3.20
1 H-NMR
of MAME
NMR analyses easily allow the identification of the structural
groups typifying mycolic acids such as the cyclopropanes, double
bonds, ketone, epoxy, hydroxyl, mid-chain wax ester.
1 H-NMR is
useful to identify most characteristic signals and to quantify them.
1 H1 H-COSY-NMR spectra may help to distinguish characteristic
spin system “off-diagonal,” particularly in mixture samples.
1 H-NMR spectra of purified MAME (see Subheading 3.14) were
recorded at 298 K using a 600-MHz Bruker Avance III spectrometer equipped with a TCI cryoprobe. Chemical shift values
(in ppm) are relative to the internal CHCl 3 at 7.26 ppm.
1. Dissolve roughly 1 mg of purified MAME in CDCl 3
(99.9% D).
2. Transfer MAME in an NMR 5-mm-diameter glass tube with a
Pasteur pipette.
3. Temperature can vary from 293 to 298 K.
4. Bruker zg and cosygpmfqf are used to analyze mycolates,
respectively, by 1D1 H- or 2D1 H1 H-COSY-NMR. If integration of 1D1
H-NMR spectra is required, set the relaxation
delay at 3 s.
5. Data analysis: Integration signals of CH 3 are related to the
methoxy signal of ester at 3.70 ppm, which is calibrated for
3H. Table 2 indicates the proton resonance signals for specific
structural groups of MAME with diagnostic values (δ ppm)
observable on
1 H-NMR spectra.
Interestingly, two methoxy-mycolic acids have been characterized in mycobacteria: the mid-chain methoxy-MA (i.e., methoxymycolate of M. tuberculosis) and the ωÀ1 methoxy-MA (M. alvei).
The position of the methoxyl group in the long chain is easily
elucidated by the examination of the
1 NMR spectra (Table 2).
Notably, if the same resonance (δ ¼ 3.35 ppm) is observed for
both methoxy-MAs, the presence of a signal (δ ¼ 1.1 ppm) in
ωÀ1 methoxy-MA is indicative of a methyl group attached to the
same tertiary carbon [55]. This allows the location of the methoxyl
group at the subterminal carbon of the long-chain and explains the
different TLC migrations of this MA (migration near ω-carboxylic
MA) compared to mid-chain methoxy-MA (near keto-MA region).
Moreover, the distinction of the configuration cis or trans of epoxy
groups is easily differentiated by resonance at 2.90 ppm for cis and
2.71 for the trans epoxy ring [11, 12].
When overlapped signals are observed, it is necessary and possible to identify the off-diagonal spin systems on
2D1
H1 H-COSY-NMR spectra, as indicated in Table 3.
For more data, refer to the careful analyses of individual mycolic acids in representative mycobacteria by NMR [56].
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Marie-Antoinette Lane ´ elle et al.
1 H-NMR
of MAME
NMR analyses easily allow the identification of the structural
groups typifying mycolic acids such as the cyclopropanes, double
bonds, ketone, epoxy, hydroxyl, mid-chain wax ester.
1 H-NMR is
useful to identify most characteristic signals and to quantify them.
1 H1 H-COSY-NMR spectra may help to distinguish characteristic
spin system “off-diagonal,” particularly in mixture samples.
1 H-NMR spectra of purified MAME (see Subheading 3.14) were
recorded at 298 K using a 600-MHz Bruker Avance III spectrometer equipped with a TCI cryoprobe. Chemical shift values
(in ppm) are relative to the internal CHCl 3 at 7.26 ppm.
1. Dissolve roughly 1 mg of purified MAME in CDCl 3
(99.9% D).
2. Transfer MAME in an NMR 5-mm-diameter glass tube with a
Pasteur pipette.
3. Temperature can vary from 293 to 298 K.
4. Bruker zg and cosygpmfqf are used to analyze mycolates,
respectively, by 1D1 H- or 2D1 H1 H-COSY-NMR. If integration of 1D1
H-NMR spectra is required, set the relaxation
delay at 3 s.
5. Data analysis: Integration signals of CH 3 are related to the
methoxy signal of ester at 3.70 ppm, which is calibrated for
3H. Table 2 indicates the proton resonance signals for specific
structural groups of MAME with diagnostic values (δ ppm)
observable on
1 H-NMR spectra.
Interestingly, two methoxy-mycolic acids have been characterized in mycobacteria: the mid-chain methoxy-MA (i.e., methoxymycolate of M. tuberculosis) and the ωÀ1 methoxy-MA (M. alvei).
The position of the methoxyl group in the long chain is easily
elucidated by the examination of the
1 NMR spectra (Table 2).
Notably, if the same resonance (δ ¼ 3.35 ppm) is observed for
both methoxy-MAs, the presence of a signal (δ ¼ 1.1 ppm) in
ωÀ1 methoxy-MA is indicative of a methyl group attached to the
same tertiary carbon [55]. This allows the location of the methoxyl
group at the subterminal carbon of the long-chain and explains the
different TLC migrations of this MA (migration near ω-carboxylic
MA) compared to mid-chain methoxy-MA (near keto-MA region).
Moreover, the distinction of the configuration cis or trans of epoxy
groups is easily differentiated by resonance at 2.90 ppm for cis and
2.71 for the trans epoxy ring [11, 12].
When overlapped signals are observed, it is necessary and possible to identify the off-diagonal spin systems on
2D1
H1 H-COSY-NMR spectra, as indicated in Table 3.
For more data, refer to the careful analyses of individual mycolic acids in representative mycobacteria by NMR [56].
136
Marie-Antoinette Lane ´ elle et al.