The pioneering work of Stenhagen and collaborators [13–15]
using electron-impact (EI) MS coupled to gas chromatography
(GC) remains the reference in the field of characterization of volatile FAME, by allowing the determination of the precise structure
of specific multi-methyl-branched FA of Mycobacterium tuberculosis, resulting from the hydrolysis of PDIM, SGL, or polyphthienoyl
trehalose (PPT), the so-called mycocerosic, phthioceranic, or phtienoic acids (Figs. 1 and 2), even in a mixture.
Applied to MA, the method has been very informative for fine
structure characterization, such as the localization of functional
groups on the meromycolate chain. Both the α-alkyl and the
mero-aldehyde chains are easily identified (Fig. 1): characteristic
peaks corresponding to pyrolysis fragments (C 22 , C 24 , or C 26 ), due
to the temperature of volatilization around 300
C used in the
method, and mero-aldehydes are observed (Fig. 1). MAME containing one or more cyclopropane rings and a function generating
an aldehyde by pyrolysis give specific cleavages of the meromycolic
chain at the level of the rings. From these fragments, the number of
methylenes m 1 , m 2 , and m 3 (Fig. 1) can be deduced, the intensity
CH 3 -(CH 2 ) m1 -CH-CH-(CH 2 ) m2 -CH-CH-(CH 2 ) m3 -CH-CH-COOH
CH 2
CH 2
(CH 2 ) m4
OH
CH 3
distal
proximal
alpha-mycolic acid
CH 3 -(CH 2 ) m4 -CH 2 -COOH
acid
CH 3 -(CH 2 ) 19 -C*H-CH 2 -C*H-CH 2 -C*H-CH 2 -C*H- COOH
CH 3
CH 3
CH 3
CH 3
CH 3 -(CH 2 ) 17 -C*H-CH 2 -C*H-CH=C-COOH
CH 3
CH 3
CH 3 -(CH 2 ) m1 -CH-CH-(CH 2 ) m2 -CH-CH-(CH 2 ) m3 CHO
CH 2
CH 2
CH 3
meroaldehyde
phthienoic acid P27
mycocerosic acid M32
Fig. 1 Structures of representative fatty acids of M. tuberculosis. Mycocerosic or mycolipanolic acid (M32).
Phthienoic acid (P27), also named C27 mycolipenic acid, characteristic of virulent strains. * indicate chiral
centers. P27 and M32 refer to the GC/MS profile (Fig. 2). Dicyclopropanic mycolic acid (α-mycolic acid, type I)
of M. tuberculosis. m 1 , m 2 , m 3 , m 4 are the methylene numbers. The arrow indicates the pyrolytical cleavage
generating the ester and mero-aldehyde
Lipid and Lipoarabinomannan
111
using electron-impact (EI) MS coupled to gas chromatography
(GC) remains the reference in the field of characterization of volatile FAME, by allowing the determination of the precise structure
of specific multi-methyl-branched FA of Mycobacterium tuberculosis, resulting from the hydrolysis of PDIM, SGL, or polyphthienoyl
trehalose (PPT), the so-called mycocerosic, phthioceranic, or phtienoic acids (Figs. 1 and 2), even in a mixture.
Applied to MA, the method has been very informative for fine
structure characterization, such as the localization of functional
groups on the meromycolate chain. Both the α-alkyl and the
mero-aldehyde chains are easily identified (Fig. 1): characteristic
peaks corresponding to pyrolysis fragments (C 22 , C 24 , or C 26 ), due
to the temperature of volatilization around 300
C used in the
method, and mero-aldehydes are observed (Fig. 1). MAME containing one or more cyclopropane rings and a function generating
an aldehyde by pyrolysis give specific cleavages of the meromycolic
chain at the level of the rings. From these fragments, the number of
methylenes m 1 , m 2 , and m 3 (Fig. 1) can be deduced, the intensity
CH 3 -(CH 2 ) m1 -CH-CH-(CH 2 ) m2 -CH-CH-(CH 2 ) m3 -CH-CH-COOH
CH 2
CH 2
(CH 2 ) m4
OH
CH 3
distal
proximal
alpha-mycolic acid
CH 3 -(CH 2 ) m4 -CH 2 -COOH
acid
CH 3 -(CH 2 ) 19 -C*H-CH 2 -C*H-CH 2 -C*H-CH 2 -C*H- COOH
CH 3
CH 3
CH 3
CH 3
CH 3 -(CH 2 ) 17 -C*H-CH 2 -C*H-CH=C-COOH
CH 3
CH 3
CH 3 -(CH 2 ) m1 -CH-CH-(CH 2 ) m2 -CH-CH-(CH 2 ) m3 CHO
CH 2
CH 2
CH 3
meroaldehyde
phthienoic acid P27
mycocerosic acid M32
Fig. 1 Structures of representative fatty acids of M. tuberculosis. Mycocerosic or mycolipanolic acid (M32).
Phthienoic acid (P27), also named C27 mycolipenic acid, characteristic of virulent strains. * indicate chiral
centers. P27 and M32 refer to the GC/MS profile (Fig. 2). Dicyclopropanic mycolic acid (α-mycolic acid, type I)
of M. tuberculosis. m 1 , m 2 , m 3 , m 4 are the methylene numbers. The arrow indicates the pyrolytical cleavage
generating the ester and mero-aldehyde
Lipid and Lipoarabinomannan
111