The attenuated strain of Mycobacterium bovis, Bacille Calmette-Guérin (BCG), used worldwide to prevent tuberculosis and leprosy is also clinically used as an immunotherapeutic agent against superficial bladder cancer. An anti-tumor polysaccharide has been isolated from the boiling-water extract of the Tice substrain of BCG and tentatively characterized as consisting primarily of repeating units of 6-linked-glucosyl residues. M. tuberculosis and other mycobacterial species produce a glycogen-like α-glucan, composed of repeating units of 4-linked-glucosyl residues substituted at some positions 6 by short oligoglucosyl units, that also exhibits an anti-tumor activity. Therefore, the impression prevails that mycobacteria synthesize different types of anti-neoplastic glucans or, alternatively, the BCG substrains are singular in producing a unique type of glucan that may confer to them their immunotherapeutic property. The present study addresses this question through the comparative analysis of α-glucans purified from the extracellular materials and boiling-water extracts of three vaccine substrains. The polysaccharides were purified and their structural features were established by mono-and two-dimensional NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the enzymatic and chemical degradation products of the purified compounds. The glucans isolated by the two methods from the three substrains of BCG were shown to exhibit identical structural features shared with the glycogen-like α-glucan of M. tuberculosis and other mycobacteria. Incidentally, we observed an occasional release of dextrans from Sephadex columns that may explain the reported occurrence of 6-substituted-α-glucans in mycobacteria.

structure, NMR spectroscopy, Mycobacterium, vaccine, glucan, tuberculosis

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
6	aDGlcp	99.8	73.5	75.5	71.8	72.3	67.9
4	aDGlcp	100.1	72.0	73.4	70.0	?	61.3
	aDGlcp	98.5	?	?	?	?	?


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
6	aDGlcp	5.05	3.65	3.80	3.57	3.98	3.85-4.04
4	aDGlcp	5.38	3.62	3.72	3.44	3.73	3.80-3.90
	aDGlcp	4.99	3.64	4.03	3.66	3.88	?


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
6	aDGlcp	99.8/5.05	73.5/3.65	75.5/3.80	71.8/3.57	72.3/3.98	67.9/3.85-4.04
4	aDGlcp	100.1/5.38	72.0/3.62	73.4/3.72	70.0/3.44	?/3.73	61.3/3.80-3.90
	aDGlcp	98.5/4.99	?/3.64	?/4.03	?/3.66	?/3.88	?/?

The spectrum also has 6 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]OC[C@H]1O[C@H](OC[C@H]2O[C@H]([*])[C@H](O)[C@@H](O)[C@@H]2O[C@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@@H](O)[C@@H]1O

486.423 g/mol (C₁₈H₃₀R₂O₁₅, R = next and previous repeats, not counted in mol weight)

The attenuated strain of Mycobacterium bovis, Bacille Calmette-Guérin (BCG), used worldwide to prevent tuberculosis and leprosy is also clinically used as an immunotherapeutic agent against superficial bladder cancer. An anti-tumor polysaccharide has been isolated from the boiling-water extract of the Tice substrain of BCG and tentatively characterized as consisting primarily of repeating units of 6-linked-glucosyl residues. M. tuberculosis and other mycobacterial species produce a glycogen-like α-glucan, composed of repeating units of 4-linked-glucosyl residues substituted at some positions 6 by short oligoglucosyl units, that also exhibits an anti-tumor activity. Therefore, the impression prevails that mycobacteria synthesize different types of anti-neoplastic glucans or, alternatively, the BCG substrains are singular in producing a unique type of glucan that may confer to them their immunotherapeutic property. The present study addresses this question through the comparative analysis of α-glucans purified from the extracellular materials and boiling-water extracts of three vaccine substrains. The polysaccharides were purified and their structural features were established by mono-and two-dimensional NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the enzymatic and chemical degradation products of the purified compounds. The glucans isolated by the two methods from the three substrains of BCG were shown to exhibit identical structural features shared with the glycogen-like α-glucan of M. tuberculosis and other mycobacteria. Incidentally, we observed an occasional release of dextrans from Sephadex columns that may explain the reported occurrence of 6-substituted-α-glucans in mycobacteria.

structure, NMR spectroscopy, Mycobacterium, vaccine, glucan, tuberculosis

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,4,4,4,4	aDGlcp	100.1	72.0	73.8	78.0	72.0	61.3
4,4,4,4	aDGlcp	100.1	72.0	73.8	78.0	72.0	61.3
4,4,4,6	aDGlcp	?	?	?	?	?	?
4,4,4	aDGlcp	98.5	?	?	?	?	?
4,4	aDGlcp	100.1	72.0	73.8	78.0	72.0	61.3
4	aDGlcp	100.1	72.0	73.8	78.0	72.0	61.3
	aDGlcp	100.1	72.0	73.8	78.0	72.0	61.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,4,4,4,4	aDGlcp	5.38	3.66	3.98	3.66	3.85	?
4,4,4,4	aDGlcp	5.38	3.66	3.98	3.66	3.85	?
4,4,4,6	aDGlcp	?	?	?	?	?	?
4,4,4	aDGlcp	4.99	3.64	4.03	3.66	3.88	?
4,4	aDGlcp	5.38	3.66	3.98	3.66	3.85	?
4	aDGlcp	5.38	3.66	3.98	3.66	3.85	?
	aDGlcp	5.38	3.66	3.98	3.66	3.85	?


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,4,4,4,4	aDGlcp	100.1/5.38	72.0/3.66	73.8/3.98	78.0/3.66	72.0/3.85	61.3/?
4,4,4,4	aDGlcp	100.1/5.38	72.0/3.66	73.8/3.98	78.0/3.66	72.0/3.85	61.3/?
4,4,4,6	aDGlcp	?/?	?/?	?/?	?/?	?/?	?/?
4,4,4	aDGlcp	98.5/4.99	?/3.64	?/4.03	?/3.66	?/3.88	?/?
4,4	aDGlcp	100.1/5.38	72.0/3.66	73.8/3.98	78.0/3.66	72.0/3.85	61.3/?
4	aDGlcp	100.1/5.38	72.0/3.66	73.8/3.98	78.0/3.66	72.0/3.85	61.3/?
	aDGlcp	100.1/5.38	72.0/3.66	73.8/3.98	78.0/3.66	72.0/3.85	61.3/?

The spectrum also has 11 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@@H](CO)O[C@H](O[C@@H]3[C@@H](CO[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O)O[C@H](O[C@@H]4[C@@H](CO)O[C@H](O[C@@H]5[C@@H](CO)O[C@H](O[C@@H]6[C@@H](CO)O[C@H]([*])[C@H](O)[C@H]6O)[C@H](O)[C@H]5O)[C@H](O)[C@H]4O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O

1134.987 g/mol (C₄₂H₇₀R₂O₃₅, R = next and previous repeats, not counted in mol weight)

The attenuated strain of Mycobacterium bovis, Bacille Calmette-Guérin (BCG), used worldwide to prevent tuberculosis and leprosy is also clinically used as an immunotherapeutic agent against superficial bladder cancer. An anti-tumor polysaccharide has been isolated from the boiling-water extract of the Tice substrain of BCG and tentatively characterized as consisting primarily of repeating units of 6-linked-glucosyl residues. M. tuberculosis and other mycobacterial species produce a glycogen-like α-glucan, composed of repeating units of 4-linked-glucosyl residues substituted at some positions 6 by short oligoglucosyl units, that also exhibits an anti-tumor activity. Therefore, the impression prevails that mycobacteria synthesize different types of anti-neoplastic glucans or, alternatively, the BCG substrains are singular in producing a unique type of glucan that may confer to them their immunotherapeutic property. The present study addresses this question through the comparative analysis of α-glucans purified from the extracellular materials and boiling-water extracts of three vaccine substrains. The polysaccharides were purified and their structural features were established by mono-and two-dimensional NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the enzymatic and chemical degradation products of the purified compounds. The glucans isolated by the two methods from the three substrains of BCG were shown to exhibit identical structural features shared with the glycogen-like α-glucan of M. tuberculosis and other mycobacteria. Incidentally, we observed an occasional release of dextrans from Sephadex columns that may explain the reported occurrence of 6-substituted-α-glucans in mycobacteria.

structure, NMR spectroscopy, Mycobacterium, vaccine, glucan, tuberculosis

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,4,4,4,4	aDGlcp	100.1	72.0	73.8	78.0	72.0	61.3
4,4,4,4	aDGlcp	100.1	72.0	73.8	78.0	72.0	61.3
4,4,4,6,6	aDGlcp	?	?	?	?	?	?
4,4,4,6	aDGlcp	?	?	?	?	?	?
4,4,4	aDGlcp	98.5	?	?	?	?	?
4,4	aDGlcp	100.1	72.0	73.8	78.0	72.0	61.3
4	aDGlcp	100.1	72.0	73.8	78.0	72.0	61.3
	aDGlcp	100.1	72.0	73.8	78.0	72.0	61.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,4,4,4,4	aDGlcp	5.38	3.66	3.98	3.66	3.85	?
4,4,4,4	aDGlcp	5.38	3.66	3.98	3.66	3.85	?
4,4,4,6,6	aDGlcp	?	?	?	?	?	?
4,4,4,6	aDGlcp	?	?	?	?	?	?
4,4,4	aDGlcp	4.99	3.64	4.03	3.66	3.88	?
4,4	aDGlcp	5.38	3.66	3.98	3.66	3.85	?
4	aDGlcp	5.38	3.66	3.98	3.66	3.85	?
	aDGlcp	5.38	3.66	3.98	3.66	3.85	?


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,4,4,4,4	aDGlcp	100.1/5.38	72.0/3.66	73.8/3.98	78.0/3.66	72.0/3.85	61.3/?
4,4,4,4	aDGlcp	100.1/5.38	72.0/3.66	73.8/3.98	78.0/3.66	72.0/3.85	61.3/?
4,4,4,6,6	aDGlcp	?/?	?/?	?/?	?/?	?/?	?/?
4,4,4,6	aDGlcp	?/?	?/?	?/?	?/?	?/?	?/?
4,4,4	aDGlcp	98.5/4.99	?/3.64	?/4.03	?/3.66	?/3.88	?/?
4,4	aDGlcp	100.1/5.38	72.0/3.66	73.8/3.98	78.0/3.66	72.0/3.85	61.3/?
4	aDGlcp	100.1/5.38	72.0/3.66	73.8/3.98	78.0/3.66	72.0/3.85	61.3/?
	aDGlcp	100.1/5.38	72.0/3.66	73.8/3.98	78.0/3.66	72.0/3.85	61.3/?

The spectrum also has 17 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@@H](CO)O[C@H](O[C@@H]3[C@@H](CO[C@H]4O[C@H](CO[C@H]5O[C@H](CO)[C@@H](O)[C@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)O[C@H](O[C@@H]4[C@@H](CO)O[C@H](O[C@@H]5[C@@H](CO)O[C@H](O[C@@H]6[C@@H](CO)O[C@H]([*])[C@H](O)[C@H]6O)[C@H](O)[C@H]5O)[C@H](O)[C@H]4O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O

1297.128 g/mol (C₄₈H₈₀R₂O₄₀, R = next and previous repeats, not counted in mol weight)