrum, yeast, and horse heart porphyrins c.

Porphyrins c have been obtained from Rhodospirillum rubrum cytochrome c2, yeast cytochrome c, and horse heart cytochrome c and compared using proton magnetic resonance and circular dichroism. Identity of the spectra establishes that chemically and stereochemically the three porphyrins c are identical. Since the stereochemistry of the porphyrin alpha-thioether linkage is not affected in the conversion to porphyrin c, the stereochemistry at the porphyrin alpha-thioether bonds among the corresponding cytochromes c also must be the same. Differences between the proton magnetic resonance of R. rubrum cytochrome c2 and horse heart cytochrome c which were rationalized by invoking an opposite stereochemistry at these condensation sites (Smith, G. M., and Kamen, M. D. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 4303) must therefore be attributed to other factors.     

A novel lepidopteran sex pheromone produced by females of a Lithosiinae species, Lyclene dharma dharma, in the family of Arctiidae

Female moths of Lyclene dharma dharma (Arctiidae, Lithosiinae) produced three pheromone components (I-III), which strongly stimulated male antennae. Using GC-MS analysis and chemical derivatizations, the following structures were estimated: 6-methyl-2-octadecanone (I), 14-methyl-2-octadecanone (II), and 6,14-dimethyl-2-octadecanone (III). While the stereochemistry of the chiral centers could not be determined because it was difficult to collect a sufficient amount of the natural pheromone, the plain structures of I and II were confirmed by synthesis of the racemic mixtures starting from diols. These methyl-branched ketones have not been identified as a natural product, indicating that they constitute a new chemical group of lepidopteran female sex pheromones.     

Interspecies differences in the enantioselectivity of epoxide hydrolases in Cryptococcus laurentii (Kufferath) C.E. Skinner and Cryptococcus podzolicus (Bab'jeva & Reshetova) Golubev

Isolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heathland indigenous to South Africa, were screened for the presence of enantioselective epoxide hydrolases for 2,2-disubstituted epoxides. Epoxide hydrolase activity for the 2,2-disubstituted epoxide (+/-)-2-methyl-2-pentyl oxirane was found to be abundantly present in all isolates. The stereochemistry of the products formed by the epoxide hydrolase enzymes from isolates belonging to the two species (11 isolates representing C. laurentii and 23 isolates representing C. podzolicus) was investigated. The enantiopreferences of the epoxide hydrolases for 2,2-disubstituted epoxides of these two species were found to be opposite. All strains of C. laurentii preferentially hydrolysed the (S)-epoxides while all C. podzolicus isolates preferentially hydrolysed the (R)-epoxides of (+/-)-2,2-disubstituted epoxides. These findings indicate that the stereochemistry of the products formed from 2,2-disubstituted epoxides by the epoxide hydrolase enzymes of these yeasts should be evaluated as additional taxonomic criterion within the genus Cryptococcus. Also, the selectivity of some epoxide hydrolases originating from isolates of C. podzolicus was high enough to be considered for application in biotransformations for the synthesis of enantiopure epoxides and vicinal diols.     

Biphenyl dioxolanes as circular dichroism probes for the assignment of absolute configuration to aliphatic diols: Extending the scope to anti 1,n-diols and cyclic syn 1,2-diols

We describe herein the use of a flexible biphenyl moiety as efficient chirality probe in the assignment of the absolute configuration (AC) of aliphatic, non-chromophoric diols. The diols are transformed in the corresponding biphenyl dioxolanes in which the biphenyl system has either a P or M torsion depending on the chirality of the diol. As the correlation between biphenyl torsion and diol AC has been established and the sense of torsion is revealed by the sign of the biphenyl A band at 250 nm in the CD spectrum of the dioxolane, then the diols AC can be assigned simply looking at the CD spectra of these derivatives. This approach proved to be general, straightforward, and reliable for anti 1,2- 1,3-, and 1,4-diols bearing both one and two stereogenic centers and for cyclic syn 1,2-diols.     

Lipase-catalyzed polycondensations: effect of substrates and solvent on chain formation,dispersity, and end-group structure

The effects of substrates and solvent on polymer formation, number-average molecular weight (M(n)), polydispersity, and end-group structure for lipase-catalyzed polycondensations were investigated. Diphenyl ether was found to be the preferred solvent for the polyesterification of adipic acid and 1,8-octanediol giving a M(n) of 28 500 (48 h, 70 degrees C). The effect of varying the alkylene chain length of diols and diacids on the molecular weight distribution and the polymer end-group structure was assessed. A series of diacids (succinic, glutaric, adipic, and sebacic acid) and diols (1,4-butanediol, 1,6-hexanediol, and 1,8-octanediol) were polymerized in solution and in bulk. It was found that reactions involving monomers having longer alkylene chain lengths of diacids (sebacic and adipic acid) and diols (1,8-octanediol and 1,6-hexanediol) give a higher reactivity than reactions of shorter chain-length diacids (succinic and glutaric acid) and 1,4-butanediol. The bulk lipase-catalyzed condensation reactions were feasible, but the use of diphenyl ether gave higher M(n) values (42,400 g/mol in 3 days at 70 degrees C). The polydispersity varied little over the conditions studied giving values 相似文献   

α-Carbonic anhydrases are sulfatases with cyclic diol monosulfate esters

Carbonic anhydrases (CA) catalyze activated ester hydrolysis in addition to the hydration of CO(2) to bicarbonate. They also show phosphatase activity with 4-nitrophenyl phosphate as substrate but not sulfatase with the corresponding sulfate. Here we prove that the enzyme is catalyzing the synthesis of cyclic diols from sulfate esters. 5-, 6- and 8-membered ring cyclic sulfates incorporating a neighboring secondary alcohol moiety were treated with CA II and yielded the corresponding cyclic diols. Inhibitory properties of obtained cyclic and original sulfate esters were then investigated on human carbonic anhydrase I (hCA I), hCA II, hCA IV and hCA VI (h?=?human isoform). K(I)-s of these compounds ranged between 32.7-423 μM against hCA I, 2.13-32.4 μM against hCA II, 13.7-234 μM against hCA IV and 76-278 μM against CA VI, respectively. The sulfatase activity of CA with such esters is amazing considering the fact that 4-nitrophenyl-sulfate is not a substrate of these enzymes.     

The free lipids of Mycobacterium leprae harvested from experimentally infected nine-banded armadillos

The free lipids of a sample of Mycobacterium leprae were extracted by a procedure designed to produce separate non-polar and polar fractions. The composition of these lipids was analysed semi-quantitatively by five special thin-layer chromatographic systems covering the total range of mycobacterial lipid polarities. In order of increasing polarity, the major lipids were dimycocerosates of phthiocerol A, phthiocerol B and phthiodiolone A, glycosyl phenolphthiocerol dimycocerosates and phospholipids, including monoacylphosphatidylinositol di- and pentamannosides. The diacylated forms of these latter lipids, found in most mycobacteria, were not present. The composition of the free lipids of the leprosy bacillus, surveyed over the total polarity range for the first time, showed that the patterns were particularly related to those of Mycobacterium bovis, Mycobacterium kansasii and Mycobacterium marinum.     

聚乳酸基热固性聚氨酯及其形状记忆性能

通过聚乳酸二元醇和聚乳酸-聚己内酯共聚物二元醇与六亚甲基二异氰酸酯(HDI)三聚体交联反应合成了一系列生物基热固性聚氨酯(Bio-PUs)。利用傅里叶红外(FTIR)、差示扫描量热分析(DSC)、热失重分析(TGA)、万能拉伸机和细胞毒性等测试方法对获得的聚乳酸基聚氨酯进行了表征。结果表明,与聚乳酸二元醇相比,聚乳酸-聚己内酯共聚物二元醇降低了生物基热固性聚氨酯的玻璃化温度(Tg),提高了热固性聚氨酯的热稳定性;且聚乳酸-聚己内酯型聚氨酯的力学性能和形状记忆性能更为优异。其中,聚乳酸-聚己内酯共聚物二元醇分子量为3 000时得到的热固性聚氨酯(Bio-PU2-3000)的杨氏模量为277.7 MPa,伸长率为230%;聚乳酸-聚己内酯共聚物二元醇分子量为1 000得到的热固性聚氨酯(Bio-PU2-1000)在人体体温下的形变回复时间仅为93 s。另外,通过显微镜观察到细胞在含聚乳酸基热固性聚氨酯的培养液中生长状态良好,表明制备得到的生物基聚氨酯无细胞毒性。     

α-Carbonic anhydrases are sulfatases with cyclic diol monosulfate esters

Carbonic anhydrases (CA) catalyze activated ester hydrolysis in addition to the hydration of CO₂ to bicarbonate. They also show phosphatase activity with 4-nitrophenyl phosphate as substrate but not sulfatase with the corresponding sulfate. Here we prove that the enzyme is catalyzing the synthesis of cyclic diols from sulfate esters. 5-, 6- and 8-membered ring cyclic sulfates incorporating a neighboring secondary alcohol moiety were treated with CA II and yielded the corresponding cyclic diols. Inhibitory properties of obtained cyclic and original sulfate esters were then investigated on human carbonic anhydrase I (hCA I), hCA II, hCA IV and hCA VI (h?=?human isoform). K_I-s of these compounds ranged between 32.7–423 μM against hCA I, 2.13–32.4 μM against hCA II, 13.7–234 μM against hCA IV and 76–278 μM against CA VI, respectively. The sulfatase activity of CA with such esters is amazing considering the fact that 4-nitrophenyl-sulfate is not a substrate of these enzymes.     

Identification of cis-diols as intermediates in the oxidation of aromatic acids by a strain of Pseudomonas putida that contains a TOL plasmid.

Pseudomonas putida BG1 was isolated from soil by enrichment with p-toluate and selection for growth with p-xylene. Other hydrocarbons that served as growth substrates were toluene, m-xylene, 3-ethyltoluene, and 1,2,4-trimethylbenzene. The enzymes responsible for growth on these substrates are encoded by a large plasmid with properties similar to those of TOL plasmids isolated from other strains of Pseudomonas. Treatment of P. putida BG1 with nitrosoguanidine led to the isolation of a mutant strain which, when grown with fructose, oxidized both p-xylene and p-toluate to (-)-cis-1,2-dihydroxy-4-methylcyclohexa-3,5-diene-1-carboxylic acid (cis-p-toluate diol). The structure of the diol was determined by conventional chemical techniques including identification of the products formed by acid-catalyzed dehydration and characterization of a methyl ester derivative. The cis-relative stereochemistry of the hydroxyl groups was determined by the isolation and characterization of an isopropylidene derivative. p-Xylene-grown cells contained an inducible NAD+-dependent dehydrogenase which formed catechols from cis-p-toluate diol and the analogous acid diols formed from the other hydrocarbon substrates listed above. The catechols were converted to meta ring fission products by an inducible catechol-2,3-dioxygenase which was partially purified from p-xylene-grown cells of P. putida BG1.     

Cytotoxic cytochalasins from the endozoic fungus Phoma sp. of the giant jellyfish Nemopilema nomurai

Four new cytochalasin derivatives (1-4), together with cytochalasin B (5), were isolated from the fungus Phoma sp. obtained from the giant jellyfish Nemopilema nomurai. The planar structure and relative stereochemistry were established by analysis of 1D and 2D NMR data. The absolute configuration was defined by the modified Mosher's method. The compounds showed significant cytotoxicity against a small panel of human solid tumor cell lines (A549, SK-OV-3, SK-MEL-2, XF 498, and HCT15) with IC(50) values in the range of 0.5-30 μM. The cytochalasin B (5) showed obvious cytotoxicity with IC(50) of 7.9 μM against HeLa human cervical carcinoma cells.     

A Novel Lepidopteran Sex Pheromone Produced by Females of a Lithosiinae Species,Lyclene dharma dharma,in the Family of Arctiidae

Female moths of Lyclene dharma dharma (Arctiidae, Lithosiinae) produced three pheromone components (I–III), which strongly stimulated male antennae. Using GC–MS analysis and chemical derivatizations, the following structures were estimated: 6-methyl-2-octadecanone (I), 14-methyl-2-octadecanone (II), and 6,14-dimethyl-2-octadecanone (III). While the stereochemistry of the chiral centers could not be determined because it was difficult to collect a sufficient amount of the natural pheromone, the plain structures of I and II were confirmed by synthesis of the racemic mixtures starting from diols. These methyl-branched ketones have not been identified as a natural product, indicating that they constitute a new chemical group of lepidopteran female sex pheromones.     

Metabolism and mutagenic activation of benzo(a)pyrene by subcellular fractions from mussel (Mytilus galloprovincialis) digestive gland and sea bass (Discenthrarcus labrax) liver

1. The formation of B(a)P diols, phenols and bacterial mutagens by mussel subcellular fractions is dependent on NADPH whereas B(a)P quinones, the major metabolites, appear to be produced by radical reactions and chiefly in the absence of NADPH.2. B(a)P metabolism in sea bass liver fractions is totally dependent on NADPH and insensitive to radical scavengers excepted tocopherol inhibition of B(a)P mutagenesis.3. The 9–10 and 7–8 epoxides formed by sea bass microsomes have a high affinity for EH which readily metabolized all those epoxides to diols.4. Alpha-naphtoflavone inhibits sea bass B(a)P metabolism at high concentration (100 μM) whereas it increases it at low concentration (20 μM).     

Synthesis and characteristics of allylic 4-pregnene-3,20-diols found in gonadal and breast tissues

Recently several allylic steroids have been found in gonadal and breast tissues. In order to establish their presence and identity in tissues and determine the possible biological properties, a method for the synthesis of 4-pregnene-3 alpha,20 alpha-diol, 4-pregnene-3 alpha, 20 beta-diol, 4-pregnene-3 beta,20 alpha-diol, and 4-pregnene-3 beta,20 beta-diol was developed using 4-pregnene-3,20-dione (progesterone) as substrate and freshly-prepared aluminum isopropoxide in isopropyl alcohol as reducing agent. The yields were about 19%, 30%, 13%, and 38% for the 3 alpha,20 alpha-, 3 alpha,20 beta-, 3 beta,20 alpha-, and 3 beta,20 beta-diols, respectively. The structures and stereochemistry of these diols were established using proton and carbon NMR spectroscopy and infrared and mass spectrometry.