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Background: mycobacterial cells contain complex mixtures of mycolic acid esters. These can be used as antigens recognised by antibodies in the serum of individuals with active tuberculosis, caused by Mycobacterium tuberculosis. In high burden populations, a significant number of false positives are observed; possibly these antigens are also recognised by antibodies generated by other mycobacterial infections, particularly ubiquitous ‘environmental mycobacteria’. This suggests similar responses may be observed in a low burden TB population, particularly in groups regularly exposed to mycobacteria. Methods: ELISA using single synthetic trehalose mycolates corresponding to major classes in many mycobacteria was used to detect antibodies in serum of individuals with no known mycobacterial infection, comprising farmers, abattoir workers, and rural and urban populations. Results: serum from four Welsh or Scottish cohorts showed lower (with some antigens significantly lower) median responses than those reported for TB negatives from high-burden TB populations, and significantly lower responses than those with active TB. A small fraction, particularly older farmers, showed strong responses. A second study examined BCG vaccinated and non-vaccinated farmers and non-farmers. Farmers gave significantly higher median responses than non-farmers with three of five antigens, while there was no significant difference between vaccinated or non-vaccinated for either farmer or non-farmer groups. Conclusions: this initial study shows that serodiagnosis with mycobacterial lipid antigens can detect antibodies in a population sub-group that is significantly exposed to mycobacteria, in an assay that is not interfered with by vaccination. Given the links between mycobacterial exposure and a range of immune system diseases, further understanding such responses may provide a new opportunity for monitoring public health and directing treatment.

Complex mixtures of natural dimycoloyl diarabinoglycerols isolated from mycobacteria have been shown to be both potent immune signalling agents and potentially valuable antigens in the serodiagnosis of mycobacterial infections. We now report the highly stereocontrolled synthesis of diacyl L-glycerol-(1′→1)-β-D-arabinofuranosyl-α-D-arabinofuranosides based on simple fatty acids and single defined synthetic mycolic acids. NMR analysis confirmed that the synthetic core was identical to that in natural mixtures.

Cells of Mycobacterium alvei are known to contain a unique set of mycolic acids with a (ω-1)-methoxy group; although the various enzymes in the biosynthesis of other types of mycolic acid have been widely studied, the biosynthetic route to this substituent is unclear. We now define the stereochemistry of the (ω-1)-methoxy fragment as R, and describe the synthesis of a major R-(ω-1)-methoxy-mycolic acid and its sugar esters, and of two natural M. alvei diene mycolic acids.

R- and S-Glycerol mycolates derived from single synthetic α-, keto- and methoxy-mycolic acids are described.

The differentiation of macrophages into lipid-filled foam cells is a hallmark of the lung granuloma that forms in patients with active tuberculosis (TB). Mycolic acids (MAs), the abundant lipid virulence factors in the cell wall of Mycobacterium tuberculosis (Mtb), can induce this foam phenotype possibly as a way to perturb host cell lipid homeostasis to support the infection. It is not exactly clear how MAs allow differentiation of foam cells during Mtb infection. Here we investigated how chemically synthetic MAs, each with a defined stereochemistry similar to natural Mtb-associated mycolates, influence cell foamy phenotype and mycobacterial proliferation in murine host macrophages. Using light and laser-scanning-confocal microscopy, we assessed the influence of MA structure first on the induction of granuloma cell types, second on intracellular cholesterol accumulation, and finally on mycobacterial growth. While methoxy-MAs (mMAs) effected multi-vacuolar giant cell formation, keto-MAs (kMAs) induced abundant intracellular lipid droplets that were packed with esterified cholesterol. Macrophages from mice treated with kMA were permissive to mycobacterial growth, whereas cells from mMA treatment were not. This suggests a separate yet key involvement of oxygenated MAs in manipulating host cell lipid homeostasis to establish the state of TB.

We report the synthesis of three stereoisomers of a mycolic acid from Mycobacterium tuberculosis containing a di-cis-cyclopropane and of two stereoisomers of a mycolic acid containing a proximal trans-cyclopropane and a distal cis-cyclopropane.

An efficient synthetic approach to tri-arabino di-mycolates, using structurally defined synthetic α-, keto and methoxy mycolic acids is described.

Fragments of mycobacterial cell walls such as arabinoglycerol mycolate and dimycoloyl diarabinoglycerol, comprising complex mixtures of mycolic acids, have immunostimulatory and antigenic properties. A related di-mycoloyl tri-arabinofuranosyl glycerol fragment has been isolated from cell wall hydrolysates. An effective stereoselective synthesis of tri-arabinofuranosyl glycerol, followed by coupling with stereochemically defined mycolic acids of different structural classes, to provide unique di-mycoloyl tri-arabinofuranosyl glycerols is now described.

The cell wall of mycobacteria is characterised by glycolipids composed of different classes of mycolic acids (MAs; alpha-, keto-, and methoxy-) and sugars (trehalose, glucose, and arabinose). Studies using mutant Mtb strains have shown that the structure of MAs influences the inflammatory potential of these glycolipids. As mutant Mtb strains possess a complex mixture of glycolipids, we analysed the inflammatory potential of single classes of mycolate esters of the Mtb cell wall using 38 different synthetic analogues. Our results show that synthetic trehalose dimycolate (TDM) and trehalose, glucose, and arabinose monomycolates (TMM, GMM, and AraMM) activate bone marrow-derived dendritic cells in terms of the production of pro-inflammatory cytokines (IL-6 and TNF-α) and reactive oxygen species, upregulation of costimulatory molecules, and activation of NLRP3 inflammasome by a mechanism dependent on Mincle. These findings demonstrate that Mincle receptor can also recognise pentose esters and seem to contradict the hypothesis that production of GMM is an escape mechanism used by pathogenic mycobacteria to avoid recognition by the innate immune system. Finally, our experiments indicate that TMM and GMM, as well as TDM, can promote Th1 and Th17 responses in mice in an OVA immunisation model, and that further analysis of their potential as novel adjuvants for subunit vaccines is warranted.

Routes to single enantiomers of hydroxy, methoxy and ketomycolic acids containing an α-methyl-trans-alkene unit as well as related trehalose, glucose, arabinose and glycerol esters are described. In serodiagnostic assays to detect antibodies to mycobacterial lipids, the trehalose esters give higher responses to the serum of patients with culture positive tuberculosis than to that of culture negative patients.

The synthesis of mycobacterial mycolic acid related wax esters and of their trehalose di- and mono-esters is described. The trehalose dimycolates (TDMs) synthesised activated bone marrow derived dendritic cells (BMDCs) in vitro more strongly than trehalose dibehenate or the trehalose monomycolates (TMMs). The inflammatory effects were similar to those of TDM from either a synthetic keto- or methoxy-mycolic acid, but somewhat stronger than those of a TDM from an α-mycolic acid. In vivo, the effects of one wax ester TDM were similar to those of the methoxy-MA and α-mycolic acid TDMs and trehalose dibehenate.

The synthesis of single mono-arabino mycolates, important lipid antigens from mycobacteria is described, using structurally defined synthetic mycolic acids. Preliminary assays indicate that these are differentially antigenic to antibodies in the serum of people with active tuberculosis.

Toxoplasmosis is a serious disease caused by Toxoplasma gondii, one of the most widespread parasites in the world. Lipid metabolism is important in the intracellular stage of T. gondii. Stearoyl-CoA desaturase (SCD), a key enzyme for the synthesis of unsaturated fatty acid is predicted to exist in T. gondii. Sterculic acid has been shown to specifically inhibit SCD activity. Here, we examined whether sterculic acid and its methyl ester analogues exhibit anti-T. gondii effects in vitro. T. gondii-infected Vero cells were disintegrated at 36 hr because of the propagation and egress of intracellular tachyzoites. All test compounds inhibited tachyzoite propagation and egress, reducing the number of ruptured Vero cells by the parasites. Sterculic acid and the methyl esters also inhibited replication of intracellular tachyzoites in HFF cells. Among the test compounds, sterculic acid showed the most potent activity against T. gondii, with an EC50 value of 36.2 μM, compared with EC50 values of 248-428 μM for the methyl esters. Our study demonstrated that sterculic acid and its analogues are effective in inhibition of T. gondii growth in vitro, suggesting that these compounds or analogues targeting SCD could be effective agents for the treatment of toxoplasmosis.

Mycolic acid methyl esters were extracted from Mycobacterium avium by a mild saponification protocol, designed to preserve labile components. The resulting mixture of α-, keto- and wax ester mycolates was accompanied by some degraded ω-carboxymycolic acid dimethyl esters, whose overall structures were found to support previous studies. Chromatography of the mono-carboxylic mycolates gave an inseparable mixture of keto- and wax ester mycolates and separate α-mycolates. Reduction of the ketomycolate components allowed isolation and characterisation of intact wax ester mycolates for the first time. Minor α- and ω-carboxymycolates were detected in which methyl branches were located on either the proximal or distal sides of trans-alkene groups.

The preparation of 6-O-mycolylglucoses (GMMs) from single synthetic mycolic acids matching the overall structure of some of the major natural glucose monomycolates of Mycobacterium tuberculosis and other mycobacteria is reported.

The synthesis of an α'-mycolic acid of Mycobacterium smegmatis and other mycobacteria, containing a cis,cis-diene, and of the trehalose mono- and di-mycolates of this, and of a related α'-mycolic acid containing one cis-alkene, is reported.

Synthesis of single enantiomers of mycolyl arabino glycerols, glycolipids previously isolated from Mycobacterium avium–Mycobacterium intracellulare complex, is described using a modified glycosidation method and esterification with structurally defined synthetic mycolic acids. The l-glycero-compound has an essentially identical NMR spectrum to the arabino-glycerol fragment of the natural material.

Synthetic mycolic acids matching the overall structure of the major mycolic acids of Mycobacterium tuberculosis are coupled to trehalose to generate the corresponding synthetic trehalose monomycolate (TMM) and trehalose dimycolate (TDM; cord factor), either with two identical or two different mycolic acids.

Funding for this work was provided by theWelsh Government A4B Programme, grant HE06 POC1022 to MSB and CDG. Background: During pulmonary tuberculosis (PTB) antibodies are generated to trehalose esters of mycolic acids which are cell wall lipids of Mycobacterium tuberculosis (Mtb). Attempts have been made to use these complex natural mixtures in serological tests for PTB diagnosis. Aim: The aim of this work was to determine whether a serological test based on a panel of defined individual trehalose esters of characteristic synthetic mycolic acids has improved diagnostic accuracy in distinguishing patients with culture positive PTB from individuals who were Mtb culture negative. Method: One hundred serum samples from well-characterized patients with presumptive tuberculosis, and diagnosed as having pulmonary smear and culture positive TB, or being culture and smear negative were evaluated by ELISA using different combinations of synthetic antigens and secondary antibodies. Using cut-off values determined from these samples, we validated this study blind in samples from a further 249 presumptive TB patients. Results: With the first 100 samples, detailed responses depended both on the precise structure of the antigen and on the secondary antibody. Using a single antigen, a sensitivity/specificity combination for smear and culture positive PTB detection of 85 and 88% respectively was achieved; this increased to 96% and 95% respectively by a statistical combination of the results with seven antigens. In the blind study a sensitivity/specificity of 87% and 83% was reached with a single antigen. With some synthetic antigens, the responses from all 349 samples were significantly better than those with the natural mixture. Combining the results for seven antigens allowed a distinction between culture positive and negative with a ROC AUC of 0.95. Conclusion: We have identified promising antigen candidates for serological assays that could be used to diagnose PTB and which could be the basis of a much-needed, simple, rapid diagnostic test that would bring care closer to communities. Publisher PDF

We report the synthesis of single enantiomers of epoxy-mycolic acids containing an α-methyl- trans -alkene or a cis -cyclopropane with structures that match those of major isomers of such molecules present in complex mixtures in Mycobacteria such as Mycobacterium fortuitum or Mycobacterium smegmatis

Ozonolysis of Z,Z,Z-cylonona-1,4,7-triene leads to a 1,9-difunctionalised Z,Z-3,6-nonadiene which is readily converted into a range of polyunsaturated pheromones and fatty acids.

Reaction of the bis-dibromocarbene adduct of cyclonona-1,4,7-triene with methyllithium leads to a mixture of meso and racemic cycloundeca-1,2,5,6,9-pentaenes; the former rearranges at ambient temperature through a set of pericyclic reactions.

The syntheses of a number of methoxy and keto-mycolic acids containing an α-methyl-trans-cyclopropane unit and with chain lengths identical to those reported in major homologues in natural samples are reported.

Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry. Oxygenated (methoxy- and keto-) mycolic acid was found to be more antigenic than alpha-mycolic acids. Synthetic methoxy-mycolic acids were the most antigenic, one containing a trans-cyclopropane apparently being somewhat more antigenic than the natural mixture. Trans-cyclopropane-containing keto- and hydroxy-mycolic acids were also found to be the most antigenic among each of these classes. However, none of the individual synthetic mycolic acids significantly and reproducibly distinguished the pooled serum of TB positive patients from that of TB negative patients better than the natural mixture of MA. This argues against the potential to improve the specificity of serodiagnosis of TB with a defined single synthetic mycolic acid antigen from this set, although sensitivity may be facilitated by using a synthetic methoxy-mycolic acid.

We report the synthesis of single enantiomers of two epoxy-mycolic acids containing an α-methyl-trans-alkene.

We report routes to single enantiomers of hydroxy and ketomycolic acids containing an α-methyl-trans-alkene unit.

We report the syntheses of a single enantiomer of an unprotected ketomycolic acids containing a cis-cyclopropane and of related hydroxy-mycolic acids.

Synthetic mycolic acids matching the overall structure of the major α- and methoxy-mycolic acids of Mycobacterium tuberculosis are coupled to trehalose to generate the corresponding synthetic trehalose dimycolate (TDM; cord factor) and trehalose monomycolate (TMM).

The synthesis of three stereoisomers of a major homologue of the methoxymycolic acids present in Mycobacterium tuberculosis is described.

Some single enantiomers of epoxy-mycolic acids containing an α-methyl-trans-alkene or a cis-cyclopropane are synthesized.

Three stereoisomers of a wax ester meromycolate have been prepared starting from mannitol. A detailed comparison of their NMR spectra with those reported for a homologous series of natural wax esters allows the relative configurations of the α-methyl group and adjacent trans-cyclopropane to be determined.

We report the synthesis of a single enantiomer of an α-methyl-trans-cyclopropane unit present in a number of mycolic acids and its incorporation into a reported 1,2-dialkylcyclopropane meromycolate that contains one cis-1,2-dialkylcyclopropane and one α-methyl-trans-1,2-dialkylcyclopropane.

We report a synthesis of a single enantiomer of a mycolic acid from Mycobacterium tuberculosis containing a di-cis-cyclopropane. The method can be simply varied to modify the chain lengths or the absolute stereochemistry of either cyclopropane.

3-(2 S -Heptylcycloprop-1 S -yl)propanoic acid 2-phenylethanamide was synthesised from cis -cyclopropan-1,2-dimethanol via enzymatic desymmetrisation of the dibutyrate; it gave identical NMR spectroscopic data to those reported for grenadamide but had an equal and opposite absolute rotation, indicating that the latter is the 2 R ,1 R -enantiomer.

During growth of Pseudomonas putida strain TW3 on 4-nitrotoluene (4NT) or its metabolite 4-nitrobenzoate (4NB), the culture medium gradually becomes yellow-orange with a λ max of 446 nm. The compound producing this color has been isolated and identified as a new phenoxazinone, 2-aminophenoxazin-3-one-7-carboxylate (APOC). This compound is formed more rapidly and in greater quantity when 4-amino-3-hydroxybenzoate (4A3HB) is added to growing cultures of strain TW3 and is also formed nonbiologically when 4A3HB is shaken in mineral salts medium but not in distilled water. It is postulated that APOC is formed by the oxidative dimerization of 4A3HB, although 4A3HB has not been reported to be a metabolite of 4NT or a product of 4NB catabolism by strain TW3. Using the cloned pnb structural genes from TW3, we demonstrated that the formation of the phenoxazinone requires 4-hydroxylaminobenzoate lyase (PnbB) activity, which converts 4-hydroxylaminobenzoate (4HAB) to 3,4-dihydroxybenzoate (protocatechuate) and that 4-nitrobenzoate reductase (PnbA) activity, which causes the accumulation of 4HAB from 4NB, does not on its own result in the formation of APOC. This rules out the possibility that 4A3HB is formed abiotically from 4HAB by a Bamberger rearrangement but suggests that PnbB first acts to effect a Bamberger-like rearrangement of 4HAB to 4A3HB followed by the replacement of the 4-amino group by a hydroxyl to form protocatechuate and that the phenoxazinone is produced as a result of some misrouting of the intermediate 4A3HB from its active site.

3-Oxa-2,4-dioxobicyclo[3.1.0]hexane provides a flexible starting material for the preparation of single enantiomers of a meromycolic acid, a long chain fatty acid containing two remote cis-cyclopropanes.

A number of 1- and 1,2-disubstituted cyclopropenes undergo [4+2]-cycloaddition to methyl vinyl ketone or acrolein at ambient temperature to produce 2-oxabicyclo[4.1.0]hept-3-enes. When 1-phenyl-2-trimethylsilyl-cyclopropene is treated with 0.4 mol. equiv. of m-chloroperbenzoic acid, the derived ring-opened enone undergoes [4+2]-cycloaddition to the remaining starting material at ambient temperature.

1,1-Dibromocyclopropanes are converted into the corresponding monobromocyclopropanes (as mixtures of stereoisomers where appropriate) by reaction with 1.0–1.3 mol equiv. of ethylmagnesium bromide and 2–10 mol% titanium isopropoxide for 90%). With ethylmagnesium bromide, the reaction occurs very slowly in the absence of catalyst; with methylmagnesium bromide, the reaction does occur in the absence of catalyst, but is only slightly promoted in the presence of titanium isopropoxide. Reactions with a number of other Grignard reagents are also discussed. In the case of phenethylmagnesium bromide, the major product containing the phenethyl-group is ethylbenzene, together with small amounts of styrene and ethyl 4-phenyl-2-butyl ether, a product of trapping of the solvent, ether. In other cases, relatively large amounts of a diether, formally derived by hydrogen abstraction adjacent to the ether oxygen followed by dimerisation, are isolated. No products were identified incorporating the cyclopropane and either the Grignard alkyl group or the solvent. Labelling studies indicate that the hydrogen introduced into the cyclopropane is not derived from either the α- or β-positions of the Grignard reagent. When the reduction is carried out with phenethylmagnesium bromide in d8-tetrahydrofuran both monobromides contain deuterium.

Cyclopropene fatty acid esters (5) and (6) and (7) have been synthesised as potential structure-based inhibitors of Δ6-, Δ12- and Δ15-desaturases

Both diastereomers of the tris-allene, cyclododeca-1,2,5,6,9,10-hexaene have been obtained using a triple cyclopropylidene–allene rearrangement. On the NMR timescale, one has D3 symmetry, and is the smallest hydrocarbon synthesised to have this symmetry, and the second has C2 symmetry.

1,1-Dibromo- and 1,1-dichlorocyclopropanes are converted into the corresponding monohalo-cyclopropanes (as mixtures of stereoisomers where appropriate) by reaction with 1.2–1.3 mol. equiv. of ethyl magnesium bromide and a small amount of titanium isopropoxide in ether. In the presence of an excess of ethylmagnesium bromide the product from the dibromide is the non-halogenated cyclopropane.

Analogues of the pheromones of three insect species (Musca domestica L., Plutella xylostella L. and Ephestia elutella Hbn.) in which a Z-alkene has been replaced by a 1,2-disubstituted cyclopropene have been synthesised. The analogues interfere with normal mating behaviour for each species.

While the reactions of a number of functionalised cyclopropenes with (diisopropoxy-phosphoryl)nitrile oxide lead to 2-oxa-3-azabicyclo[3.1.0]hex-3-enes. 1-bromo-3.3-dimethyl-cyclopropene leads to an isoxazole and 3.3-dimethyl-1.2-dichlorocyclopropene leads to an oxazine

Methyl 1,1,2-tribromocyclopropanecarboxylate is readily available by dibromocyclopropanation of methyl α-bromoacrylate. Reaction with methyllithium at low temperature provides a simple route to methyl 2-bromocyclopropene carboxylate. while modification of the ester group followed by reaction with methyllithium leads to a series of related four-carbon cyclopropenes. The tribromo-ester is also readily converted into 1,1,2,2-tetrabromocyclopropane, a valuable three-carbon cyclopropene synthon.

Patient serum antibodies to mycolic acids have the potential to be surrogate markers of active tuberculosis (TB) when they can be distinguished from the ubiquitously present cross-reactive antibodies to cholesterol. Mycolic acids are known to interact more strongly with antibodies present in the serum of patients with active TB than in patients with latent TB or no TB. Examples of single stereoisomers of mycolic acids with chain lengths corresponding to major homologues of those present in Mycobacterium tuberculosis have now been synthesised with a sulfur substituent on the terminal position of the α-chain; initial studies have established that one of these binds to a gold electrode surface, offering the potential to develop second generation sensors for diagnostic patient antibody detection.

1,2-Dibromocyclopropene ring-opens in solution at 0 – 20 °C to give 1,2-dibromoprop-2-en-1-ylidene, which is trapped by alkenes; the derived cyclopropanes react efficiently with methyl lithium to produce eth-1-en-1-ylidenecyclopropanes. Reaction of the cyclopropene with methyl lithium in ether in the presence of an alkene leads to the same allenes by trapping of the parent allenic carbene, propa-1,2-dienylidene, albeit in low yield

The major product of degradation of ciprofibrate (1), 2-[4-(2,2-dichlorocyclopropyl)phenoxy]-2-methylpropanoic acid, in aqueous sodium hydroxide under reflux is 2-[4-(3-hydroxypropynyl)-phenoxy]-2-methylpropanoic acid (11). A further product, 2-(4-ethynylphenoxy)-2-methylpropanoic acid (12) is derived from 11 under the reaction conditions. A third degradant is identified as 2-[4-(2-carboxyethyl)phenoxy]-2-methylpropanoic acid (13). Under similar conditions, but at pH 7, the products of degradation were found to be 2-[4-(2-chloro-1-hydroxyprop-2-en-1-yl)phenoxy]-2-methylpropanoic acid (9) and (Z)-2-[4-(2-chloro-1-hydroxyprop-2-en-3-yl)-phenoxy]-2-methylpropanoic acid (10). Treatment of 10 with aqueous sodium hydroxide under reflux afforded a mixture of products in which 11 and 12 predominated, whereas similar treatment of 9 led to compound 13 among other products. A labelling study indicates that the acid 21 derived from base treatment of 17 is labelled only at C-2 of the propanoic acid side chain; the same labelling pattern is observed in the acid 21 derived by base treatment of the labelled allylic alcohol 18. Mechanisms are suggested which may explain these observations.