Further specific triglycosyl phenol phthiocerol diester from Mycobacterium tuberculosis

Mono- and two-dimensional 1H-NMR allowed the structural elucidation of a glycolipid belonging to the phenolic mycosides series: 2,3,4-tri-0-methyl fucopyranosyl (alpha 1----3) rhamnopyranosyl (alpha 1----3) rhamnopyranosyl (alpha 1----dimycocerosyl) phenol phthiocerol. It shares with the major phenolic glycolipid the two terminal sugar residues, suggesting its potential antigenicity. The glycolipid may also represent an intermediate in the biosynthesis of the major one.     

The phenolic mycoside of Mycobacterium ulcerans: structure and taxonomic implications.

Mycobacterium ulcerans and some pathogenic mycobacterial species elaborate wax A consisting of related long-chain beta-diol components (phthiocerol and related compounds) esterified by multimethyl-branched fatty acids. With the exception of M. ulcerans, wax A-containing mycobacteria also synthesize glycosylated phenol phthiocerol diester and related compounds: the so-called phenolic mycosides. In a deliberate effort to characterize this latter class of compounds in M. ulcerans, 20 strains were examined. Phenolic mycosides were found in two strains. Application of chemical analyses, including one- and two-dimensional NMR spectroscopy, allowed the structural elucidation of glycolipids identified as 3-O-methyl-alpha-L-rhamnosyl phenol phthiocerol diphthioceranate investigators. As phenolic mycosides are highly species-specific molecules, this finding stresses the close phylogenetic link between M. marinum and M. ulcerans. Incidentally, a survey of the mycolate content of M. ulcerans showed that methoxymycolate could not be detected in three strains.     

Antioxidant properties of flavonol glycosides from tea

We have determined the antioxidant activity of the major flavonols found in tea: a monoglycoside, a diglycoside and two triglycosides of kaempferol and three monoglycosides, a diglycoside and two triglycosides of quercetin. The Trolox equivalent antioxidant capacity (TEAC) and inhibition of iron/ascorbate-induced lipid peroxidation of phosphatidyl choline vesicles were measured. In the aqueous phase TEAC assay, the quercetin monoglycosides and diglycoside were approximately half as effective as quercetin aglycone. The quercetin triglycosides were much less effective than the monoglycosides and the diglycoside. The kaempferol glycosides were 32-39% less effective in the aqueous phase antioxidant assay compared to the kaempferol aglycone. Quercetin monoglycosides and diglycoside were potent inhibitors of lipid peroxidation, in contrast to the triglycoside which was much less effective. All the kaempferol glycosides were significantly less potent inhibitors of lipid peroxidation compared to the kaempferol aglycone. The compounds described herein demonstrate the antioxidant activity of the major flavonols in tea and indicate the effect of substituting a range of sugar moieties in the phenolic C ring.     

Structure and antigenicity of the major specific glycolipid antigen of Mycobacterium leprae

Earlier we reported on the presence of a specific phenolic glycolipid (Phenolic Glycolipid-I) in Mycobacterium leprae, and in infected armadillo tissues (Hunter, S. W., and Brennan, P. J. (1981) J. Bacteriol. 147, 728-735). It had an inherent oligosaccharide, composed of 3-O-Me-rhamnose, 2,3-di-O-Me-rhamnose, and 3,6-di-O-Me-glucose, glycosidically linked to the phenol substituent. The structure of the oligosaccharide has now been determined, by partial acid hydrolysis, permethylation, 1H NMR, and 13C NMR as: 3,6-di-O-Me-Glcp(1 beta leads to 4)2,3-di-O-Me-Rhap(1 alpha leads to 2)3-O-Me-Rhap1 alpha leads to phenol (assuming that the glucose substituent is in the D-enantiomeric configuration, and the two methylated rhamnoses are L). Acid hydrolysis of deacylated Phenolic glycolipid-I yielded a phenolic phthiocerol "core," and mass spectrometry and proton NMR of the permethylated core suggested the following structure: (formula, see text) Combined gas-liquid chromatography-mass spectrometry showed three tetramethyl branched "mycocerosic" acids, C30, C32 and C34, with molecular weights (as methyl esters) of 466, 494, and 522, respectively. These are esterified to the hydroxyl functions of the branched glycolic chain. Evidence is also presented that the glycolipid is immunologically active, reacting with rabbit antisera to M. leprae and with sera from lepromatous leprosy patients.     

Antioxidant properties of flavonol glycosides from tea

Abstract

We have determined the antioxidant activity of the major flavonols found in tea: a monoglycoside, a diglycoside and two triglycosides of kaempferol and three monoglycosides, a diglycoside and two triglycosides of quercetin. The Trolox equivalent antioxidant capacity (TEAC) and inhibition of iron/ascorbate-induced lipid peroxidation of phosphatidyl choline vesicles were measured. In the aqueous phase TEAC assay, the quercetin monoglycosides and diglycoside were approximately half as effective as quercetin aglycone. The quercetin triglycosides were much less effective than the mono-glycosides and the diglycoside. The kaempferol glycosides were 32–9% less effective in the aqueous phase antioxidant assay compared to the kaempferol aglycone. Quercetin monoglycosides and diglycoside were potent inhibitors of lipid peroxidation, in contrast to the triglycoside which was much less effective. All the kaempferol glycosides were significantly less potent inhibitors of lipid peroxidation compared to the kaempferol aglycone. The compounds described herein demonstrate the antioxidant activity of the major flavonols in tea and indicate the effect of substituting a range of sugar moieties in the phenolic C ring.     

Structural elucidation of the major phenolic glycolipid from Mycobacterium kansasii. I. Evidence for tetrasaccharide structure of the oligosaccharide moiety

Data from the literature indicate the presence, in the Mycobacterium kansasii wall, of a phenolic glycolipid called mycoside A. A tentative trisaccharide structure was proposed for the oligosaccharide part, whereas the aglycone was found to correspond to a phenol phthiocerol residue esterified by two mycocerosic acids. In the present work, structural information mainly arising from fast atom bombardment-mass spectrometry, tandem mass spectrometry, and 1H NMR of native and chemically degraded phenolic glycolipid indicates a tetrasaccharide structure for the inherent oligosaccharide. This structure is now determined as: 2,6-dideoxy-4-O-methyl-arabino-hexopyranosyl(1 alpha----3)2-O-methyl-4-O- acetylfucopyranosyl(1 alpha----3)2-O-methyl-rhamnopyranosyl(1 alpha----3)2-4-di- O-methylrhamnopyranosyl-1 alpha----phenolglycol. Evidence for the structure of the dideoxyhexose, unique in mycobacteria, is presented in the following paper (Fournié, J.-J., Rivière, M., Papa, F., and Puzo, G. (1987) J. Biol. Chem. 262, 3180-3184).     

Iridoid and phenolic diglycosides from Canthium berberidifolium

An iridoid diglycoside, 6-O-beta-D-apiofuranosyl-mussaenosidic acid, and four phenolic diglycosides, canthosides A-D, were isolated from the aerial part of Canthium berberidifolium, along with seven known compounds. Structural elucidations were based on analyses of spectroscopic data.     

Glycolipids of recent clinical isolates of Mycobacterium tuberculosis: chemical characterization and immunoreactivity

Five distinct glycolipids were readily detected in isolates of Mycobacterium tuberculosis. Spectroscopic methods and chemical degradation techniques allowed the structural identification of four of these glycolipids. The specific phenolic glycolipid antigen previously characterized from the Canetti strain was found in all the strains examined, with identical structural features (triglycosyl phenol phthiocerol dimycocerosate). The other three glycolipids identified were acylated trehaloses: penta-acyl trehalose (containing phthienoyl substituents), tetra-acyl trehalose 2'-sulphate (with C40-C50 hydroxyphthioceranoyl substituents) and diacyl trehalose 2'-sulphate (with C16 and C18 substituents). The two latter glycolipids as well as the phenolic glycolipid immunoreacted with whole-cell antiserum, indicating their surface location. The occurrence of these glycolipid antigens in recent clinical isolates suggests their possible utilization in the serodiagnosis of tuberculosis and the rapid identification of M. tuberculosis with specific antisera.     

Further specific extracellular phenolic glycolipid antigens and a related diacylphthiocerol from Mycobacterium leprae

Mycobacterium leprae in infected armadillo tissue produces extracellular phthiocerol-containing lipids in amounts well in excess of the bacterial mass. The principal component (1.38 mg in 1 g of liver, wet weight, containing 3.7 X 10(10) M. leprae bacilli) consists of a mixture of two phthiocerol homologs, 3-methoxyl-4-methyl-9, 11-dihydroxyoctacosane and 3-methoxyl-4-methyl-9, 11-dihydroxytriacontane, (formula: see text); in which the hydroxyl functions are acylated by a mixture of three 'mycocerosic acids': 2,4,6,8-tetramethylhexacosanoate, 2,4,6,8-tetramethyloctacosanoate, and 2,4,6,8-tetramethyltriacontanoate. The structures were established by saponification of the native lipid, direct probe electron impact- or chemical ionization-mass spectrometry of the phthiocerol or its permethylated derivative, and gas-liquid chromatography-electron impact-mass spectrometry of the methyl esters of the fatty acids. In addition to the previously reported M. leprae-specific triglycosylphenolicdiacyl phthiocerol (Hunter, S. W., Fujiwara, T., and Brennan, P. J. (1982) J. Biol. Chem. 257, 15072-15078), the extracellular products contain small amounts (about 60 micrograms/g of infected liver, wet weight) of two other phenolic glycolipids, one of which (Phenolic Glycolipid III) has been structurally elucidated, (formula: see text); assuming certain enantiomeric configurations for the sugar substituents; the R-acyl functions are identical with those in the diacylphthiocerol. Phenolic Glycolipid-III reacts in enzyme-linked immunosorbent assays with sera from patients with leprosy and with rabbit antisera raised against whole M. leprae. The phthiocerol-containing lipids may be synonymous with the electron transparent capsules of M. leprae, and their unreactive state may confer on them the role of passive protectors of the bacillus.     

Distribution of phthiocerol diester, phenolic mycosides and related compounds in mycobacteria

Among 28 mycobacterial species studied, only Mycobacterium tuberculosis, M. bovis, M. africanum, M. marinum, M. kansasii, M. gastri and M. ulcerans produced waxes yielding long-chain beta-diol components (called phthiocerol and companions) and polymethyl-branched fatty acids on saponification. The same mycobacterial species also produced diesters of phenol phthiocerol and companions. Fatty acids esterifying these fatty alcohols in M. marinum and M. ulcerans were found to belong to the phthioceranic series (dextrorotatory fatty acids), in contrast to those of the other species (laevorotatory fatty acids called mycocerosic acids), both groups having the same chain length and methyl-branched positions. M. kansasii and M. gastri contained the same waxes with identical structures, as did M. tuberculosis, M. bovis and M. africanum. Neither the type strain of M. tuberculosis, nor that of M. bovis or M. marinum accumulated the strain-specific phenolic glycolipids.     

Structural elucidation and antigenicity of a novel phenolic glycolipid antigen from Mycobacterium haemophilum

The structure of a novel antigenic glycolipid that distinguishes the opportunistic pathogen Mycobacterium haemophilum from all other mycobacteria was established by a series of degradation reactions leading to products that were analyzed by gas/liquid chromatography-mass spectrometry. The complete structure of the oligosaccharide unit was determined as 2,3-di-O-CH3-alpha-L-Rhap(1----2)3-O-CH3-alpha-L-Rhap(1----4 )-2,3-di-O-CH3-alpha-L-Rhap(1----. The lipid portion of the phenolic glycolipid was composed of two component phenolphthiocerols differing by two methylene groups, as determined by analysis of their per-O-trideuteriomethylated derivatives. The diol unit of the phenolphthiocerols has a threo relative configuration. The absolute stereochemistry of the asymmetric centers of the phenolphthiocerols is uncertain, but the centers are probably 3R, 4S, 9R, and 11R as found for phthiocerol A from Mycobacterium tuberculosis. The hydroxyl functions of the branched glycolic chain are esterified to a complex mixture of multi-methyl branched mycocerosic acids, C27, C30, C32, C34, and C37 with molecular weights (as methyl esters) of 424, 466, 494, 522, and 564, respectively. The stereochemistry of the methyl branches of the mycocerosates have R absolute configuration. The glycolipid is highly antigenic and appears to be specific for M. haemophilum. There are intriguing similarities between the product from M. haemophilum and the well-known phenolic glycolipid I of Mycobacterium leprae, a matter that is discussed.     

Cyanogenic glycosides from the rare Australian endemic rainforest tree Clerodendrum grayi (Lamiaceae)

The cyanogenic diglycoside lucumin ((R)-mandelonitrile-β-d-primeveroside) and monoglucoside prunasin ((R)-mandelonitrile-β-d-glucoside) were isolated from the foliage of the rare Australian rainforest tree species Clerodendrum grayi (Lamiaceae). This is the first reported isolation of the diglycoside lucumin from vegetative tissue (foliage), and the first reported co-occurrence of lucumin and prunasin. Furthermore, unusually, the diglycoside lucumin was the most abundant cyanogen accounting for approximately 60% of total cyanide in a leaf tissue.     

Hydroquinone diglycoside acyl esters from the stems of Glycosmis pentaphylla

Four hydroquinone diglycoside acyl esters, glypentosides A-C (1-3) and seguinoside F (4), were isolated from the stems of Glycosmis pentaphylla. Glypentosides A-B (1-2) were identified as compounds and designated as methoxyquinol 4-O-[(5-O-trans-p-coumaroyl)-beta-d-apiofuranosyl-(1-->2)-beta-d-glucopyranoside] (1) and 4-demethylantiarol 4-O-[(3-methoxy-4-hydroxy-benzoyl)-beta-d-apiofuranosyl-(1-->2)-beta-d-glucopyranoside] (2). Glypentoside C (3) is a hydroquinone diglycoside acyl ester with a neolignan moiety in the acyl unit. Their structures were elucidated by the combination of one- and two-dimensional NMR analysis, mass spectrometry and chemical evidences.     

Identification of phthiodiolone ketoreductase, an enzyme required for production of mycobacterial diacyl phthiocerol virulence factors

Diacyl phthiocerol esters and their congeners are mycobacterial virulence factors. The biosynthesis of these complex lipids remains poorly understood. Insight into their biosynthesis will aid the development of rationally designed drugs that inhibit their production. In this study, we investigate a biosynthetic step required for diacyl (phenol)phthiocerol ester production, i.e., the reduction of the keto group of (phenol)phthiodiolones. We utilized comparative genomics to identify phthiodiolone ketoreductase gene candidates and provide a genetic analysis demonstrating gene function for two of these candidates. Moreover, we present data confirming the existence of a diacyl phthiotriol intermediate in diacyl phthiocerol biosynthesis. We also elucidate the mechanism underlying diacyl phthiocerol deficiency in some mycobacteria, such as Mycobacterium ulcerans and Mycobacterium kansasii. Overall, our findings shed additional light on the biosynthesis of an important group of mycobacterial lipids involved in virulence.     

A novel phenolic glycolipid from Mycobacterium leprae possibly involved in immunogenicity and pathogenicity.

A phenolic glycolipid was obtained in high amounts (2% of dry weight) from Mycobacterium leprae isolated from infected armadillo liver. Infrared and nuclear magnetic resonance spectroscopy showed that it is closely related to "mycoside A" from Mycobacterium kansasii and is therefore a glycosylphenolic phthiocerol diester. The crucial difference between the two products is in the composition of the attached trisaccharide. Gas-liquid chromatography-mass spectroscopy showed that the product from M. kansasii is composed of 2,4-di-O-methylrhamnose, 2-O-methylrhamnose, and 2-O-methylfucose, whereas that from M. leprae contains 2,3-di-O-methylrhamnose, 3-O-methylrhamnose, and 3,6-di-O-methylglucose. The distinct composition of the oligosaccharide segment of the glycolipid from M. leprae may make it useful for the chemical and serological differentiation of this organism from other mycobacteria. Surprisingly large quantities (2.2 mg/g of dry liver) of the glycolipid were also found in infected liver residue freed of M. leprae, suggesting that it may be responsible for the electron-transparent "foam" surrounding the organism in infected lepromatous tissue.     

Urinary hydroxylysine and hydroxylysyl glycosides excretion in patients with Turner's syndrome

Urinary excretion of 5-hydroxylysine (Hyl) and its two glycosides, a monoglycoside (Gal-Hyl) and a diglycoside (Glc-Gal-Hyl), and the ratio of the diglycoside to monoglycoside have been studied in 30 patients with Turner's syndrome and in 38 healthy controls. In patients, the urinary excretion of Hyl and its diglycoside was similar to that obtained in the controls, while the excretion of the monoglycoside was significantly lower before the age of 17 years. As a consequence, between 6 and 17 years of age the Glc-Gal-Hyl/Gal-Hyl ratio is significantly higher in patients with Turner's syndrome than in normal subjects. The results of our study seem to indicate a disturbance in the turnover of collagen in Turner's syndrome.     

Suppression of monocyte oxidative response by phenolic glycolipid I of Mycobacterium leprae

Mycobacterium leprae synthesizes a unique phenolic glycolipid (PGL-I) in abundant quantities. We studied the effect of PGL-I on the generation of superoxide anion (O2-) by stimulated human monocytes. Peripheral blood monocytes pretreated with PGL-I released less O2- when stimulated with M. leprae than did control monocytes. Monocytes pretreated with dimycocerosyl phthiocerol, mycoside A of Mycobacterium kansasii, or mycoside B of Mycobacterium microti, on the other hand, released O2- in quantities comparable to control monocytes in response to M. leprae stimulation. Monocyte O2- release in response to other stimuli of the oxidative metabolic burst, such as PMA, zymosan, Mycobacterium bovis Bacille Calmette-Guérin, or M. kansasii, was unaffected by lipid pretreatment. These findings demonstrate that PGL-I has a direct effect on monocyte O2- generation in response to M. leprae and suggest that PGL-I is a modulator of phagocytic cell function.     

A spirostanol saponin from the underground parts of Ruscus aculeatus

A new spirostanol saponin was isolated from the underground parts of Ruscus aculeatus and the structure was assigned as (23S,25R)-spirost-5-ene-3 beta,23-diol 23-O-[O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside] on the basis of spectroscopic analysis, including two-dimensional NMR spectroscopic techniques and the result of acid hydrolysis. The saponin is unique in structure having a diglycoside unit at C-23 of the spirostanol skeleton.     

Identification of a naturally‐occurring 8‐[α‐D‐glucopyranosyl‐(1→6)‐β‐D‐glucopyranosyl]daidzein from cultivated kudzu root

Introduction – Kudzu root (Radix puerariae) is a rich source of isoflavones that are effective in preventing osteoporosis, heart disease and symptoms associated with menopause. The major isoflavonoids in kudzu root extracts were reported as puerarin, daidzin and daidzein. Recently, an unknown isoflavonoid (compound 1) was detected from one‐year‐old kudzu root cultivated in Vietnam. Objective – To identify a novel compound 1 in kudzu root extract and determine the structure of the compound by ESI⁺ TOF MS‐MS, ¹H‐, ¹³C‐NMR and enzymatic hydrolysis. Methodology – Samples were prepared by extraction of one‐year‐old kudzu root with 50% ethanol and the isoflavonoids were purified using recycling preparative HPLC. Unknown compound 1 was detected using UV‐light at 254 nm in TLC and HPLC analyses. The molecular weight of 1 was determined using a TOF mass spectrometer equipped with an electrospray ion source. The structure of 1 was determined from the ¹³C and ¹H NMR spectra recorded at 100.40 and 400.0 MHz, respectively. Results – ESI⁺ TOF MS‐MS analysis shows that 1 is a puerarin diglycoside. The interglycosidic linkage of diglycoside determined by ¹H‐, ¹³C‐NMR, and enzymatic hydrolysis suggests that 1 has a glucosyl residue linked to puerarin by an α‐1,6‐glycosidic bond. This compound is the first naturally‐occurring 8‐[α‐D ‐glucopyranosyl‐(1→6)‐β‐D ‐glucopyranosyl]daidzein in kudzu root. The concentration of glucosyl‐α‐1,6‐puerarin in kudzu root was 2.3 mg/g as determined by HPLC. Conclusion – The results indicate that puerarin diglycoside is one of the major isoflavonoids in kudzu root and has a significant impact on the preparation of highly water‐soluble glycosylated puerarin. Copyright © 2009 John Wiley & Sons, Ltd.     

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.