Cord-factor analogs: synthesis of 6,6'-di-O-mycoloyl- and -corynomycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside)

Appropriate solvolysis of 2,3,2',3'-tetra-O-benzyl-4,6,4', 6'-tetra-O-mesyl-alpha,alpha-trehalose gave 2,3,2',3' -tetra-O-benzyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) (2). Selective tosylation or mesylation of 2 respectively gave the 6, 6'-ditosylate (3) and 6,6'-dimesylate (4), the structures of which were confirmed by the 1H-n.m.r. spectra of the corresponding 4,4'-di-O-acetyl derivatives. Treatment of 3 with potassium mycolate in toluene, and subsequent hydrogenolysis, gave the 6'-mycolate 6-tosylate derivative. Treatment of 3 with potassium mycolate or potassium corynomycolate in hexamethylphosphoric triamide, followed by catalytic hydrogenolysis, yielded the respective cord-factor analogs 6,6'-di-O-mycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) and 6,6'-di-O-corynomycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside). The same 6,6'-diesters were obtained from the 6,6'-dimesylate 4. Putative 4,6-anhydro-6'-monomycolates are also described.     

Synthesis of 6,6'-di-O-mycoloyl- and corynomycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) via triflates

Tritylation of 2,3,2',3'-tetra-O-benzyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) (4) (A. Liav, H.M. Flowers and M.B. Goren (1984) Carbohydr. Res. 133, 53-58) followed by benzylation and acid hydrolysis gave 2,3,4,2',3',4'-hexa-O-benzyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) (6). Triflation of 6 with triflic anhydride gave the ditriflate 7. Treatment of 7 with potassium mycolate or potassium corynomycolate in toluene, followed by catalytic hydrogenolysis afforded the respective cord-factor analogs 6,6'-di-O-mycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) (10) and 6,6'-di-O-corynomycoloyl (alpha-D galactopyranosyl alpha-D-galactopyranoside) (11). An alternative approach, based on the debenzylation of 2,3,2',3'-tetra-O-benzyl-6,6'-di-O-p-tolylsulfonyl- (alpha-D-galactopyranosyl alpha-D-galactopyranoside) (1) and conversion of the latter into the corresponding 3,4,3',4'-diisopropylidene derivative 3 failed to yield satisfactory results.     

Selective replacement of primary hydroxyl groups in carbohydrates: preparation of some carbohydrate derivatives containing halomethyl groups

Selective halogenation of hydroxymethyl groups in sugars and nucleosides has been achieved by use of triphenylphosphine and carbon tetrahalides (chloride, bromide, or iodide) in pyridine. Methyl α-d-glucopyranoside, 1,2,-O-isopropylidene-α-d-glucofuranose, inosine, and uridine give almost quantitative yields of their primary halomethyl analogs. Similarly, 6,6′-dichloro-6,6′-dideoxysucrose is prepared from sucrose. Chlorination and bromination of 5,6-anhydro-1,2-O-isopropylidene-α-d-glucofuranose by these reagents give 6-chloro-6-deoxy-1,2-O-isopropylidene-α-d-glucofuranose and 6-bromo-6-deoxy-1,2-O-iso-propylidene-α-d-glucofuranose, respectively.     

Synthesis and biological evaluation of trehalose analogs as potential inhibitors of mycobacterial cell wall biosynthesis

Analogs of trehalose are reported that were designed to interfere with mycolylation pathways in the mycobacterial cell wall. Several derivatives of 6,6'-dideoxytrehalose, including N,N'-dialkylamino and 6,6'-bis(sulfonamido) analogs, were prepared and evaluated for antimycobacterial activity against Mycobacterium tuberculosis H(37)Ra and a panel of clinical isolates of Mycobacterium avium. 6,6'-Diaminotrehalose and its diazido precursor were both inactive, but significant activity apparently related to aliphatic chain length was found among the sulfonamides, N-alkylamines, and one of the amidines.     

Synthesis of mono- and dideoxygenated alpha,alpha-trehalose analogs

In this work, we describe the synthesis and NMR characterization of four mono- and four dideoxygenated analogs of alpha,alpha-D-trehalose. The symmetrical (2,2'-, 3,3'-, 4,4'- and 6,6'-) dideoxy analogs were obtained via selective protection and subsequent radical deoxygenation of the desired hydroxyl group set. The unsymmetrical (2'-, 3'-, 4'- and 6'-) monodeoxy analogs were synthesized by desymmetrization of alpha,alpha-trehalose and subsequent deoxygenation under radical conditions. Complete assignment of all (1)H and (13)C resonances in the spectra of these deoxytrehaloses was achieved through the extensive use of 2D [(1)H,(1)H] and [(1)H,(13)C] correlation NMR experiments. The synthesis of these trehalose analogs sets the stage for future biochemical and NMR-based studies to probe the substrate interactions of trehalose with the recently identified mycobacterial sulfotransferase Stf0.     

Inhibition of Streptococcus mutans 6715 glucosyltransferases by sucrose analogs modified at positions 6 and 6'

Sucrose derivatives modified at position 6 (6-deoxysucrose, 6-thiosucrose, 6,6'-dithiodisucrose, and 6,6'-dideoxy-6,6'-difluorosucrose) were tested as inhibitors of the two Streptococcus mutans 6715 glucosyltransferases. 6-Deoxysucrose was the best inhibitor studied, competitively inhibiting the soluble-D-glucan forming enzyme (GTF-S) and the insoluble-D-glucan forming enzyme (GTF-I) with Ki values one order of magnitude lower than the sucrose Km values. 6-Thiosucrose was also a competitive inhibitor for both enzymes. 6,6'-Dithiodisucrose and 6,6'-dideoxy-6,6'-difluorosucrose only inhibited GTF-I; 6,6'-dithiodisucrose gave mixed inhibition and 6,6'-dideoxy-6,6'-difluorosucrose gave uncompetitive inhibition. 6-Thiosucrose was a substrate for both enzymes to produce acceptor products when acceptors were present. GTF-I synthesized de novo a water-insoluble, (1----3)-6-thio-alpha-D-glucan from 6-thiosucrose.     

Untersuchungen zur modifikation der cellobiose und synthese von methyl-2,6-didesoxy-4-O-(6-desoxy-β-D-glucopyranosyl)-α-D-arabino-hexopyranosid (methyl-4-O-β-D-chinovosyl-α-D-olivosid)

Starting with cellobiosides, several different procedures were employed to prepare 6,6′-dichloro-6,6′-dideoxy, 6,6′-dibromo-6,6′-dideoxy, and 6,6′-dideoxy-6,6′-diiodo derivatives. Reduction with lithium aluminum hydride or nickel boride afforded peracetyl derivatives of methyl, phenyl, and benzyl 6-deoxy-4-O-(6-deoxy-β-D-glucopyranosyl)-β-D-glucopyranoside. Following acetolysis or hydrogenolysis, the glycosyl halide and the corresponding-glycal 40 were prepared. Iodomethoxylation of 40 and subsequent reduction gave the title compound. Alternatively, the halomethoxylation products of cellobial hexaacetate gave, by various procedures, the 2,6,6′-trideoxy-2,6,6′-trihalo derivatives, which, in turn, could be reduced to the title compound. The structures of the derivatives prepared were unequivocally assigned by n.m.r. spectroscopy. The various reaction sequences were compared with respect to the number of steps and the yields obtained.     

Comparative specificities of trehalases from various species.

1. Using derivatives or non-symmetrical analogs of alpha,alpha-trehalose, we studied the catalytic specificities of trehalases from various species: Pseudomonas fluorescens, Melolontha vulgaris, porcine and human kidneys. 2. alpha,Beta-trehalose, beta,beta-trehalose, 6,6'dideoxy alpha,alpha-trehalose, alpha-D-xylopyranosyl alpha-D-xylopyranoside were shown to be neither substrates nor inhibitors. 3. 6'deoxy alpha,alpha-trehalose, alpha-D-glucopyranosyl alpha-D-xylopyranoside, alpha-D-allopyranosyl alpha-D-glucopyranoside and alpha-D-galactosyl alpha-D-glucopyranoside, which all possess an intact alpha-D-glucopyranosyl residue, were split by all these trehalases. 4. alpha-D-glucopyranosyl alpha-D-mannopyranoside, alpha,alpha-trehalosamine are competitive inhibitors. 5. These results show the importance of the primary alcohol group at C-6, of the equatorial configuration of the OH groups at C-2, C-3 and C-4 and of the modification of the structure at C-2 of the substrate for the catalytic activity.     

6,6′-Aryl trehalose analogs as potential Mincle ligands

6,6′-Aryl trehalose derivatives have been synthesized with a view towards identifying novel Th-17-inducing vaccine adjuvants based on the high affinity Mincle ligand Brartemicin. The initial structure-activity relationships of these novel trehalose-based compounds were investigated. All compounds have been evaluated for their ability to engage the Mincle receptor and induce a potential pro-Th17 cytokine profile from human peripheral blood mononuclear cells based on IL-6 production in human peripheral blood mononuclear cells. The preliminary biological characterization of the designed analogs presented in this paper should aid in the future design and testing of more affine ligands that may foster the discovery of novel adjuvants with improved pharmacological properties.     

Stereoselective chlorination of methyl β-lactoside with mesyl chloride in N,N-dimethylformamide

Treatment of methyl β-lactoside with mesyl chloride in N,N-dimethylformamide under a variety of conditions gave complex mixtures of chlorinated products, of which nine were isolated and characterised. Chlorination at a secondary position always occurred with inversion of configuration. When the reaction was conducted at 94° for 9 days, a mixture of the 3,3′,4′,6,6′-pentachloride, the 3,3′,6,6′- and 3,4′,6,6′-tetrachlorides, and the 3,6,6′- and 3′,6,6′-trichlorides was obtained together with the 3′,4′-epoxide of the 6,6′-dichloride, which was an artefact. Under milder conditions, the 6,6′-dichloride was encountered, together with methyl 6-chloro-6-deoxy-β-D-glucopyranoside which had arisen by hydrolysis of the interglycosidic bond. It is particularly noteworthy that displacement occurred at C-3′ of the lactoside, in spite of the vic-axial group at C-4′ which should hinder nucleophilic displacement at C-3′. The cause of this anomaly is discussed.     

2,2',6,6'-Tetrachlorobiphenyl is estrogenic in vitro and in vivo

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants whose effects on biological systems depend on the number of and the positions of the chlorine substitutions. In the present study we examined the estrogenicity of the fully ortho-substituted PCB, 2,2',6,6'-tetrachlorobiphenyl (2,2',6,6'-TeCB). This PCB was chosen as the prototypical ortho-substituted PCB to test the hypothesis that ortho-substitution of a PCB with no para- or meta-chlorine-substitutions results in enhanced estrogenic activity. The results indicate that 2,2',6,6'-TeCB is estrogenic both in vitro, in the MCF-7 cell focus assay, and in vivo, in the rat uterotropic assay. The estrogenic activity elicited by the addition of 5 microM 2,2',6,6'-TeCB to the medium of MCF-7 cultures was inhibited by the estrogen receptor (ER) antagonist, LY156758, suggesting that 2,2',6,6'-TeCB or a metabolite is acting through an ER-dependent mechanism. Results from competitive binding assays using recombinant human (rh) ER indicate that 2,2',6,6'-TeCB does not bind rhERalpha or rhERbeta. A metabolite of 2,2',6,6'-TeCB, 2,2',6,6'-tetrachloro-4-biphenylol (4-OH-2,6,2',6'-TCB), does bind rhERalpha and rhERbeta and is also 10-fold more estrogenic than 2,2',6,6'-TeCB in the MCF-7 focus assay; however, this metabolite is not detected in the medium of MCF-7 cultures exposed to 2,2',6,6'-TeCB. Taken together, the results suggest that the estrogenicity observed in human breast cancer cells and the rat uterus may be due to 1) an undetected metabolite of 2,2',6,6'-TeCB binding to the ER, 2) 2,2',6,6'-TeCB binding directly to a novel form of the ER, or 3) an unknown mechanism involving the ER.     

Two classes of cAMP analogs which are selective for the two different cAMP-binding sites of type II protein kinase demonstrate synergism when added together to intact adipocytes

Twenty-five cyclic nucleotide analogs were tested individually to act as lipolytic agents and to activate adipocyte protein kinase. The lipolytic potency of individual analogs correlated better with their Ka for protein kinase and their lipophilicity rather than with either parameter alone. Some of the most potent lipolytic analogs had I50 values for the particulate low Km cAMP phosphodiesterase suggesting that their effect was not due to raising endogenous cAMP levels through inhibition of phosphodiesterase. The most potent lipolytic analogs contained a thio moiety at the C-8 or C-6 position. These analogs exhibited concave upward dose-response curves. At high concentrations, some analogs were as effective as optimal concentrations of epinephrine in stimulating glycerol release. The regulatory subunit of protein kinase has two different intrachain cAMP-binding sites and cAMP analogs modified at the C-8 position (C-8 analogs) are generally selective for Site 1 and analogs modified at the C-6 position (C-6 analogs) are generally selective for Site 2 (Rannels, S. R., and Corbin, J. D. (1980) J. Biol. Chem. 255, 7085-7088). Thus, C-8 and C-6 analogs were tested in combination to stimulate lipolysis in intact adipocytes and to activate protein kinase in vitro. Each process was stimulated synergistically by a combination of a C-6 and C-8 analog. Two C-8 analogs or two C-6 analogs added together did not cause synergism of either process. For both lipolysis and protein kinase activation, C-8 thio analogs acted more synergistically than C-8 amino analogs when incubated in combination with C-6 analogs, a characteristic of type II protein kinase. It is concluded that the observed synergism of lipolysis is due to binding of cAMP analogs to both intrachain sites and that it is the type II protein kinase isozyme which is responsible for the lipolytic response.     

The covalent modification of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate. Reaction at a binding site other than the active site.

A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfide bonds between the 6 thiol group on the purine ring and certain key cysteines on myosin, heavy meromyosin, and subfragment one. The EDTA ATPase activities of myosin and heavy meromyosin were completely inactivated when 4 mol of thiopurine nucleotide was bound. When similarly inactivated, subfragment one, depending on its method of preparation, incorporated either 1 or 2 mol of thiopurine nucleotide. Modification of a single cysteine on subfragment one resulted in an inhibition of both the Ca2+ and the EDTA ATPase activities, but the latter always to a greater extent. Modification of two cysteines per head of heavy meromyosin had the same effect suggesting that the active sites were not blocked by the thiopurine nucleotides. Direct evidence for this suggestion was provided by equilibrium dialysis experiments. Heavy meromyosin and subfragment one bound 1.9 and 0.8 mol of [8-3H]adenylyl imidodiphosphate per mol of enzyme, respectively, with an average dissociation constant of 5 X 10(-7) M. Heavy meromyosin with four thiopurine nucleotides bound or subfragment one with two thiopurine nucleotides bound retained 65-80% of these tight adenylyl imidodiphosphate binding sites confirming the above suggestion. Thus previous work assuming reaction of thiopurine nucleotide analogs at the active site of myosin must be reevaluated. Ultracentrifugation studies showed that heavy meromyosin which had incorporated four thiopurine nucleotides did not bind to F-actin while subfragment one with one thiopurine nucleotide bound interacted only very weakly with F-actin. Thus reaction of 6,6'-dithiobis(inosinyl imidodiphosphate) at nucleotide binding sites other than the active sites reduces the rate of ATP hydrolysis and inhibits actin binding. It is suggested that these second sites may function as regulatory sites on myosin.     

Reverse transformation of Harvey murine sarcoma virus-transformed NIH/3T3 cells by site-selective cyclic AMP analogs

Eighteen site-selective cAMP analogs modified at either the C-8 position or the C-6 position were tested for their growth regulatory effects on the Harvey murine sarcoma virus-transformed NIH/3T3 clone 13-3B-4 cells grown in a serum-free defined medium. All 18 analogs, when tested individually, exhibited an appreciable growth inhibitory effect at micromolar concentrations. The most potent growth inhibitory analogs contained a thio moiety at the C-8 position. In general, C-6 analogs required 5-10-fold greater concentrations than C-8 analogs to produce the same degree of growth inhibition. The growth inhibition induced by these analogs was accompanied by a change in cell morphology; cells treated with the analogs exhibited the morphology characteristic of untransformed fibroblasts, while untreated cells retained a transformed phenotype. The regulatory subunit of cAMP-dependent protein kinase, the cAMP receptor protein, has two different intrachain cAMP binding sites, and cAMP analogs modified at the C-8 position (C-8 analogs) are generally selective for Site 1, while analogs modified at the C-6 position (C-6 analogs) are generally selective for Site 2. Thus, C-8 and C-6 analogs were tested in combination to enhance the growth regulatory effect. Both growth inhibition and morphological change were enhanced synergistically by a combination of the C-6 and C-8 analogs. Two C-6 analogs or two C-8 analogs added together did not cause synergism. For both growth inhibition and phenotypic change, C-8 thio analogs acted far more synergistically than C-8 amino analogs when cells were treated in combination with C-6 analogs, suggesting a response of the RII rather than the RI cAMP receptor protein. DEAE-cellulose chromatography revealed that the growth inhibition, in fact, correlates with an increase of the RII cAMP receptor protein and a decrease of the RI receptor protein. The growth inhibitory effect of the site-selective analogs was not due to the cytotoxic effect of adenosine metabolites as shown by the different behavior of 8-Cl-cAMP compared with 8-Cl-adenosine in 1) cell cycle effects and 2) release from growth inhibition. It is concluded that the observed growth inhibition and phenotypic reversion of 13-3B-4 cells is most likely mediated through the cellular effector, the RII cAMP receptor protein.     

The synthesis of 2,1′-anhydro-,2,1′:3,6-dianhydro-, and 2,1′:3,6: 3′,6′-trianhydro-sucrose

1′-O-Mesyl-6,6′-di-O-tritylsucrose and the corresponding 1′-O-tosyl derivative were prepared from 6,6′-di-O-tritylsucrose by selective sulphonylation. Both sulphonates underwent intramolecular cyclisation reactions, to give 2,1′-anhydrosucrose in high yields rather than the isomeric 1′,4′-anhydride. Sequential benzoylation, detritylation, and mesylation of the 2,1′-anhydride afforded 2,1′-anhydro-6,6′-di-O-mesylsucrose tetrabenzoate which, in the presence of base, gave 2,1′:3,6:3′,6′-trianhydrosucrose that was not identical with the product previously claimed to have this structure. Several derivatives of 2,1′-anhydrosucrose were prepared possessing different functional groups at either the 6,6′- or 4,6′-positions. Dimolar mesitylene-sulphonylation of 3,3′,4′6′-tetra-O-acetylsucrose gave the 6,1′-disulphonate, which, in the presence of alkali, gave 2,1′:3,6-dianhydrosucrose, which was transformed into the 2,1′:3,6:3′,6′-trianhydride by sequential bromination at C-6′ (carbon tetrabromide-triphenylphosphine) and base-catalysed cyclisation. Treatment of 3,3′,4′,6′-tetra-O-benzoylsucrose with sulphuryl chloride furnished the 4,6,1′-trichloro derivative, which, on alkaline hydrolysis, was converted into 2,1′:3,6-dianhydro-4-chloro-4-deoxy-galacto-sucrose.     

Synthesis and evaluation of trehalose-based compounds as anti-invasive agents

Brartemicin is a trehalose-based inhibitor of tumor cell invasion produced by the actinomycete of the genus Nonomuraea. In order to explore the preliminary structure-activity relationship and obtain more potent inhibitors, a series of brartemicin analogs were synthesized through the Mitsunobu coupling of the secondary hydroxyls benzyl protected α,α-d-trehalose with benzoic acid derivatives, followed by modification of functional groups and deprotection. These compounds were evaluated for their inhibitory activity against invasion of murine colon 26-L5 carcinoma cells in vitro. Among the synthetic analogs tested, 6,6′-bis(2,3-dimethoxybenzoyl)-α,α-d-trehalose (5e) was found to be the most potent anti-invasive agent, exhibited a 2.6-fold improvement with regard to the parent natural product brartemicin, and it is considered to be a promising lead molecule for the anti-metastasis.     

Study on anti-proliferative effect of benzoxathiole derivatives through inactivation of NF-κB in human cancer cells

To investigate the anti-proliferative effect of a newly discovered NF-kB inhibitor, 6,6-dimethyl-2-(phenylimino)-6,7-dihydrobenzo[d][1,3]oxathiol-4(5H)-one (1a), a series of its analogs (1b-n) were prepared and evaluated for their NF-κB inhibition and anti-proliferative activity against various human cancer cell lines. Slight variation of hydrophobicity by replacement of dimethyl group of 1a at 6-position with bulky isopropyl group and introduction of para-fluoro substitution on 2-phenyl group showed good NF-κB inhibitory activity and anti-proliferative activity. However, excessive increase in hydrophobicity with 2,4,6-trichloro substituents on phenyl group resulted in the loss of both the activities. From the SAR results, 2-phenylimino-6,7-dihydrobenzo[d][1,3]oxathiol-4(5H)-one was identified as the lead scaffold for investigating new anticancer agent through inactivation of NF-κB.     

Incorporation of Exogenous Purines and Pyrimidines by Methanococcus voltae and Isolation of Analog-Resistant Mutants

Methanococcus voltae incorporated exogenous adenine, guanine, hypoxanthine, and uracil, but not thymine. Growth of M. voltae was also sensitive to purine and pyrimidine analogs. Of the 20 analogs tested, 12 were inhibitory at 1 mg/ml. The most effective inhibitors were purine analogs with endocyclic substitutions. Nucleoside analogs and analogs with exocyclic substitutions or additions were less effective. Four purine analogs, 8-aza-2,6-diaminopurine, 8-azaguanine, 8-azahypoxanthine, and 6-mercaptopurine and one pyrimidine analog, 6-azauracil, were especially toxic. The MICs were 20, 0.5, 2.0, 80, and 10 μg/ml, respectively. Spontaneous resistance mutants were isolated for these five analogs. The MICs for these mutants were 20.5, 8.2, >65, >41, and 20.5 mg/ml, respectively. These concentrations far exceeded the solubilities of the analogs and represented an increase in resistance of at least three orders of magnitude. In addition to demonstrating cross resistance to several of the analogs, four of these mutants lost the ability to incorporate exogenous bases. These appeared to be mutations in the salvage pathways for purines and pyrimidines. In contrast, the mutant resistant to 6-mercaptopurine was not defective in purine uptake. Instead, it degraded 6-mercaptopurine. In the presence or absence of high concentrations of the analogs, the growth rates of the resistant mutants were no less than one-half of the growth rate of the wild type in the absence of the analog. The high level of resistance and rapid growth are very desirable properties for the application of the mutants in genetic experiments.     

Possible Regulatory Roles of Cytokinins : NADH Oxidation by Peroxidase and a Copper Interaction

Apparently free-base cytokinins can interact with cupric ions in a specific manner. Oxidation of NADH by a horseradish peroxidase system was strongly promoted by such cytokinins provided cupric ions were present. Oxidation was promoted by 5 micromolar kinetin, zeatin, 6-benzylaminopurine (BA), or 6-(Δ²-isopentenylamino)purine (2iP) but not by adenine, 6-methylaminopurine or 6,6-dimethylaminopurine. The 6-methylaminopurine promoted oxidation at 500 micromolar but adenine and 6,6-dimethylaminopurine did not. Activity of the free-base purines correlated well with their activity in cell-division assays. However, addition of methoxymethyl-, cyclohexyl-, or tetrahydropyranyl- at N-9 of BA or of ribosyl- at N-9 of BA, 2iP, kinetin, or zeatin eliminated activity in the peroxidase system. In a nonenzymic system containing cupric ions, all of the bases, including adenine, inhibited the Cu²⁺ -stimulated oxidation of ascorbic acid. As in the peroxidase system, the N-9 derivatives were inactive. The cytokinin promotion of NADH oxidation by peroxidase may result from an interaction of the hormones with copper, with peroxidase conferring a specificity similar to the cytokinin specificity observed in growth and development.