THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255, Nu. 1, lswe of January 10, pp. 182-189, 1980 Printed in U S A .

Purification of Fatty Acids from Mycobacterium tuberculosis H37Ra*

(Received for publication, July 16, 1979)

Nilofer Qureshi and Kuni Takayama From the Tuberculosis Research Laboratory, William S. Middleton Memorial Veterans Administration Hospital, Madison, Wisconsin 53705 a n d the Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin 53706

Heinrich K. Schnoes From the Department of Biochemistry, Colleze ofArrricultura1 a n d Life Sciences, University of Wisconsin, Madison, Wisconsin 53706

- . -

A complex mixture of nonmycolate c 3 0 - 6 6 fatty acids from Mycobacterium tuberculosis H37Ra were resolved into their individual components. Initially, two size ranges of long chain fatty acids, c 3 0 - 4 0 and c41-56 , were obtained and derivatized as p-bromophenacyl esters. The C30-40 esters were further fractionated by argenta- tion-thin layer chromatography to remove the unsatu- rated esters. The c 3 0 - 4 0 and c 4 1 - 5 6 esters were separated into their structural classes by thin layer chromatog- raphy using Silica Gel G plates and the solvent system of hexane/p-dioxane (49:1, v/v). Final separation into individual components was achieved by reverse-phase high performance liquid chromatography on a Cis- bonded silica column with the solvent system of p - dioxane/acetonitrile: 3:7, v/v, for the c 3 0 - 4 0 esters, 1:1, v/v, for the c 4 1 - 5 6 esters; and 9:11, v/v, for the methoxy- containing C41-56 esters.

Nuclear magnetic resonance, mass spectral, and chemical analyses of these purified fractions revealed the presence of the following: (a) a series of C34, C35, C37, C38, C39, and c 4 0 fatty acids containing two cis-cyclopro- pane rings; (b) a series of Ca4, C3B, and C S 8 fatty acids also containing two cis-cyclopropane rings, but differ- ing in structure from the previously mentioned series; (c) a larger series of C41, c 4 3 - 5 6 fatty acids containing two cis-cyclopropane rings; (d) a series of C44, c 4 6 - 4 9

fatty acids containing a methoxy group and either one or two cis-cyclopropane rings; and (e) several series of fatty acids containing the a, f l unsaturation.

The structure of one of the major components was a cis,cis-3,4- and 15,16-dimethylenetetratriacontanoic acid ( c 3 6 ) . This and many of the other long chain fatty acids appear to be structurally related to the a- and methoxymycolic acids.

The Mycobacterium tuberculosis species contains a wide range of nonmycolate fatty acids from C R - ~ , of which palmitic acid (C,,) is the most abundant (1, 2). The relatively short

* This work was supported in part by the Medical Service of the Veterans Administration, by Public Health Service Research Grant AI-11297 from the National Institute of Allergy and Infectious Dis- eases, and by the Wisconsin Alumni Research Foundation. A prelim- inary report of some of this work was presented at the annual meeting of the American Society of Biological Chemists/The American As- sociation of Immunologists in Atlanta, Ga., on June 4 to 8, 1978. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

chain fatty acids (c16 to c26) are associated with the neutral lipids, phospholipids, and glycolipids. The longer chain fatty acids (>cZ6) are recent discoveries and their function and association with lipids are not yet known. Takayama et al. (1) and Winder and Collins (3) showed that low concentrations of isoniazid (0.2 pg/ml), a drug widely used for the prevention and treatment of tuberculosis, inhibited the synthesis of my- colic acids, saturated fatty acids greater than CZ6, and unsat- urated fatty acids greater than c24 in M. tuberculosis H37Ra. The long chain fatty acids were suggested to be precursors of mycolic acids. Mycolic acids are a-alkyl, ,&hydroxy fatty acids, which are the major constituent of the mycobacterial cell wall

In our efforts to study the biosynthesis of mycolic acids in M . tuberculosis H37Ra and to locate the exact site of action of isoniazid, we established the structures of the a-mycolic acids (6) and the monounsaturated Cz4 fatty acids (7). The structure of the a-mycolic acids of the H37Ra strain is shown in Structure 1 where a = 17, b = 10, c = 15, 17, 19, 21; and d = 21,23. The most abundant components of the monounsat- urated C24-32 fatty acids had the double bond at the w-19 position; thus, these acids were structurally related to the a- mycolic acids. We then wanted to relate the structures of the long chain fatty acids (c30-56) to the mycolic acids in the H37Ra strain.

(4,5).

/c,“z /“\“2 T r : CH,(CH,)o-CH-CH-(CH,)bCH-CH-CH-(CH~)~-CH-CH-C”OH

I

I CH3

It is now technologically PO ible to separate complex mix- tures of high molecular weigh r fatty acids and determine their structures. Gas-liquid chromatography can feasibly be applied only to methyl esters of fatty acids about Caz or lower and, therefore, the Cw-% acids found in M. tuberculosis H37Ra are not amenable to such fractionation. Recently however, we used reverse-phase HPLC’ to purify a homologous series of mycolic acids (C76-w) and unsaturated fatty acids (6, 8, 9).

We have now fractionated a complex mixture of nonmyco- late c30-56 fatty acids from M. tuberculosis H37Ra by applying a combination of TLC and reverse-phase HPLC. We have determined the structure of the most abundant of the long chain fatty acids, a C36 acid containing two cis-cyclopropane

The abbreviations used are: HPLC, high performance liquid chro- matography; TLC, thin layer chromatography; Me& (TMS in struc- tures), trimethylsilane.

182

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Long Chain Fatty Acids 183

rings, and have structurally related i t to the a-mycolic acids. We also isolated several new series of saturated long chain fatty acids, including the methoxy-containing G 4 , C46.49 acids.

EXPERIMENTAL PROCEDURES

Materials-Distilled-in-glass acetonitrile, p-dioxane, chloroform, and methanol were purchased from Burdick and Jackson Laboratory. The p-bromophenacyl ester derivatization kit and methyl cis-9,lO- methylene octadecanoate were obtained from Applied Sciences Lab- oratories, Inc. All other reagents were of analytical grade.

Growth of Bacteria-Cells of M. tuberculosis H37Ra were grown a t 37°C in glycerol/alanine/salts medium in a New Brunswick 28-liter fermentor (1).

Isolation and Purification of Fatty Acids-The scheme for the isolation and purification of fatty acids from M. tuberculosis H37Ha is shown in Fig. 1. Harvested cells (approximately 1.0 kg, drained weight) were extracted twice overnight with 8 liters of chloroform/ methanol (2:1, v/v). The pooled extract was saponified by refluxing for 4 h in 600 ml of 5% KOH (w/v) in ethanol/water (l : l , v/v). The sample was acidified with HC1 (6 N) and extracted three times with equal volumes of diethyl ether. The pooled ether extract was washed once with an equal volume of water and evaporated to dryness. About 6 g of the ether extract were combined with 133 X loti cpm of I4C- fatty acids obtained from M. tuberculosis H37Ra (prepared sepa- rately) and passed through a silicic acid column (Bio-Si1 HA, minus 325 mesh, 450 g). The column was washed with 5% diethyl ether in petroleum ether and the fatty acids were eluted with 8% diethyl ether in petroleum ether and collected as two fractions. The earlier fraction (1.65 g) was enriched with the long chain fatty acids, whereas the later fraction (1.2 g) contained mainly the shorter chain acids CI,, and ClX. The enriched long chain fatty acids then were fractionated in three portions to size on a Sephadex LH-20 column (2 X 144 cm) by using the solvent system of chloroform/methanol (4:1, v/v). Results of this fractionation are shown in Fig. 2. The C.,0.4~ (150 mg) and Cll.56 (80 mg) fatty acids obtained were derivatized to thep-bromophenacyl esters (IO). The C.~o_lu fatty acid esters were fractionated into a saturated and unsaturated series using Silica Gel G-AgNO.I plates and developed with chloroform. Since the C.11-5ti esters only contained trace amounts of unsaturation, they were not fractionated on a Silica Gel G-AgNO:, plate.

The saturated C,3,,.4,, and Ct1-56~ p-bromophenacyl esters were ap- plied to 250-pm Silica Gel G thin layer plates (20 X 20 cm) at a load of 3 to 4 mg/plate and developed in hexane/p-dioxane (491, v/v) six times. The C.I~,.,~ esters were separated into four TLC fractions: A (6 mg), B (15 mg), C (25 mg), and D (25 mg) (Fig. 3). The C.21..*i esters were separated into five fractions: E (16 mg), F (7.2 mg), G (10.4 mg), H (3.2 mg), and I (<I mg) (Fig. 3). All nine fractions were separated into their individual components by HPLC.

HPLC Fractionation-HPLC was performed with two Waters model 6OOOA solvent delivery systems, a Waters model 660 solvent programmer, Waters model U6K universal liquid chromatograph injector, and a Perkin-Elmer variable wavelength detector (model LC-55). A reverse-phase pBondapak CI" column (4 mm X 30 cm; Waters Associates Inc.) was used a t a flow rate of 1 ml/min. Isocratic solvent system used wasp-dioxane/acetonitrile: 3:7, v/v, for the Ca,-4, esters; 1:1, v/v, for the Cll.~ti esters; and 9:11, v/v, for the methoxy- containing C41"1ti esters.

Preparation of Keto Derivatives of the Methyl Ester of the CXF, Fatty Acid- The p-bromophenacyl ester of the C:,, fatty acid (TLC Fraction C) was hydrolyzed back to the free acid by being dissolved in chloroform/acetic acid (2:1, v/v), adding zinc dust and stirring a t room temperature for 2 h (11):'' This acid then was methylated with diazomethane and derivatized to the keto esters by controlled oxida- tion with Cr0.3 as previously described (6, 12). The mono- and dike- tones were separated on a Silica Gel G thin layer plate with the solvent system of petroleum ether/diethyl ether (7:3, v/v). RF values for the monoketones were 0.58 for the 17- and 14-keto derivatives and 0.50 for the 5-keto derivative. The yellow band of the TLC dye mixture (The Anspec Co., Inc.) migrated with an RF of 0.44. A portion of the ketones was reduced to the alcohol with sodium borohydride in methanol and purified by TLC using the solvent system mentioned above. The resulting RF values were 0.39 and 0.25 for 17- and 14-

~~ ~

' These fractions were later shown to contain A'-fatty acids. ' Attempts to hydrolyze the p-bromophenacyl of the C:,i fatty acid

(TLC Fraction D) by this procedure resulted in the formation of an alcohol and the free acid.

HARVESTED CELLS OF

X . t u b e r c u l o s i s H37Ra

-"- CHLOROFOR"METHAN0L EXTRACT

S a p o n i f i c a t i o n

2 . S i l i c i c a c i d c o l u m n

ENRICHED FATTY ACIDS

S e p h a d e x LH-20 c o l u m n

1. D e r i v a t i z a t i o n 1. D e r i v a t i z a t i o n

2 . A r g e n t a t i o n - T L C

5- J .-. TLC

SATLlRATED -BROMOPHENACYL ESTERS

I 2. HPLC 1 2 . HPLC

COElPONENTS OF COIQONENTS OF

DICYCLOPROPYL- AND DICYCLOPROPYL

METHOXY ESTERS ESTERS

FIG. 1. Scheme for the isolation and purification of long chain fatty acids from M. tuberculosis H37Ra.

'r

4-

3-

n I 0 X

I u

ELUTION VOLUME (ml)

FIG. 2. Sephadex LH-20 column chromatography of en- riched I4C-fatty acids. Solvent system used was chloroform/meth- anol (4:l. v/v). The second peak eluting from the column was the G I - 5 6 acids.

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184 Long Chain Fatty Acids

3 2 I

of the bromide ion, and a M-213/215 fragment at m/e 515 due to the loss of thep-bromophenacyl group (Structure 2). Cleav- age at the cyclopropane ring was not observed. Fraction D showed one major and four minor peaks. The major compo- nent (HPLC Fraction 3, Fig. 40 ) was a C37 ester (Table 11) which contained three apparent unsaturations. HPLC Frac- tion 5 was a C39 ester of the same series.

2

NMR Analysis of the Methyl Ester of C.XI" Fatty Acids- Results of the NMR analyses of the TLC Fractions A + B, C, and D of the C30-40 methyl esters are summarized in Table 111. They revealed chemical shifts characteristic of cis-cyclopro- pane rings a t T = 9.4 and 10.3 for all three fractions. NMR spectra of TLC Fractions A + B and C were similar, whereas the spectrum of TLC Fraction D showed peaks a t T = 3.3,3.4, 4.0, and 4.2. Gunstone et al. (13) reported chemical shifts at T = 3.9, 3.72, and 4.3 for the olefinic protons of a cis-AL-CIH I

fatty acid. Low field triplet produced by an a-methylene group adjacent to the carbonyl group of TLC Fractions A + B and C was absent. These results suggest the presence of a cis-A2 double bond in TLC Fraction D.

H Q I O 0

FIG. 3. TLC ofp-bromophenacyl esters of Cso-ro (Lane 2) and C41.56 (Lune 3) fatty acids. Lane I , TLC dye. Solvent system was hexane/p-dioxane (98:2, v/v) developed six times. Spots were visual- ized by spraying the plate with dichromate/H2S04 reagent and char- ring.

hydroxy derivatives, and 0.31 and 0.21 (minor band) for the 2- and 5- hydroxy derivatives, respectively. The yellow band of the TLC dye mixture migrated with an RF of 0.43. The purified alcohols were silylated with N,O-bis(trimethylsily1)-trifluoroacetamide containing 1% trimethylchlorosilane as the catalyst (Pierce Chemical Co.) a t 60°C for 60 min.

Instrumental Analyses-Fourier transform NMR spectra were determined with a Bruker model HX-WE spectrometer a t 90 MHz. Mass spectra were obtained on an AEI model MS 902 mass spectrom- eter using an ionization potential of 70 e.v. and source temperature of 150-170°C. Samples were introduced at the inlet with a probe.

RESULTS

Purification of C.?o-ro Fatty Acids-The initial mass sepa- ration was achieved using a Sephadex LH-20 column (Fig. 2). After derivatization to the p-bromophenacyl esters, the satu- rated Can-4, esters were separated by TLC into four fractions, (Fig. 3, A through D). The results of HPLC of the saturated c30-40 esters and the subsequent mass spectral analyses of their individual components are shown in Fig. 4 and Tables I and 11. TLC Fraction A resolved into two major components, dicyclopropyl CJ5 (Peak 3) and CS7 (Peak 5) (Fig. 4A). Three minor components of dicyclopropyl Cas, C3e, and Cas esters (Peaks 4, 6, and 7, respectively) were also observed. Fraction B resolved into a similar series (predominantly the odd-num- bered dicyclopropyl C35, C37, and G ) , but it also contained CZa and (240. Fraction C resolved into one major and five minor peaks which were identified as HPLC Fraction 1 (a saturated CZ6 ester), and HPLC Fractions 2 to 6 (dicyclopropyl C34-3" esters). Fraction 4 (a major component) was identified as a C36 ester whose mass spectrum showed a molecular ion M at m/e 728/730, a M-79/81 fragment at m/e 649 due to the loss

C

I 4

E

D

FIG. 4. HPLC fractionation of the p-bromophenacyl esters of fatty acids. A, TLC Fraction A; B, TLC Fraction B; C, TLC Fraction C; and D, TLC Fraction D. Column used was pBondapak CIR. Mobile phase was p-dioxane/acetonitrile (3:2, v/v).

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Long Chain Fatty Acids 185

TABLE I Mass spectral analysis of purified HPLC fractions of TLC Bands

A, B, and C, p-bromophenacyl C~O.K, esters (Fig. 4, A to C)

HPLC No. of No' Of cy- fraction carbon"

pane ring

m/e clopro-

81 (-Br) 213/215 M - ?9/ M -

592,594 700,702 62 1 487 714,716 635 50 1 728,730 649 515 742,744 663 529 756,758 677 54 3 770,772 69 1 557 784,786 705 571

Major component is in Italics.

TABLE I1 Mass spectral analysis of purified HPLC fractions of TLC band 0,

p-bromophenacyl C:J,K, esters (Fig. 40) No. of ap- m/e

HPLC No. of parent un- fraction carbon" satura-

tionh M - 79,' M - 81 (-Br) 213/215

1 C I5 3 712,714 633 499 2 C 16 3 726,728 64 7 513

C.17 3 740,742 661 527 4 C.," 3 754,756 675 54 1

C,l3 3 768,770 689 555

3

5

I' Major component is in italics. Results indicate that there are two &-cyclopropane rings and a

single double bond at the a$ carbons.

Characterization of the Dicyclopropyl Fatty Acid-The methyl ester of the dicyclopropyl CSC; fatty acid (TLC Fraction C, Fig. 3, and HPLC Peak 4 of Fig. 4C) was derivatized to the keto esters by oxidation with CrOs and an aliquot was frac- tionated by TLC to yield two monoketone products. The mass spectra of these two products are shown in Fig. 5. Both fractions revealed molecular ions ( M ) a t m/e 560 and M - 32 at m/e 528. The 17- and 14-keto mixed derivatives (Fig. 5A) showed cleavage around the keto functional group, giving fragments at m/e 267 and 321 (Structures 3 and 4). Other major fragments were at m/e 336, 349, 487, and 500. The 5- keto derivative (Fig. 5B) gave two prominent fragments at m/e 141 and 447 (Structure 5) with other major fragments a t m/e 156, 169,487, and 500.

( 267' 335 + I 3 500 I

267 r' 335+1

3211 349) 4

,163 ,141

155+1 ( 441'

5

TABLE I11 NMR analysis of TLC fractions of C.JO," methyl esters

Silica Gel G TLC of methyl esters of CX.,O fatty acids with the solvent system of hexane/p-dioxane (98:2, v/v) developed three times was similar to the corresponding fractionation of thep-bromophena- cy1 esters (shown in Fig. 3). Bands A and B were not resolved.

Functional group proton Chemical shift (7) of TLC fraction

A t B C D

cis-Cyclopropane ring 10.30, 9.40 10.30, 9.41 10.30, 9.41 Terminal methyl 9.12 9.12 9.12 Saturated paraffin chain 8.74 8.73 8.74 OL carbon of fatty acid es- 7.60, 7.70 7.64, 7.75 Absent

Methyl ester a$ unsaturation of fatty Absent Absent 4.28, 4.00, 3.44,

ter 6.33 6.31 6.29

acid ester 3.33

A model compound, methyl-cis-9,10-methylene octadeca- noate, was derivatized to the 8- and 11-keto esters, reduced to alcohol with sodium borohydride, and separated by TLC to yield methyl-8-hydroxy- and methyl-ll-hydroxy-czs-9,10- methylene octadecanoates. The mass spectra of the Me:{Si derivatives of the two esters (Fig. 6) showed the molecular ion at m/e 398. I t also showed the expected M-15 (minus hle:,Si methyl), "31 (minus ester methoxy), "47, and "90 (minus (CHd,SiOH) fragments at m/e 383,367,351, and 308, respec- tively (14). Other peaks which were present were at m/e 73 ((CH&Si+f, m/e 75 ((CH&SiOH+) and m/e 129

(CH2=CH"CH==OSi (CH,3):l). Major fragments arose from cleavage at the Me,Si group on the side away from the ring a t m/e 299 and X55 for the ll-Me:lSi and 8-MetISi derivatives, respectively, as shown in Structures 6 and 7 (Fig. 6, A and B, respectively).

+

(2% OTMS CH, I I \

0 II

CH-CH-CH-(CH,),-C-OCH,

6

CH, OTMS 0 II

255

?

The remaining monoketones and diketones of the dicyclo- propyl C:rti ester were reduced with sodium borohydride to the alcohols and fractionated by TLC into three major (17-, 14-, and 2-hydroxy derivatives) and one minor (5-hydroxy deriv- ative) monohydroxy bands. The six possible derivatives of the diols were separated into two bands. The MelSi derivatives were prepared from each band and analyzed by mass spec- trometry. The spectra of 17-, 14-, and 2-OMe:lSi derivatives revealed molecular ions at m/e 634 and peaks at m / e 73, 75, and 129, which are characteristic of MeySi ethers of aliphatic esters (Fig. 7). The upper end of the spectra of the 17- and 14- OMeBSi derivatives showed fragments at m/e 619 ( M - 15), 603 ( M - 31), and 544 (M - 90). Intense ion peaks due to cleavage at the group appeared at m/e .?95 for both 17- and 14-OMe3Si derivatives (Structures 8 and 9). These frag- mentation patterns were similar to chose for the derivatives of the model compounds (Fig. 6; Structures 6 and 7).

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186 Long Chain Fatty Acids

- 6

r

s

m/e FIG. 5. Mass spectra of methyl esters of: A, a mixture of 17- and 14-keto derivatives; and B, 5-keto derivative of cis,cis-3,4-

and 15,16-dimethylenetetratriacontanoic acid.

I'" f 29 (254) 255(296)

s

360 I 400

m /e FIG. 6. Mass spectra of MeaSi derivatives of (A) 11- and (B) 8-hydroxymethylene Cls fatty acid methyl esters.

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Long Chain Fatty Acids 187

r" OTMS CH, I / \ / \ II CHZ 0

CH3-(CH2),5-CH2 CH -CH-CH-(CHZ),~-CH-CH-CH,"OCH3

8 I

0

9 The spectrum of the 2-OMe3Si derivative showed fragments

at m / e 103 (CHFO Si(CH3)3) and m / e 575 (Structure l o ) , which are considered characteristic of the a-hydroxymethyl esters, Me3Si esters (14) . It showed a fragment a t m / e 590 ( M - 44) due to the loss of Con. This fragment subsequently lost HOMenSi, giving rise to a fragment at m/e 500. The loss of C- 1 and C-2 plus proton by a McLafferty rearrangement gave rise to a fragment at m / e 472. The 5-OMesSi derivative was produced in minute quantities. Its spectrum showed a molec- ular ion at m / e 634 and a cleavage near the MeaSi group to yield a fragment a t m / e 215 (Structure 1 1 ) .

IO 575

The Me3Si derivatives of the two diol bands were analyzed by mass spectrometry and the results were consistent with the previous positional assignments of the two cyclopropane rings. The CR6 acid was thus identified as cis,cis-3,4- and 15,16- dimethylenetetratriacontanoic acid (Structure 1 2 ) .

12 Characterization of the Dicyclopropyl Cj7 Fatty Acid-The

methyl ester of the C:37 fatty acid (TLC Fraction D, Fig. 3) could not be acetylated with acetyl chloride or reduced with sodium borohydride, suggesting the absence of hydroxy and keto groups. NMR analysis showed the absence of methoxy protons but the presence of an a-/3 double bond and cis- cyclopropane rings (Tables I1 and 111). Mass spectral analysis revealed the molecular ion peak a t m/e 740/742, suggesting a CS7 acid with three apparent unsaturations. Our interpretation was that it contains two cyclopropane rings and a single double bond. I t migrated like a saturated acid on AgN03- Silica Gel G TLC. When the methyl ester of the dicyclopropyl Csi acid (TLC Fraction D, Fig. 3) was oxidized with Cr03, only one monoketone product was obtained. The mass spec- trum revealed a molecular ion at m / e 572, a M - 32 fragment at m / e 540, and a M - 60 fragment at m / e 512. Cleavage at the keto group resulted in fragments at m/e 321,336,349, and 267 similar to the ones observed for the major monoketone product of the CX ester (Fig. 5A). This suggests that one cyclopropane ring is at the 0-19 position. The proposed struc- ture for the CR7 ester is shown by Structure 13 where b + c = 10.

13

, s S b I Q 5 l c 10

8 . - s i

a Y

- 6 :

- ' d

- 2 x

- 0 r

Y I " , . I . , , , I 1 , , I ,

200 300 l ' " " ' ' ~ ~ l " ' ' " ' ' ~ ' , ' " ' 400 600 600

c t' c - 0 ",- - 3 z Y r - w

I - -

Y ya - 2 0 - 2 a

0 t c - 2 ,.. = 590 . I -

w 4R

L L 4 b 1 1 , , I , I , ~ , I I . I , , , , I , , , '

300 k00 I > ' ' ' 600 " " I " " "

m/e FIG. 7. Mass spectra of MetSi derivatives of 17- (A), 14- (B), and 2- (0 hydroxy-cis,cis-3,4- and IjJ6-dimethylenetetratria-

contanoic acid methyl esters.

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188 Long Chain Fatty Acids

A

5

12 24 36 4 8

5

12 24 36 4 8 MINUTE

1 12 24 36 4 8 60

MINUTE

FIG. 8. HPLC fractionation of the p-bromophenacyl esters of C40-so fatty acids. A, TLC Fraction E, mobile phase p-dioxane/ acetonitrile (l:l), B, TLC Fraction G , (methoxy) mobile phasep-dioxane/acetonitrile (9:ll).

TABLE IV Mass spectral analysis ofpurified HPLC fractions of TLC band E,

p-bromophenacyl C 4 , . ~ ~ ; esters (Fig. SA)

1 C4 3 2 798,800 719 585 2 CLJ 2 812,814 733 599 3 C43 2 826,828 747 613 4 c44 2 840,842 761 627 5 (24.5 2 854,856 775 64 1

7 c47 2 882,884 803 669

9 C49 2 910,912 83 1 697 10 CY, 2 924,926 845 711 11 C5l 2 938,940 859 725 12 C i2 2 952,954 873 739

6 C4b 2 868,870 789 655

8 C4H 2 896,898 817 683

13 C5.r 2 966,968 887 753 14 C 54 2 980,982 901 767

" Major components are in italics.

Purification of the C41-,56 Fatty Acids-The C4," fatty acids were derivatized to thep-bromophenacyl esters and separated by preparative TLC into five fractions (TLC Fractions E through I , Fig. 3) . NMR analyses of these fractions revealed chemical shifts characteristic of a cis-cyclopropane ring at T = 9.4 and 10.3. Like the C37 esters (TLC Fraction D), TLC fractions F and H revealed the presence of an a, p unsatura- tion. TLC fractions G and H showed the presence of methoxy protons (T = 6.66). Results of the HPLC fractionation of the two major TLC Fractions E and G are shown in Fig. 8, A and B, and their mass spectral data are summarized in Tables IV and V, respectively. TLC Fraction E revealed a series of dicyclopropyl C41.56 esters that could be structurally related to the C X fatty acid (Structure 12). TLC Fraction G showed a series of esters containing a methoxy group and either one or two cyclopropane rings. NMR revealed no unsatura- tion.

TABLE V Mass spectral analysis ofpurified HPLC fractions of TLC band G,

p-bromophenacyl GLW, esters (Fig. 8B) No. of appar-

HPLC fraction No. of carbon" ent unsatura-

- tion

1 C45

2 CJB 3 C47 4 C4"

5 C46

C4r C 4 7

6 CSO

7 C48 CSt C4.9

8 CSL C50

9 C.5.3

10 C51 CS, c 52

11 C5r,

12 C53 CW CS,

Major components are in italics.

2 2 2 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

m / e M - 32

838,840 852,854 866,868 880,882 854,856 894,896 868,870 908,910 882,884 922,924 896,898 936,938 910,912 950,952 924,926 964,966 938,940 978,980 952,954 992,994 966,968

TLC Fractions F and H showed CWS dicyclopropyl and C49-56 cyclopropyl plus methoxy esters containing a, /? unsat- uration similar to the C37 ester. TLC fraction I was a minor fraction that appeared to contain a keto group which was reduced to the alcohol with sodium borohydride.

DISCUSSION

The complexity of the C30-,56 fatty acids mixture in M . tuberculosis H37Ra required that a structural class separation be made. We found that TLC using the hexane/p-dioxane system would achieve this. The subsequent separation of the

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Long Chain Fatty Acids 189

fatty acids into their individual components by reverse-phase HPLC was generally simple and effective. However, the sep- aration of fatty acids greater than C:a required the application of a new HPLC method which we recently developed (8). We successfully fractionated and analyzed these purified, non- mycolate, long chain fatty acids from the H37Ra strain. This work extends previous studies done by other investigators on the fatty acids of mycobacteria (1, 7, 9, 15-17).

We found several series of fatty acids which were structur- ally classified into the following categories: ( a ) dicyclopropyl, ( 6 ) methoxy, ( c ) keto, and (d) a, /3 unsaturated fatty acids. The dicyclopropyl fatty acids appeared to exist in at least two structurally distinct forms. We do not know the difference, but it must be very subtle, i.e. the presence or absence of a branch methyl group or differences in the position of the internal ring. The dicyclopropyl series containing C34, CJs, (237,

Cex, C:39, Cqu acids differed from the dicyclopropyl series con- taining C3, C:j6, C:IH acids (the most abundant series). We do not know how the higher series of dicyclopropyl Cql, C4:1-R6 fatty acids relates to these two lower series.

The methoxy fatty acids appeared to be a complex mixture even after the TLC class separation. Mass spectral analyses showed that the mixture of fatty acids contained one and two cyclopropane rings.‘ The size range was from Cq4 to C4n-49. These fatty acids might be related to the methoxymycolic acids. We also found fatty acids of the size range of C41-sl; containing a keto functional group, as well as cyclopropane rings. Trace amounts precluded further study. These acids might be related to the ketomycolic acids.

Several series of fatty acids contained an a, /3 unsaturation. The most prominent of these was the C:j7 acid containing two cyclopropane rings. We also found a larger series of CRo-56 acids containing two cyclopropane rings which must be structurally different from the lower series. The methoxy-fatty acids had their own series of a, /3 unsaturated acids. The size range of these acids was C49-rS. The unsaturated fatty acids from M. tuberculosis H37Ra were previously found to contain several series of a, /3 unsaturated acids (9). The significance of these fatty acids is not known. They could be products of /3 oxida- tion. Structurally, they appeared to be related to the phthien- oic acids that are found only in virulent strains of M . tuber- culosis (18).

We have described a simple and effective method to deter- mine the location of cyclopropane rings and have character- ized the C I , ; fatty acid to be cis,cz.s-3,4- and 15,16-dimethyl- enetetratriacontanoic acid (Structure 12). A structural rela- tionship now exists among the prominent monounsaturated CLI. fatty acids (7), the above-mentioned C.,R fatty acid, and the u-mycolic acids (6). The positions of the double bond in the monounsaturated fatty acids (a”-C,, ,,Ai-CZn ,, A!’-C2H,,, A”- C.,U (, and A1”-C,,2 ,) and in the alkyl terminal cyclopropane ring in cis,ci,s-3,4- and 15,16-dimethylenetetratriacontanoic acid and u-mycolic acid are identical (w-19). The positions of the

’ We have now developed a HI’LC method to separate such difficult mixtures using the microparticulate silica column (l’akayama, K., Qureshi, N., .Jordi, H. C., and Schnoes, H. K. (1979) J. Liquid Chromcctog. 2, 861-873).

internal cyclopropane ring in both of the latter two fatty acids are also identical (w-31), as are the distances between the two rings (10 carbons). The structural evidence is consistent with a piecursor-product relationship. Larger dicyclopropyl fatty acids, such as the C,,, C4:~-:,ti series, might represent precursors along the pathway from G,,, leading to the synthesis of a- mycolic acids. Potential precursors spanning the pathway from CZ4 to C:ln have also been isolated (9).

This study is a necessary first step in our efforts to elucidate the pathway to the biosynthesis of mycolic acids. We must also show: ( a ) that these potential precursors are products of synthesis and ( 6 ) the precursor-product relationship, either by pulse-chase experiments or by using a cell-free enzyme system in following the partial reactions. By studying the time course of long chain fatty acid synthesis in M. tuberculosis H37Ra using [I4C]acetate as the metabolite, Takayama et al. (1) showed that these acids are probably products of synthesis and not of mycolic acid degradation. Inhibition studies with isoniazid suggested that these long-chain fatty acids are pre- cursors of mycolic acids (1). We are now studying the precur- sor-product relationship of long chain fatty and mycolic acids, both at the whole cell and cell-free levels.

Acknourledgments-We thank Mr. Richard W. Boyle for his ex- cellent technical assistance and Ms. Cynthia Birch for her valuable editorial assistance on this manuscript.

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N Qureshi, K Takayama and H K SchnoesPurification of C30-56 fatty acids from Mycobacterium tuberculosis H37Ra.

1980, 255:182-189.J. Biol. Chem. 

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