(E) double bond is less retained than the corresponding cis
(Z) double bond. As binding force is weak, it is easily broken
followed by the extraction of the compounds from the silica gel
and, accordingly, is well adapted to purification by preparative
TLC. Alternatively, step 2 can be substituted by immersion of
the plates in the 10% solution of NO 3 Ag for 30 min.
18. Esters obtained after periodate-permanganate oxidation are
identified by their GC/MS fragmentation pattern:
m/z 74 McLafferty rearrangement for linear monoesters,
results from cis distal double-bond cleavage.
m/z 88 for α-methyl branch monoesters, resulting from the
cleavage of trans distal double bond.
m/z 98 for diesters in the case of diethylenic MA.
The molecular ion weight of monoesters being observed at
M-31 (see Fig. 2 and Subheading 3.5 for GC/MS).
19. The contribution to the molecular rotation of the chiral centers
2 and 3 bearing hydroxyl and carboxyl groups (mycolic unit) is
known: Molecular rotation [M] D ¼ +30
for corynomycolic
acid. Consequently, all mycolic acids exhibit a positive molecular rotation. However, additional chiral centers present in the
meromycolic chain could interfere: this exception is illustrated
by epoxy mycolic acids where the carbon bearing the methyl
branch adjacent to the oxirame brings a negative contribution
to the molecular rotation leading to a [M] D ¼ À7
.
20. MALDI-TOF/TOF-MS and MS/MS analyses are conducted
in the positive ionization and reflectron mode by accumulating
10 spectra of 250 laser shots, using the 5800 MALDI TOF/TOF Analyser (Applied Biosystems/Absciex) equipped with an
Nd:Yag laser (349-nm wavelength). For MS and MS/MS data
acquisitions, uniform, continuous, and random stage motion
was selected at a fixed laser intensity of 4000
(instrument-specific units) and 400 Hz pulse rate and 6000
(instrument-specific units) and 1000 Hz, respectively. For
MS/MS data acquisition, the fragmentation of selected precursor ions was performed at collision energy of 1 kV.
21. An alternative method for the analysis of glycosyl composition
is through the use of capillary electrophoresis monitored by
laser-induced fluorescence, which is more sensitive than GC
[64]. It is based on the tagging of monosaccharides by reductive amination using 1-aminopyrene-3,6,8-trisulfonate (APTS)
that can be detected with high sensitivity by LIF. Hydrolyze
LAM (1 μg) in the presence of mannoheptose (1 nmol) as an
internal standard using 200 μL of 2 M TFA at 110
C for 2 h.
Allow the solution to cooldown and evaporate to dryness.
Incubate with 0.4 μL of 0.2 M APTS in 15% acetic acid and
Lipid and Lipoarabinomannan
147
(Z) double bond. As binding force is weak, it is easily broken
followed by the extraction of the compounds from the silica gel
and, accordingly, is well adapted to purification by preparative
TLC. Alternatively, step 2 can be substituted by immersion of
the plates in the 10% solution of NO 3 Ag for 30 min.
18. Esters obtained after periodate-permanganate oxidation are
identified by their GC/MS fragmentation pattern:
m/z 74 McLafferty rearrangement for linear monoesters,
results from cis distal double-bond cleavage.
m/z 88 for α-methyl branch monoesters, resulting from the
cleavage of trans distal double bond.
m/z 98 for diesters in the case of diethylenic MA.
The molecular ion weight of monoesters being observed at
M-31 (see Fig. 2 and Subheading 3.5 for GC/MS).
19. The contribution to the molecular rotation of the chiral centers
2 and 3 bearing hydroxyl and carboxyl groups (mycolic unit) is
known: Molecular rotation [M] D ¼ +30
for corynomycolic
acid. Consequently, all mycolic acids exhibit a positive molecular rotation. However, additional chiral centers present in the
meromycolic chain could interfere: this exception is illustrated
by epoxy mycolic acids where the carbon bearing the methyl
branch adjacent to the oxirame brings a negative contribution
to the molecular rotation leading to a [M] D ¼ À7
.
20. MALDI-TOF/TOF-MS and MS/MS analyses are conducted
in the positive ionization and reflectron mode by accumulating
10 spectra of 250 laser shots, using the 5800 MALDI TOF/TOF Analyser (Applied Biosystems/Absciex) equipped with an
Nd:Yag laser (349-nm wavelength). For MS and MS/MS data
acquisitions, uniform, continuous, and random stage motion
was selected at a fixed laser intensity of 4000
(instrument-specific units) and 400 Hz pulse rate and 6000
(instrument-specific units) and 1000 Hz, respectively. For
MS/MS data acquisition, the fragmentation of selected precursor ions was performed at collision energy of 1 kV.
21. An alternative method for the analysis of glycosyl composition
is through the use of capillary electrophoresis monitored by
laser-induced fluorescence, which is more sensitive than GC
[64]. It is based on the tagging of monosaccharides by reductive amination using 1-aminopyrene-3,6,8-trisulfonate (APTS)
that can be detected with high sensitivity by LIF. Hydrolyze
LAM (1 μg) in the presence of mannoheptose (1 nmol) as an
internal standard using 200 μL of 2 M TFA at 110
C for 2 h.
Allow the solution to cooldown and evaporate to dryness.
Incubate with 0.4 μL of 0.2 M APTS in 15% acetic acid and
Lipid and Lipoarabinomannan
147