~~oc~~~~cuet ~iop~ysicu Acta, 116.5( 1992153-60 0 1992 Elsevier Science Publishers B.V. All rights reserved 0~5-2~40/92/$05.~

53

BBALIP 54052

A new glycolipid from Mycobacterium aviumMycobacterium l’ntracellulare complex Motoko Watanabe a, Sukeyoshi Kudoh a, Yasuji Yamada b, Kazuo Iguchi ’ and David E. ~inn~kin ’ ’ Research Institute of BCG, Tokyo Napani, ’ Tokyo College uf Pharmacy, Tokyo (Japan) and ’ Department of Chemistry, University of Newcastle upon Tyne, Newcastle upon Tyne (UK)

(Received 16 June 1992)

Key words: Glycolipid; 5-Mycoloylarabinofuranosyl-5-mycoloylarabinofuranosyig~ycerol; Mycobacterium arium-M. intracellulare complex

From a nonpolar lipid fraction of ~y~~ba~ter~~~~ aLium-IWycobacterium iizt~a~ellu~are complex cell mass, a new glycoli~id was obtained, which was shown to be ~-mycoloyl-~-arabinofuranosyl-(1 + ~)-~-myco~oyl~-arabjnofuranosyl-~1 -+ l’)-giycerol. When examined by TLC, all the 12 strains of this species tested, incIuding clinical isolates, were found to contain this glycolipid. But the glycolipid was not detected in ~ycobac~er~u~ bovis BCG or ~yc~bac~eri~~ tubrculosis H37Rv.

Introduction Mycobacterial

cell envelopes

contain

quantities

of

both covalently bonded lipids and free lipids. So-called free lipids are the lipids easily extractable with organic solvents, affixed loosely to the cell matrices to form part of cell envelope. From such a free lipid layer, a number of complex lipids of a range of polarity have been isolated and chemically characterized 111, and their possible arrangement in the cell envelope matrix has been suggested [Z-4]. Those complex lipids are often antigenic. Some of them, such as phenolic glycolipids from M. leprae [5], M tuberculosis [6-S], M. boLlis BCG [9], M. kansasii [lO,l I] and M. haemophilium [I21 and lipooligosaccharides based on trehalose 18,131 are reported to be species-specific antigens, and their usefulness in the clinical serodiagnosis has been suggested 114,151. The ~yc5bac#e~~m album-~ycobacterium ~~t~ace~~~Z~recomplex, generally caIled MA1 complex, inciudes mild opportunistic pathogens of increasing clinical importance as the old age population increases and HIV infections prevail. The most intensively studied lipid antigens in the MA1 complex are the serotype-specific complex glycopeptidolipids (Mycoside-C) which are said to characterize MA1 complex [16-181.

Correspondence to: M. Watanabe, Research Institute of BCG, 3-l-5 Matsuyama, Kiyose. Tokyo 204, Japan.

In the present work, from a hexane extract of a clinical isolate of MA1 complex, we isolated a new glycolipid, named it GI-ai and determined its structure as 5-mycoloyl-p-arabinofuranosyl-(1 -+ 2)-5-mycoloyl-cuarabinofuranosyl-(1 + l’)-glycerol. This glycolipid, which immunologically reacts with rabbit antisera raised to freeze-dried MA1 complex cell mass, was detected in all the 12 strains of the MA1 compIex tested, (IX41-05, 10, 11, 18-24, K-5 and 7 from the Culture Collection of the Research Institute of Tuberculosis; all were identified as either Ma&m or Mintrace&law by the method using the Genprobe Rapid Diagnostic System), but not in M.bo~‘is BCG (Glaxo FlO, Pasteur 1173132, Tokyo 172) or ALtuberculosis H37Rv. The procedures of derivatizations used for the structural elucidation are briefly summerized in a flow sheet in Fig. 1.

Cells

Cells of a clinical isolate of MA1 complex, KK-41-24, obtained from the Research Institute of Tuberculosis Wyose, Tokyo) were shake cultured at 37°C for 3-4 weeks in Sauton’s medium supplemented with 4 ppm Zn”+, 2 ppm Ca2+, 2 ppm Cu2+ (added as ZnSO,’ 7H,O, CaCl, .2H,O and CuSO, * SH,O, respectively), 0.08% sodium pyruvate and 1% soluble starch. The cells were collected by centrifugation, resuspended in 1% formalin, centrifuged, washed once with water by

54 Hexane extract of cell mass

1 G"i

-p)

Gl-ai acetate

__l:-_..-II-.;'._ Deacyl Gl-ai CHzN2 I Methyl mycolates

Alcohols

~AQO,P~I-\,A~_ Deacyl Gl-ai peracetate

Fig. I. Brief description underlined were obtained

Deacyl Gl-ai permethylate

of derivatization used. The compounds in pure form and their spectral data were taken.

centrifugation and finally freeze-dried. The yield of freeze-dried cell mass was 2.5-2.8 g/200 ml medium per 500 ml conical flask. NMR and mass spectra ‘H- and “C-NMR spectra were taken with a Bruker model AM 400 spectrometer (400 MHz for ‘H-NMR and 100 MHz for “C-NMR). For 2D C-H and H-H COSY NMR spectra, a Bruker AM 500 spectrometer (500 MHz for ‘H-NMR and 125 MHz for ‘“C-NMR) was used. The solvent used was CdCl,. Electron impact mass spectra were taken with a Hitachi M-80 mass spectrometer. A VG Auto Spec mass spectrometer was used for the GC/MS measurement. I and was placed on a column of silica gel (Fuji-Davidson’s micro bead silica gel 4B, 200-350 mesh) (2.5 x 35 cm) prepared with hexane. The column was successively washed with a 1: 1 mixture of hexane-CHCl, (300 ml) and CHCl, (100 ml), and the washings were discarded. Then, the column was eluted with 6% MeOH in CHCl,. The fractionated eluates (5 ml each) were tested for their glycolipid contents by TLC, and the fractions containing Gl-ai (20-35) were pooled, concentrated under reduced pressure to give a waxy residue of crude Gl-ai (400 mgl, which was applied to two preparative silica gel plates (20 x 20 cm). The plates were sequentially

A

B

6

C

Fig. 2. TLC profiles of Cl-ai and its ester components. (A) 2 D TLC of hexane extract of freeze-dried MA1 complex cell mass. Solvent: first direction, 6% MeOH in CHCI,; second direction, 25% acetone in toluene. Detection: heating after PMA spray. Only the spot marked G appeared as dark wine-red spot when sprayed with I-naphthol reagent and heated. (BI TLC of three alcohols derived from mycolates in Cl-ai by LiAlH, reduction of Gl-ai acetate. 1, 2 and 3 are Alcohols 1-3, respectively. Solvent; 1.2% MeOH in CHCI,. Detection; PMA spray and heating. [Cl TLC of four compounds derived from alkaline hydrolysis of Gl-ai. 1, 2, 3 and 4 are compounds l-3 (methyl mycolates) and compound 4 (alcohol), respectively. Solvent; 5% acetone in toluene. Detection: PMA spray and heating. Compounds 1 and 2 are hetter separated on TLC triple developed with 10% diethyl ether in hexane.

55 developed with CHCl, and then with 5% MeOH in CHCl, to give about 100 mg of crude Gl-ai which was further purified by TLC using 25% acetone in toluene as developing solvent. The yield of pure Gl-ai was about 80 mg (0.23% w/w of freeze-dried cell mass). Acetylation of Gl-ai

Gl-ai (100 mg) was treated with Ac,O (1 ml) and pyridine (1 ml) at 80°C for 1 h and then at 37°C overnight. The mixture was cooled in ice water, to which cold MeOH (2 ml) was added. After cooling in ice water, the resulting waxy precipitate was separated, which was almost pure Gl-ai acetate. For the NMR measurement, Gl-ai acetate thus obtained was further subjected to TLC using 1.5% AcOEt in CHCI, to remove a trace of impurities. The yield of pure Gl-ai acetate was 100 mg. Alkaline hydrolysis

To a test tube containing Gl-ai (100 mg) was added 10% tetrabutylammonium hydroxide (8 ml). By using a sonic bath, the waxy lipid was dispersed to give an emulsion, which was kept at 100°C overnight. Then, the mixture was cooled in ice water, and the white waxy mass produced and the clear aqueous layer were separated. The waxy mass was dissolved in diethyl ether (5 ml), which was washed with ice-cold water (1 ml). The washing and the aqueous layer were combined, which was then passed through a small cation exchange resin column (1 X 3 cm>. After careful adjustment of pH to neutral with Na,CO,, the eluate was freeze-dried to give deacyl Gl-ai. The ether layer was sequentially washed with dilute H,SO, and then water, dried over Na,SO, and treated with CH,N, to give a methyl ester mixture. Acetylation of deacyl Gl-ai

Deacyl Gl-ai from the above alkaline hydrolysis of 100 mg of Gl-ai was treated with Ac,O (0.5 ml) and pyridine (0.5 ml) at 37°C overnight. To the cooled mixture was added cold MeOH (1 ml) and the solvent was removed under reduced pressure. The waxy residue of deacyl Gl-ai acetate was then purified by TLC using 1.8% MeOH in CHCI, to give about 10 mg of pure deacyl GI-ai peracetate. Methylation of deacyl Gl-ai

Deacyl Gl-ai obtained from alkaline hydrolysis of Gl-ai (100 mg) was vacuum dried over P,O, at 60°C overnight. To the dried residue, Ag,O (300 mg) which had also been vacuum dried at 60°C over P,O, for 2 days, dry dimethyl formamide (1 ml> and methyl iodide (0.4 ml) were added. The mixture was stirred under anhydrous condition for 2 days at 37°C. The mixture was centrifuged and the residue was repeatedly extracted with CHCl, (3 ml X 4). The supernatant was

evaporated under reduced pressure to give a residue which was also extracted with CHCI, (5 ml X 3). All the CHCl, extracts were washed with saturated aqueous KI solution and dried over Na,SO,. The solvent was removed to give crude deacyl Gl-ai permethylate, which was purified by TLC using 3% MeOH in CHCl, to give 7 mg of pure deacyl Gl-ai permethylate. LiAlH, reduction of Gl-ai acetate

GI-ai acetate (100 mg) was treated with LiAlH, (20 mg> in dry diethyl ether (20 ml) for 1 h at room temperature. After the addition of a drop of acetone, the solvent was removed. The residue was extracted repeatedly, first with diethyl ether (10 ml x 3) and then with water (3 ml X 5). The diethyl ether extract gave a mixture of alcohols derived from acyl moieties of Gl-ai. After careful pH adjustment to neutral with cation exchange resin, the aqueous layer was freeze-dried to give a residue, which was treated with Ac,O and pyridine to give deacyl Gl-ai acetate, the yield of which, after purification by TLC with 1.8% MeOH in CHCl,, was 10 mg. Acid hydrolysis of deacyl GI-ai

Deacyl Gl-ai prepared from 30 mg of Gl-ai was heated in 2 N H,SO, (0.2 ml) at 100°C for 3 h. Then the reaction mixture was treated with a small amount of anion exchange resin and concentrated to give a residue, which was dissolved in H,O to be used for the identification of its sugar component by TLC on NaH,PO,-treated silica gel plate as described by Hansen [19], and by HPLC. HPLC for identification of sugars

The above aqueous solution prepared from the acid hydrolysate of deacyl Gl-ai was subjected to HPLC performed according to the method described by Kinoshita [20]. Briefly, the sample was introduced into a TSKgel Sugar AXG column, 4.6 X 150 mm (Toso Co Ltd, Japan). The column temperature was 68°C. The solvent introduced at a flow rate of 0.4 ml/min was 0.5 M borate buffer, pH 8.7, containing 3% of borateethanolamine complex which had been prepared by adding 1 mol of ethanolamine to 700 ml of warm (60°C) EtOH in which 1 mol of boric acid had been dissolved and by collecting the resulting precipitate after several hours at room temperature. The eluate was introduced into a 12 m stainless steel tube of 0.4 mm diameter, the first 10 m of which was kept in a silicone oil bath at 130°C and the following 2 m in ice water, before being introduced into a fluorescence detector. (Excitation at 353 nm, emission at 436 nm) GC-MS of alditol acetate

Pure deacyl Gl-ai permethylate (5 mg) was heated in a sealed tube with 2 N H,SO, (0.5 ml) at 100°C for 4 h.

56

The hydrolysate was neutralized with anion exchange resin, freeze-dried, treated with NaBH, in MeOH at room temperature for 3 h, treated with AcOH-containing MeOH under reduced pressure and then acetylated with Ac,O (0.1 ml) and pyridine (0.1 ml). Results

On TLC, the acid hydrolysate of deacyl GI-ai gave a single spot, the R, of which was about the same as that of arabinose (0.48). By HPLC, the sample gave a single peak of arabinose at a retention time of 18 min. In the ‘H-NMR spectra of Gl-ai, its acetate (Fig. 31 and other derivates are observed two anomeric proton signals, one at 4.9-5.0 and the other at 5.0-5.4. Accordingly, Cl-ai was shown to contain 2 mol of arabinose as its sugar moiety. Acid companent

The methylatio~ product of the diethyl ether-soluble fraction from alkaline hydrolysate of GI-ai gave four compounds (Compounds l-4, Fig. 20, when separated by two TLC systems, one using 5% acetone in toluene and the other using triple development with 10% diethyl ether in hexane. In the ‘H-NMR spectra of Compounds I-3, the signals at 3.67 (3 H, s, CHOCO-1, 3.66 (1 H, br, -CH(OH)CHRCOO-) and 2.41-2.45 (1. H, m, -CI$OH)CHRCOO-1 were commonly observed, showing that they were mycoiic acid methyl esters. In addition, the signals at -0.34 (2 H, m), 0.54 (2 H, ml

and 0.63 (4 H, m), ascribable to c&substituted cyclopropane ring protons, were observed in the spectrum of Compound 1, whereas the signals at 0.09, 0.14, 0.19 and 0.45 (1 H each, m) assigned to ~~~~s-substituted cyclopropane ring protons and at 0.66 (1 H, m, cyclopropyl-CH(CH,)-1 were in those of Compounds 2 and 3. The spectrum of Compound 2 showed a multiplet of three protons at 2.38-2.40 (-CI-$CH,)COCH,-1 and a doublet of three protons at 1.05 (-COCH(Cr-J-1. The spectrum of Compound 3 gave a signal at 2.29f2 H, t, -CH,CH,COOCH,) and another methyl ester signal at 3.74.-n the basis of the ‘H-NMR data, Compounds l-3 were identified as methyl esters of di-cis-substituted cyclopropyl mycolic acid (cu-mycolic acid), ~~u~~-substituted cycIopropy1 beak-mycolic acid (ketomycolic acid) and w-carhoxy ~~~~~-substituted cyclopropyl mycolic acid (hydrolysed wax ester mycolic acid), respectively. Compound 4 was identified as the alcohol moiety of wax ester mycolate on the basis of the proton signals at 3.80 (1 H, m, -CH,CI-J(CH,)OH), 1.18 (3 H, d, -CH(C~~OH) and 0.88 C3I-I t, CH,CH 2-). In some experiments, unhydrolysed wax ester mycolic acid methyl ester with its R, value on TLC between those of a-mycolate and ketomycolate was obtained, whose ‘H-NMR spectrum was characterized by the signals of trans-cyclopropyl protons and those at 1.19 (3 H, d, -CH~CH~~OCOCH~-~, 2.28 (2 H, t, -CH(CH,lOCOCH2-) and 4.88 (1 H, m, -CE_I(CH,)OCOCH2-1 in addition to those commonly observed in the spectra of Compounds I-3, showing that this intramolecular ester bonding is fairly resistant to alkaline hydrolysis.

Oi7

4.0

2.5

2.0

016

015

1.5

Fig, 3. ’ H-NMR spectrum of C&i acetate EnCdCI,.

014

0.3 ' 012

0.7

0:o -0.1 ‘012 -0!3 -0i4

57 TABLE

I

Areas of some characteristic proton signals obsewed in ‘HEWER spectrum of Gl-ai acetate in CdCl, Groups

cis-

transcyclopropyl

cyclopropyl Protons -?-IH-

-C”-“\-?+ Me

CH

I

wax ester mycolate

keto mycolate

all mycolates

arabinose-A

glycerol

-CHOCOCH,-

-CCHCOCH,-

-CHCHCOO-

Cl H

C2’ H

CH,

Me

Me

Acb

k

FI Chemical shifts Signal

-0.34

areas

2.0

0.45

2.25

2.50

2.40

5.08

5.35

5.20

1.5

1.9

0.5

1.1

2.5

3.2

1.3

Mycolic acids (mol) in Gl-ai cY-mycolic acid

ketomycolic

0.80

The a-akyl The product

acid

wax ester mycolic acid

0.45

0.75

mass spectra of those esters showed that their chains are CH,-(CH,),,-, as reported [21]. ether soluble fraction of LiAiH, reduction of Gl-ai acetate gave, when separated by TLC

5.0 Fig, 4.2 D C-H COSY NMR spectrum

using 1.2% MeOH in CHCI,, three alcohols (Alcohols 1-3, Fig. 2B). In the ‘H-NMR spectra, in addition to the signals at 3.92 (1 H, m, -CI-_I(OH)CHRCH,OI-I) and 3.66 (2 H in Akohols 1 and 2, and 4 H in AIcohol

4.5 of Gi-ai acetate

3.5

4.0 in CdCI,

showing

the presence

of glycerol.

wm

3, m, -C&OH) observed in all the three alcohols, the signals of &-substituted cyclopropyl protons at -0.34 (2 H, m>, 0.54 (2 H, m> and 0.63 (4 H, m> were observed in the spectrum of Alcohol 1, and those of truns-substituted cyclopropyl protons at 0.09-0.19 (3 H, ml and 0.45 (1 H, m) were in the spectra of Alcohols 2 and 3. The signals at 3.43 (0.5 H, ml and 3.49 (0.5 H, ml ascribable to the isomeric proton (-CH(CH),CEJ (OH)-) produced by the reduction of the carbonyl group of ketomycolic acid were observed in the spectrum of Alcohol 2. On the basis of the ‘H-NMR data, Alcohols 1-3 were identified as the alcohols derived from a-mycolic acid, ketomycolic acid and wax ester mycolic acid, respectively.

oxymethylene protons at 4.18 and 4.28, all duly assigned by the C-H COSY NMR spectrum, were forming one independent group. In the ‘H-NMR spectrum of Gl-ai acetate (Fig. 31, as shown in Table I, the ratio between the signal area of arabinose-A Cl proton at 5.40 and that of glycerol C2’ proton at 5.20 (or of glycerol Cl’ proton at 3.56 or 3.80) is 1: 1, showing that Gl-ai contains 1 mol of glycerol. Chemical structure of GI-ai

The relationship between each molecule constituting Gl-ai may be elucidated by the comparison of the chemical shifts of the ‘H- and ‘“C-NMR signals of Gl-ai and its derivatives shown in Table II, in which all the signals have all been duly assigned by the H-H and C-H COSY NMR spectra or by the decoupling technique. The chemical shift of glycerol Cl’ proton is about the same in all the four compounds, suggesting that the linkage involving glycerol Cl’ remains the same in all these compounds and that, therefore, Cl’ hydroxyl is engaged in the formation of glycoside linkage. On acetylation, the signals of glycerol C2’ and C3’ protons of Gl-ai shifted to lower field, suggesting that the C2’ and C3’ hydroxyls in Gl-ai are free, whereas neither of the proton signals of the two arabinose C5 methylenes showed any lower field shift, suggesting that both of the arabinose C5 hydroxyls of Gl-ai are esterified in Gl-ai with mycolate residues. On the basis of the chemical shift values of arabinose carbons [22] and the fact that the chemical shifts

Mycolic a&d composition

The ratio between the three mycolic acids constituting the ester component of Gl-ai may be estimated by comparing the areas of some well-isolated characteristic signals in the ‘H-NMR spectrum of Gl-ai acetate (Fig. 3). Thus, Table I shows that the mol ratio between arabinose-A and mycolic acid is 1: 2, and that the 2 mol of mycolic acids consist of 0.80, 0.45 and 0.75 mol, respectively, of cy-, keto- and wax ester mycolic acids. Glycerol moiety

The presence of glycerol was clearly shown by the 2 D H-H COSY NMR spectrum (Fig. 4) of Gl-ai acetate, in which one pair of oxymethylene protons at 3.56 and 3.80, one oxymethin proton at 5.20 and another pair of TABLE

II

Chemical shifts of ‘-‘C- and ‘H-NMR signals of Cl-ai and its dericatir’es in CdCI, GI-ai ‘H Glycerol Cl’

69.8

C2’ C3’

70.6 63.7

Arabinose-A Cl 102 c2 77.3 C3 76.5 c4 79.7 c5 65.5 Arabinose-B Cl c2 c3 c4 C5

106 88.4 77.6 80.4 63.5

GI-ai acetate

Deacyl GI-ai peracetate

Deacyl GI-ai permethylate

“C

UC

17C

3.62 3.75 3.85 3.62-3.65 3.72-3.75

65.2

5.03 (d) 4.05 4.05-4.10 4.05 4.25 4.40

99.0 77.5 75.8 79.2 65.7

5.02 (s) 4.09 4.07 4.18 4.22 4.38

104.7 83.3 77.5 80.5 64.3

69.8 62.5

J values (Hz) given only were reasonably

‘H 3.56 3.80 5.20 4.18 4.28

(dd, J 4.8, 10.6) (dd, J 5.9, 10.6)

5.35 4.93 5.29 4.08 4.27 4.31

(d, J 4.5) (dd, J 4.5, 6.4) (dd, J 4.9, 6.4) (dt, J 4.9, 4.9, 8.2) (dd, J 4.9, 11.3) (dd, J 8.2, 11.3)

(dd, .I 6.3, 11.7) (dd, J 3.4, 11.7)

4.89 (s) 4.25 4.92 4.18 4.20-4.25

(2 H, m)

easily calculated.

s, singlet;

65.3 69.8 62.8

99.5 77.2 75.4 79.0 65.2

105 84.0 77.5 80.8 63.8

d, doublet;

‘H

3.60 3.80 5.20 4.17 4.25

(dd. J 4.5, 11.0) (dd, J 5.2, 11.0)

66.2

(dd, J 5.2, 11.7) (dd. J 4.0, 11.7)

5.39 (d, J 4.7) 4.98 (dd, J 4.7, 6.6) 5.34 (dd, J 5.1, 6.6) 4.12(dt, J4.6,5.1, 7.8) 4.22 (dd, J 7.8, 11.6) 4.38 (dd, J 4.6, 11.6) 4.91 (s) 4.22 4.98 4.17 4.18 4.30 (dd, J 2.7, 10.3) dd, double

doublet;

‘H

79.0 72.3

3.56 3.79 3.48 3.47 (2 H. m)

99.5 85.8 84.5 80.2 75.0

5.14 (d, J 4.2) 3.80 (dd, J 4.2, 7.0) 3.77 (dd, J 6.1, 7.0) 4.00 (dt, J 5.7, 5.7, 6.1) 3.50-3.53 (2 H, m)

107.0 85.2 85.9 80.9 72.8

m, multiplet.

5.02 4.25 3.75 4.10 3.58 3.59

(d, J 1.2) (dd, J 1.2, 3.2) (dd, J 3.2, 6.2) (dt, J 4.0, 6.0, 6.2) (dd, J 6.0, 10.6) (dd, J 4.0. 10.6)

MYcol-OHqC

o

Myco~-oH2c&a~;H OH Ara-S AM-A

Fig. 5. Structure

&H,OH

of GI-ai.

of both the arabinose C4 protons remain at about the same value in all the compounds, these two arabinose molecules were identified as furanosides. On acetylation, the ‘H-NMR signals of C2 and C3 protons of arabinose-A and C3 proton of arabinose-B shifted to lower field, showing that C2 and C3 of arabinose-A and C3 of arabinose-B bear free hydroxyls in Gl-ai. The chemical shift of C2 carbon of arabinose-B remains in lower field in Gl-ai and its acetylation derivatives, and that of arabinose-B C2 proton of Gl-ai does not shift on acetylation, showing that arabinose-B C2 is the location where arabinose-A links. Of the two arabinose Cl proton signals, the signals caused by Cl proton of arabinose-A are always doublets with J values of about 4.5 Hz, whereas those due to Cl proton of arabinose-B are singlets, suggesting that arabinose-A is p, whereas arabinose-B is (Y[231. On the basis of the data given above, GI-ai was shown to be 5-mycoloyl-P-arabinofuranosyl-(1 + 2)-5mycoloyl-a-arabinofuranosyl-(1 + l’)-glycerol (Fig. 5). The results of mass spectrum of deacyl Gl-ai acetate (Fig. 6) and of GC/MS of the alditol acetate mixture

03

CHzOAc 117 I

MeO&H I 181

HCOMe

I 117

191-MeOH--+ ’ - AcOH---,

101

HCOAc ~H,OM~

129.03

1:29.c )3

;H,OAc AcOCH 189-AcOH+129

161- MeOH+ 100

i9

i9

AcOH-+lOl

161.05

90 *5

SO 70 60

Fig. 7. GC/MC of alditol acetates derived from deacyl GI-ai permethylate. (A) GC profile. (B) MS of peak 7.51, (C) MS of peak 9.01.

50 40 30

derived from the acid hydrolysate of deacyl Gl-ai permethylate (Fig. 7) further confirm this structure.

20 10

Discussion

0 J

r

100

Fig. 6. Mass spectrum

of deacyl Gl-ai acetate.

The new glycolipid, Gl-ai, isolated from a hexane extract of MA1 complex cell mass consists of 2 mol of

sugars, 2 mol of fatty acids and 1 mol of glycerol. But in contrast to glycosyl d~acylglycerol-ape lipids commonly found in plants, Gram-positive bacteria or algae [24,25], in Gl-ai, these units are arranged as acylglycosyl- acylglycosylglycerol, or 5-mycoloylarabinofuranosyl-(1 --$2)-5-mycoloylarabinofuranosyl-(1 -+ l’>glycerol, Whether these arabinose residues are D- or L.was not examined. However, since n-arabinose is a major sugar of mycobacterial cell wall components, the arabinose residues are shown in D form in Fig. 5. According to Daffe et al. [26], the structure of the end portion of arabinogalactan, commonly present in mycobacterial cell walls, is the ~-arabinofuranosyl-~1 + 2)-ff-arabinofuranosyl group and, according to McNeil et al. 1271, two thirds of the C5 hydroxyls of the P-arabinofuranosyl-(1 -+ 2&-arabinofuranosyl end group are esterified with mycolic acid residues to form the 5-mycoloyl-/3-arabinofuranosyl-(1 + 2)-5-mycoloyla-arabinofuranosyl end unit. It is interesting that in Gl-ai, the 5-mycoloyl-P-arabinofuranosyl-(1 -+ 2)-5mycoloyl-ru-arabinofuranosyl moiety forms a glycoside linkage with glycerol Cl’ hydroxyl, whereas the same unit forms a glycoside Iinkage with another arabinose residue in mycoIoy1 arabinogalactan. However, it is not likely that Gl-ai is an in vitro product of MAI complex cells produced by the incubation of the cells in glycerol-rich Sauton’s medium because only in MA1 patients’ sera were very high anti-Gl-ai titers detected (unpublished data). According to Rouanet and Laneelle 1281, 5mycoloyldiarabinofuranoside and synthetic 5-mycoloyl(2’-hydroxy ethyl)-arabinofuranoside have about the same effect as 6,6’-dimycoioyltrehalose on the oxidative phospho~lation and respiration of mitochondria, and therefore, 5-mycoloyldiarabinofuranoside, possibly derived from cell wall arabinogalactan during the long course of mycobacterial disease, may function in the same way as 6,6’-dimycoloyltrehalose detached from the ceil walls. Then, Gl-ai, a 5-mycoloyldiarabinofuranoside derivative of a relatively small molecular weight, readily available on the cell wall, may be expected to contribute to the mobidity of MA1 diseases by acting on mitochondria in a similar way, as it is or by providing some active derivatives.

We thank Dr. Yasuo Shida of the Analytical Centre of Tokyo College of Pharmacy for the measurements of ‘H-NMR, ‘“C-NMR and mass spectra.

References 1 Minnikin, D.E. (19821 Lipids: Complex lipids, their chemistry, biosynthesis and role, in The Biology of Mycobacteria, (Ratledge. C. and Stanford. J., eds), Vol. 1, pp. 95-184, Academic Press. London. 2 McNeil, M.R. and Brennan, P.J. (19911 Res. Microbial. 142, 45 l-463. 3 Minnikin, D.E. (19911 Res. Microbial. 142, 423-427. 4 Rastogi, N., Hellio, IX. and David, H.L. (1991) Zentralbl. Bakteol. 275. 287-302. s Hunter, SW., Fujiwara, T. and Brennan, P.J. (1082) J. Biol. Chem. 257, 15072-15078. 6 Daffe, M., Lacave, C., Laneelle, M.A. and Lanieile, G. (1957) Eur. J. Biochem. 167, lS5-160. 7 Papa, F., Laszlo, A. and David, H.L. (19881 Ann. Inst. Pasteur 139, 535-545. 8 Papa, F., Cruaud, P. and David, H.L. (1989) Res. Microbial. 140, 569-578. 9 Vercellone, A. and Puzo, Cl. (1989) J. Biol. Chem. 264,7447-7454. 10 Papa, F.. Riviere, M., Foarnie, J.J., Puzo, G. and David, H. (1987) J. Clin. Microbial. 25, 2270-2273. 11 Papa, F., Vaquero, M. and David, H.L. (1988) Ann. Inst. Pasteur 139. 149-157. 12 Besra, G.S., McNeil, N., Minnikin, D.E., Portaels, F., Ridell, M. and Brennan, P.J. (19911 Biochemistry 30, 7772-7777. 13 Brennan, P.J. (198% Mycobacterium and other actinomycet~s, in Microbial Lipids (Ratledge, C. and Wilkinson, S.G.. eds.), Vol. 1, pp. 203-298, Academic Press, London. 14 Torgal-Garcia, J.. David, H.L. and Papa, F. 11988) Ann. Inst. Pasteur 139, 289-294. 15 Praputtaya, K., Suriyanor, V., Hirunpetcharat, C., Rungruenp manakit, K. and Suphawilai, C. (19901 Asian Pac. J. Allergy Immunol. 8, 19-2.5. 16 Brennan. P.J. and Goren, M.B. (19791 J. Biol. Chem. 254, 420% 4211. 17 Brennan, P.J., Mayer, H.. Aspinell, G.O. and Nam Shin, J.E. (19811 Eur. J. Biochem. 115, 7-15. 18 Brennan, P.J. (19811 Rev. Inf. Dis. 3. 905-913. 19 Hansen, S.A. f 1975) J. Cbromatogr. 107, 224-226. 20 Kinoshita, T. (1986) Report for the Ministry of Education Special Resarch Fund, No. 59570926, Japan. 21 Kaneda. K., Imaizumi, S. and Mizuno, S. (1988f J. Gen. Microbiol. 134, 2213-2229. 22 Bock, K. and Pederscn, C. (1983) Adv. Carbohydr. Chem. Biochem. 41, 27-h6. 23 Mizutani, K., Kasai, R., Nakamura, R. and Tanaka, 0. (1989) Carbohydr. Res. 185, 27-38. 24 Kitagawa, I., Hayashi, K. and Kobayashi. M. (19HYl Chem. Pharm. Bull. 37, 849-851. N., Imamura, H., Sakakibara, J. and Yamada, N. 25 Murakami. (1990) Chem. Pharm. Bull. 38, 3497-3499. 26 Daffe, M., Brennan, P.J. and McNeil, M. (1990) J. Biol. Chem. 265, 6734-6743. 27 McNeil, M., Daffe. M. and Brennan, P.J. (1991) J. Biol. Chem. Zhh, 13217-23223. J.M. and Lan&lle, G. (19831 Ann. Microbial. (Inst. 28 Rouanet, Pasteur) 134B, 233-239.

A new glycolipid from Mycobacterium avium--Mycobacterium intracellulare complex.

From a nonpolar lipid fraction of Mycobacterium avium--Mycobacterium intracellulare complex cell mass, a new glycolipid was obtained, which was shown ...
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