3.21 Purification of
Lipoarabinomannan
(LAM)
LAM is a water-soluble molecule and is not solubilized by organic
solvents used to extract lipids from cells. Obtaining LAM thus
requires a subsequent aqueous extraction of delipidated cells.
LAM is a macroamphiphilic molecule that shows a high intrinsic
molecular heterogeneity. Moreover, the mycobacterial cell envelope contains structurally related molecules such as the lipomannan
(LM), the PIM or the arabinomannan (AM), and mannan. Obtaining homogenous fractions of LAM thus requires these critical steps
following the aqueous extraction from delipidated cells: (1) elimination of protein and nucleic acid contaminants, (2) separation of
Table 2
Chemical shifts of
1
H resonance for specific structural groups of MAME
Group
δ H (ppm)
Mycolic unit
(Hallmark of mycolic
acids)
2.43 (1H, m) proton of carbon 2 bearing branch
3.58 (1H broad) proton of carbon 3 bearing hydroxyl
Cyclopropane (CP) cis
– 0.34 (1H ddd) highly shielded cyclopropane ring proton
0.56 (1H ddd) methylene of cyclopropane ring proton
0.64 (2H, m) methine of CP ring protons
Cyclopropane (CP) trans 0.09/0.13/0.21 (3H, m) three trans CP ring protons
Double bonds cis
5.36 (2H, t) ethylenic protons
2.05 (4H, m) protons of the two methylene adjacent to the double bond
Double bonds trans
5.22 (1H, dd) and 5.35 (1H, dt) for trans double bond protons
2.03 (1H, m) methine group adjacent to the trans double bond
1.99 (2H, dt) protons for the methylene adjacent to the double bond
Ketone
2.35 (2H) protons of the methylene adjacent to the ketone carbonyl
2.46 (1H m) methine group adjacent to the keto carbonyl
Methoxyl (mid chain)
3.35 (3H s) methoxy-methyl protons
2.95 (1H broad) proton on the methoxy bearing carbon
0.84 (3H d) protons methyl branch adjacent to the methoxyl group
Hydroxyl
3. 43 (1H broad) proton on the carbon bearing the additional hydroxyl
group
Epoxyde cis
2.90 protons of the cis epoxyde ring
Epoxyde trans
2.71 (broad) protons of the trans epoxyde ring
1.1 (doublet 3H) protons methy branch adjacent to the epoxyde ring
Wax-ester
a
2.27 (2H t) methylene adjacent to the mid-chain ester
4.90 (m) proton methylene
1. 19 protons of methyl branch (3H)
ωÀ1 methoxyl
b
3.35 (3H, s) methoxy-methyl proton
1.1 (3H, d) protons of methyl group
Data in parentheses are coupling constants in hertz: s, singlet; d, doublet; m, multiplet.
a
Intact wax-ester is observed only in mild alkaline conditions.
b
ωÀ1 methoxy-MA has a more polar migration on TLC than mid-chain methoxy-MAME on TLC
Lipid and Lipoarabinomannan
137
Lipoarabinomannan
(LAM)
LAM is a water-soluble molecule and is not solubilized by organic
solvents used to extract lipids from cells. Obtaining LAM thus
requires a subsequent aqueous extraction of delipidated cells.
LAM is a macroamphiphilic molecule that shows a high intrinsic
molecular heterogeneity. Moreover, the mycobacterial cell envelope contains structurally related molecules such as the lipomannan
(LM), the PIM or the arabinomannan (AM), and mannan. Obtaining homogenous fractions of LAM thus requires these critical steps
following the aqueous extraction from delipidated cells: (1) elimination of protein and nucleic acid contaminants, (2) separation of
Table 2
Chemical shifts of
1
H resonance for specific structural groups of MAME
Group
δ H (ppm)
Mycolic unit
(Hallmark of mycolic
acids)
2.43 (1H, m) proton of carbon 2 bearing branch
3.58 (1H broad) proton of carbon 3 bearing hydroxyl
Cyclopropane (CP) cis
– 0.34 (1H ddd) highly shielded cyclopropane ring proton
0.56 (1H ddd) methylene of cyclopropane ring proton
0.64 (2H, m) methine of CP ring protons
Cyclopropane (CP) trans 0.09/0.13/0.21 (3H, m) three trans CP ring protons
Double bonds cis
5.36 (2H, t) ethylenic protons
2.05 (4H, m) protons of the two methylene adjacent to the double bond
Double bonds trans
5.22 (1H, dd) and 5.35 (1H, dt) for trans double bond protons
2.03 (1H, m) methine group adjacent to the trans double bond
1.99 (2H, dt) protons for the methylene adjacent to the double bond
Ketone
2.35 (2H) protons of the methylene adjacent to the ketone carbonyl
2.46 (1H m) methine group adjacent to the keto carbonyl
Methoxyl (mid chain)
3.35 (3H s) methoxy-methyl protons
2.95 (1H broad) proton on the methoxy bearing carbon
0.84 (3H d) protons methyl branch adjacent to the methoxyl group
Hydroxyl
3. 43 (1H broad) proton on the carbon bearing the additional hydroxyl
group
Epoxyde cis
2.90 protons of the cis epoxyde ring
Epoxyde trans
2.71 (broad) protons of the trans epoxyde ring
1.1 (doublet 3H) protons methy branch adjacent to the epoxyde ring
Wax-ester
a
2.27 (2H t) methylene adjacent to the mid-chain ester
4.90 (m) proton methylene
1. 19 protons of methyl branch (3H)
ωÀ1 methoxyl
b
3.35 (3H, s) methoxy-methyl proton
1.1 (3H, d) protons of methyl group
Data in parentheses are coupling constants in hertz: s, singlet; d, doublet; m, multiplet.
a
Intact wax-ester is observed only in mild alkaline conditions.
b
ωÀ1 methoxy-MA has a more polar migration on TLC than mid-chain methoxy-MAME on TLC
Lipid and Lipoarabinomannan
137