Branched fatty acids from mycobacterium aurum

266

Rioc~hrn~icu et Rwplywu

Ac,ru. 7 I I ( 19X2) 266-27 I Elscvicr Biomedical Press

BBA 51096

BRANCHED

FAm

ACIDS

ELIE RAFIDINARIVO, ARLETTE JEAN-CLAUDE PROME

FROM

MYCOBACTERIUM

SAVAGNAC,

CHARLOTTE

LACAVE

Centre de Recherche de Biochimre et de G&nPlique Celluluires du CNRS. (Received

November

AURUM and

118, route de Norhonne. 3106? Toulou.w Cedew (Frunw)

23rd, 198 1)

KQJ words: Futty acrd cornpositron; Brunched

chain; h(vc’osunoicucid; (M. uurunz)

New methyl-branched fatty acids were isolated from the lipids of Mycobacterium aurum, belonging to both saturated and non-saturated series. The most abundant component of the former series was identified as a C,,-mycosanoic acid (2-L, 4-L-dimethyleicosanoic acid). The unsaturated fraction contained a mixture of 2-L, 4-L-dimethyl-ll-eicosenoic acid and 2-L, 4-L-dimethyl-14-eicosenoic acid. The biosynthetic precursors of these, according to the hypothesis of elongation by propionate units, were found in the non-branched hexadecenoic fraction. The lipidic fraction containing mycosanoic acid was a partially acylated oligosaccharide devoid of sulfate or phosphate groups.

Introduction

Isolation of the non-hydroxylated fatty acid fraction The cells were extracted by a mixture of chloroform/methanol (2 : 1, v/v) and the dry extract was saponified. After acidification and extraction by diethyl ether, the fatty acids were methylated by ethereal diazomethane and chromatographed on neutralized silicic acid using a gradient of diethyl ether in hexane. The non-hydroxylated fractions, as identified by thin-layer chromatography and infrared spectroscopy, were pooled and analyzed by gas-liquid chromatography.

During our investigation on the action of some drugs on the fatty acid metabolism in mycobacteria, we have studied the lipidic composition of Mycobacterium aurum strain At. The growth of this strain is inhibited by very low amounts of isoniazid. In the present paper, we reported the existence of unusual branched fatty acids in the lipids of this mycobacterium. Materials and Methods

Isolation of the branched fatty acids The fatty acid methyl esters possessing a normal aliphatic chain were eliminated by coprecipitation with urea [I]. The separation between saturated-branched and unsaturated fatty acid methyl esters was achieved by silver nitrate/silicic acid column chromatography [2]. Each fraction was then fractionated by high-performance reverse-phase chromatography (PC ,,-Bondapak). The saturated fatty acid esters were eluted by a mixture of acetonitrile/acetone (4:6, v/v) with a

Bacteria and growth conditions Strain A+ of M. aurum was a gift from Professor Hugo L. David, Institut Pasteur, France. This bacteria was maintained on Lowenstein-Jensen slants. Bacteria for lipid analysis were grown in 2-l Erlenmeyer flasks containing 500 ml Middlebrook medium 7H9 (Difco, supplemented with 0.5% casitone and 1% glucose) under agitation at 37°C. The bacteria were collected by centrifugation at the end of the exponential growth period. 0005-2760/82/OOOS0000/$02.75

0 1982 Elsevier Biomedical

Press

261

flow rate of 1 ml/min. The capacity factor values of palmitic, tuberculostearic and C,, -mycosanoic (IA) methyl esters were 0.45, 0.64 and 0.96, respectively. The unsaturated fatty acid esters were separated using acetonitrile as mobile phase with a flow rate of 0.5 ml/min. The capacity factor of hexadecenoate, octadecenoate and C,, mycosenoate (V and VI) were 0.73, 1.15 and 2.28, respectively. Pure fractions isolated by HPLC were analyzed directly by mass spectrometry or transformed chemically as described below. Analysis of unsaturated fatty acids The fractions containing hexadecenoic or octadecenoic acid methyl esters were analyzed chemically by oxidative cleavage according to the method of Lemieux and Von Rudloff [3]. The resulting fatty acids were esterified by diazomethane and identified by gas-liquid chromatography by analogy with authentic samples of mono- and I-o-diacid methyl esters. Results are given in Table I. The fraction containing an unsaturated branched C,,-fatty acid, as detected by mass spectrometry, was hydrogenated using platinum as catalyst. The resulting saturated fatty acid was found to be identical to compound I,: identical mass spectrum, same GC retention time, dextrorotation.

TABLE

I

CLEAVAGE PRODUCTS PERIODATE/PERMANGANATE UNBRANCHED FATTY ACIDS

ARISING OXIDATION

Carbon number of the unsaturated fatty acid

Cleavage Monocarboxylic acids

Dicarboxylic acids

CM

C6 C, C9 C9

Cl,

CM

products

C9 C, C9

FROM OF THE

Relative proportion (%)

75 15

10 100

In another experiment carried out on products extracted from bacteria grown on a medium supplemented with [l-‘4C]acetate, dihydroxy mycosanoic acids were obtained from the unsaturated-branched C,, fraction by the following successive operations [4]: formation of epoxide by reaction with peracids; opening of the epoxide ring by refluxing with acetic acid; alkaline hydrolysis. Thus, 1 mg esters were mixed with 6 mg pnitroperbenzoic acid in 0.6 ml anhydrous ether and the solution was left to react 1 day at room temperature. The solution was then separated by thin-layer chromatography (silica gel G, hexane/diethyl ether, 9: 1 v/v) and the radioactive band (R, = 0.4) was scraped off and eluted. These epoxyesters were dissolved into 0.3 ml acetic acid and the mixture was refluxed during 3 h. After evaporation of acetic acid, the residue was saponified by 5% potassium hydroxide. The resulting hydroxy acids were extracted with diethyl ether and methylated by ethereal diazomethane. The methyl esters were chromatographed on thin-layer plates (silica gel G, hexane/ether, 1: 1 v/v) and the radioactive band (R, = 0.3) was eluted. After formation of TMS-derivatives by the method of Sweeley et al. [5], the product was analyzed by GC/MS. Two slightly separated GC peaks (shoulder) on 3% OVl, 1 m length column, were observed. Alternatively, a mass spectral analysis was realized by direct inlet on the unseparated mixture of TMS-derivatives which gave readily interpretable spectra, indicating the superimposition of the spectra of the two products, III and IV. Instrumentation A Varian Mat 3 11 A mass spectrometer instrument was used for direct inlet measurements. The GC/MS spectra were obtained by means of a NERMAG R-1010 instrument equipped with a PDP-8 computer. The GC analyses were performed on a GIRDEL F30 gas chromatograph equipped with OVl support-coated glass columns. The location of radioactive TLC bands was achieved by a Panax radioscanner. The separations by high-performance chromatography were obtained by a Waters M 6000 A instrument equipped with a differential refractometer and a 0.39 X 30 cm PC, 8-Bondapak column.

268

<

Results Saturated

CH,-_(CH,),tCH+CH,-~‘CH-COOCH, I

methyl branched fatty acids

The gas chromatographic profiles of the nonhydroxylated fatty acid esters of M. aurum are presented in Fig. 1. By comparison with the wellknown profile of the esters from M. phlei, some extra peaks were detected. The major represents roughly 15% of the total of the non-hydroxylated fatty acids. Other minor compounds were detected. As described in the experimental section, this major component, named compound I,, was concentrated in saturated fractions that did not form urea-adducts. Thus, it seemed to be a branched fatty acid. Its isolation was obtained by high-performance liquid chromatography. An electron-impact mass spectrometric analysis indicated the following features. The molecular weight is 354 (methyl ester of a C,,-fatty acid). An

t 0

5

10 Retention

15 time

20

I CH,

i j

I ~ CH,

L.....-m/z101

:----,m/z129 compound

IA: n = 15

compound

1~: n = 13

Scheme

1

intense fragment ion at m/z 88 indicates a-methyl branching. An increase of the intensity of m/z 101 and m/z 129 comparative to the spectra of straight chain fatty acid methyl esters, together with a low intensity of m/z 115, suggested y-methyl branching. The presence of methyl branches on carbons (Y and y was confirmed by the examination of ions in the high mass range: the loss of the C,-C, and C&,-C, part of the chain a characteristic feature of fatty esters - produces ions at m/z 311 and 283. The former corresponds to the loss of a propyl group (which indicates a methyl branch on the first neutral fragment). The latter is due to the loss of a pentyl group (indicating two methyl branches on the corresponding fragment). No particularly intense ion characteristic of other fragmentations of the aliphatic chain can be observed. Thus, compound I, is a 2,4-dimethyleicosanoic acid methyl ester (SchemeI). Its specific optical rotation is [a]20n = + 7” 1’ (C = 0.96 isooctane). By GC/MS analysis of the saturated-branched fraction, two minor components corresponding to homologues or analogues were detected. Thus, an homologous 2,4-dimethyloctadecanoic acid methyl ester (In) was identified, together with single branched 2-methyl octadecanoic acid methyl ester II (Scheme 2).

(min)

Fig. I. Gas chromatographic profile of the non-hydroxylated fatty acid methyl esters of M. crurum. The peaks were identified as: I, myristic (C14:O); 2, hexadecenoic (Cl6: I); 3, palmitic (C16:O); 4, unidentified; 5. oleic (Cl8: I); 6, steak (C18:O); 7, tuberculostearic; 8, mycosenoic (compounds V and VI); 9. mycosanoic (compound I,); IO, docosenoic (C22 : I): I I, behenic (C22 : 0); 12, tetracosenoic (C24 : I); 13. lignoceric (C24 : 0).

CH,-(CH,),,-CH-COOCH, I compound

LH,

Scheme

2

II

269

OTMS OTMS

+.

I II cHJ(cH,),-CHGkcH-(CH,),-CH-CH,-CH-COOCH, I I i I

transfer I

L

of TMS group

1 A

AH,

AH,

\

(III)

+O-TMS

\

!-OCH

I m/z

&H-

iMS

215

;)=cH-(cH,)~-cH-cH,-CH-C~~CH~ I cH3

I TMS m/z

3 CH,

CH, 1 cb

CH-CH,

315

(kH& CH-OTMS ‘OdH

‘CH-(CH&-CH-CH,-CH-C=i--TMS OTMS

CH,

I CH,

I OCH,

(CH,), CH3

m/z 388

Scheme

3

Unsaturated methyl branched fatty acids In the unsaturated fraction, unusual GC peaks were also observed. GC/MS analysis indicated that the major product was a 2,4-dimethyl eicosenoic acid methyl ester (molecular ion at m/z 352; characteristic and intense fragment ions at m/z 88, 101, 129; very low intensity of m/z 115). A minor homologue was characterized as 2,4-dimethyl octadecenoic acid methyl ester. Lastly, a very low amount of a methyl ester of a branched monounsaturated C&fatty acid was found. The mass spectrum of this latter also indicates a 2,4-dimethyl branching. Due to the biosynthetic rules for this class of compounds (see below) and because of the odd carbon atom number of the latter homologue, a third methyl branch on position6 is likely. The expected characteristic features are observed in the mass spectrum but the intensity of the corresponding ions is not sufficient to allow a firm assignment. A complete structural study was achieved on the major component of this series, after isolation

by reverse-phase high-performance liquid chromatography. Firstly, the unsaturated ester was hydrogenated. It gives a saturated methyl ester identical to compound I, by its mass spectrum and GC retention time. Moreover, this saturated compound is dextrorotatory. Next, the location of the unsaturation was determined by mass spectrometric analysis of the TMS derivative of the a-diol obtained after dihydroxylation of the double bond. Since a direct study indicated the superimposition of two different spectra, a GC/MS analysis was performed and two different mass spectra were differentiated by the observation of the decrease or increase of major fragment peaks during the repetitive scanning over the partially resolved GC peak. The trailing edge of the peak gives a spectrum characteristic of the major component with the following intense ions: m/z 215, 315 and 388. According to the known fragmentation pathway of such derivatives [4], these ions correspond to the cleavage of the C-C bond between the two tri-

270

“‘My

I

CH,p(CH,),pCH

yb4s

+CH WCH,),CH-CH,p-CHMOOCH, I CH, ,’

CH,

\\ >\

//’

‘,

(IV) ml2

173

m/z

357

Scheme

CH,m

(CH,),mpCH

=CH

4

-(CH,),-CH-

XH,-CHI CH,

CH,

~~(CH,),~--pCH=C~-(~~,&~pC~-C~i-~~H--CoOC~,

‘3 Scheme

COOCH, I CH,

I ‘3

(V)

(VI)

5

methylsilyloxy groups. The former contains the methyl end of the molecule, the second one contains the carboxyl end and the latter is due to trimethylsilyl transfer to the carboxyl group prior to fragmentation (Scheme3). Thus, this product possesses structure III. The minor component included in the leading edge of the chromatographic peak possesses structure IV (Scheme 4). Hence, the structure of the corresponding unsaturated methyl branched fatty acids are V and VI (Scheme 5). The specific optical rotation of the mixture of the two unsaturated compounds is [ (Y]*‘IJ = + 9” 1’ (C = 0.34, isooctane). Discussion Structural analogy with other branched mycobacterial fatty acids Saturated fatty acids containing a and y branches have been described in mycobacteria. The first known were called mycocerosic acids. Their chain length was longer than the present one. In human and bovine strains of M. tuberculosis, the three main components were a &-acid with three branches, a (&-acid with four branches, and a &-acid with four branches (major compound) [6,7]. In M. phlei, a C,,-acid with five branches was also described [8]. However, the methyl mycocerosates were levorotatory and the D configuration for each assymetric carbon was determined [9].

Several dextrorotatory 2,4-dimethyl-branched fatty acids were also reported from the tubercle bacillus, Cason et al. [lo] have isolated a saturated 2,4-dimethyl-branched C,, fatty acid for which they proposed a 2-L, 4-L configuration. However, the optical rotation of the natural sample (+2”2’) was significantly lower than the value of the synthetic 2-L, 4-L-dimethyl docosanoic acid (+ 7O4’). It is interesting to notice that the optical rotation of compound I, isolated from M. aurum possesses a very close value (+ 7” 1’). Since a slightly lower aliphatic chain would not modify significantly the specific optical rotation, it seems highly probable that compound I, possesses a 2-L, 4-L configuration. Thus, according to the nomenclature of Cason et al. [ 111, the corresponding acid should be called C,,-mycosanoic acid. Another class of dextrorotatory 2,4-dimethyl branched fatty acids was described more recently. These acids, called phthioceranic acids [ 121, possess a much higher degree of branching (from 5 to 10 methyl branches) and a larger carbon number (from C3, to C,,). Comparisons with metabolic relations It was demonstrated that the (2,4...)-branched fatty acids are biosynthesized by successive condensations of propionate units with common fatty acids of the bacteria. Thus, biosynthetically, all the branched fatty acids appeared to be also closely related (Scheme 6). If the unsaturated 2,4-dimethyl-branched acids were synthesized by a similar process, the unbranched unsaturated precursors would be present in the bacteria. Hence, according to structures V and VI, the presence of A7- and AlO-hexadecenoic acids was expected. To verify this hypothesis the structure of the components of the hexadecenoic acid fraction was investigated after a permanganate-periodate oxida-

cH,-

(CH,),-(TH-CH,),-YH-CO~H kH,

&H,

n :17

m:l

” : 15

m:l

” z15

mz4to

cason s

ca

nycosano~

C?Z -mycosano~c 9

Scheme

Pht,ocetanlC

6

acid

acids

acid from

M

auwm

271

CL) Cli-(CH,),,-CH-CH,-CH-CH=C-COOH

CZI phttenolc

ac,d

Scheme

(VII)

7

tive cleavage according to the procedure of Lemieux and Von Rudloff. The results are presented in TableI, which indicates that the expected fatty acids are effectively present since the major component was AlO-hexadecenoic acid (75%). The A7- and A9-hexadecenoic acids were present in lower amounts: respectively 10 and 15%. The cis geometrical isomerism can be deduced by the lack of formation of urea adducts. In contrast, the octadecenoic fraction was found to only contain A9-octadecenoic acid (oleic acid). The presence of the A10 unusual isomer of palmitoleic acid in mycobacteria has been reported already. It represents about 80% of the hexadecenoic fraction in A4. phfei [ 131 and small amounts were detected in the tubercle bacillus

the latter were not found, particularly the loss of 87 and 100 mass units from the molecular ion [IS]. So, the double bond was not between carbons 2 and 3 in the former. Thus, M. aurum seems to not contain any phtienoic acid. A structural similarity between mycosanoic acid and phtioceranic acids could also indicate a similar physiological role. Hence, an analogy between their lipidic complex structures could be expected. It has been mentioned already [14] that phtioceranic acids are components of a trehalose lipid containing a sulfate group (sulfolipid). In preliminary experiments, we found that the C,,mycosanoic acid from M. aurum is linked to an oligosaccharide fraction different from clr-trehalose. Moreover, when bacteria were grown on a medium supplemented with [ 35S]sulfate or [ 32P]phosphate, no radioactivity was incorporated into the lipid. Thus, C,,-mycosanoic and phthioceranic acids were linked to largely different structures and a similar physiological role seems improbable. References

[141Since such close similarities in the structures of mycosanoic acids from M. aurum and tubercle bacillus have appeared, one can suggest that they possess similar properties in the metabolism of the bacteria. Cason et al. [lo] have postulated a relationship between the C,,-mycosanoic acid and the C,,-phtienoic acid VII (Scheme 7) because the latter would be biosynthesized by addition of a propionate unit on a 2,4-dimethyl-branched fatty acid possessing an L configuration. If a similar relation exists in M. aurum, the C,,-mycosanoic acid would be the precursor of a C,,-phtienoic acid. We have already mentioned that we have detected a 2,4-dimethyl-branched unsaturated C,,-fatty acid (possibly 2,4,6-trimethyl-branched) as a minor compound. However, a thorough comparison between the mass spectra of this compound and the spectra of methyl phtienoate revealed that some characteristic fragmentations of

10 I1 12 13

Asselineau, C. and Asselineau, J. (1964) Ann. Chim. 9, 441-479 De Vries, B. (1963) J. Am. Oil. Chem. Sot. 40, 184- 186 Lemieux, R.U. and Von Rudloff, E. (1955) Can. J. Chem. 33, 1701-1709 Mint&in, D.E. (1978) Chem. Phys. Lipids 21, 3 13-347 Sweeley. CC., Bentley, C., Makita, M. and Wells, W.W. (1963) J. Am. Chem. Sot. 85, 2497-2507 Gastambide, B. (1954) Thesis Dr. Sci. Paris Polgar, N. and Smith, W. (1963) J. Chem. Sot. 3,3081-3085 Prome, J.C. (I 968) Thesis Dr. Sci. Toulouse Asselineau, C., Asselineau, J., Ryhage, R., StallbergStenhagen, S. and Stenhagen, E. (1959) Acta Chem. Stand. 13, 822-824 Cason, J., Lange, G.L. and Urscheler, U.R. (1964) Tetrahedron 20, 1955- 196 I Cason, J., Lange, G.L., Miller, W.T. and Weiss, A. (1964) Tetrahedron 20, 9 I- 106 Goren, M.B., Brokl, O., Das, B.C. and Lederer, E. (1971) Biochemistry 10, 72-81 Lennarz, W.J., Scheuerbrandt, G. and Bloch, K. (1962) J. Biol. Chem. 237, 664-671

14 Cason, J. and Miller, W.T. 883-887 I5 Ryhage, R., Stallberg-Stenhagen, Arkiv. Kemi. 18, 179-187

(1963)

J. Biol.

Chew.

S. and Stenhagen,

238,

E. (196 1)