Characterization of a specific arabinosyltransferase activity involved in mycobacterial arabinan biosynthesis

Mycobacterium smegmatis strains that contain inactivated EmbA or EmbB proteins are unable to synthesize terminal Arabeta1-->2Araalpha1-->5(Arabeta1-->2Araalpha1-->3)Araalpha1-->5Araalpha1-->(Ara(6)) motif in the cell wall polysaccharide arabinogalactan. Instead, the mutants contain a linear Arabeta1-->2Araalpha1-->5Araalpha1-->5Araalpha1-->(Ara(4)) motif, suggesting that these proteins are involved in the synthesis or transfer of the disaccharide Arabeta1-->2Araalpha1--> to an internal 5-linked Ara. Therefore, we synthesized a linear Arabeta1-->2Araalpha1-->5Araalpha1-->5Araalpha1-->5Araalpha1--> with an octyl aglycon as an arabinosyl acceptor in cell-free assays. A facile assay was developed using the chemically synthesized glycan, membrane, and particulate cell wall as the enzyme source, and 5-phosphoribose diphosphate pR[(14)C]pp as the arabinose donor. The results unequivocally show that two arabinofuranosyl residues were added at the tertiary -->5Araalpha1--> of the synthetic glycan. This activity was undetectable in strains of M. smegmatis where embB or embA had been genetically disrupted. Normal activity could be restored only in the presence of both EmbA and EmbB proteins.     

Synthesis of docosasaccharide arabinan motif of mycobacterial cell wall

Mycobacterial arabinan is a common constituent of both arabinogalactan (AG) and lipoarabinomannan (LAM). In this study, synthesis of β-Araf containing common arabinan docosasaccharide motif (22 Araf monomer units) of mycobacterial cell wall was achieved. Our synthetic strategy toward arabinan involves (1) the stereoselective β-arabinofuranosylation using both 3,5-O-TIPDS-protected and NAP-protected arabinofuranosyl donors for straightforward intermolecular glycosylation and intramolecular aglycon delivery (IAD), respectively, and (2) the convergent fragment coupling with branched fragments at the linear sequence using thioglycoside donor obtained from the corresponding acetonide at the reducing terminal of each fragment through a three-step procedure. Because the acetonide at the reducing terminal of all fragments would be converted to thioglycoside as the glycosyl donor, and mainly Bn ether protections were used, our strategy will be readily applicable to the synthesis of more complex arabinan, arabinogalactan, and arabinomycolate derived from mycobacterial CWS.     

Synthesis and antituberculosis activity of C-phosphonate analogues of decaprenolphosphoarabinose,a key intermediate in the biosynthesis of mycobacterial arabinogalactan and lipoarabinomannan

The cell wall complex in mycobacteria, including the human pathogen Mycobacterium tuberculosis, is comprised in large part of two polysaccharides that contain a significant number of arabinofuranose residues. Both polysaccharides are assembled by a family of arabinosyltransferases that use decaprenolphosphoarabinose (3) as the donor species. In this paper, we describe the synthesis of a panel of C-phosphonate analogues of 3, which were designed to inhibit these arabinosyltransferases and thus block the biosynthesis of mycobacterial cell wall polysaccharides. A number of routes were explored for the preparation of the targets. The successful approach involved the synthesis of a protected C-phosphonate allyl ester 16, which was then coupled to an alkene via an olefin cross metathesis reaction. Subsequent reduction of the alkene with diimide and deprotection afforded the targets. Screening of these compounds in vitro against Mycobacterium tuberculosis revealed that one of the compounds, 15f, possessed antituberculosis activity, with an MIC value of 3.13 microg/mL.     

Versatile polyketide enzymatic machinery for the biosynthesis of complex mycobacterial lipids

The cell envelope of Mycobacterium tuberculosis (Mtb) is a treasure house of a variety of biologically active molecules with fascinating architectures. The decoding of the genetic blueprint of Mtb in recent years has provided the impetus for dissecting the metabolic pathways involved in the biosynthesis of lipidic metabolites. The focus of the Highlight is to emphasize the functional role of polyketide synthase (PKS) proteins in the biosynthesis of complex mycobacterial lipids. The catalytic as well as mechanistic versatility of PKS. in generating metabolic diversity and the significance of recently discovered fatty acyl-AMP ligases in establishing "biochemical crosstalk" between fatty acid synthases (FASs) and PKSs is described. The phenotypic heterogeneity and remodeling of the mycobacterial cell wall in its aetiopathogenesis is discussed.     

Synthesis of 2′,3′-dihydrosolanesyl analogues of β-d-arabinofuranosyl-1-monophosphoryldecaprenol with promising antimycobacterial activity

Two new hydrolytically stable analogues of β-d-arabinofuranosyl-1-monophosphoryldecaprenol, the donor substrate for mycobacterial arabinosyltransferase, have been prepared. Biological evaluation of these compounds in vitro against Mycobacterium tuberculosis H₃₇Rv strain revealed a promising activity.     

Stereoselective synthesis of a fragment of mycobacterial arabinan

Strategies for the stereoselective synthesis of mycobacterial arabinan were explored. Arabinofuranosyl donors with various protective groups were screened in terms of suitability for beta-(1,2-cis)-selective glycosylation. The protective group was found to affect the stereoselectivity of arabinofuranosylation. Beta-selectivity was drastically enhanced by using donors protected with 3,5-TIDPS, possibly due to conformational constraints on the furanose ring. Synthesis of heptaarabinofuranoside was then performed to demonstrate the practicality of this methodology. [reaction: see text].     

Synthesis of the docosanasaccharide arabinan domain of mycobacterial arabinogalactan and a proposed octadecasaccharide biosynthetic precursor

Two major components of the cell wall in mycobacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), are polysaccharides containing arabinofuranose residues. In one of these polysaccharides, arabinogalactan, this arabinan domain consists of three identical motifs of 22 arabinofuranose residues, which are in turn attached to an underlying galactofuranan backbone. Recent studies have proposed that this docosanasaccharide motif, and a structurally related arabinan present in another cell wall polysaccharide, lipoarabinomannan, are biosynthesized from a common octadecasaccharide precursor. To facilitate the testing of this hypothesis, we report here the first total syntheses of these 18- and 22-residue oligosaccharides both functionalized with an aminooctyl linker arm. The route to the target compounds involved the preparation of four tri- to heptasaccharide building blocks possessing only benzoyl protecting groups that were coupled in a highly convergent manner via glycosyl trichloroacetimidate donors. Each of the targets could be prepared in only six steps from these intermediates, and in both cases more than 10 mg of material was obtained. These compounds are expected to be useful tools in probing the biosynthesis of these arabinan-containing polysaccharides. Such studies are essential prerequisites for the identification of novel anti-TB agents that target arabinan assembly.     

Loss of a mycobacterial gene encoding a reductase leads to an altered cell wall containing beta-oxo-mycolic acid analogs and accumulation of ketones

Mycolic acids are essential components of the mycobacterial cell wall. In this study, we show that a gene encoding a reductase involved in the final step of mycolic acid biosynthesis can be deleted in Mycobacterium smegmatis without affecting cell viability. Deletion of MSMEG4722 (ortholog of Mycobacterium tuberculosis Rv2509) altered culture characteristics and antibiotic sensitivity. The DeltaMSMEG4722 strain synthesized alpha-alkyl, beta-oxo intermediates of mycolic acids, which were found esterified to cell wall arabinogalactan. While the precursors could not be isolated directly due to their inherent instability during base treatment, their presence was established by prior reduction of the beta-oxo group by sodium borohydride. Interestingly, the mutant also accumulated unsaturated ketones, similar to tuberculenone from M. tuberculosis, which were shunt products derived from spontaneous decarboxylation of alpha-alkyl, beta-oxo fatty acid precursors of mycolic acids.     

CD1c presentation of synthetic glycolipid antigens with foreign alkyl branching motifs

Human CD1c is a protein that activates alphabeta T cells by presenting self antigens, synthetic mannosyl phosphodolichols, and mycobacterial mannosyl phosphopolyketides. To determine which molecular features of antigen structure confer a T cell response, we measured activation by structurally divergent Mycobacterium tuberculosis mannosyl-beta1-phosphomycoketides and synthetic analogs with either stereorandom or stereospecific methyl branching patterns. T cell responses required both a phosphate and a beta-linked mannose unit, and they showed preference for C(30-34) lipid units with methyl branches in the S-configuration. Thus, T cell responses were strongest for synthetic compounds that mimicked the natural branched lipids produced by mycobacterial polyketide synthase 12. Incorporation of methylmalonate to form branched lipids is a common bacterial lipid-synthesis pathway that is absent in vertebrates. Therefore, the preferential recognition of branched lipids may represent a new lipid-based pathogen-associated molecular pattern.     

Mycobacterial phenolic glycolipid virulence factor biosynthesis: mechanism and small-molecule inhibition of polyketide chain initiation

Phenolic glycolipids (PGLs) are polyketide-derived virulence factors produced by Mycobacterium tuberculosis, M. leprae, and other mycobacterial pathogens. We have combined bioinformatic, genetic, biochemical, and chemical biology approaches to illuminate the mechanism of chain initiation required for assembly of the p-hydroxyphenyl-polyketide moiety of PGLs. Our studies have led to the identification of a stand-alone, didomain initiation module, FadD22, comprised of a p-hydroxybenzoic acid adenylation domain and an aroyl carrier protein domain. FadD22 forms an acyl-S-enzyme covalent intermediate in the p-hydroxyphenyl-polyketide chain assembly line. We also used this information to develop a small-molecule inhibitor of PGL biosynthesis. Overall, these studies provide insights into the biosynthesis of an important group of small-molecule mycobacterial virulence factors and support the feasibility of targeting PGL biosynthesis to develop new drugs to treat mycobacterial infections.     

1,3-Dipolar cycloaddition of nitrile oxides to (R)-1-(1-phenylethyl)-3,5-bis[(E)-arylmethylidene]tetrahydro-4(1H)-pyridinones: synthesis and antimycobacterial evaluation of novel enantiomerically pure di- and trispiroheterocycles

Enantiomerically pure (R)-(1-phenylethyl)-3,5-bis[(E)-arylmethylidene]tetrahydro-4(1H)-pyridinones were synthesized for the first time, and their 1,3-dipolar cycloaddition with nitrile oxides affording di- and trispiroheterocycles regio- and stereoselectively in moderate yields was investigated. These compounds were evaluated against Mycobacterium tuberculosis H37Rv (MTB) and multi-drug-resistant M. tuberculosis (MDR-TB). Among the compounds screened, the dispiroheterocycle, namely, (5R,6R,10S)-3,9-bis(4-chlorophenyl)-10-(2,4-dichlorophenyl)-14-[(E)-(2,4-dichlorophenyl)methylidene]-12-[(R)-1-phenylethyl]-1,4,7-trioxa-2,8,12-tri-azadispiro[4.0.4.4]tetradeca-2,8-diene 5m was found to possess the maximum activity with MIC of 0.49 μM against MTB, being 9.6 and 15.6 times more potent than ciprofloxacin and ethambutol, respectively. Against MDR-TB, 5m displayed maximum activity with an MIC of 0.49 μM, with it thus being more active than rifampicin, isoniazid, ciprofloxacin and ethambutol by 7.8, 23, 77 and 124 times, respectively.     

Combinatorial lead optimization of [1,2]-diamines based on ethambutol as potential antituberculosis preclinical candidates

Despite relatively modest potency, ethambutol (EMB, (S,S)-[N,N-di-2-amino-1-butanol]ethylenediamine) is a mainstay of contemporary chemotherapy for the treatment of tuberculosis. We have developed a solid-phase synthesis of 1,2-diamine analogues of EMB using a novel acylation-reduction sequence that is compatible with high-throughput 96-well format chemistry. Using this procedure, we have synthesized 63 238 diamine analogues in pools of 10 that are suitable for testing. MIC and a target-based reporter assay were used to direct deconvolution of 2796 individual compounds from these mixtures, and the 69 most potent molecules were resynthesized in milligram quantities for hit confirmation. Purification of these individual active diamine analogues allowed the identification of 26 compounds with activity equal to or greater than EMB. Amines which occurred most frequently in active compounds included many with large hydrophobic moieties, suggesting that optimization was perhaps selecting for the isoprenoid binding site of the arabinosyltransferase target of EMB. N-Geranyl-N'-(2-adamantyl)ethane-1,2-diamine (109), the most active of these diamines, displayed a 14-35-fold improvement in activity in vitro against Mycobacterium tuberculosis, as compared to EMB.     

Synthesis of C-disaccharide analogues of the α-d-arabinofuranosyl-(1→5)-α-d-arabinofuranosyl motif of mycobacterial cell walls via alkynyl intermediates

Two C-glycosides related to the recurrent α-d-arabinofuranosyl-(1→5)-α-d-arabinofuranosyl structural motif of mycobacterial arabinan have been prepared by routes involving acetylenic intermediates.     

当归多糖ASP3及其水解产物的NMR光谱分析

对当归多糖ASP3的糖链结构进行分析. 分别采用0.2 mol/L三氟乙酸(Trifluoroacetic acid, TFA)和内切-α-(1→4)-聚半乳糖醛酸酶(EndoPG)对ASP3进行部分酸水解和酶水解, 并对水解前后多糖组分的1D和2D NMR光谱特征进行分析. 实验结果表明, ASP3是一种果胶多糖. GalpA和Rhap位于多糖分子的主链, 由1→4-D-GalpA相连形成的“光滑区”(半乳糖醛酸聚糖)是其主要组成部分; 由α-(1→4)-GalpA通过O4位与α-(1→2)-和α-(1→2,4)-Rhap的O2位交替连接所形成的重复单元[→4)-α-GalpA-(1→2)-α-Rhap-(1→]构成具有较高分支的“毛发区”(富含中性糖侧链的鼠李半乳糖醛酸聚糖). Galp和Araf是中性糖侧链的主要组成, 通过Rhap残基的O4位与主链相连. 非还原性末端T-β-Galp, β-(1→3)-, β-(1→3,6)-, β-(1→4)-, β-(1→4,6)-Galp聚合形成以β-(1→3,6)-Galp为分支点的β-(1→6)-半乳聚糖和以β-(1→4,6)-Galp为分支点的β-(1→4)-半乳聚糖. T-α-Araf, α-(1→5)-Araf和α-(1→3,5)-Araf聚合形成以α-(1→3,5)-Araf为分支点的α-(1→5)-阿拉伯聚糖. 此外, 由α-(1→5)-阿拉伯聚糖通过α-(1→3)连接与β-(1→6)-半乳聚糖末端聚合形成阿拉伯半乳聚糖.     

Assembly and characterization of 8-arm and 12-arm DNA branched junctions

Branched DNA molecules can be assembled into objects and networks directed by sticky-ended cohesion. The connectivity of these species is limited by the number of arms flanking the branch point. To date, the only branched junctions constructed contain six or fewer arms. We report the construction of DNA branched junctions that contain either 8 or 12 double-helical arms surrounding a branch point. The design of the 8-arm junction exploits the limits of a previous approach to thwart branch migration, but the design of the 12-arm junction uses a new to principle achieve this end. The 8-arm junction is stable with 16 nucleotide pairs per arm, but the 12-arm junction has been stabilized by 24 nucleotide pairs per arm. Ferguson analysis of these junctions in combination with 3-, 4-, 5-, and 6-arm junctions indicates a linear increase in friction constant as the number of arms increases; the 4-arm junction migrates anomalously at 4 degrees C, suggesting stacking of its domains. All strands in both the 8-arm and 12-arm junctions show similar responses to hydroxyl radical autofootprinting analysis, indicating that they lack any dominant stacking structures. The stability of the 12-arm junction demonstrates that the number of arms in a junction is not limited to the case of having adjacent identical base pairs flanking the junction. The ability to construct 8-arm and 12-arm junctions increases the number of objects, graphs, and networks that can be built from branched DNA components. In principle, the stick structure corresponding to cubic close-packing is now a possible target for assembly by DNA nanotechnology.     

Arabinofuranose disaccharide analogs as inhibitors of Mycobacterium tuberculosis

Several octyl 5-O-(α-d-arabinofuranosyl)-α-d-arabinofuranoside disaccharide analogs substituted at the 5-position of the non-reducing end sugar were synthesized and tested in vitro against Mycobacterium tuberculosis (M.tb.), Mycobacterium avium complex (MAC) as well as in a cell free assay system for arabinosyltransferase acceptor/inhibitor activity. A few compounds showed interesting inhibitory activity in the cell free assay as well as against the whole microorganism in vitro.     

Differences in electrophoretic behavior between linear and branched PEG‐conjugated proteins

The objective of this study was to characterize the differences in electrophoretic behavior between linear and branched PEG‐conjugated proteins. Human growth hormone and alpha‐lactalbumin modified by linear or branched PEGs with molecular weight of 10 kDa were analyzed by SEC, MALDI‐TOF MS, SDS‐PAGE, and microchip CGE (MCGE). Chromatographic and mass spectrometric differences between the linear and branched PEG‐proteins on SEC and MALDI‐TOF MS were small, but their electrophoretic behaviors on SDS‐PAGE and MCGE were significantly different. In particular, MCGE showed significant differences in the peak width and the migration times of linear and branched PEG‐proteins, in which the branched PEG‐proteins exhibited a narrower peak and longer migration time than the linear PEG‐proteins. This phenomenon may explain the longer circulation half‐life for the branched PEG‐proteins observed in previously reported in vivo studies. Consequently, this study indicates that MCGE may be a valuable tool for differentiating linear and branched PEG‐proteins.     

Synthesis of arabinofuranose branched galactofuran tetrasaccharides, constituents of mycobacterial arabinogalactan

Mycolyl-arabinogalactan (mAG) complex is a major component of the cell wall of Mycobacterium tuberculosis, the causative agent of tuberculosis disease. Due to the essentiality of the cell wall for mycobacterium viability, knowledge of the biosynthesis of the arabinogalactan is crucial for the development of new therapeutic agents. In this context, we have synthesized two new branched arabinogalactafuranose tetrasaccharides, decenyl β-D-Galf-(1→5)-β-D-Galf-(1→6)[α-D-Araf(1→5)]-β-D-Galf (1) and decenyl β-D-Galf-(1→6)-[α-D-Araf-(1→5)]-β-D-Galf-(1→5)-β-D-Galf (2), as interesting tools for arabinofuranosyl transferase studies. The aldonolactone strategy for the introduction of the internal d-Galf was employed, allowing the construction of oligosaccharides from the non-reducing to the reducing end. Moreover, a one-pot procedure was developed for the synthesis of trisaccharide lactone 21, precursor of 2, which involved a glycosylation-deprotection-glycosylation sequence, through the use of TMSOTf as catalyst of the trichloroacetimidate method as well as promoter of TBDMS deprotection.     

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.     

Preparation, properties, and applications of n-pentenyl arabinofuranosyl donors

[reaction: see text] The development of n-pentenyl furanosyl donors has been tested using arabinose as a model. The readily prepared ortho ester (NPOE) is converted into disarmed (NPG(AC)) and thence armed (NPG(ALK)) n-pentenyl arabinofuranosides. The reactivities of these furanosyl donors and pyranosyl counterparts have been assessed by allowing pairs of both to compete for an acceptor. For the NPOE and armed (NPG(ALK)) pairs, coupling products were obtained from donors, whereas for the disarmed (NPG(AC)) pair, only the arabinofurano coupled product was obtained. To probe their synthetic utility, the NPOE was shown to be the only precursor needed to prepare an alpha-1,5-linked arabinan segment of the complex lipoarabinomannan cell wall array of Mycobacterium tuberculosis.