The major mycolic acids of Mycobacterium smegmatis

Prog,LipidRes. Vol. 21. pp. 91 107, 1982

0163 7827/82/020091 17S08.50/0 Copyright ~) 1982 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

THE MAJOR MYCOLIC ACIDS OF M YCOBACTERI UM SMEGMA TIS GARY R. GRAY, MARGARETY. H. WONG and SUSANJ. DANIELSON Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, U.S.A. CONTENTS |. INTRODUCTION II. ISOLATION AND DERIVATIZAT1ON III. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A. Normal phase B. Reverse phase IV. MASSSPECTROMETRY

91 92 92

92 94 95

V. PROTON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY VI. LOCALIZATION OF DOUBLE BONDS

97 100

A. Reductive ozonolysis B. Oxidative ozonolysis VII. CONCLUSIONS

100 101 105

REFERENCES "

107

I. I N T R O D U C T I O N

Mycolic acids are complex, high molecular weight fatty acids produced by all Mycobacteria. They were first isolated from a human strain of M. tuberculosis, I'19 but they have since been found in a wide variety of pathogenic and nonpathogenic Mycobacteria. 2 All of the known mycolic acids have the basic structure (1), wherein R 1 is either a C22 or C24 linear alkane and R 2 is a more complex structure, OH

I

R2--CHCHCOzH

I

(1)

R1 comprised of as many as 60 carbon atoms and containing a variety of functional groups. The isolation of the "mycolic acid fraction" from the bacterium is relatively simple, since their alkali salts are soluble in ether. Because of this property, they were referred to by Anderson I as "unsaponifiable wax." The further fractionation of these fatty acids is, however, a much more difficult task. Procedures developed earlier involved initial chromatography of the free acids on alumina, a followed by conversion of the fractions so-obtained to their methyl esters, and subsequent chromatography on silica gel, magnesium silicate or alumina. 2,9 Despite the carefully executed chromatographic procedures employed, it was recognized that the mycolic acids so-obtained were "... always isolated in the form of more or less complex mixtures. ''2 Moreover, analytical methods suitable for the evaluation of the homogeneity of a given preparation were not available, so the further chemical characterization of these fatty acids was an extraordinarily difficult undertaking. Recently, the development of high performance liquid chromatographic procedures for the fractionation of mycolic acids 16.~a has prompted a re-examination of the structures proposed previously. Through the combined use of normal phase silica gel chromatography and reverse phase partition chromatography on columns containing octadecylsilyl bonded phases, Is it is now possible to fractionate complex mixtures of mycolic acids into components containing discrete sets of functional groups, as well as unique molecular weights. In addition to these newly developed fractionation procedures, based on high J,P.L.R. 21/2--A

91

92

Gary R. Gray, Margaret Y. H. Wong and Susan J. Danielson

performance liquid chromatography (HPLC), new characterization procedures have been developed which utilize chemical ionization mass spectrometry (CIMS). These newly developed techniques, combined with high field nuclear magnetic resonance (NMR) spectroscopy and chemical degradation procedures, have been used to fractionate and characterize the major mycolic acids from Mycobacterium smegmatis. 8"2°'21 This work represents the first instance where the individual homologs of the major mycolic acids present in a Mycobacterium have been characterized, and will be the subject of this review. II. I S O L A T I O N

AND DERIVATIZATION

Mycolic acids are not found as their free carboxylate anions within the bacterium, at least not in significant quantities, but are present esterified to carbohydrates. They can be isolated by saponification of the chloroform--extractable lipids, ~9 the intact bacterial cell wall, 8 or the organic-solvent-extracted cell wall. ~8 We prefer isolation from the mycobacterial cell wall, or organic-solvent--extracted cell wall, because of the convenience and the amount of material obtained (usually approximately 25~ of the weight of the dry cell wall). If the organic-solvent-extracted cell wall is desired, extraction with neutral organic solvents is accomplished as described by Azuma et al. 4 Saponification is accomplished by suspension in 2.5~ (w/v) KOH in methanol:benzene (1:1) as described by Kanetsuna and Bartoli. 14 After acidification, the reaction mixture is extracted with ether. When the intact bacterial cell wall was subjected to this saponification procedure, considerable amounts of lower molecular weight fatty acids were present in the ether extract, but these were readily removed after derivatization (see below). The extracted mycolic acids are subsequently converted to their phenacyl esters (2) in order to provide a sensitive, quantitative OH

O

O

(2) R' method for detection. If greater sensitivity is desired, p-bromophenacyl esters are used because of their greater extinction coefficient at 254 nm (e = 16,500 vs E = 6000 for phenacyl esters). Both ester derivatives are formed in quantitative yields by treatment with the respective phenacyl bromides and an amine base (triethylamine or N,N'-diisopropylethylamine) in chloroform8 or chloroform:methanol (1:1, v/v). ~8 Separation of the mycolic acid esters from derivatizing reagents and lower molecular weight fatty acid esters is accomplished by gel permeation chromatography on Sephadex LH-20 (Fig. 1). In all cases, mycolic acid phenacyl or p-bromophenacyl esters are found to elute at the column void volume, well-separated from lower molecular weight fatty acid esters and excess derivatizing reagents. 8'1s'2~ In cases where significant amounts of lower molecular weight fatty acids are present in the reaction mixture, Sephadex LH-20 chromatography is best carried out on a 2 x 100 crn column in order to achieve complete separation from the mycolic acid esters. III. H I G H

PERFORMANCE

LIQUID

CHROMATOGRAPHY

A. Normal Phase

For all the Mycobacteria that we have examined to date, 1a'2~ HPLC of their mycolic acid phenacyl or p-bromophenacyl esters on silica has been found to fully resolve those types containing discrete sets of polar functional groups (e.g., hydroxyl, methoxyl, carbonyl, alkenyl). As illustrated herein for M. smegmatis mycolic acids, however, normal

The major mycolic acids of Mycobacterium smegmatis ¢

T

!

93

,

i0.

¢j

ii

[ $

2~

v >I-I .¢

i hl r.J Z 0.~

........ J

i

et,

QC

~ N L I M I B E R

FiG. 1. Purification of M. smegmatis mycolic acid p-bromophenacyl esters by chromatography on Sephadex LH-20 (2.5 x 55 cm) in chloroform:methanol (1:I, v/v). Mycolic acids were obtained from cells grown in the presence of [UJ4C]glycerol. Fractions (2.9 mL) were collected at a flow rate of approximately 60 m L / E

phase HPLC does not resolve chain length homologs, or isomers differing only by the presence or absence of a nonpolar functional group such as a C-methyl branch. The derivatized mycolic acids were dissolved in a minimum amount of 16% chloroform in hexane and injected onto a 10-gM particle-size #Porasil column (Water's Associates), eluted isocratically with the same solvent. The column effluent was monitored for absorbance at 254 nm with a Water's Associates model 440 ultraviolet detector. Under these conditions, two major and two minor components were observed, and are denoted as Peaks B, C and A, D, respectively (Fig. 2). The preparative isolation of Peaks B and C was accomplished under the same conditions, With 1-2 mg of the mycolic acid mixture being applied to the column in each run. Although Peaks B and C are not completely resolved under these conditions, the pure components can be obtained in pure form after separate reapplication to the column. Equally satisfactory resolution, and somewhat greater loading capacity, was achieved by chromatography on a "semi-preparative" #Porasil column (0.78 x 30 cm), obtained from the same manufacturer.

tic

B ¢

A

TIME, min

FxG. 2. Fractionation of M. smegmatis mycolic acid phenacyl esters by HPLC on /~Porasil (0.39 x 90cm). The column was eluted with 16~o chloroform in hexane at a flow rate of

2 mL/min. (Reproduced from Wong et aL,2t with permisssion.)

94

Gary R. Gray, Margaret Y. H. Wong and Susan J. Danielson ,5O

B-3

40

~0

_z

20

o

j Ld C) Z nn n.o

I0 O-

I

,o

I

20

I

~o TIME,

go

~o

I

~o

rain.

FIG. 3. Fractionation of M. smeomatis mycolic acid phenacyl esters of Peak B, Fig. 2, by HPLC on pBondapak C18 (0.78 x 30 cm). Components eluting at 20-30 rain arise from a small amount of contaminating Peak C. (Reproduced from Wong et al., 2~ with permission.)

B. Reverse Phase

Reverse phase HPLC on supports containing an octadecylsilyl (ODS) bonded phase has been shown to separate chain length homologs of the phenacyl ester derivatives of mycolic acids s'ts'21 as well as short chain fatty acids. 6 As demonstrated by Danielson and Gray, s and presented herin, reverse phase HPLC is also capable of resolving mycolic acid derivatives with identical chain lengths and functional groups, but differing only by the presence or absence of a secondary C-methyl branch. Mycolic acid fractions B and C (Fig. 2) were dissolved in a minimum amount of 5 0 ~ chloroform in methanol and applied to a #Bondapak C~s column (0.78 × 30 cm, Water's Associates), equilibrated in 25~o chloroform in methanol at a flow rate of 2 mL/min. The column was eluted under slightly different conditions in order to achieve maximal resolution of the individual components. 2~ For component B, the column was eluted with a 45-min linear gradient to 4 5 ~ chloroform in methanol, to give 5 components (Fig. 3). For component C, the column was eluted with a 45-min linear gradient to 35~o chloroform in methanol, which resulted in the resolution of 4 components (Fig. 4). Further examination of these components by ammonia CIMS, as described below, demonstrated that each of the C components possessed a unique molecular weight. Each of the five B 5o ..J

C-5 --404

I-tad

I t13

~ J Ld tJ Z

0 03 ,'n

}

--

50

C-2

Z _m

hZ --IO~ C-4

Iz

6O TIME, mi~

FIG. 4. Fractionation of M. smegmatis mycoUc acid phenacyl esters of Peak C, Fig. 2, by HPLC on #Bondapak Cls (0.78 x 30 cm). Components eluting after 45 rain arise from a small amount of Peak B. (Reproduced from Wong et al., 21 with permission.)

The major mycolic acids of Mycobacterium smegmatis

95

4

I _ o

3

y

e

T &

If.

215

510

I 7 5

'-i I0 0

TIME, min. FIG. 5. Fractionation of M. smegmatis mycolic acid phenacyl esters of Peak B by HPLC on a DuPont ODS column (0.46 x 25 cm). The mycolic acids were obtained from cells grown in the presence of L-[methyl-3H]methionine. Components eluting prior to 40 min arise from a small amount of contaminating Peak C. (Reproduced from Danielson and Gray, a with permission.)

components, however, was found to be a mixture of two components differing in molecular weight by 14 amu. From spectroscopic evidence and biosynthetic arguments, it was proposed that each B component was a mixture of two components of identical chain length that differed only by the presence or absence of a secondary C-methyl branch. 21 That the above proposal concerning the B components was indeed correct was established by completely resolving all 10 components, using carefully optimized conditions (Fig. 5).8 In this case, a 5-/tM particle-size ODS column (0.46 x 25 cm, DuPont) was used. The column was equilibrated in 33~o chloroform in methanol at a flow rate of 1.0 mL/min, and was maintained at 45°C. These conditions were found to be essential to achieve the resolution shown. In the experiment shown in Fig. 5, B mycolic acids were obtained from cells grown in the presence of L-[methyl-3H]methionine, which was found to label only those components containing the secondary C-methyl branch. The preparative isolation of the 10 B-series components was accomplished under the conditions shown in Fig. 5, with approximately 0.5 mg of the B-mixture being applied in each run. The individual B-series components were re-chromatographed two additional times under the same conditions in order to obtain chromatographically pure components. The preparative isolation of the 4 C-series components (Fig. 4) was much easier, because of the greater capacity of the column and the more easily achieved resolution. In this case, 2-3 mg of the C-mixture was applied in each run, and re-chromatography of the individual components was not necessary. For all components, however, subsequent chromatography on/~Porasil (Fig. 2) was necessary in order to remove contaminants that were found to elute from reverse phase columns. 21 IV. M A S S S P E C T R O M E T R Y

The molecular weights of the individual components of the B-and C-series mycolic acids were determined by electron impact and CIMS. 8'21 When subjected to mass spectral analysis (Fig. 6), each component gave the expected aldehyde (3) and ester (4) pyrolysis products, 1° and from the molecular weights observed for these components, the molecular weights and molecular formulas of the individual components were determined. Although the molecular ions of the aldehyde (3) and ester (4) fragments were observed in the electron impact mass spectra of these derivatives, the intensities of these ions, especially the higher molecular weight aldehyde ions, were very weak due to extensive further fragmentation. The aldehyde and ester fragments were readily observed by ammonia chemical ionization mass spectrometry, however. In the positive-ion ammonia

Gary R. Gray, Margaret Y. H. Wong and Susan J. Danielson

96 H

O

;! A

CH

'

OH

O

R 2 - - t . - - H + [R - - C H ~ - - - C - - O R ]

~

RI--CH2C--OR (4)

(3)

OR

I RI (2), R = phenacyl

R2--CItO. NH~

[R2--CttO]--I e

RtCH2CO2R. N H ~

(61

(5)

[ R ~ C H 2 C O f l l ] - - 1°

17)

(8)

+ RICH2CO2 R- H ~ (9)

RICH2CO2e (10)

FIG. 6. Pyrolysis of mycolic acid esters and fragments observed in their positive- and negative-ion

chemical ionization mass spectra.

chemical ionization mass spectra of these derivatives, the base peak is the ammonium cluster ion of the ester fragment (7), and a peak of lower intensity is observed for the protonated ester (9). Aldehyde fragments are observed exclusively as their ammonium cluster ions (5). In the negative-ion spectrum, which is determined at the same time, the base peak is the carboxylate anion (10) derived from the ester. Relatively intense ions are also observed for the M-1 fragment of the ester (8) and the M-1 fragment of the aldehyde (6). In all cases, the cleavage fragments observed in the negative-ion ammonia chemical ionization mass spectra are much more intense than those observed in the positive-ion spectra. Figure 7 illustrates the spectra obtained for component B-6 (see Fig. 5), which are representative. In the positive-ion spectrum (top), only the following ions are observed: m/e 504, identified as the ammonium cluster ion of the ester (7); m/e 487, identified as the protonated ester (9); m/e 800 identified as the ammonium cluster ion of the aldehyde (5). The negative-ion spectrum (bottom) contains only the following peaks: m/e 367, identified as the carboxylate anion (10); m/e 485, identified as the M-1 peak of the ester (8); m/e 781, identified as the M-I peak of the aldehyde (6). The results of these studies (Table 1) established that each of the C-components (Fig. 4)

POS. 504 3,IZ I..Z

800

k_

W >

NEG. B67 I

..I

300

485

400

500

600

700

800

mlt

FIG. 7. Positive- and negative-ion ammonia chemical ionization mass spectra of mycolic acid B-6

phenacyl ester. (Reproduced from Danielson and Gray: with permission.)

The major mycolic acids of Mycobacterium smeomatis

97

TABLE 1. Molecular Formulas and Molecular Weights of B- and C-series Mycolic Adds from M. smegmatis Molecular formula Mycolic acid C-1 C-2 C-3 (7"4a B-I B-2 B-3 B-4 B-5 B-6 B-7 B-8 B-9 B-10

Rt C22H*s C22H,s C22Ha.s C22H45 C2~H4.s C22H4s C2~H,s C2,H,s C22H,5 C22H4s C22H45 C22H4$ C22H4.5 C22H4s

R2 C3sHe9 C37H73 C39H77 C(tHst C(9H95 CsoH97 CstH99 C~,Htot Cs3Ht03 Cs(Hto, CssHto~ Cs6Hto9 Cs7Htlt CssHlt 3

Free acid

M.W. Free acid

C6oHtlsO3 C62H12203 C64H12603 C66H13oO3 C74H14403 CTsHt,eO3 C76Ht(sO3 CT~HtsoO3 CTsHj$203 C79Hts40 3 CsoHls603 CslHlssO3 Cs2H16oO3 C83H16203

886 914 942 970 1080 1094 1108 1122 1136 1150 1164 1178 1192 1206

• Component C-4 was found to contain a small amount of a component with a molecular weight of 996. (From Wong et al. zt and Danieison and GrayS).

and each of the B-components (Fig. 5) possessed a unique molecular weight. Components of the C-series were found to form an homologous series with the empirical formula C.H2.-203, where n = 60, 62, 64 and 66. For each component, R ~ was found to be only C22H45, and R 2 was found to contain a single unit of unsaturation. The B-series mycolates were found to comprise two homologous series, both with the empirical formula C.H2.-403. In the B-series where n = 74, 76, 78, 80 and 82, R 1 was found to be C22H, s and R 2 was found to contain two units of unsaturation. In members of the second B-series (n = 75, 77, 79, 81 and 83), R 1 was also C22H45 and R 2 was also found to contain two units of unsaturation. Each member of this series was found to contain a secondary C-methyl branch which was absent in homologs of the series containing an even number of carbon atoms (see below). V. P R O T O N NUCLEAR M A G N E T I C RESONANCE SPECTROSCOPY

Highfield IH nuclear magnetic resonance (NMR) spectroscopy has proven to be an extremely valuable technique for the structural characterization of mycolic acids. All of the mycolic acids that we have examined so far, including those derived from many pathogenic as well as saprophytic species, have given characteristic and readily interpretable spectra. The most intense resonance in all mycolic acid spectra is, of course, that assigned to the many aliphatic methylene protons. For some mycolic acids, in fact, if the spectrum is plotted with this resonance on-scale, no other resonances are observed. Considerable amplification of the spectrum is therefore required in order t o identify resonances attributable to other functionalities. Shown in Figs. 8-10 are the 270 MHz tH NMR spectra of the phenacyl esters of mycolic acid C-3 (Fig. 8), a mixture of mycolic acids B-3, B-5 and B-7 (Fig. 9), and a mixture of mycolic acids B-4, B-6, and B-8 (Fig. 10), which are representative of the spectra obtained for the three major types of M. smegmatis mycolic acids. The three spectra contain many resonances in common: 3 0.91 (t, J = 6 Hz), assigned to the terminal methyl groups of the aliphatic chains; ~ 1.29 (broad s), assigned to the aliphatic methylenes; 3 2.03 (multiplet), assigned to the methylene protons allylic to a carboncarbon double bond; b 2.61 (multiplet), H-2; 3 3.50 (d, J = 5 Hz), C-3 hydroxyl; ~ 3.80 (multiplet), H-3; ~ 3.37 (complex), vinylic protons; ~ 5.57 (center of AB pattern), phenacyl methylene; 6 7.28 (s), CHC13; ~ 7.52 (t, J = 7.5 Hz), H-3,3 phenacyl; ~ 7.66 (t, J = 7.5 Hz), H-4 phenacyl; ~ 7.94 (d, J = 7,5 Hz), H-2,6 phenacyl. These spectra contain two very important differences, however. In the spectrum of the B-4, 6, 8 mixture (Fig. 10, inset), a

98

Gary R. Gray, Margaret Y. H. Wong and Susan J. Danielson

M srnegrno//s C - 3 Phenocyt Ester

•

I

IJ

/rF

jl

J

i

I0

9

8

7

6

5

4

3

2

O

PPM FIG. 8, The 270-MHz proton magnetic resonance spectrum of mycolic acid C-3 phenacyl ester in deuteriochloroform (equilibrated with D:O). Before exchange with D20, the C-3 hydroxyl group appeared as a doublet {J = 6 Hz) at 63.32. (Reproduced from Wong et al., 21 with permission.)

doublet (J = 6.5 Hz) is present at 6 0.96, which can be assigned to a secondary C-methyl group. This resonance is clearly absent in the spectrum of the B-3, 5, 7 mixture (Fig. 9, inset) which confirms the conclusion drawn from the labeling experiment with L-[methyl3H]methionine, i.e. that only the even-numbered B-series homologs possessed a C-methyl branch. It can also be demonstrated that the C-methyl group is allylic to a carbon-carbon double bond, since irradiation of the allylic methylene proton resonances at 6 2.03 results in collapse of the 6 0.96 doublet to a singlet. The other important difference in these spectra is the integral value obtained for the vinylic proton resonances. Using the integral value of the five aromatic ring hydrogens as an internal standard, the integral value of the vinylic proton region in the spectrum of C-3 (Fig. 8) was found to correspond to four hydrogens, whereas the integral value of the vinylic proton region in

I.I 1.0 0,9 0.8 O.7 PPM ~j~

'q

i

i

i

i

i

I0

9

B

"?

6

5

i

1

i

u

4

3

2

I

PPM FIG. 9. The 270-MHz proton magnetic resonance spectrum of a mixture of mycolic acids B-3, B-5 and B-7 phenacyl esters in deuteriochloroform. (Reproduced from Danielson and Gray. s with permission.)

0

The major mycolic acids of Mycobacterium smegmatis

i

i

I.I

1.0

i

i

i

0.9

O. i

0.7

99

I

PPM

, I.,3

6

.

"f

.

.

.

.

5

6

.

4

.

3

2

I

0

PPM FIG. 10. The 270-MHz proton magnetic resonance spectrum of a mixture of mycolic acids B-4, B-6 and B-8 in deuteriochloroform. (Reproduced from Danielson and Gray, s with permission.)

the spectra of both B-3, 5, 7 and B-4, 6, 8 was found to correspond to six hydrogens. Subtracting the two ester methylene hydrogens also present in this region gives two vinylic hydrogens for C-3 and four vinylic hydrogens for each component of the B-series. These results are in perfect agreement with the results of mass spectrometry which demonstrated that one unit of unsaturation was present in each C-component and two units of unsaturation were present in each B-component. The results of these studies, therefore, established that the C-series homologs possess the basic structure (11) where x + y = 32, 34, 36 and 38. Members of the odd-numbered B-series homologs possess the

OH

I CH3(CH2)xCH=CH(CH2)yCHCHCO2H

(11)

I n-C22H45

basic structure (12), where x + y + z = 44, 46, 48, 50 and 52, and even-numbered B-series homologs possess the basic structure (13), also where x + y + z = 44, 46, 48, 50 and 52. The exact position of the OH

I

CH 3(CH2)xCH---~CH(CH2)yCH~CH(CH2)zCHCHCO2H

(12)

J n-C22H45

OH

I

CH3(CH2)xCH-~-CH(CH 2)yCH=CHCH(CH2)z_ tCHCHCO2H

i CH3

(13)

I n-C22H45

C-methyl branch as shown in (13) is arbitrary; NMR studies indicate only that it occupies one of the four allylic positions.

100

Gary R. Gray, Margaret Y. H. Wong and Susan J. Danielson VI. L O C A L I Z A T I O N O F DOUBLE BONDS

Localization of the double bonds in homologs of the monoalkene series (11) and the two different dialkene series (12) and (13) was accomplished by reductive 2° and oxidative 8 ozonolysis procedures, respectively, and the fragments that were generated were separated and characterized by mass spectrometry. In contrast to direct mass spectral procedures, these procedures allowed both quantification and identification of the various cleavage fragments, and therefore a somewhat quantitative description of the content of positional isomers in each of the homologs. In addition, a procedure involving the physical separation and the subsequent mass spectral characterization of the cleavage fragments seemed to be particularly desirable for double bond localization in dialkene homologs of structure (13) because of problems presented by the presence of the C-methyl branch, i.e. physical separation of the ozonolytic fragments allows the straightforward identification of those containing the C-methyl branch merely by determining which fragments are radiolabeled when ozonolysis is performed using mycolic acids labeled by growth of the bacterium in the presence of L-[methyl-3H] methionine.

A. Reductive Ozonolysis The positions of localization of the single carbon-carbon double bond in homologs of the C-series were established by combined ammonia, positive-ion CIMS gas-liquid chromatography of aldehyde ozonolysis products. 2° Ozonolysis of the phenacyl esters (14), followed by decomposition of the ozonide with zinc dust in 509/0 aqueous acetic acid gave aldehydes (15) and (16) (Fig. 11), which were analyzed either directly or after trimethylsilylation. Trimethylsilylation of (16) prevented its pyrolysis to dialdehyde (17) and ester (18) when injected into the gas chromatograph. The analysis of these fragments was accomplished with and without trimethylsilylation in order to distinguish between monoaldehydes of structure (15) and dialdehydes of structure (17); from the nomainal mass determined for the molecular weight of an aldehyde, it is not possible to distinguish between the structure of a monoaldehyde or a dialdehyde shorter in chain length by one methylene unit. As an example of the data obtained in these experiments, when the mixture of C-series mycolic acid phenacyl esters was ozonolyzed and the products were trimethylsilylated, gas-liquid chromatography revealed the presence of two major monoaldehydes (components 4 and 5, Fig. 12) which were identified by positive-ion ammonia CIMS as C19- and C2t-aldehydes, respectively. If, however, the ozonolysis products were examined without silylation (Fig. 13), Clv-, C19- and C21-dialdehydes CH3(CH2LCHO OH ]

CH 3(CH2)xCH=CH(CH2LCHCHCO2R • I C22H,~

(15) 1) O~

~

2~z...oA°

+

OH j OHC(CH2)yCHCHCO2R

r

"~/

(14)

:

C22Ha,s (16) OSiMe3

OHC(CH2)yCHO + C2sH4~COzR (17) (18)

CHa~---NSiMe3

OSiMe3 I OHC(CH2)yCHCHCO2R I C22H,,5 FtG. 1I. Fragments formed by reductive ozonolysis of C-series mycolic acid phenacyl esters ([14] R = phenacyl), and their pyrolytic or trimethylsilylatedderivatives.

The major mycolic acids of Mycobacteriumsmegmatis

rr Q:

101

4

Z

_o

_.1

I 2

TIME, rain.

FIG. 12. Gas-liquid chromatogram of the trimethylsilylated ozonolysis products derived from the mixture of C-series mycolic acid phenacyl esters. The column (3% Dexil 300 on 100/120 Supelcoport, 1/8 inch x 6 feet) was programmed from 150 to 320°C at 4°C/rain. Components eluting from the column were detected by the total ion current in their positive-ion ammonia chemical ionization mass spectra. (Reproduced from Wong and Gray, t° with permission.)

(components 5, 7 and 9, respectively, Fig. 13) were observed in addition to the C19- and C2~-aldehydes (components 3 and 6, respectively, Fig. 13). In these experiments, not all of the components eluting from the gas chromatography column gave role values corresponding to the ammonium cluster ions of aldehydes or dialdehydes. The presence of these impurities really does not interfere, however, with characterization of the aldehyde ozonolysis fragments.

3 A

,

H,

6

A,

I0

.

A

T I M E , rain

FIO. 13. Gas-liquid chromatogram of the ozonolysis products derived from the mixture of C-series mycolic acid phenacyl esters. The column conditions were the same as in Fig. 12 except that the temperature was programmed at a rate of 6°C/rain. (Reproduced from Wong and Gray, "° with permission.)

In a similar manner, the individual C-series homologs were ozonolyzed and the products were analyzed, except for component C-1, for which insufficient amounts of material were available for study. Each of the components gave a small amount of the C17-aldehyde ([15], x = 15) and much larger amounts of C19- and C21-aldehydes ([151 x = 17 and 19, respectively). In addition, the dialdehyde (17) fragments observed were those expected based on the known molecular fomula of each homolog. The results of these studies (Table 2) demonstrated that each C-series homolog (11) was composed of two major positional isomers with x = 17 and 19.

B. Oxidative Ozonolysis An oxidative ozonolysis procedure was chosen for localization of the double bonds in homologs of the two dialkene series (12) and (13)8 because of the inherent inability to distinguish between monoaldehydes and dialdehydes by molecular weight analysis, as noted above. The individual B-series phenacyl esters were first de-esterified with activated zinc in glacial acetic acid and hexane. Ozonolysis of the free acids, followed by decomposition of the ozonides with silver oxide in ethanol, ~T yielded a series of monoacids and diacids which were converted to their phenacyl monocsters and diesters, respectively (Fig. 14). Separation of the monoesters (19) and diesters (20) and (21) from

102

Gary R. Gray, Margaret Y. H. Wong and Susan J. Danielson TABLE 2. Structures of the Major Positional Isomers of the Monoalkene Mycolic Acids from M. smegmatis OH

I

CHa(CH2),CH~CH(CH 2)yCHCHCO2H

i

C22H45 Component C-2 C-3 C-4

x

y

17 19 17 19 17 19

17 15 19 17 21 19

(From Wong and Gray2°).

derivatizing reagents was accomplished by HPLC on a 10-#M particle-size #Porasil column (Fig. 15). The monoesters (Peak A) and diesters (Peak B) were combined and chromatographed on a 10-#M particle-size #Bondapak column. Shown in Figs. 16 and 17, respectively, are the elution profiles for fragments derived from components B-5 and B-6, OH /

CH 3(CH 2),CH~-----CH(CH 2), CH~---CH (CH 2),~HCHCO2 H

I

C22H4~ (12) II O5 12) Ag20. EtOH 31 BrCH2COCt, H s. iPr2EtN

OH CH3(CH2),CO2R

RO2CICH2),CO2R

RO2C(CH2),~HCHCO2R

I

C22H45

(19)

(20)

(21)

FXG. 14. Fragments formed by oxidative ozonolysis of B-series mycolic acids (12) and their subsequent derivatization. R = phenacyl.

B

100

ao ~ z

6 0 '-

~ 2og ,o

/

~)

20

410

610

1

80

TI ME, min.

FIo. 15. Separation of the monoester (19) and diester (20, 21) oxidative ozonolysis products from excess pbenacyl bromide and other impurities by HPLC on #Porasil (0.78 × 30 cm). The column was equilibrated in 5% chloroform in hexane, then eluted with a linear gradient over 70 rain to 1000~, chloroform at a flow rate of 2mL/min. (Reproduced from Danielson and Gray, s with permission.)

The major mycolic acids of Mycobacterium smegmatis

cE

-

-

I00

g

90

Od

; /

103

.S

o

70

z

m " o

60

m '~

;

u Z

20

50

o'o

20 T I M E . rain.

FIG. 16. Fractionation of the combined monoesters and diesters (Peaks A and B. respectively, Fig. 15)derived from mycolic acid B-5 by HPLC on #Bondapak Cjs (0.39 x 30cm). The column was equilibrated in 50% acetonitrile: 95% ethanol (70:30) in water then eluted over a 45 rain

period to 100% acetonitrile:95% ethanol (70:30) at a flow rate of 2 mL/min. (Reproduced from Danielson and Gray) with permission.)

which are representative. The numbered components were collected separately, and their identities were established by negative-ion ammonia CIMS, wherein characteristic M-1 and carboxylate anion fragments were observed (see Section IV). For the evennumbered components of the B-series, the ozonolysis experiments were repeated with mycolic acids labeled by growth of the bacterium in the presence of L - [ m e t h y l - 3 H ] methionine in order to identify ozonolysis fragments containing tritium, and therefore, a C-methyl branch. The presence of the C-methyl branch in a given ester was confirmed by electron impact mass spectrometry, wherein characteristic McLafferty rearrangement fragment ions were observed. ~ Under the elution conditions shown in Figs 16 and 17, low molecular weight diesters (20) were found to elute first (components 1-I0, 1Z Fig. 16 and components 1-9, Fig. 17), monoesters (19) were eluted next (components .11, 13-17, Fig. 16 and components 10-15, Fig. 17), and high molecular weight diesters (21) and (22) were eluted last (components

16

[

/

~r

I00

.c_

90

3: o bJ

/

o

80 70

o

~S i i?

1819

,'o TI

M

50

20

4 6

;

z

60

t2

2

g

6'0

8]0

E, min.

FIG. 17. Fractionation of the combined monoesters and diesters (Peaks A and B, respectively, Fig. 15) derived from mycolic acid B-6 by HPLC on/zBondapak CI8. The column and elution conditions are the same as in Fig. 16. (Reproduced from Danielson and Gray, s with permission.)

Gary R. Gray, Margaret Y. H. Wong and Susan J. Danielson

104

18-21, Fig. 16 and components 16-21, Fig. 17). In OH

I

RO2CCH(CH2)

I CHa

(22)

z- ICHCHCO2R

I C22H45

Fig. 16, for example, components 5 and 7 are C12- and C~4-diesters ([20] y = 10, 12), respectively, component 16 is a C19-monoester ([19] x = 17) and component 20 is a C45-diester ([21] z = 19). In Fig. 17, component 7 is a C15-diester ([20] y = 13), component 14 is a C19-monoester ([19] x = 17) and component 20 is a C4s-diester ([22], z = 18).

The above oxonolysis procedure was applied to each of the unmethylated B mycolic acids, except for B-9 which was not isolated in a quantity sufficient to allow characterization. For each mycolic acid, the mole fraction of each ozonolysis fragment was determined by integration of the elution profile on HPLC (as in Fig. 16). 8 A wide variety of monoesters (19), low molecular weight diesters (20) and high molecular weight diesters (21) were found to comprise the ozonolysis product of each unmethylated B mycolic acid, but a few of these were found to predominate. The only low molecular weight diesters (20) present in significant quantity were those with y = t0 and y = 12. Monoesters (19) where x = 15, x = 17 and x = 19 were predominant, and four high molecular weight diesters (21) were present in significant quantities, those with z = 15, z = 17, z = 19 and z = 21. All of the above fragments were not present in each of the homologs, however. The predominant fragments derived from B-l, for example, were monoesters (19) where x = 15 and x = 17, diesters (20) where y = 10 and y = 12, and diesters (21) where z = 15 and z = 17. Since B-1 has the discrete molecular formula C74H14403 (free acid) where x + y + z = 44, it follows that B-1 is comprised mainly of the positional isomers where x = 17, y = 12, z = 15; x = 15, y = 12, z = 17 and x = 17, y = 10, z = 17. Similar analysis of the other components reveals the major positional isomers present in each unmethylated B mycolic acid (Table 3). The isomers listed are the allowed combinations of monoesters (19) and diesters (20, 21) present in greater than 10Yo relative abundance.

TABLE 3. Structures of the Major Positional Isomers of the Unmethylated Dialkene Mycolic Acids from M. smegmatis OH

I

CH 3(CH2)~CH~CH(CH2)~CH~-~-CH(CH 2)zCHCHCO2 H

Component

x

y

B-1

13 15 15 17 17 15 17 17 15 17 17 19 19 17 19 19

12 10 12 10 12 12 10 12 12 10 12 10 12 12 10 12

B-3 B-5

B-7

(From Danielson and GrayS).

C22H4~ z 19 19 17 17 15 19 19 17 21 21 19 19 17 21 21 19

The major mycolic acids of Mycobacterium smegmatis

105

TAaLE 4. Structures of the Major Positional Isomers of the Methylated Dialkene Mycolic Acids from M. smeomatis OH

f

CH3(CH2)~CH~--~CH(CH2)yCH~CHCH(CH2)~_ l CHCHCO2H Component

x

y

B-4

15 17 17 15 17 17 17 19

13 11 13 13 13 13 15 13

B-6 B-8

C22H4.5 z 18 18 16 20 18 20 18 18

(From Danieison and GrayS).

In addition to the isomers present in greatest abundance, as listed, there are also several other possible isomers for each mycolic acid based on the many additional monoester and diester fragments observed in smaller amounts. The mole fractions of each of the fragments arising from ozonolysis of the methylated B mycolic acids (B-4, B-6 and B-8 were examined) were also determined and similar complexity was observed. The major fragments observed, however, were monoesters (19) where x = 15, 17 and 19, diesters (20) where y = 11 and 13 and diesters (22) where z = 16, 18 and 20. Again, using the allowed combinations of monoesters and diesters present in greater than 10~ relative abundance, the methylated B mycolic acids were found to be comprised predominantly of the positional isomers in Table 4. VII. C O N C L U S I O N S

It is interesting to compare the structures determined for the individual homoiogs of the three major homologous series of M . smegmatis mycolic acids with each other as well as with the structures previously derived using the unseparated mixtures of homologs. With regard to previous work, mycolic acids of general structures (13) and (11) were known to be present, and were referred to as ~- and ~'-mycolic acids, respectively. 5'11.15 The major constituent of the monoalkene mycolic acids was reported 15 to possess structure (11), where x = 17 and y = 17, which is now known to be one of the major positional isomers in mycolic acid C-2 (Table 2). Minor components were observed, however, with x = 15 and y = 19, but components were not observed with x = 19. In the present study, components with x = 17 and 19 were found to predominate, and components with y = 15 and 21 were observed in addition to those with y = 17 and 19. The structures established herein for the methylated dialkene mycolic acids (13) are also in good agreement with the structures previously proposed. The structure established for the major positional isomer of mycolic acid B-6 ([13] x = 17, y = 13, z = 18, Table 4) is identical to that proposed by Et6madi, et al. 1 ~ for the major dialkene mycolic acid. Although these workers did not separate the dialkene homologs, they did identify several other oxidative ozonolysis fragments, including monoesters where x = 15-19, low molecular weight diesters where y = 12-16 and one high molecular weight diester where z = 18. In our work, monoesters (19) with x = 15, 17 and 19, low molecular weight diesters (20) with y = 11 and 13, and high molecular weight diesters (22) with z = 16, 18 and 20 were found to predominate, however. The structures previously reported for the methylated dialkene mycolic acids (13) were composites not only of their five chain-length homologs, but also of the five chain-length homologs of the unmethylated dialkene mycolic acids (12). Although dialkene mycolic acids of structure (12) had been proposed to be intermediates in the biosynthesis of the methylated dialkene mycolic acids (13), 13 the presence of the

106

Gary R. Gray, Margaret Y. H. Wong and Susan J. Danielson

unmethylated dialkene homologs (12) had gone undetected due to the inability to chromatographically resolve the two types. From an inspection of the structures determined for the major positional isomers of each of the homologs (Tables 2-4), some interesting speculation can be advanced with regard to the biosynthetic relationships among the three major mycolic acid types. For the monoalkene homologs (11), the major positional isomers present are those with x = 17 and 19 and y = 15, 17, 19 and 21 (Table 2). These values are identical to the xand z-values, respectively, determined for the major positional isomers present in homologs of the unmethylated dialkene mycolic acids (l-12] Table 3). From these results, it can be proposed that the unmethylated dialkene mycolic acids (12) and the monoalkene mycolic acids (11) share a common biosynthetic pathway in which the dialkene mycolates are synthesized from monoalkene mycolate precursors by the splicing-in of an internal "y-fragment." The possible biosynthetic relationship between the methylated (13) and unmethylated (12) dialkene mycolic acids also merits scrutiny. In view of the structures actually determined for these two series of mycolates (Tables 3 and 4), a previous proposal as to their route of biosynthesis can be evaluated. Although not observed in their initial studies, 11 Et6madi et al., did report the isolation of small quantities of an unmethylated dialkene mycolic acid in a later study. 12 Based on the structure determined for this mycolate (23), Jaurequiberry et al. 13 OH

I

CH3(CH2)I 7CH~CH(CH2)14CH~-~CH(CH2)17 CHCHCO2H

(23)

I C22H45

OH

I

CH3(CH2)ITCH--~CH(CH2)l 3CH~CHCH(CH2)I 7 CHCHCO2H

(24)

I CH3

C22H45

proposed a pathway for its conversion to the major methylated dialkene mycolate (24) as summarized in the partial structures below: --(CH2)13--CH2--CH~CH--(CH2)l 7--

, --(CH2h 3--CH2---CHCH(CH2)x 7--

/

,

CH3

--(CH2)13--CH~--~CH--CH(CH2)17--

I

CH3 In this proposed transformation, electrophilic addition of the methyl group to the carbon-carbon double bond, presumably from S-adenosylmethionine, is followed by proton abstraction to regenerate the double bond at a position one carbon f a r t h e r from the carboxyl terminal. This proposed mechanism for incorporation of the C-methyl branch may not be correct, as can be seen by inspection of the structures actually observed for methylated and unmethylated dialkene homologs of the same chain length. Analysis of the distribution of positional isomers in each of the unmethylated-methylated pairs (B-5 vs B-6, etc.) leads to the conclusion that /f the unmethylated dialkene mycolates are indeed precursors of the methylated dialkene mycolates, incorporation of the methyl

The major mycolic acids of

Mycobacterium smegmatis

107

group must be accomplished by a route involving rearrangement of the double bond to a position one carbon nearer to the carboxyl terminal. In each unmethylated-methylated pair of identical overall chain length, the distribution of positional isomers is such that "y-values" of 10 and 12 predominate in the unmethylated homolog whereas "y-values" of 11 and 13 predominate in the methylated homolog. These conclusions, of course, are only speculative. Based on the distributions observed for the major positional isomers in each of the homologs, however, they seem plasusible. More insight into the nature of the biosynthetic mechanisms operating in M. smegmatis is needed if the pathways leading to the synthesis of these high molecular weight fatty acids are to be understood. (Received 12 November 1981)

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

ANDERSON,R. J. d. biol. Chem. 85, 351-354 (1929). ASSELINEAU,J. In: The Bacterial Lipids, pp. 122-143, Holden-Day, Inc., San Francisco, 1966. ASSF.LINEAU,J. and LEDERER,E. C. R. Acad. Sci. 22~ 1892-1894 (1949). AZUMA,I., Rmi, E, E., MEYER,T. J. and ZBAR, B. J. Natl. Cancer Inst. 52, 95-101 (1974). I]ARBIER,M. and LEDERER,E. Biochim. Biophys. Acta 14, 246-258 (1954). BORCH,R. F. Anal. Chem. 47, 243%2439 (1975). BUDZIKIEW1CZ,H., DJERASSI,C. and WILLIAMS,D. H. In: Mass Spectrometry of Organic Compounds, p. 183, Holden-Day, Inc., 1967. DANIELSON,S. J. and GRAY, G. R. (submitted for publication). DEMARTEAU,H. C. R. Acad. Sci. 232, 2494-2496 (1951). ET~MAm,A. H. Bull. Soc. Chim. Fr. 1537-1541 (1964). ETI~MADI,A. H., OKUDA, R. and LEDERER,E. Bull. Soc. Chim. Fr. 868-870 (1964). ETI~MADI,A. H., PINTE, F. and MARKOVlTS,J. C. R. Acad. Sci. Paris 262, 1343-1346 (1966). JAUREQUIBERRV,G., LENFANT,M,, DAS, B. C. and LEDERER,E. Tetrahedron, Suppl. 8, Part 1, 27 32 (1966). KANETSUNA,F. and BARTOLi,A. J. Gen. Microbiol. 70, 209-212 (1972). KREblBEL,J. and ETt~MAm,A. H. Tetrahedron 22, 1113-1119 (1966). QURESHI,N., TAKAYAMA,K., JORDI, H. C. and ScaNo~s, H. K. J. biol. Chem. 253, 5411-5417 11978). Sn~MA, M. and RODRIGUEZ,H. R. Tetrahedron 24, 6583-6589 (1968). S'rECK,P. A., SCHWARTZ,B. A., ROSENDAHL,M, S. and GRAY,G. R. J. biol. Chem. 253, 5625-5629 (1978). STODOLA,F. H., LF..SUK,A. and ANDERSON,R. J. J. biol. Chem. 126, 505-513 (1938). WONG, M. Y. H. and GRAY, G. R. J. biol. Chem. 254, 5741-5744 (1979). WONC, M. Y. H., STECK, P. A. and GRAY, G. R. J. biol. Chem. 2,~1, 5734-5740 (1979).

I.P,L.R.21/2--S