the chemistry of some native constituents of the

THE CHEMISTRY OF SOME NATIVE CONSTITUENTS OF THE PURIFIED WAX OF MYCOBACTERIUM TUBERCULOSIS

BY HANS NOLL*

(From the Division of Tuberculosis, The Public Health Research Institute of The City of New York, Inc., New York, New York)

(Received for publication, June 18, 1956)

“Purified wax,” as isolated by Anderson, is a complex lipide extracted from cultures of acid-fast bacilli with chloroform. It is characterized by its insolubility in methanol (1). Anderson investigated its composition by chemical identification of the fragments after hydrolytic degradation. The results indicated that the purified wax was not chemically homogeneous, and the question of how the hydrolytic fragments were originally combined has not been answered.

Progress toward the isolation of the native constituents of the purified wax was made by Asselmeau (2). Extracting the purified wax with boiling acetone, he obtained a soluble portion termed “Wax C” and an in- soluble residue, the “Wax D” fraction. Wax C was characterized by its low melting point and the absence of nitrogen and high molecular carbohydrate components, and Wax D consisted chiefly of high molecular lipopolysaccharides containing small amounts of nitrogen and phosphorus

(2,3). Introducing the technique of chromatographic partitioning, Asselineau

was able to isolate from Wax C two components in a pure state, which he identitled as mycolic acid and an ester of phthiocerol. Later, Philpot and Wells (4) identified the acid part of this ester as mycocerosic acid.

A continuation and extension of the work on composition and chemistry of the native constituents of Wax C are reported in the present paper. An improved method of solvent partitioning of the purified wax resulted in a better defined separation of Wax C and Wax D. Several new compounds were isolated from the Wax C of various virulent strains of Mycobuctium tuberculosis, among these a triglyceride of a long chain fatty acid (C,, to C26) and a monoglyceride of mycolic acid.

EXPERIMENTAL

Isolation of Wax C

The production of the bacterial cultures and the strains used in this study have been described in a previous paper (5). The preparation of the

* Present address, Department of Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.

149

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150 PURIFIED WAX OF M. TUBERCULOSIS

purified wax and Wax C has been improved in the following manner. The chloroform extracts are concentrated in vacua to about 300 ml. and then clarified through a Seits filter. If the chloroform extracts are markedly turbid, Seitz filtration often becomes difficult, and this difficulty is over-

TABLE I Chromatogram I. Chromatography of 8.10 Gm. of Wax C Br6vannes Dissolved

in Petroleum Ether, Adsorbed on 60 Cm. of Magnesium Silicate-Celitc

l-6 7

8

9

10

11

12

13

14 15 16-19 29-24 25 26 27 28-31

Eluted with 50 ml. of

Petroleum ether “ ether -benzene

1:l Petroleum ether-benzene




1:l Petroleum ether-benz .le


1:l Benzene-ether 1: 1

‘I 1:l I‘ 1:l “ 1:l

Ether + 5% MeOH “ + 5% “ “ + 5% “ I‘ + 10% “

Recovered . . . . . . . . . . . . . . . . . . . . . 1718.7 = 72%

mg. Traces

177.6

162.7

81.2

61.9

47.9

44.9

22.2

676.5 39.8 48.2 7.6

192.3 119.2 13.7 33.0

T

M.P.

OC.

59-52

4546

42-43

40

36-37

37-39

49-41 42-43

c.mm. 0.1 N [eONa permg

0

0

0.3

0.5

Infrared spectNm

NO.

2 (Fig. 1)

3 (Fig. 1)

3 ((5)* Fig.1)

* From the literature.

come by adding to the concentrated chloroform extracts 1 part ether, followed by 1 part acetone before filtration. The clear filtrate is taken to dryness, the residue dissolved in ether, and the purified wax precipitated by adding an excess of methanol. The purified wax is then separated into Wax C and Wax D by dissolving it in a small amount of benzene to which 1.5 parts of acetone are added slowly with stirring. A gummy precipitate results, which adheres to the bottom of the glass. The clear supernatant fluid is decanted and Wax C is precipitated by the addition of an excess


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H. NOLL 151

of methanol. After cooling, the precipitate is filtered on a fritted glass filter, washed with methanol, and dried. It forms a white powder, m.p. 40-50”.

The yield of Wax C obtained by this method ranged from 60 to 90 per cent of the total purified wax. In contrast to this, extraction of the puri-

TABLE II Chromatogram II. Chromatography of 1.60 Gm. of Wax C VallBe Dissolved in

Petroleum Ether, Adsorbed on 100 Gm. of Magnesium Silicate-Celite

1 2 3 4 5 6 7-12

13

14

15-24

25 26 27 28-38 39 40 41 42-46

-

--

-

Eluted with 50 ml. of

Petroleum ether “ “ ‘I ‘I I‘ “ I‘ ‘I I‘ “ “ “ “ ether-benzene



1:l Benzene-ether 1:l

‘I 1:l I< 1:l “ 1:l

Ether + 5% MeOH “ + 5% “ “ + 5% “ “ + 5% “

Recovered . . . . . . . . . . . . . 1352.5 = 900%

w. 33.9

417.3 156.7 86.2 36.3 22.9 57.5

104.4

20.1

26.7

47.5 170.8

15.3 35.7 59.6 29.3

9.3 23.0

-_

--

-

l&p.

“C.

48-51 48-51 51-K

39-4(

27-3: 35-3( 36-3

38-3! 39

c.mm. 0.1 N leONa permg

0

0.3

0.4

0.6

2 (Fig. 1)

5 (Fig. 1)

4 ((5)* Fig. 1)

* From the literature.

fied wax with boiling acetone (2) yielded only 10 to 30 per cent Wax C. Extraction with boiling acetone also leaves a large part of the cord factor in the Wax D fraction, while fractional precipitation assures a high yield of cord factor in Wax C.

Clasti~tion of Main Non-Toxic Constituents of Wax C

Chromatographic partitioning of Wax C yields a series of neutral fractions (Tables I to III; (5) Table II) characterized by their melting points


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TABLE III Chromatogram IZI. Chromatography of 1 JO Gm. of Waz C of Isonicotinic Acid

Hydra&de-Resistant Strain Y%ba”* Dissolved in Petroleum Ether,

l-9 10

11

12

13

14

15

16

17 18 19 20 21 22 23 24 25 26 27

Adsorbed on 7’6 Gm . ‘JJ f Magnesium Silicate-Celite

Eluted with 50 ml. of IQ. “.mm. 0.1 N ‘eoNa perme

Petroleum ether “ ether-benzene






1:l Petroleum et,her-benzene

1:l Benzene-ether 1: 1

“ 1:l ‘I 1:l “ 1:l “ 1:l “ 1:l “ 1:l

Ether + 5yo MeOH “ + 5% “ “ + 5% (‘ “ + 5% “

w.

12.9 7.5

T.

537.8 39-31 0

65.3

41.6 31 0

31.7

22.0

7.8

123.8 9.3

18.9 12.5 5.9 4.3 3.8

180.0 20.2 3.4 2.8

28-29 0.9

49-51 6.0

4247 42

Recovered . . . 1111.5 = 93%

3.5

1 (Fig. 1)

5 ((5) Fig. 1)

* A variant of H37Rv, resistant to isonicotinic acid hydrazide, obtained through the courtesy of Dr. R. L. Mayer, Ciba Pharmaceutical Products, Inc., Summit, New Jersey.

and, more specifically, by their infrared spectra.l The spectra of the main fractions listed in Chromatograms I to IV are reproduced in Fig. 1. According to their infrared absorptions, they can be classified as follows:

1 The infrared spectra were taken at the American Cyanamid Company, Stamford, Connecticut, with a Perkin-Elmer model No. 21 double beam infrared spectropho- tometer. The author wishes to express his thanks to Dr. R. C. Gore and Mr. N. B. Colthup for invaluable assistance in preparing and interpreting the spectra.


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H. NOLL 153

saturated aliphatic long chain esters (Compounds Al, AZ); saturated aliphatie long chain hydroxy esters (Compound B1) ; unsaturated aliphatic long chain hydroxy esters (Compound C,) ; and aliphatic-aromatic long chain hydroxy esters (Compound DJ.

3i-m :

FIG. 1. Infrared spectra of Wax C compbnents (taken from melts). Spectrum 1, Compound Al; Spectrum 2, Compound AZ; Spectrum 3, Compound B1; Spectrum 4, Compound Cl; Spectrum 5, Compound D1.

Compound A1 (Fig. 1, Spectrum I)

This is the most abundant component occurring in Wax C of the strains H37Rv and PN, DT, C. It has never been found in Wax C of Brevannes and of the bovine strain Vallee. In view of the melting point and chromatographic adsorption ((5) Table II, Fractions 1 to 5), it was suggested (56) that this compound is identical with an ester of mycocerosic acid and phthiocerol, described by Philpot and Wells (4). This has now been confirmed by reductive cleavage with LiAlH* and subsequent chromatography of the reaction products on magnesium silicate-Celite. Two fractions were obtained. Fraction 1 (m.p. 35-36”), eluted with petroleum ether-benzene 1: 1, was identified as mycocerosic alcohol by comparing its infrared spectrum (Fig. 2, Spectrum 2) with the spectrum of mycocerosic


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alcohol obtained after reducing a sample of mycocerosic acid2 with LiAlH4. Fraction 2 (m.p. 683 was eluted with benzene-ether 1: 1. Its infrared spectrum (Fig. 2, Spectrum 1) was identical with that of an authentic sample of phthiocerol.8 From the absence of hydroxyl bands in the infrared spectrum of Compound A1, as well as from the relative amounts of the two components obtained after reductive cleavage, it is concluded that both hydroxyl groups of phthiocerol are esterified with a mycocerosic acid residue. This is in agreement with previous findings (6, 7).

I-

/-

6ol 1 1400 1200 loot

Y’l

IJ’

T J

f d

FIG. 2. Infrared spectra of degradation products of Wax C components (taken frommelts). Spectrum 1, phthiocerol; Spectrum 2, mycocerosic alcohol; Spectrum 3, mycocerosic acid; Spectrum 4, As acid.

Polgar (8) has isolated an acid, apparently identical with Anderson’s mycocerosic acid, which he termed mycoceranic acid. By a series of stepwise degradations, he arrived at Structure I, which was later corrob-

CHa-(CH&--CH-CHs-CH-CHz---CH-COOH I I I CHs C& CHa

(1)

orated by synthesis of some of the structural units of this acid (9). The infrared spectrum of Anderson’s mycocerosic acid (Fig. 2, Spectrum 3)

* A sample of myeocerosic acid prepared by Dr. R. J. Anderson wa8 obtained from Dr. R. C. Gore.

* Kindly supplied by Dr. R. J. Anderson.


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H. NOLL 155

seems to be in agreement with the structure worked out by Polgar. The presence of several terminal CHs groups is indicated by the stroxig CHo band at 1380 cm.-l in the spectrum of the acid as well as in the spectra of its ester and its alcohol derivatives. The infrared spectra of long chain fatty acids show two strong C-O bands near 1285 and 1235 cm,-‘, the first band being usually somewhat stronger. Freeman found a reversal of intensities occurring in this pair of bands in the spectra of single branched fatty acids with a methyl group in the 2 position, and in the spectra of higher branched fatty acids with several methyl groups close to the car- boxy1 group (10). The occurrence of this reversal in the spectrum of mycocerosic acid is in agreement with the structure of Polgar’s mycoceranic acid (Structure I) and thus further indicates the identity of the two acids (3).

The structure of phthiocerol has been the subject of studies by several workers (2,11-15). Polgar and coworkers (14, 15) concluded that phthiocerol is 4-methoxy-4-methyl-n-tetratriacontane-9,11-diol (Structure II).

OH OH OCHa I I I

CHa-(CH&--CH-CHs-CH-(CH,)r-C-CHI-CH~-CH~ I

CHs (II)

The presence of a 1,3-glycol structure had previously been established by Demarteau-Ginsburg and Lederer (13). There is some difference of opinion between Polgar and Lederer as to the position of the methoxyl group. In contrast to the Structure II proposed by Polgar, the French workers assigned the methoxyl to position 5 on the carbon next to the methyl branching (13). Because of recent results, however, Lederer and Stenhagen appear to have definite evidence4 that the methoxyl group is attached to carbon 3. A characteristic feature of the infrared spectrum of phthiocerol (Fig. 2, Spectrum 1) is the presence of two sharp bands at 830 and 925 cm.+. Strong absorptions in this region have been associated with the group

;: c-c-o

I C

in tertiary alcohols and ethers (16). The fact that the two bands are lacking in the dimycoceranate of phthiocerol indicates that they are related

4 E. Lederer, personal communication.


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to the free secondary hydroxyls rather than to the presence of a tertiary methoxyl group in phthiocerol.

The structure of phthiocerol-dimycoceranate (Compound A1) is pictured in Structure III, on the basis of the available chemical information concerning its acidic and alcoholic components.

CH: I

CHa

CH-OCH, I

CH-CHI

(C&J, I

CH-O-CO- CH-CHs -(CH&-CH, I

CHs [i 1 CHa a

CH-O-CO- CH-CHe -(CH&,-CHs

(C&ha [I 1 CHa * CHs

(III)

Compound AZ (Fig. 1, Spectrum 2)

This ester fraction is relatively abundant in Wax C of the strains Brevan- nes, Vallee, BCG, and H37Ra but occurs only irregularly and in much smaller proportions in the strains H37Rv and PN, DT, C, which in contrast are rich in the Compound A1 component (phthiocerol-dimycoceranate).

Compound AZ (eluted with petroleum ether-benzene 1: 1 (Table I, Fraction 7)) has been characterized by the following data:

M.p. 50-58”. C 78.37, H 12.32 “ 78.23, (‘ 12.29

The infrared spectrum taken from a melt shows the general absorption pattern described for triglycerides of long chain fatty acids by Shreve et a.!. (17), a strong band at 1175 flanked by weaker bands at 1250 and 1105 cm.+. The band at 1420 has been associated with CH2 next to C=O (18), and thus is common to esters of straight chain fatty acids but absent in the spectra of esters of fatty acids having a branching at the a-carbon.

Saponification400 mg. of Compound AZ were saponified with KOH in moist isopropyl alcohol, as described for cord factor (5). The solution


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H. NOLL 157

was then acidified with HCl and extracted with ether. The ether extracts yielded 390 mg. of white powder which were subsequently chromatographed on magnesium silicate-Celite. Practically all of the material was eluted with petroleum ether-benzene 1: 1 (v/v) in a series’of fractions forming a broad peak. The infrared spectra of all these fractions were identical, indicating that the material was homogeneous. After recrystallization from hot acetone, a microcrystalline powder was obtained with the following properties :

M.p. 79-76”; acid equivalent 389. C 77.94, H 12.68

Infrared Spectrum (Fig. 2, Spectrum 4)-The infrared spectrum of the AZ acid taken as a melt shows the characteristic absorptions of long chain fatty acids in the solid state. According to Jones et al. (19), a typical feature of such spectra useful for identification is a progression of bands of uniform intensity and spacing between 1180 and 1350 cm.-‘. Lengthen- ing of the carbon chain increases their number with a concurrent displace- ment to lower frequencies. The position of these progression bands in the spectrum of the Compound As acid6 is at 1190, 1200, 1212, 1225, 1238, 1250, 1265, and 1280 (shoulder) cm.-‘.

Elementary analysis and titration indicate for AS acid a chain length of C&o C&S. Its infrared spectrum does not match any of the spectra of the saturated long chain fatty acids published by Jones et al., which include members up to Czl. Its high melting point is in accordance with that of a saturated and straight chain structure. This is supported by its infrared spectrum. The C-Cl& band at 1380 cm.-’ is very weak, reflecting the small molecular proportion contributed by the single terminal CHa group in straight long chain fatty acids. Although the available data are not considered sufficient for an exact identification, we may tentatively assign to the AZ acid the structure of tetracosanoic acid:

CzrHlsOa. Calculated. C 78.19, H 13.12 (mol. wt., 368.6; m.p. 84”) Found. “ 77.94, “ 12.68 ( “ “ 389; m.p. 79-76”)

The results from infrared spectroscopy and saponification suggest for Compound AZ the structure of a triglyceride of tetracosanoic acid:

CVSHUSOS (f6CHt). Calculated. C 78.74, H 12.86 Found. “ 78.37, “ 12.32

“ 78.23, “ 12.29

6 After this manuscript was completed, we received, through the courtesy of Dr. Lederer, pure samples of tetracosanoic and hexacosanoic acid. Comparison of- their infrared spectra showed an extremely close similarity between the An acid and tetracosanoic acid. A final identification, however, has to await a-more extensive study upon a larger series of homologous acids, including pentacosanoic acid.


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Compound B1 (Fig. 1, Spectrum 3)

This hydroxy ester is a major constituent of Wax C! of the strains B&an- nes and BCG. The material is elutd from the column with benzene- ether 1: 1 (Table I, Fractions 14 to 24). After precipitation from ether with methanol, it forms a white powder with the following characteristics:

C 79.54, H 12.33, N 0 “ 79.69, “ 12.55, “ 0

M.p. 38-39”. [a]: +6.4” f 2.5” (C = 0.7 in benzene)

Infrared Spectrum (Fig. 1, Spectrum S)-The strong bonded OH band at 3350 indicates the presence of several free hydroxyl groups. The bands in the C-O stretching region at 1050, 1100, ad 1170 cm.-’ agree well in position with the three bands which have been found to be characteristic for monoglycericles by Matutano and Martin (20). The bands at 1050 and 1100 have been associated with the C-O stretching of primary and secondary OH, respectively, and the strong absorption at 1170 cm.+ with the stretching of the ester linkage,

II -c-o-c-

Saponifiation-800 mg. of Compound B1 were saponified in moist isopropyl alcohol as described (5). The solution was then acidified with 3.3 ml. of 1 N HCI and, after addition of 10 ml. of HzO, extracted with petroleum ether. The petroleum ether extracts yielded 733 mg. (92 per cent) of a waxy residue which was obtained as a white powder after precipitation from ether with methanol. This material was identical in all its properties (melting point, acid equivalent, C,H analysis, infrared spectrum) with mycolic acid Bdvannes (C187H17401) (21), as obtained from saponification of cord factor (5).

The aqueous portion of the hydrolysate was deionized by filtering through Amberlite MB-3 and then lyophilized. A colorless viscous liquid was obtained which was identified as glycerol by its infrared spectrum. It follows from the yield of mycolic acid after saponification (92 per cent) as well as from the C,H analysis of the original material that Compound B1 is a monomycolate of glycerol:

CpdH1~oOe. Calculated. C 79.57, H 13.36 Found. “ 79.54 “ 12.33

“ 79.59, “ 12.55

This finding is corroborated by the infrared spectrum of Compound B1, which shows the characteristic bands of a monoglyceride. Since the optical rotation of the ester (+6.4’) is considerably stronger than that of mycolic acid (+l.S”), a new asymmetrical center must have been added.


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H. NOLL 159

Consequently, mycolic acid must be linked to one of the a-carbons of glycerol which renders the P-carbon asymmetrical, as shown in Structure IV.

H&h0-C-CH-CH-C~~HI~~OH f 5CH, I I

H*C$OH Cd340

I H&OH

(IV)

Compound Cl (Fig. 1, Spectrum 4) This neutral component, m.p. 28”, has occasionally been found in the

petroleum ether-benzene’eluates during chromatography of Wax C Brevan-

FIQ. 3. Ultraviolet absorption spectrum of Compound DI. 0.1 per cent solution in isooctane.

nes. Its main spectral characteristics, apart from its bonded -OH and C--O absorptions, are three sharp bands at 1650, 1385, and 750 cm.+ associated with cis double bonds of the C-CH=CH-C type.

Compound DI (Fig. I, Spectrum 6)

This component, m.p. 27-32” (Table III, Fraction 25), has been isolated only from Wax C of the bovine strain Vallee. Smith et, al. (22) have published an infrared spectrum which is identical to Spectrum 5 presented in Fig. 1. They concluded from an extensive study of a great number of bovine and other strains that this component was characteristic of the bovine type.

The infrared spectrum of Compound D1 shows three characteristic sharp bands at 1620, 1590, and 1515 cm.+ which are correlated with a substituted benzene. Its ultraviolet spectrum (Fig. 3) shows the charac-


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160 PURIFIED w.4x 0~ M. ~BERCUL~SIS

teristic absorptions of a disubstituted aromatic ether. On the other hand, the strong CH2 absorptions in the infrared spectrum suggest that the aromatic part of the molecule is combined with a long chain aliphatic residue. These characteristics indicate t,hat Compound D1 is related to

TABLE IV Percentage Range of Wax C Components

Strain A eof k cu tures

wks.

PN, DT, C .......... 12 H37Rv.. ........... 2-3 “Ciba”*. ........... 2-3 Brbvannes .......... 2-3 Vall6e. .............. 2-3

* See Table III, footnote.

T

Al

SC-90 60-70 60-70

0 0

Compound No.

A, B1 --

0 0 0 0 0 0

20-30 40-M) 60-70 0

- Wxdic acid Cord factor

10-20 l-3 l-5 10-15 5-10 5-10 0 8-20 0 S-15

CORI- “N”,“d

A1

AZ

B,

MA*

CFt

TABLE V Adsorption Sequence and Chemical Constitution of Wax C Components

Observed elution sequence, in order of

Functional groups, in order of increasing adsorption

Eluted with

I 1 No. of groups per molecule 1

WdLuOd 1410 1 W7sH1440~J 1144 0

G&w,04) 1358 0 K~HI~IOI) 1284 0 (Cd3ieaOrr) 2903 3

2 0 0 Petroleum ether 3 0 0 “ ether-benzene

1:1 1 4 0 Benzene-ether 1: 1 0 2 1 2 10 0 Ether + 5% MeOH

* MA = mycolic acid. t CF = cord factor.

leprosols of Crowder et al. (23), which have been shown to be alkyl derivatives of resorcinol (24).

Relative Proportions of Constituents of Wax C in Various Strains of Virulent Tuber& Bacilli

Table IV summarizes the reIative proportions of the main components contained in the Wax C of five different bact.erial strains. It can be seen


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H. NOLL 161

that strain differences are clearly reflected in qualitative and quantitative variations of the non-toxic ester components, while cord factor is the only Wax C constituent present in all of these strains. The lower relative yield in the strains PN, DT, C is probably due to the age of the culture, since it has been shown that the yield of cord factor decreases as the cultures grow older (5, 6).

Chromatographic Adsorption and Chemical Constitution

Within a given class of compounds, the adsorption affinity is determined by the nature and number of polar groups present in the molecule. The order of elution is therefore a direct reflection of the chemical constitution. The functional groups relevant to this study have been classified in order of increasing adsorption affinity for alumina and magnesia as C. 0 *C < CO-OR < OH < CO *OH (25). When the main constituents of Wax C are arranged according to the number and relative adsorption affinity of their functional groups (Table V), the resulting sequence coincides with the observed chromatographic elution order. Thus, the findings concerning the chemical. constitution of these compounds are in excellent agreement with their chromatographic behavior.

DISCUSSION

In recent years conflicting reports have been published concerning the biological activity of Anderson’s purified wax and similar preparations (26-29). These contradictory findings indicate the need for better methods for the resolution of complex lipide fractions and the chemical characterization of the individual components. A combination of chromatography with infrared spectroscopy provided an efficient tool toward this end (5, 6, 30). Since chemically distinct lipide components have characteristic infrared spectra, it was possible to control, spectroscopically, the efficacy of the partitioning process and the purity of the resulting fractions. The infrared spectrum then served as a means of identification prior to structural analysis by chemical degradation methods.

The main difficulty in the purification of Anderson’s purified wax arises from the fact that it contains a certain proportion of high molecular lipopolysaccharides (Wax D) which are difficult to separate from the lower molecular lipides (Wax C). In addition, Wax D, because of its high molecular structure, defied attempts at purification by the methods effective for the partitioning and characterization of the Wax C constituents (31). The present study, therefore, was limited to an investigation of the chemical composition of Wax C. For the isolation of this fraction, a method of solvent fractionation was developed which resulted in a more complete separation of Wax C from Wax D, and gave considerably higher


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yields of Wax C than previous methods did. A clear separation of Wax C from Wax D is important for biological studies, since both fractions contain active components, causing essentially different phenomena.

The chemical study of Wax C has shown that it consists mainly of unusually long chain fatty acids which are esterified with polyhydroxy compounds such as phthiocerol, glycerol, and trehalose to form lipides of a relatively high molecular weight, ranging from 1000 to 3000. The molecular size and predominantly paraffinic structure account for the similar solubility of these compounds in organic solvents, in particular their insolubility in methanol, on which the isolation of Wax C is based. An analysis of the chemical composition of Wax C obtained from different virulent strains revealed significant qualitative and quantitative variations. The only constituent uniformly present in Wax C of all the strains analyzed so far was cord factor, the structure of which has recently been determined as trehalose-6,6’-dimycolate (32). Another ester of mycolic acid has been isolated from the strains Brevannes and BCG in the form of an Ly-monoglyceride. Free mycolic acid did not occur regularly in the Wax C of young bacterial cultures. Only the H37Rv strain consistently yielded small quantities of the free fatty acid. The presence of large amounts of free mycolic acid (5) in the wax of old, steam-killed cultures probably results from autolytic processes, at least part of it being liberated from cord factor, as indicated by a corresponding reduction in cord factor.

It has often been emphasized that the tubercle bacillus waxes contain little (3) or no (33) glycerides. This general statement is obviously not correct. The present results indicate that the relatively low glycerol values found by previous authors in hydrolysates of crude wax fractions are due to the unusually large molecular size of the constituent fatty acids rather than to the absence of glycerides. In some bacterial strains glycerides constitute as much as 70 per cent of the total Wax C (Table IV). It is an open question whether this large proportion of glycerides is related to the composition of the culture medium which contains glycerol as the chief source of carbon.

It is suggestive to consider the compounds isolated from Wax C as representatives of a wider family of structurally related components occurring throughout the lipide fractions of the tubercle bacillus. Thus, structural analogues of lower molecular weight, i.e. similar combinations of polyhydroxy compounds with fewer or shorter paraffinic chains, would be considerably more soluble in methanol and hence more likely to occur in some fraction other than Wax C. Such considerations are of particular interest with regard to fatty acid esters of trehalose because of the possible biological activity of structural analogues of cord factor. This view is supported by earlier reports on esters of trehalose with shorter fatty acids


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H. NOLL 163

occurring in Anderson’s acetone-soluble fat (21, 34), as well as in certain lipides of diphtheria bacilli (35).

SUMMARY

The chemical composition of Wax C from various virulent strains of Mycobacterium tuberculosis has been investigated by chromatographic partitioning, combined with infrared spectroscopy. A series of compounds designated as Compounds AI, AZ, B1, C1, and D1 has been isolated in purified form and characterized by their infrared spectra. The chemical structure of the following compounds has been determined by hydrolytic degradation: Compound A1 as phthiocerol-dimycoceranate, Compound AZ as a triglyceride of a straight chain fatty acid CZZ to CZ6, and Compound B1 as an cu-monoglyceride of mycolic acid.

The significance of these findings and their relation to previous results are discussed.

The technical assistance of Mr. Eugene Jackim in this research is grate- fully acknowledged.

BIBLIOORAPHY

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Hans NollTUBERCULOSIS

WAX OF MYCOBACTERIUMCONSTITUENTS OF THE PURIFIED

THE CHEMISTRY OF SOME NATIVE

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