176

3i~~im;cu

et ~~u~~.ysjc~ Acfa, 1042 (1990) 176-181 Elsevier

BBALIP

53305

Characterisation of phenolic ~lyc~lipids from ~yc~~~c~eri~~ ~~~~n~~ Gary Dobson

‘J f

David E. Minoan ‘, Gurdyal S. Besra I, Anthony and Mogens Magnusson 4

1. Mallet 3

’ Department of Chemistry and 2 Department of Mcrobioiogx The Vniuersify, Newcastle upon Tyne, ’ Institute of Dermatologv, St. Thomas’ Hospitui, London (U.K.) and 4 Tuberculin DeFartme~f, Sratens Seruminsf~tut,Copenhagen ~Denmark~ (Received

Key words:

Phenolic

glycolipid;

Ph~nolpht~ocerol;

26 May 1989)

Phenolphthiot~ol;

~etbyl-branched

fatty acid; ( M, marinum).

The phenolic glycolipids from two strains of Mycobaczerium markurn have been isolated and characterised. The ~yeoIipi~ from &f. merinum MNC 170 were princip~ly glyeosides of diacyl C3,, C, and C,, phenolph~i~erols A, but in M. murinum MNC &12, these lipids were accompanied by glycosides of diaql phenoiph~i~io~ones A and novel ~h~io~ols A with the same overall c~~n-Ien~~. Ttrr! main acyl com~nen~ of the phendie ~ycol~pids from M, marimtm MNC 170 were C, dimetbyl and C,, and C, trimethyl-branched fatty acids, but in the lipids of 84. mar&mm MNC 842, the C,, ~i~thy1 acid was the oniy principal eminent. The sugar ~om~sition of all these glycolipids had been previously shown to correspond to IT-O-methylrhamnose.

Introduction

Certain species of clinically significant mycobacteria produce characteristic glycosyl diacyl, phenolphthiocerols (Table I) which are considered to be potential cell-surface lipid antigens [l-8]. These so-called phenolic glycolipids are usually based on phenolphthiocerol A (Table I, I) but the presence of other members of the phenolphthiocerol family (Table I, II-IV) should be considered. A recent survey of the distribution of these glycolipids [9], revealed the presence of major amounts of a polar type of phenolic glycolipid in extracts of M. marinum MNC 842. This report demonstrates that this lipid is based on a novel phenolpht~ot~ol A (Table I, II) and that the less polar component from this strain is a mixture based on both phenolpht~~erol A (Table I, I) and phe~olphthiodiolone A (Table I, III). The phenolic glycolipid from M. marinum MNC 170 was found to be based principally on phenoipht~ocerol A (Table I, I). Materials and Methods 44. marinum MNC 170 and MNC 842 were grown to stat~ona~ phase in Sauton’s medium (180 ml) in 250 ml

Correspondence: D.E. Minnikin, Department of Chemistry, versity, Newcastle upon Tyne, NE1 7RU, U.K. ~5-2760/~/$03,50

0 1990 Ekevier

Science Publishers

The Uni-

B.V. (Biomedical

flasks at 38OC and harvested as described previously [9,10]. Non-polar lipids were extracted using a biphasic mixture of equal amounts of methanolic saline (30 ml 0.3% aqueous NaCl added to 100 ml methanol) and petroleum ether (b.p. 60-80” C). The upper petroleum ether layer was evaporated to dryness and the presence of phenolic glycolipids investigated by two-dimensional thin-layer chromato~aphy (TLC) [3,9]. Glycolipids were purified by preparative TLC. The sugars were removed from the purified glycolipids by acid methanolysis, heating a solution of the lipid in toluene (0.25 ml) overni~t at 60°C with a solution of ethanoyl chloride (0.08 ml) in dry methanol (2 ml). The cooled reaction mixture was extracted with petroleum ether (3 ml followed by 1 ml} and the combined extracts were evaporated to dryness. The crude diacylated members of the phenolphthi~erol family were dissolved in dichloromet~ane (1 ml) containing iodomethane (0.025 ml) and mixed for 30 min with 0.1 M tetrabutylammonium hydrogen sulphate in 0.2 M aqueous sodium hydroxide (1 ml) to produce methylated diacylated phenolphthiocerols by phase-transfer catalysis [3]. These derivatives were examined and purified by TLC using a triple development with petroleum ether/acetone (99 : 1). The isolated products were cleaved with lithium aluminium hydride to methyl phenolpht~~erols and long-chain alcohols and these were converted to ~-butyldimethyIsily1 (TBDMS) ethers, as described previously [11,12]. Division)

177 the least polar glycolipid G (Fig. lb) is based on a mixture of both phenolphthiocerol A (Table I, I) and phenolpht~odiolone A (Table I, III), but the most polar glycolipid G,, derived from a novel phenolphthiotriol A (Table 1, II). The spectrum of the glycolipid G from h4. marinum MNC 170 (Fig. la) showed that the lipid was based principally on phenolphthiocerol A (Table I, I), but the presence of a multiplet at 1.03 ppm (signal B) indicated the presence of a small amount of a lipid based on phenolphthiodiolone A (Table I, III). A notable point is that the signal (I) at 4.89 ppm for the > CH.OOC-protons (Fig. 1) is significantly downfield from that (4.83 ppm) recorded for other phenolic glycolipids from A4. bouis and M. kansusii Dobson, G., unpublished results). The very small coupling ( J,,z < 2 Hz) of the anomeric protons (Fig. 1) is in agreement with previous studies on the

Nuclear magnetic resonance (NMR) spectra were recorded for ‘H in CDCl, solution at 360.13 MHz (Edinburgh University WH-360 NMR Service). Sequential decoupling experiments were performed at 200 MHz. Mass spectra were determined on a VG Analytical Ltd 305 spectrometer, interfaced to a 2025 data system [ll]. Results and Discussion

Examination of the non-polar lipids of M. marinum MNC 842 by two-dimensional TLC revealed the presence of two closely related phenolic glycolipids G and as G,, as previously recorded [9]. A single phenolic glycolipid (G) was detected in the lipids of M. marinum MNC 170 [9]. The NMR spectra of the individual purified glycolipids are shown in Fig. 1. It is apparent that, for the glycolipids from M. marinum MNC 842,

a)

1 tvl

I

6

4

t

t

ppm

2

0

Fig. 1. Proton NMR spectra (360 MHz) of phenolic glycolipids: (a), glycolipid (G) from M. marinum MNC 170; (b), least polar lipid (G) and (c), most polar lipid (G,) from M. marinum MNC 842. The signals are assigned as follows: A, methyl groups; B, (1.03 ppm), C% -CH,. CO. CH(CH,-; C, (0.95), C~.CH,.Cn(OH).CH(CH,)-; D, (1.13, 1.15), -CH(CH,).COO-; E, (2.54) CH,.CH,.CO.CH.(CH,)and -CH(CH,)~COO-; F, (2.84). CHs.CH,.CH(OCHs)-; G, (3.32). CH-,.CHa.CH(Os)-; H, (3.54). -0CHs) (sugar); I, (4.89), z CH.OOC-; J. aromatic ring; K, -CH,-; L, H&k M, CHCI,; N, (5.52) sugar C-l; 0, (4.25) sugar C-2; P, (3.80), sugar C-5; Q, (3.59), sugar C-3 and C-4; R. -OH (removed with 40). Starting from the anomeric proton (N), the other sugar ring protons (O-Q) were assigned by successive decoupling.

178 TABLE I

Taxon

Sugar unit (R)

M. boois, M. a~ricon~m, M. microti M. marinum ikl. kansarii

2~~-Me-rhamnose 3-O-Me-rhamnose 2,6-didcoxy-4-0-Me-arabi~ohexose, 2-0-Me-fucose, 2-~“Me-rhamnose. 2,4+Me&amnose 3,6-U-Me&ucose, 2,3-~Me~~rhamnose, 3-O-Me-rhamnose 2,3,4-O-Me,-fucose, rhamnose, 2-O-Me-rhamnose

M. @rue M. tuberculosis (Canetti strains)

Phe~oipht~~ero~

A

ocn,

CN,-CH&Pbenoi~ht~ot~oI

A

OH yzf

ye

7”3

CH,-CH,-&I-fPhenolpht~~iolone A 0

-CH-[CH&-CH-CH,-CH-[CH&

CH,--CH,-:Phenolpb~i~roI B OCH, CH,-

X = 16-22

AH73

Diesters

y = 16-20, z = 2-5

R’- CH,-[CH,],-[CH,-CH],C~-

glycolipids from &_i.~~~~~~~ [13-151 and suggests LY linkages in accordance with those in other phenolic glycolipids [4,6]. After removal of the sugars, the methylated diacyl phe~olpht~o~erols were analysed by TLC. For M. marinwn MNC 842, the least polar glycolipid G, showed two major components (RF 0.56 and 0.34) on TLC with a trace of an intermediate component (RF 0.46). The derivative from the most polar glycolipid G,, appeared as only a single component with R, 0.10. Only one major component (RF 0.56), was produced on degradation of the glycolipid G from iM. ~~r~~~~ MNC 1’70, but minor components (R, 0.34 and 0.46) were also present.

No.

ofcarbonsa

Value of y a Valueof 2 * [M-57]+ (m/z) M. marinum MNC 170 G JW.mar&m MNC 842 G hf. ma&mm MNC 842 0,

23 18 1 397

24 16 2 411

0.1 0.6

if In methyl-branched acid (Table 1, V). b Data normal&d to largest peak; tc =

trace.

3.2 b 3.2 1.9

25 17 2 425 0.5 0.1 0.x

Analysis by TLC of the reduction products from the derivatives of the least polar g~ycolipid G, showed again two components (RF 0.59 and 0.27), the latter corresponding to the o&y reduction product from the most polar glycolipid, G,. Components corresponding to long-chain alcohols ( RF 0.92) were seen in the reduction products from both glycolipids. These results suggest that glycolipid G is a mixture of lipids based on phen~lpht~~roi A (Table I, I), phenoipht~~iolone A {Table I, III) and G, is a ~henolphthiot~ol A (Table I, II) derivative. Examination by TLC in petroleum ether~toluene (98 : 2), of the TBDMS ethers of both reduced products showed the presence of long-chain alcohol derivatives (RF 0.90) and methyl phenol-

26 18 2 439

3 453

100 1.1 0.4

47.6 100 100

27 16

28 17 3 467 1.6 0.3 0.2

29 18 3 481 76.1 0.7 0.5

30 16 4 495 4.3 0.8 0.8

31 17 4 509 0.1 tc

32 18 4 523 1.6 tc tc

179 10 0

l-

I

14 7 I I

173 0,. R ^_^

J I

189 H

?K? L-u

E _I_ ^ YYY

I I

, I

269 ._

0

M-57 1

600

I

800

Fig. 2. Mass spectrum of total tri-TBDMS ethers of methyl phenolphthiotriol from glycolipid G, of M. marinum MNC 842. Peak assignments: A, M - 57 - 132 (57 = r-butyl, 132 = TBDMS-OH); B, M - 263 ((2 X 132) - 1); C, M - 395 ((3 X 132) - 1); D, cleavage between carbons 10 and 11; E, loss of 132 (TBDMS-OH) from D; F and G, cleavage between carbons 9 and 10 with loss of 132 and 2X 132, respectively; H, C(CH,),.Si(CH,),. O+ = Si(CH,),; I, Si(CH,),.O+ = Si(CH,),; J, CH,.CH,.CH+.OTBDMS; K, C,H, = 0+.CH3; R’ and R”, ions formed by rearrangement after loss of 57 from either TBDMS group [ll] (for example, m/z 605 = TBDMS-0.Si(CH3)2.0+ = CH.[CHI],o.C,H,.OCH,); R”‘, loss of TBDMS-OH from R”.

phthiotriol tri-TBDMS ethers (R, 0.10). Using a petroleum ether/toluene (50: 50) TLC system, the products from the least polar glycolipid G, also showed the presence of a spot (RF 0.31) corresponding to diTBDMS ethers of methyl phenolphthiocerol A. Multimethyl-branched long-chain alcohol TBDMS ethers produce, on mass spectrometry, characteristic ions due to loss of the t-butyl group (57 mass units) [ll]. Homologous [A4 - 57]+ ions, from m/z 397 to 523, were examined by selected ion monitoring GC-MS of the alcohol derivatives (Table II). Mass spectrometry of di-TBDMS ethers of the phthiocerol family do not produce intense [M - 57]+ ions; the initial loss of 57 mass units is followed by a rearrangement to produce a characteristic pair of intense ions [ll]. It was expected that the mass spectra of TBDMS ethers of the methyl phenolphthiocerol family would also contain such diagnostic rearrangement ions. The mass spectrum of the tri-TBDMS ether of the suspected methyl phenolphthiotriol is shown in Fig. 2 and a similar spectrum was recorded for the product derived from the phenol-

phthiodiolone glycolipid. It was not possible to record mass spectra of the individual components of the triTBDMS ether derivatives by GC-MS, since these compounds were too large for the available instrumentation and data system. The methyl phenolphthiocerol A diTBDMS ether derivatives were, however, amenable to GC-MS and the results of selected ion monitoring experiments are shown in Table III.

TABLE III Selected ion monitoring

of methylphenolphthiocerol A di-TBDMS

No. of carbons ’ Value of x a R’ ion (m/z) M. marinum MNC 170 M. marinum MNC 842

35 16 549 0.9 = 1.6

37 18 577

39 20 605

35.5 100

loo 97.3

’ In parent phenolphthiocerol A (Table I, I). b Rearrangement ions identified previously [ll]; see Fig. 2. ’ Data normalised to largest peak.

ethers

41 22 633 55.5 71.4

180 The data presented above show that the phenolic gly~olipids from M. marinum MNC 842 are composed of three major components even though the original [9] TLC chromatogram indicated two lipids. The least polar lipid (G), is composed of glycolipids based on both phenolphthi~erol A (Table I, I), ph~nolphthiodioio~e A (Table I, II) and the most polar glycolipid (G,), which was based on a novel phenolphthiotriol (Table I, II). The presence of trace amounts of a glycolipid based on phenolphthio~erol B (Table I, IV) was suggested by TLC of the methyl phenolpht~ocerol diesters but no structural determinations were possible. The sugar composition of all the glycolipids appeared to be the same by previous TLC analysis f9] which was consistent with a 3-O-methylrhamnose structure. The principal components of the phenolpht~ocerol A units in the least polar glycolipids (G) from M, ma&urn MNC 170 and MNC 842 have 37, 39 and 41 carbons (Table III) with x = 18, 20 and 22 (Table I, I), in general agreement with previous studies {13-151. The phenolphthi~iolone A component of the least polar glycolipid (G) from M. marjnum MNC 842 was also shown by mass spectrometry to have the same chainlengths (Table I, III, x = 18, 20 and 22). The phenolpht~otriol A from the polar glycoIipid G, from M. marinum MNC 842 also had the same overall range of components (Fig. 2; Table I, II, x = 18, 20 and 22). Phenol& glycolipids based on phthiodiolone A have been characterised previously [13-151 but those with a phenolpht~ot~ol core are reported here for the first time. A minor variant (mycoside G’) of the phenolic glycolipid from M. mar~~urn [14,15] is esterified with mycolic acids but its chromatographic mobility is very different from that of the more polar major variant (G,) described here. The multimethyl-branched acyl components gave slightly different profiles for the phenolic glycolipids of the two strains under study (Table II). For iM. marinum MNC 170, C,, dimethyl and C,, and C,, trimethyl acids were all present in major proportions (Table II). In contrast, the Cz7 trimethyl fatty acid was the only prominent component in both glycolipids from M. rn~r~~~rn MNC 842 (Table II). These results are in general agreement with the composition of the fatty acid components of the phthiocerol-based waxes, determined previously [12,16]. It has been shown [ld] that the multimethyl branched acids esterified to pht~o~erols and phenolpht~o~erols in M. marinum have a stereochemistry opposite to that of the mycocerosic acids, characteristic of M. fubercufosis. Four out five of strains examined previously showed a single component on TLC [9] and one of these from MNC 170 is shown here to be based mainly on phenolphthiocerol A. M. mar&urn MNC 842, though showing only two components on TLC, produces major glyeolipids based on three core units, phenolpht~ocerol A,

phenolpht~~iolone A and pht~otrio1 A. The sugar composition of all these glycolipids is the same, suggesting that MNC 842 is a genuine strain of 1M.marinum. It has been noted previously [9] that all four representatives of M. gastri and two out of eleven isolates of IV. ~a~~~i~ appeared to contain small proportions of a more polar phenolic glycolipid perhaps also based on a phenolphthiotriol. The previous semi-quantitative TLC analyses of these ~ycolipids [3,9] show that they all occur in significant amounts in different strains of M. marinum . It is considered [l-8] that the phenolic glycolipids from the taxa listed in Table I are potenti~ surface lipid antigens whose dist~bution is of value in classification and identification [2,3,9]. Although the TLC pattern of phenolic glycolipids is atypical in N. marin~m MNC 842, the glycolipids give the same characteristic colour on spraying the chromatograms with l-naphthol/sulphuric acid [3,9]. The similar composition of the multimethyl-branched fatty acids in the phenolic glycolipids (Table II) and pht~ocerol-based waxes 1121 from both M. ~n~ri~urn MNC 170 and MNC 842 suggests that these lipids are anchored in a similar location in an outer membrane 111. In this respect the waxes are considered to be inert defensive lipids among which are dispersed the biologically active phenolic glycolipids [l].

This study was supported by studentships (for CD. and GSB) from the British Leprosy Relief Association (LEPRA), high-field NMR analyses (II-I. Sadler, Edinburgh University) were provided on a Science and Engineering Reseach Council allocation to D.E.M.. Proton decoupling NMR experiments were carried out by Ian McKeag. References Minnikin, DE. (1982) in The Biology of the Mycobacteria, Vol. I (Ratledge, C. and Stanford, J.L.. eds.), pp. 95-184, Academic Press, London. Brennan, P.J. (1988) in Microbial Lipids, Vol. 1 (Ratledge, C. and Wilkinson, S.G., eds.), pp. 203-298, Academic Press. London. Dobson, G., finning, D.E., iviinnikin, SM., Pariett, J.H., Goodfellow, M., Ridell, M. and Magnusson, M. (1985) in Chemical Methods in Bacterial Systematics (Goodfellow, M. and Minnikin, D.E., eds.), pp. 237-265, Academic Press, London. Fournit, J.J., Riviere, M. and Puzo, G. (1987) 3. Biol. Chem. 262, 3174-3179. Fournie, J.J., Riviere, M., Papa, F. and Puzo, G. (1987) J. Biol. Chem. 262, 3280-3184. Daffe, M,, Lacave, C., LanAelle, M.A. and LanCeBe, G. (1987) Eur. J. B&hem. 16?,155-160. McNeil, M., Chatterjee, D., Hunter, S.W., Bozic, C., Jardine, I. and Brennan, P.J. (1987) Proc. Jap. Sot. Med. Mass Spectrom. 12, 3-22. Rivitre. M., Foumiti, J.J. and Puzo, C. (1987) J. Biol. Chem. 262, 14879-14884.

181 9 Minnikin, D.E., Dobson, G., Parlett, J.H., Goodfellow, M. and Magnusson, M. (1987) Eur. J. Clin. Microbial. 6, 703-707. 10 Minnikin, D.E., Minnikin, S.M., Parlett, J.H., Goodfellow, M. and Magnusson, M. (1984) Arch. Microbial. 139, 225-231. 11 Mallet, A.1.. Minnikin, D.E. and Dobson, G. (1984) Biomed. Mass Spectrom. 11, 79-86. 12 Minnikin, D.E., Dobson, G., Goodfellow, M., Magnusson, M. and Ridell, M. (1985) J. Gen. Microbial 131, 1375-1381.

13 Gastambide-Odier, M., Sarda, P. and Lederer, E. (1967) Bull. Sot. Chim. Biol. 49, 849-864. 14 Gastambide-Odier, M. (1973) Org. Mass Spectrom. 7, 845-860. 15 Gastambide-Odier, M. (1973) Eur. J. B&hem. 33, 81-86. 16 Daffe, M. and Laneelle, M.A. (1988) J. Gen. Microbial. 134, 2049-2055.