PURIFICATION AND CHARACTERIZATION OF MYCOLIC ACIDS AND THEIR METHYL ESTERS

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History and properties of mycolic acid

An ‘unsaponifiable’ wax was first isolated from the human tubercle bacillus by Anderson and co-workers in 1938. He proposed to call this ether-soluble, unsaponifiable, high-molecular weight hydroxy acid ‘mycolic acid’. Mycolic acid (MA) was described as an acid-fast, saturated acid with a low dextrorotation that contained hydroxyl and methoxy-groups. It was difficult to purify and impossible to crystallize. Pyrolytic cleavage yielded hexacosanoic acid and a non-volatile residue which showed no acidic properties. Together it showed an empirical formula of C88H172O4 or C88H176O4 (11, 140). In 1950, Asselineau proved that MAs contained a long alkyl branch in the α-position and a hydroxyl group in the β-position, thereby explaining the pyrolysis as a reversed Claisen reaction (14, 16). As the ability to elucidate the structure of such molecules advanced, the complexity of MA became more apparent. What was once described as a single component that was isolated from M. tuberculosis is now recognized as a family of over 500 related chemical structures. This amazing number of MA types that make up the major part of the cell wall of a single bacterium is an indication of the biological importance of these molecules (19).

Cholesterol in Tuberculosis

It appears that both the entry and the survival of the pathogen in the macrophage host cell depend on its attraction to cholesterol. Gatfield and Pieters (66) demonstrated that cholesterol concentrated at the specific site where mycobacteria enter the macrophage and that cholesterol depletion of the macrophage membrane prevented entry of the mycobacteria into the macrophage. This implies a mechanism of infection whereby the Mycobacterium, after docking to the macrophage, accrues cholesterol from the host membrane to the parasite cell wall and then penetrates the macrophage. The cholesterol enriched endosomic membrane then attracts and holds Tryptophan-Aspartate Coat (TACO) protein to prevent fusion of the infected organelle with the destructive lysosome (2, 58) (Figure 1.5). M. tuberculosis may facilitate its uptake into the host macrophage via the cholesterol binding Scavenger Type A receptor (159). 20 This molecular association between membrane cholesterol and mycobacterial MAs may also explain why the membrane of the phagosome appears to be tightly associated with the engulfed pathogen (46, 47). Av-gay and Sobouti (17) observed that pathogenic mycobacteria uniquely accumulates, but does not consume cholesterol from the growth medium, in contrast to the non-pathogenic mycobacteria that could rely on cholesterol as a major carbon source. Kaul et al. (85) discovered an infection-facilitating role for a « Human Receptor Ck-like » protein expressed in M. tuberculosis, of which the human equivalent is known for its function as a cholesterol sensor to regulate various genes for cholesterol homeostasis (84, 86). In addition, Korf et al. (89) showed that intraperitoneal MA administration into mice affectes mainly the macrophages and convert them into cholesterol-rich foam cells.

Esterification of natural mycolic acids

According to Laval et al. (48, 93, 100, 101), MA may be separated into its different subgroups with TLC after it has been esterified with trimethylsilyl diazomethane (TMDM). The unmethylated MAs have a polar carboxylic acid group which strongly associates with the silica on the TLC plate causing the sample to smear, thus making separation incomplete. By methylesterification of the carboxylic acid, the MA subclasses separate simply due to the differences in functional groups in the meromycolate chain. The different subclasses were separated using a mobile phase containing 9:1 (v/v) petroleum ether:diethyl ether (147). Twenty microlitres of the MA samples (10 mg/ml chloroform, M. tuberculosis MA batch A0698, MB – synthetic α-MA, kindly provided by Prof M.S. Baird (University of Wales, Bangor, UK); MA from Sigma; M. tuberculosis MA batch A1198) were incubated with 5µl of n-hexane-dissolved TMDM (2 M) at room temperature for one hour. The total volume of the TMDM incubated samples was loaded onto a silica plate that had been dried for two hours at 26 110 °C. After loading, the plate was dried briefly at 80 °C and then developed five times consecutively in a mobile phase containing 9:1 (v/v) petroleum ether:diethyl ether. Between developments, the plate was dried at 80 °C for ten minutes. Visualisation was done by sulfuric acid charring. Results are shown in Figure 2.2.

Experimental

General considerations All chemicals were purchased form Lancaster Synthesis Ltd. (UK), Aldrich Chemical Co. Ltd. (UK), or Avocado Chemical Co. Ltd (UK). Diethyl ether and THF were distilled over sodium and benzophenone under a nitrogen atmosphere, while dichloromethane was distilled over calcium hydride. Distilled solvents were used within one day. Organic solutions were dried over anhydrous magnesium sulfate. Bulk solvents were removed under vacuum at 14 mm Hg and residual traces of solvent were finally removed at 0.1 mm Hg. All glassware used in anhydrous reactions were dried for not less than 5 hours at 250 °C. Column chromatography was done under medium pressure using silica gel (particle size 33-70 µm) from DBH Chemicals (UK); thin layer chromatography (TLC) was carried out on pre-coated Kieselgel 60 F254 (Art. 5554, Merck, UK) plates. Routine gas liquid chromatography (GLC) was performed using a temperature programmable Hewlett-Packard (Agilent) 5890 Gas Chromatograph with manual injection. The carrier gas was 5.0 grade helium with a column head pressure of 100 KPa supplied by Air Products plc (UK). The column was Rtx-5 supplied by Restek Corporation (USA). The phase thickness was 25 µm, the column internal diameter 0.31 mm and the column length 15 m. Optical rotations of compounds were measured in solutions of 78 chloroform at known concentration using a Polar 2001 Automatic Polarimeter, with the assistance of Dr J. Al Dulayymi (University of Wales, Bangor, UK). Infra-red spectra were recorded as KBr disc (solid) or thin films (liquid) on NaCl windows using a Perkin Elmer 1600 series FT-IR spectrometer. NMR spectra were recorded either on a Bruker AC 250 spectrometer with 5 mm dual probe or on Bruker Advance 500 spectrometer with 5 mm BBO probe. Compounds analysed were solutions in denatured chloroform (CDCl3), unless indicated differently. All chemical shifts are quoted in δ relative to the trace resonance of protonated chloroform (δ 7.27 ppm) and CDCl3 (δ 77.0 ppm). Low resolution mass spectra using electron impact (EI) were measured at 70 eV on a Hewlett-Packard (Agilent) 5970 quadrupole mass selective detector where the Gas Chromatograph was a Hewlett-Packard (Agilent) 5890 Gas Chromatograph with a 5975 auto sampler. It contained a Rtx-5 column supplied by Restek Corporation (USA). The phase thickness was 25 µm, the column diameter 0.25 mm and the column length 25 m.

Preparation of (2S,3R)-3-methyl-henicosane-1,2-diol (11)

17 OH OH p-Toluenesulfonic acid (0.48 g, 2.51 mmol, 1.90 mol eq.) was added to a stirring solution of (S)-2,2-dimethyl-4-((R)-1-methyl-nonadecyl)-[1,3]-dioxolane (10) (5.04 g, 13.19 mmol) in THF (35 ml), methanol (50 ml) and water (5 ml) at RT. The reaction mixture was refluxed for 3½ hours. When TLC showed that no starting material was left, the solvent was evaporated and the residue diluted with petroleum ether/diethyl ether (1:1, 150 ml). Then a solution of saturated sodium bicarbonate (50 ml) was added, the organic layer separated and the aqueous layer re-extracted with diethyl ether (2 x 300 ml). The combined organic layers were washed with brine (250 ml), dried and evaporated to give a white solid of (2S,3R)-3-methylhenicosane-1,2-diol (11) (4.58 g, >99%).

Index :

• List of Abbreviations
• List of Figures
• List of Tables
• Acknowledgements
• CHAPTER 1: GENERAL INTRODUCTION
  • 1.1 Introduction
  • 1.1.1 Tuberculosis
  • 1.1.2 Mycobacterium tuberculosis, infection and immunology
  • 1.1.3 Diagnosis of TB
  • 1.1.4 Association with HIV/AIDS
  • 1.1.5 Diagnosis of TB in patients co-infected with HIV
  • 1.1.6 Other mycobacteria causing diseases
  • 1.1.7 Mycolic acids
    • 1.1.7.1 History and properties of mycolic acid
    • 1.1.7.2 Mycolic acid and the cell wall
    • 1.1.7.3 Immunological properties of cord factor and mycolic acids
    • 1.1.7.4 Cholesterol in Tuberculosis
  • 1.2 Hypothesis
  • 1.3 Aims
• CHAPTER 2: PURIFICATION AND CHARACTERIZATION OF MYCOLIC ACIDS AND THEIR METHYL ESTERS
  • 2.1 Introduction
  • 2.2 Aim
  • 2.3 Esterification and separation of mycolic acids
  • 2.3.1 Results and Discussion
    • 2.3.1.1 Preliminary experiments
    • 2.3.1.2 Esterification of natural mycolic acids
  • 2.3.2 Materials and methods
    • 2.3.2.1 General considerations
    • 2.3.2.2 Estimation of other components in natural mycolic acids
    • 2.3.2.3 NMR analysis of the natural mycolic acids
    • 2.3.2.4 Esterification of mycolic acids
    • 2.3.2.5 NMR analysis of the mycolic acids methyl esters
    • 2.3.2.6 Separation of the different subclasses of mycolic acids
    • 2.3.2.7 Estimation of different subclasses of mycolic acids
    • 2.3.2.8 NMR analysis of α-mycolic acid methyl esters
    • 2.3.2.9 NMR analysis of methoxy mycolic acid methyl esters
    • 2.3.2.10 NMR analysis of keto mycolic acid methyl ester
    • 2.3.2.11 MALDI-TOF Mass Spectrometry
  • 2.4 Biological activity/antigenicity of subclasses of mycolic acids
  • 2.4.1 Results and discussion
  • 2.4.2 Materials and methods
  • 2.4.2.1 Mycolic acids used as antigens
    • 2.4.2.1.1 Natural MA (nMA)
    • 2.4.2.1.2 Natural mycolic aicd methyl esters (mMA)
    • 2.4.2.1.3 Mycolic acids (MA)
    • 2.4.2.1.4 Alpha-MA (α-MA) and methoxy-MA (methoxy-MA)
    • 2.4.2.1.5 Natural alpha-MA from Prof Minnikin (Min α-MA)
    • 2.4.2.1.6 Cord factor (Trehalose-6,6’-dimycolate)
  • 2.4.2.2 Reagents and apparatus used in ELISA
  • 2.4.2.3 Human sera
  • 2.4.2.4 Preparation of coating solutions
  • 2.4.2.5 Blocking step
  • 2.4.2.6 Antibody binding
  • 2.4.2.7 Addition of conjugate and substrate
• CHAPTER 3: SYNTHESIS OF A METHOXY MYCOLIC ACID
  • 3.1 Introduction
  • 3.1.1 Why synthesize mycolic acids?
  • 3.1.2 Previous synthesis of mycolic acids
  • 3.1.3 Cholesterol and mycolic acids
  • 3.2 Aim
  • 3.3 Synthesis of a methoxy mycolic acid
  • 3.3.1 Results and discussion
  • 3.3.2 Experimental
  • General considerations
  • 3.3.2.1 Preparation of 1,2,5,6-di-O-isopropylidene-D-mannitol
  • 3.3.2.2 Preparation of ethyl-4,5-O-isopropylidene-(S)-4,5-dihydroxy pentanoate
  • 3.3.2.3 Preparation of (R)-3-((S)-2,2-dimethyl-[1,3]-dioxolan-4-yl)-butyric acid ethyl ester
  • 3.3.2.4 Preparation of (R)-3-((S)-2,2-dimethyl-[1,3]-dioxolan-4-yl)-butan-1-ol
  • 3.3.2.5 Preparation of (R)-3-((S)-2,2-dimethyl-[1,3]-dioxolan-4-yl) butyraldehyde
  • 3.3.2.6 Preparation of 5-hexadecylsulfanyl-1-phenyl-1H-tetrazole
  • 3.3.2.7 Preparation of 5-(hexadecane-1-sulfonyl)-1-phenyl-1H-tetrazole
  • 3.3.2.8 Preparation of (S)-2,2-dimethyl-4-((R)-1-methyl-nonadecyl)-[1,3] dioxolane
  • 3.3.2.9 Preparation of (2S,3R)-3-methyl-henicosane-1,2-diol
  • 3.3.2.10 Preparation of (S)-2-((R)-1-methyl-nonadecyl)-oxirane
  • 3.3.2.11 Preparation of 6-bromo-hexan-1-ol
  • 3.3.2.12 Preparation of 1-bromo-6-tetrahydropyranyloxynonane
  • 3.3.2.13 Preparation of (8R,9R)-9-methyl-1-(tetrahydropyran-2-yloxy) heptacosan-8-ol
  • 3.3.2.14 Preparation of 2-((8R,9R)-8-methoxy-9-methyl-heptacosyloxy) tetrahydropyran
    • 3.3.2.15 Preparation of (8R,9R)-8-methoxy-9-methyl-heptacosan-1-ol
    • 3.3.2.16 Preparation of (8R,9R)-8-methoxy-9-methyl-heptacosanal
    • 3.3.2.17 Preparation of 8-bromo-octan-1-ol
    • 3.3.2.18 Preparation of 2,2-dimethyl-propionic acid 8-bromo-octyl ester
    • 3.3.2.19 Preparation of 5-(1-octanolpivalate-8-sulfanyl)-1-phenyl-1H-tetrazole
    • 3.3.2.20 Preparation of 5-(1-octanolpivalate-8-sulfonyl)-1-phenyl-1H-tetrazole
    • 3.3.2.21 Preparation of 2,2-dimethyl-propionic acid-(16R,17R)-16-methoxy methyl-pentatriacontyl ester
  • 3.3.2.22 Preparation of (16R,17R)-16-methoxy-17-methyl-pentatriacontan-1-ol
  • 3.3.2.23 Preparation of (16R,17R)-1-bromo-16-methoxy-17-methylpentatriacontane
  • 3.3.2.24 Preparation of 5-((16R,17R)-16-methoxy-17-methyl-pentatriacontyl sulfanyl)-1-phenyl-1H-tetrazole
  • 3.3.2.25 Preparation of 5-((16R,17R)-16-methoxy-17-methyl-pentatriacontane1-sulfonyl)-1 phenyl-1H-tetrazole
  • 3.3.2.26 Preparation of cis-cyclopropane-1,2-dicarboxylic acid dimethyl ester
  • 3.3.2.27 Preparation of (cis-2-hydroxymethylcyclopropyl)-methanol
  • 3.3.2.28 Preparation of butyric acid cis-2-butyryloxymethylcyclopropylmethyl ester
  • 3.3.2.29 Preparation of butyric acid cis-2-hydroxymethylcyclopropylmethyl ester
  • 3.3.2.30 Preparation of butyric acid cis-2-bromomethylcyclopropylmethyl ester
  • 3.3.2.31 Preparation of butyric acid (1R,2S)-2-(1-phenyl-1H-tetrazol-5-ylsulfanyl methyl)-cyclopropyl methyl ester
  • 3.3.2.32 Preparation of butyric acid (1R,2S)-2-(1-phenyl-1H-tetrazole-5-sulfonyl methyl)-cyclopropyl methyl ester
  • 3.3.2.33 Preparation of 6-bromo-hexanal
  • 3.3.2.34 Preparation of butyric acid (1R,2S)-2-(7-bromo-heptyl)-cyclopropyl methyl ester
    • 3.3.2.35 Preparation of (1R,2S)-2-(7-bromo-heptyl)-cyclopropyl methanol
    • 3.3.2.36 Preparation of (1S,2R)-2-(7-bromo-heptyl)-cyclopropane carbaldehyde
    • 3.3.2.37 Preparation of (1S,2R)-1-(7-bromo-heptyl)-2-((17R,18R)-17-methoxy18-methyl hexatriacontyl)-cyclopropane
    • 3.3.2.38 Preparation of 5-(7-[1S,2R]-2-((17R,18R)-17-methoxy-18-methylhexatriacontyl)-cyclopropyl)-heptyl sulfanyl)-1-phenyl-1H-tetrazole
    • 3.3.2.39 Preparation of 5-(7-[1S,2R]-2-((17R,18R)-17-methoxy-18-methylhexatriacontyl)-cyclopropyl)-heptyl sulfonyl)-1-phenyl-1H-tetrazole
    • 3.3.2.40 Preparation of 2-(8-iodo-octyloxy)-tetrahydro-pyran
    • 3.3.2.41 Preparation of 11-(tetrahydro-pyran-2-yloxy)-undecan-1-ol
    • 3.3.2.42 Preparation of crude 2,2-dimethyl-propionic acid-11-(tetrahydro-pyran2-yloxy)-undecyl ester
    • 3.3.2.43 Preparation of 2,2-dimethyl-propionic acid-11-hydroxy-undecyl ester
    • 3.3.2.44 Preparation of 2,2-dimethyl-propionic acid-11-oxo-undecyl ester
  • 3.3.2.45 Preparation of 13-(2,2-dimethyl-propionyloxy)-tridec-2-enoic acid methyl ester
  • 3.3.2.46 Preparation of (2S,3R)-13-(2,2-dimethyl-propionyloxy)-2,3-dihydroxytridecanoic acid methyl ester
  • 3.3.2.47 Preparation of (2S,3R)-5-[10-(2,2-dimethyl-propionyloxy)-decyl]-2, dioxo-2λ
  • -[1,3,2]-dioxathiolane-4-carboxylic acid methyl ester
  • 3.3.2.48 Preparation of (R)-13-(2,2-dimethyl-propionyloxy)-3-hydroxy-tridecanoic acid methyl ester
  • 3.3.2.49 Preparation of (2R,3R)-2-allyl-[11-(2,2-dimethyl-propionyloxy)-1-hydroxy undecyl]-pent-4-enoic acid methyl ester
  • 3.3.2.50 Preparation of (1R, 2R)-1-acetoxy-11-oxoundecyl hexacosanoic acid methyl ester
  • 3.3.2.51 Preparation of (R)-2-{(R)-1-acetoxy-18-[(1S,2R)-2-((17R,18R) methoxy-18-methyl hexatriacontyl)-cyclopropyl]-octadecyl}-hexacosanoic acid methyl ester
  • 3.3.2.52 Preparation of (R)-2-{(R)-1-hydroxy-18-[(1S,2R)-2-((17R,18R) methoxy-18-methyl hexatriacontyl)-cyclopropyl]-octadecyl}-hexacosanoic acid
  • 3.4 Biological activity/antigenicity of different synthetic mycolic acids
  • 3.4.1 Results and discussion
  • 3.4.2 Materials and methods
  • 3.4.2.1 Mycolic acids used as antigens in ELISA
    • 3.4.2.1.1 Natural mycolic acids
    • 3.4.2.1.2 Synthetic acetylated alpha mycolic acid methyl ester (MB)
    • 3.4.2.1.3 Synthetic methoxy-MA (RR-RS-methoxy)
    • 3.4.2.1.4 Synthetic methoxy-MA (RR-SR-methoxy)
    • 3.4.2.1.5 Synthetic methoxy-MA (SS-SR-methoxy)
    • 3.4.2.1.6 Synthetic trans-cyclopropane-keto-MA
    • 3.4.2.1.7 Synthetic cis-cyclopropane-keto-MA
    • 3.4.2.1.8 Synthetic trans-cyclopropane-hydroxy-MA
    • 3.4.2.1.9 Synthetic cis-cyclopropane-hydroxy-MA
  • 3.4.2.2 Reagents and apparatus used in ELISA
  • 3.4.2.3 Reagents and apparatus used in Biosensor assay
  • 3.4.2.4 Preparation of liposomes containing mycolic acids, synthetic acetylated α-mycolic acid methyl ester (MB) or cholesterol
  • 3.4.2.5 Measurements of interaction between MA, MB and cholesterol
• CHAPTER 4: DISCUSSION
  • SUMMARY
  • REFERENCES

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Structure-function relationships of mycolic acids in tuberculosis