Studies on the lipids of acid fast bacilli

The lipids of human tubercle bacilli on mild alkaline hydrolysis give a methanol-insoluble product, which on prolonged hydrolysis yields three main fractions, the mycolic acids, crude phthiocerol, and the mycoceranic acids1. In the course of this work a re-investigation of the first two groups of compounds has been carried out, and synthetic analogues of methyl-branched long-chain acids occurring in tubercle bacilli have been prepared for the purpose of biological testing.
The mycolic acids - These acids have been demonstrated to be β-hydroxy acids of high molecular weight with a long alkyl side-chain in α-position. Acids containing oxygen functions in addition to the &beta-hydroxyl; group have been isolated from various strains of bacilli and a detailed description of the work up to 1961 has been given by Asselineau2. More recently, Malani and Polgar3 have described a mycolic acid containing a methoxyl group, and by oxidative degradations and vapour phase chromatography of the products were able to assign to it a partial structure (I), where n = 21 and 23, m = about 17,
(I)
OH
   |
CH3. (CH2)m. CH. CH −    ..........    CH. CH. CH. COOH
   |    |    |    |
OMe   R (CH2)nCH3 (CH2)nCH3
and R is an alkyl radical.
Gastambide - Odier et al.,sup>4
investigated mycolic acids from many sources by nuclear magnetic resonance (n.m.r.) spectroscopy, and showed that all those examined from human strains contain cyclopropane rings. Etemadi and Lederer5 applied mass spectrometry to methyl α-mycolate (Test), and in conjunction with the n.m.r. evidence proposed the general, structure (II, R=Me) for this ester.
(II)
CH2 CH2 OH
/ \ / \    |
CH3. (CH2)n1. CH    − CH. (CH2)n2. CH    − CH. (CH2)n3. CH. CH.COOR.
   |
C24H49
In the present studies unidimensional multiple chromatography6 (UMC) or crude methyl mycolate from human strains (D.T., P.N.. and C.) showed the presence of three mycolic esters which were named methyl mycolates -I, -II and -III, in order of increasing polarity. Ester-III was shown to contain a keto-group and was isolated chromatographically as its oxime. The mixture of the other two esters was acetylated, and repeated chromatography of the product gave methyl acetylmycolate -I and -II. Hydrolysis gave the corresponding hydroxy-esters which were shown to contain small amounts of the C-2 epimers. Removal of this material by chromatography on alumina gave methyl mycolate -I, m.p. 49-50°, [α]20589+ 3.7°, and methyl mycolate -II, m.p. 48-48.5°, [α]20589+ 0.1°. During the above chromatography some dehydration occurred giving methyl anhydromycolate -I, m.p. 39-41°, and methyl anhydromycolate -II, m.p. 32-35°. The material from the purification of methyl mycolate -I which contained its epimer was chromatographed repeatedly to give methyl 2-epimycolate-I, m.p. 56-57°, [α]20589-0.6°.
The oxime of methyl mycolate -III appeared on UMC to contain two components (the oxime of the corresponding acetoxy-compound behaved similarly) which were shown to be geometrical isomers in respect of the hydroxyimino grouping. Hydrolysis of the oximes of acetoxy-ester-III, and chromatography on alumina gave methyl acetylmycolate -III, m.p. 43-5-44°, [α]20589+ 6.2°. This chromatography also resulted in. some anhydromycolate-III, m.p. 52-52.5°. Methyl mycolate -III, m.p. 52-53°, [α]20589+ 5.0°, was obtained by hydrolysis of methyl acetylmycolate -III; a thioketal derivative of the latter was also prepared.
Infra-red spectra showed that cyclopropylmetnylene groups7 were present in all three esters, and ester-II contained a band due to methoxyl. The n.m.r. spectrum of methyl mycolate -I, showing two bands(6H and 2H) for the cyclopropane groups, was very similar to that reported for methyl cis-9,10-methylenehexadecanoate8 and indicated the presence of two non-adjacent cis - 1,2 - disubstituted cyclopropane groups and only two terminal methyl groups. The n.m.r. spectrum of methyl mycolate -II showed that the single cis-cyclopropane group present was in a different environment, possibly adjacent to the \/
CH-OMe grouping; a methyl branch was also detected. In the spectrum of acetoxy-ester-III three cyclopropane protons appeared in one broad band; after formation of the corresponding thioketal a single band (4H) was observed. This result suggested that the molecule contained a trans -1,2- disubstituted cyclopropane group adjacent to a carbonyl group ; the ultra-violet spectrum was in agreement. The grouping - CH(CH3).CO- was also shown to be present.
Low resolution mass spectra of all the above compounds were determined, the anhydro-esters giving the moot informative spectra. The results of these mass spectrometric studies in conjunction with the other evidence pointed to structures (III and IV) for the main components of methyl mycolate -II and methyl mycolate -III.
(III)
CH3 CH2 OH
   | / \    |
CH3. (CH2)17. CH. CH. CH    − CH. (CH2)33. CH. CH.COOMe
   |    |
OMe   cis C24H49
(IV)
CH3   O CH2 OH
   |    || / \    |
CH3− (CH2)17. CH.   C. CH    − CH. (CH2)36. CH. CH.COOMe
   |    |
OMe trans C24H49
In each case the presence of ten corresponding homologues was demonstrated suggesting a biogenetic relationship between these compounds; the trans-cyclopropane group in ester -III may have been formed via the enol during the alkaline hydrolysis of the c rude waxes. The mass spectrum of methyl mycolate -I, together with the other data described above, indicated that is essentially the same as methyl α-mycolate (Test) described by Etemadi and Lederer5. These workers supposed that a series of oxygen containing peaks at m/e 459 to 543 were due to cleavage, adjacent to cyclopropane groups, of an aldehyde fragment formed by pyrolysis in the mass spectrometer and gave the structure (II, n1 = 17, n2 = 11, n3 = 16) for the main component of this ester. however, high resolution studies of methyl mycolate -I showed that the basis for the above assignments was insecure ; moreover, recent work9 has shown that peaks due to cleavage adjacent to cyclopropane groups are not usually observed. The results available suggest that the general structure (II) is correct, with cis-cyclopropane substituents, but they do not give at present a clear indication of the exact location of the cyclopropane groups.
High-resolution infra-red spectra of methyl mycolate-I -II and -III and methyl 2-epimycolate-I showed that the epimer forms an intra-molecular hydrogen bond to a smaller extent; studies of molecular models showed that esters -I, -II and -III consequently have erythro configuration in respect of C-2 and C-3.
The phthiocerol group - Previous investigations10 indicated that phthiocerol is a mixture of two homologous β-diols having the structure (V) where n is 22 and 20,
(V)
OMe OH OH
   |    |    |
CH3. CH2. CH. CH. (CH2)4. CH. CH2. CH. (CH2)n.CH3
   |
CH3
respectively. In the present studies UMC of crude phthiocerol showed (in order of increasing polarity) the presence of the diols (V) in the following named phtniocerol A, material now designated phthiocerol B and ketonic material presumed to be essentially phthiodiolone11. The presence of several more-polar constituents was also observed. The mixture of cyclic acetals formed by reaction of crude phthiocerol with acetaldehyde and toluene-p-sulphonic acid showed on UMC the same sequence of polarity, thus indicating that phtaiocerol A and B as well as phthiodiolone contain an analogous diol system. Column chromatography of the acetals on alumina gave a mixture of the acetals of phthiocerol A and B, followed by impure acetal of phthiodiolone and by more polar acetals which latter have not been studied further. The mixture of acetals of phthiocerol A and B was chromatographed repeatedly to give phthiocerol A ethylidene acetal, m.p. 40° , and phthiocerol B ethylidene acetal m.p. 35.5°. Decomposition of the acetals gave the corresponding diols, phthiocerol A, m.p. 73-73.5° , [α]20589 -4.5° and phthiocerol B, m.p. 71-71.5°, [α]20589 -8.2°.
The infra-red spectra of both diols as well as those of the corresponding acetals showed close agreement ; all spectra included a band due to metnoxyl. The mass spectra of phthiocerol A and its corresponding acetal were in agreement with the structure (V) for phthiocerol A. The mass spectra of phthiocerol B and its acetal were analogous to those of phthiocerol A but the molecular ions were fourteen units lower in mass. A detailed comparison of the intermediate fragments in the above spectra led to the conclusion that phthiocerol B is a mixture of diols having the structure (VI), where n is 22 and 20
(VI)
OMe OH OH
   |    |    |
CH3. CH. CH. (CH2)4. CH. CH2. CH. (CH2)nCH3
   |
CH3
respectively12 . The n.m.r. spectrum of phthiocerol B confirmed that a terminal grouping -CH(CH3).CH(OMe)CH3 was present.
The impure sample of phthiodiolone ethylidene acetal contained a chromatographically similar compound, and in order to remove the impurity the acetal was heated with ethanol containing toluene-p-sulphonic acid and converted essentially to the diol. This was acetylated and the oxime prepared and purified by chromatography giving the oxime of diacetylphthiodiolone A, m.p. 38° (named by analogy with phthiocerol A). This chromatography also gave a small amount of the oxime of the etnylidene acetal of phthiodiolone A, the acetal grouping having partially survived the above hydrolysis. The diacetoxy-oxime was subjected to a two-stage hydrolysis to give phthiodiolone A, m.p. 76.5°, [α]20589-2.7° ; a portion was re-converted into phthiodiolone A ethylidene acetal, m.p. 41-42°. Mass spectra of phthiodiolone A, its acetal and the oxime of this acetal, in conjunction with n.m.r. evidence, confirmed that phthiodiolone A is a mixture of diols having the structure (VII) where n is 22 and 20, respectively.
(VII)
O OH OH
 ||    |    |
CH3. CH2. C. CH. (CH2)4. CH. CH2 CH.(CH2)n.CH3
   |
CH3
Methyl-substituted long-chain acids - Investigations on methyl-substituted acids in connection with sarcoidosis have shown (Glaxo Laboratories Ltd., private communication) that 3,12,15 - trimethyldocosanoic acid (VIII, R = H) is the only acid of those tested to give a specific response
(VIII)
CH3. (CH2)6. CH. (CH2)2. CH. (CH2)8. CH.CH2COOR
   |    |    |
CH3 CH3 CH3
when injected into sarcoid patients. In order to provide material for further biological studies, a new synthesis of thin acid has been carried out. The scheme involved the preparation of methyl 3-methyl-5-(2-thienyl) pentanoate (IX) and 3,6-dimethyltridecanoic acid (X) as intermediates.
(IX)
(CH2)2. CH. CH2COOMe
   |
CH3
(X)
CH3. (CH2)6. CH. (CH2)2. CH. CH2.COOH
   |    |
CH3 CH3
Methyl 3-methyl-5-(2-thienyl) pentanoate (IX) was prepared by a Friedel-crafts reaction of the acid chloride of methyl hydrogen β- methylglutarate with thiophen, followed by Huang-Minlon reduction of the product.
For the preparation of the intermediate (X), nonan-2-ol, obtained by a Grignard reaction between 1-bromoheptane and acetaldehyde, was oxidised to nonan-2-one. This was condensed with ethyl cyanoacetate to give ethyl 2-cyano-3-methyldec-2-enoate (XI) which, after hydrogenation,
(XI)
CH3 − (CH2)6 −   C =   C − COOEt
   |    |
CH3  CN
hydrolysis, decarboxylation, followed by hydrolysis of the resulting nitrile, gave crude 3~methyldecanoic acid. Lithium aluminium hydride reduction of the ester of this acid gave crude 3-methyldecan-l-ol; treatment of the latter with aqueous hydrogen bromide gave l-bromo-3-methyldecane. A. Grignard reaction between this compound and acetaldehyde, oxidation of the product, followed by a cyanoacetic ester condensation (as above) gave ethyl 2-cyano-3,6,-dimethyltridec-2-enoate (XII). The usual subsequent stages (as above) followed by chromatography
(XII)
CH3. (CH2)6. CH. (CH2)2. CH. = C.COOEt
   |    |  |
CH3 CH3 CN
gave 3,6-dimethyltridecanoic acid (X).
A Friedel-Crafts reaction or the acid chloride of 3,6-dimethyltridecanoic acid with the above thienyl-ester (IX), Huang-Minlon reduction of the resulting thienyl-keto-ester, desulphurisation with Raney nickel and purification of the methyl ester gave methyl 3,12,15 - trimethyldocosanoate (VIII, R=Me). A portion of this ester was hydrolised to the acid (VIII, R=H) and submitted for biological testing (Glaxo Laboratories Limited).
To obtain further information on the structural features necessary for biological activity, methyl 3,15- dimethyldocosanoate (XIII) was prepared by the following
(XIII)
CH3. (CH2)6. CH. (CH2)11. CH. CH2.COOMe
   |    |
CH3 CH3
procedure. 2-Iodothiophen was subjected to an Ullmann reaction to give 2,2-bithienyl13. Friedel-crafts reaction of the latter with the acid chloride of methyl hydrogen β-methylglutarate, Followed by Huang-Minlon reduction, gave methyl 3-methyl-5-(5-2,2-bithienyl) penitanoate (XIV). 2-Methylnonoic acid was prepared by
(XIV)
(CH2)2. CH. CH2.COOMe
   |    |
CH3 CH3
condensing l-bromoheptane with diethyl methylmalonate, followed by hydrolysis and decarboxylation. A Friedel-Crafts reaction of the acid chloride derived from this acid with the above bithienyl ester and Huang-Minlon reduction of the product, followed by esterification, gave the ester (XV).
(XV)
CH3 (CH2)6. CH. CH2 (CH2)2. CH. CH2.COOMe
   |    |
CH3 CH3
Raney nickel desulphurisation of this bithienyl ester gave methyl 3,15-dimethyldocosanoate (XIII).