Isolation and identification of poly-α-(1→4)-linked 3-O-methyl-D-mannopyranose from a hot-water extract of Mycobacterium vaccae

Article abstract of DOI:10.1016/S0008-6215(99)00248-7

A polysaccharide around 3.6 kDa has been identified as the major carbohydrate moiety of a antineoplastic protein-polysaccharide complex (PS4A) obtained by boiling intact cells of Mycobacterium vaccae in water. ¹H and ¹³C NMR spectra of this polysaccharide suggested it was a highly homogeneous polymer composed substantially of one monomer, probably an α-linked O-methylated mannose. Comparison of the COSY spectra of the original and acetylated polymer indicated that the glycosidic linkage and the methyl ether were interchangeable, at O-3 and O-4. Further study demonstrated that the benzyolated hydrolysate of the polymer was 1,2,4,6-tetra-O-benzoyl-3-O-methyl-β-mannopyranose. The hydrolysate was 3-O-methyl-α,β-mannopyranose and the polymer was therefore poly-α-(1→4)-linked 3-O-methyl-D-mannopyranose. This conclusion was further confirmed with an authentic sample of the monomer, which had spectral data identical to those of the hydrolyzate and co-eluted from an ion-exchange HPLC with the major sugar in the hydrolysate. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00248-7

www.elsevier.nl/locate/carres
Carbohydrate Research 324 (2000) 38–44
Isolation and identification of poly-a-(1“4)-linked
3-O-methyl-
D
-mannopyranose from a hot-water extract
of Mycobacterium 6accae
Xin-Xin Tian a, Afeng Li a, Isabelle V. Farrugia a, Xuesheng Mo b, David Crich b,
Michael J. Groves a,*
a Institute for Tuberculosis Research (M/C 964), Uni6ersity of Illinois at Chicago, Chicago Technology Park,
2201 W. Campbell Park Dr., c217, Chicago, IL 60612, USA
b Department of Chemistry, Uni6ersity of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607, USA
Received 23 March 1999; accepted 1 September 1999
Abstract
A polysaccharide around 3.6 kDa has been identified as the major carbohydrate moiety of a antineoplastic
1
protein–polysaccharide complex (PS4A) obtained by boiling intact cells of Mycobacterium 6accae in water. H and
13C NMR spectra of this polysaccharide suggested it was a highly homogeneous polymer composed substantially of
one monomer, probably an a-linked O-methylated mannose. Comparison of the COSY spectra of the original and
acetylated polymer indicated that the glycosidic linkage and the methyl ether were interchangeable, at O-3 and O-4.
Further study demonstrated that the benzyolated hydrolysate of the polymer was 1,2,4,6-tetra-O-benzoyl-3-O-
methyl-b-mannopyranose. The hydrolysate was 3-O-methyl-a,b-mannopyranose and the polymer was therefore
poly-a-(1“4)-linked 3-O-methyl-D-mannopyranose. This conclusion was further confirmed with an authentic sample
of the monomer, which had spectral data identical to those of the hydrolyzate and co-eluted from an ion-exchange
HPLC with the major sugar in the hydrolysate. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Protein–polysaccharide complex; Poly-a-(1“4)-3-O-methyl mannopyranose; Synthesis; Mycobacterium 6accae
bo6is BCG Tice® substrain have been found to
be associated with the extracellular integu-
ment, which is probably secreted by the bacte-
rial cells in response to environmental stress,
such as a change of oxygen tension [8]. These
materials can also be removed by digestion
with non-specific protease [9] and urea dissoci-
ation [10].
Mycobacterium 6accae, an avirulent and
fast-growing mycobacterial species, has been
found to be a potent immunostimulant. A
suspension of heat-killed M. 6accae is cur-
rently being clinically tested as an adjuvant to
tuberculosis chemotherapy and cancer im-
munotherapy [11–13]. We had previously
1. Introduction
The outmost cellular components of my-
cobacterial species have been found to consist
mainly of polysaccharides and proteins [1,2].
Several investigators have confirmed that
polysaccharides isolated from the surface of
the mycobacterial cells by water extraction
have antineoplastic activity [3–7]. The
polysaccharides isolated from Mycobacterium
* Corresponding author. Tel.: +1-312-563-0153; fax: +1-
312-563-9034.
E-mail address: groves@uic.edu (M.J. Groves)
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00248-7

X.-X. Tian et al. / Carbohydrate Research 324 (2000) 38–44
39
demonstrated that the water-soluble exocellu-
lar proteoglycans extracted by boiling M. 6ac-
cae suspensions (PS4) also had significant
antineoplastic activity [14,15]. In this report,
we identify the major carbohydrate moiety of
these antineoplastic proteoglycans as a-(1“
4)-linked 3-O-methylated mannose of molecu-
lar weight about 3.6 kDa.
buffer were digested with pronase, a type XIV
protease from Streptomyces griseus (Sigma, St.
Louis, MO) at 37 °C for 24 h in order to
release the polysaccharides. The completion of
digestion was determined with the gel-filtra-
tion HPLC system. The digestion was stopped
by adjusting the pH to about 4.5 with 2%
AcOH, followed by a brief centrifugation us-
ing a microcentrifuge (Fisher Scientific, Pitts-
burgh, PA). The supernatant was recovered
for oligosaccharide purification.
2. Experimental
Purification and NMR spectroscopy of the
oligosaccharide.—Recovered
supernatants
Bacteria and materials.—M. 6accae (ATCC
c15483, batch c93-12), purchased from
ATCC (Rockville, MD), was cultivated in
tryptic soy broth (TSB, Gibco Laboratories,
Grand Island, NJ) at 37 °C for 5–7 days. The
bacteria were collected by centrifugation and
washed with double-distilled water. Washed
cells were lyophilized in order to obtain a dry
mass. All other reagents and materials were
purchased from Fisher Scientific (Fairlawn,
NJ), Sigma Chemical Company (St. Louis,
Mo) or as noted.
Extraction and purification of the protein–
polysaccharide complex.—Approximately 0.5
g of lyophilized cells were suspended in 500
mL of double-distilled water and boiled under
reflux for 2 h. When cool, the suspension was
passed through a 0.2-mm membrane filter and
concentrated to a volume of 12–14 mL at
65 °C using a Bu¨chi Rotavapor® (Brinkmann
Instruments, Westbury, NY). The concentrate
was dialyzed against distilled water overnight
using a 3.5 kDa molecular weight cut-off dial-
ysis membrane before lyophilization. This
crude extract was labeled as PS4A.
Monosaccharide analysis of the PS4A.—
PS4A samples (2.0 mg) were hydrolyzed in 1.0
mL of 2 M trifluoroacetic acid (TFA) by
autoclave at 121 °C for 1 h in a sealed ampule.
The autoclaved samples were dried under a
flowing air stream and were analyzed with a
DX500 high-performance anion-exchange
chromatography (HPAEC) system (Dionex
Corporation, Sunnyvale, CA) equipped with a
CarboPac PA-10 column and a pulsed amper-
ometric potentiometric detector (ED40) to de-
termine the monosaccharide composition [16].
Proteolysis of PS4A.—PS4A samples (10
mg/mL) dissolved in 0.1 M Tris–acetate
were fractionated by Bio-Gel P-10 size-exclu-
sion chromatography. The columns were equi-
librated and eluted with double-distilled
water. The eluent fractions were assayed using
the phenol–sulfuric acid method for saccha-
ride [17]. The major polysaccharide-containing
fractions were recovered and deuterium-ex-
changed by lyophilization once in deuterium
1
oxide (D2O, Aldrich, Milwaukee, WI). H
NMR spectra were determined using a Bruker
Avance 500 instrument operating at 500 MHz
using D2O as solvent. All chemical shifts were
calculated from the spectrometer lock signal.
Acetylation of the oligomer.—Purified
polysaccharide (10 mg) was dissolved in 1 mL
of a 1:1 pyridine–Ac2O mixture. The reaction
was conducted at room temperature (rt) for 16
h, and the reaction mixture was dried under
vacuum at rt.
Benzoylation of the hydrolysate of the
oligomer: 1,2,4,6,-tetra-O-benzoyl-3-O-methyl-
i-D
-mannopyranose
(5).—The
purified
oligomer (ꢀ4.0 mg) was subjected to hydrol-
ysis in 1.0 mL of 2 M TFA by autoclave at
121 °C for 1 h in a sealed ampule. The recov-
ered hydrolysate, containing an excess of
sodium bicarbonate, was dissolved in pyridine
(1 mL) and treated with benzoyl chloride (120
mL, 50 equiv) then stirred for 24 h at rt, after
which thin-layer chromatography (TLC)
showed completion. The major UV-active
product (0.8 mg) was isolated by chromatog-
raphy on preparative TLC plates (eluent: 2:1
CHCl3–hexane, 5 times), and was identified
from its spectral data as 1,2,4,6,-tetra-O-ben-
zyl-3-O-methyl-b-
D
-mannopyranose (5). [h]D20
1
−28.5° (c 1.3, CHCl3); H NMR (CDCl3, 300
MHz): l 3.46 (s, 3 H, O-Me), l 3.90 (dd, 1 H,

40
X.-X. Tian et al. / Carbohydrate Research 324 (2000) 38–44
J3,4 9.5 Hz, J2,3 3.2 Hz, H-3), l 4.22 (m, 5 H,
H-5), l 4.48 (dd, 1 H, Jgem 12.2, J5,6a 4.8 Hz,
H-6a), l 4,74 (dd, 1 H, Jgem 12.2, J5,6b 4.8 Hz,
H-6b), l 5.89 (t, 1 H, J3,4=J4,5 9.5 Hz, H-4),
l 6.05 (dd, 1 H, J2,3 3.2 Hz, J1,2 1.0 Hz, H-2),
l 6.27 (d, 1 H, J1,2 1.2 Hz, H-1), l 7.10–7.65
(m, 12 H), 7.92 (d, 2 H, J 6.9 Hz), l 8.06 (d,
2 H, J 7.0 Hz), 8.06 (d, 2 H, J 7.0 Hz), l 8.16
(d, 2 H, J 7.0 Hz); 13C NMR (CDCl3, 75
MHz): 58.2 (Me), 63.1 (C-6), 67.7 (C-2), 68.1
(C-4), 73.5 (C-5), 79.8 (C-3), 91.9 (1JCH 162.1
Hz, C-1), 128.7, 130.1, 130.3, 133.2, 133.7,
164.6, 165.5, 166.2, 166.4. Anal. Calcd for
C35H30O10·0.5 H2O: C, 67.85; H, 5.04. Found:
C, 67.87; H, 5.28.
ing material. The solvent was then evaporated
in vacuo to yield a residue, which was purified
by chromatography on silica gel (eluent: 2:1
CHCl3–MeOH) to give the title product 3 as a
foam (173 mg, 97%). [h]2D0 +177.3° (c 3.0,
1
CHCl3); H NMR (CDCl3, 300 MHz): l 3.45
(m, 4 H, OMe, H-3), l 3.70 (br. d, 1 H, Jgem
12 Hz, H-6a), l 3.95 (br.d, 1 H, Jgem 12 Hz,
H-6b), l 4.00–4.20 (m, 2 H, H-4 and H-5), l
4.30 (m, 1 H, H-2), l 4.60 (br. s, 1 H, OH), l
4.70 (m, 2 H, 2×OH), l 5.58 (br. s, 1 H,
H-1), l 7.20–7.35 (m, 3 H), l 7.40–7.50 (m, 2
H); 13C NMR (CDCl3, 75 MHz): 57.3 (Me),
61.2 (C-6), 65.1 (C-4), 68.9 (C-2), 73.6 (C-5),
81.7 (C-3), 88.2 (C-1), 127.7, 129.2, 131.8,
134.0. Anal. Calcd for C13H18O5S: C, 54.53;
H, 6.34. Found: C, 54.56; H, 6.40.
Phenyl 4,6-O-benzylidene-3-O-methyl-1-thio-
h-D
-mannopyranoside (2).—A mixture of S-
-manno-
phenyl 4,6-O-benzylidene-1-thio-a-
D
3-O-Methyl- -mannose (4). Thioglycoside 3
D
pyranoside (1), prepared according to the lit-
erature method [18,19] (364 mg, 1.0 mmol)
and dibutyltin oxide (300 mg, 1.2 equiv) in
MeOH (10 mL) was refluxed with stirring
until all of the dibutylin oxide dissolved.
Methanol was then removed in vacuo, and the
residue was dissolved under N2 in dry DMF
(10 mL) and heated to 120 °C. Iodomethane
(120 mL, 2 equiv) was added with a syringe,
and the mixture was stirred at the same tem-
perature for 1 h. After cooling to rt, the
mixture was poured into water (50 mL) and
extracted with EtOAc (3×30 mL). The com-
bined organics were dried over anhyd sodium
sulfate and concentrated to a residue, which
was purified by chromatography on silica gel
with 2:1 CHCl3–hexane as eluent to give the
desired product 2 (236 mg, 63%), [h]2D0 +230°
(138 mg, 0.48 mmol) was dissolved in a mix-
ture of acetone (40 mL) and water (2 mL) and
treated with N-bromosuccinamide (188 mg,
2.2 equiv). The mixture was stirred in the
absence of light at rt until TLC indicated
complete consumption of 3 (15 h). The
volatile material was then removed in vacuo,
and the residue was purified by chromatogra-
phy on silica gel (eluent: 5:1 CHCl3–MeOH)
to afford compound 4 (78 mg, 84% yield)
1
[23,24]. Major anomer: H NMR (CD3OD,
300 MHz): l 3.42 (dd, 1 H, J2,3 3.0 Hz, J3,4 8.9
Hz, H-3), 3.45 (m, 3 H, OMe), l 3.65–3.85
(m, 4 H, H-4, H-5, 2×H-6), l 3.86 (m, 1 H,
H-2), l 5.10 (m, 1 H, H-1); 13C NMR
(CD3OD, 75 MHz): 57.5 (Me), 63.1 (C-6),
67.7 (C-4), 68.9 (C-2), 74.1 (C-5), 82.2 (C-3),
95.9 (C-1). The minor anomer was character-
1
13
(c 0.3, CHCl3); H NMR (CDCl3, 300 MHz):
ized by C NMR, l (CD3OD, 75 MHz): 57.4,
13
l 2.82 (s, 1 H, OH), l 3.60 (s. 3 H, OMe), 3.75
(dd, 1 H, J2,3 2.5 Hz, J3,4 9.5 Hz, H-3), l 3.88
(t, 1 H, Jgem =J5,6a 10 Hz, H-6a), l 4.12 (t, 1
H, J3,4=J4,5 9.5 Hz, H-4), l 4.20 (dd, 1 H,
Jgem 10 Hz, J5,6b 3.6 Hz, H-6b), l 4.40 (m, 2 H,
H-2 and H-5), l 5.65 (s, 1 H, PhCH), l 5.68
(br. s. 1 H, H-1), l 7.20–7.60 (m, 10 H). Anal.
Calcd for C20H22O5S: C, 64.15; H, 5.92.
Found: C, 63.81; H, 5.94.
63.0, 67.4, 69.2, 78.2, 85.0, 95.8; C NMR, l
(D2O, 75 MHz): 58.9, 63.6, 68.2, 69.6, 78.8,
84.8, 96.4.
1,2,4,6,-Tetra-O-benzoyl-3-O-methyl-i-
mannopyranose (5) and 1,2,4,6,-tetra-O-ben-
zoyl-3-O-methyl-h- -mannopyranose (6). To a
D
-
D
solution of tetrol 4 (30 mg, 0.15 mmol) in
pyridine (10 mL) was added benzoyl chloride
(104 mL, 0.9 mmol). The mixture was stirred
at ambient temperature overnight, then aq
sodium bicarbonate (20 mL) was added, and
the whole was extracted with EtOAc (3×20
mL). The combined organic solvents were
dried and concentrated to a residue, which
Phenyl 3-O-methyl-1-thio-h- -mannopyra-
D
noside (3).—Benzylidene acetal 2 (236 mg,
0.63 mmol) was dissolved in 1% H2SO4 in
MeOH (10 mL) at 0 °C and then stirred at rt
until TLC indicated consumption of the start-

X.-X. Tian et al. / Carbohydrate Research 324 (2000) 38–44
41
l 7.25–7.60 (m, 12 H), l 8.05–8.15 (m, 8 H);
13C NMR (CDCl3, 75 MHz): 57.9 (Me), 62.3
(C-6), 67.1 (C-2), 67.5 (C-4), 70.8 (C-5), 76.3
(C-3), 91.5 (1JCH =177.0 Hz, C-1), 128.0,
128.1, 128.2, 128.6, 128.9, 129.1, 1129.4, 129.5,
129.6, 129.7, 132.7, 133.1, 133.2, 133.7, 163.5,
165.0, 165.1, 165.8. Anal. Calcd for
C35H30O10: C, 68.85; H, 4.95. Found: C, 68.83;
H, 5.07.
was purified by chromatography on prepara-
tive TLC plates (developing system: CHCl3) to
give the two anomers 5 (55 mg, 60%, identical
to the above-described sample that originated
from M. 6accae) and 6 (18 mg, 20%). Data for
6: [h]2D0 −3.3° (c 2.6, CHCl3); 1H NMR
(CDCl3, 300 MHz): l 3.48 (s, 3 H, O-Me), l
4.13 (dd, 1 H, J2,3 3.0 Hz, J3,4 9.9 Hz, H-3), l
4.44 (m, 2 H, H-5 and H-6a), l 4.70 (br. d, 1
H, J5,6b 9.6 Hz, H-6b), l 5.85 (dd, 1 H, J2,3
3.0, J1,2 1.9 Hz, H-2), l 5.99 (t, 1 H, J3,4 =J4,5
10 Hz, H-4), l 6.58 (d, 1 H, J1,2 1.9 Hz, H-1),
3. Results
The boiling-water extract (PS4A) comprised
about 5–6% (w/w) of the dried M. 6accae cell
mass and proved to be predominantly a mix-
ture of proteins and polysaccharides with a
protein-to-sugar ratio of 1.5:1. Lipids were not
detected, and only trace amounts of phos-
phate were detected in the organic phase. The
monosaccharide profiles of PS4A were deter-
mined from the hydrolysates by a HPAEC
system with known monosaccharide standards
(Fig. 1). 3-O-Methyl- -mannose was iden-
D
tified as the most abundant sugar (37.65%),
along with glucose (25.35%), glucosamine
(18.43%), rhamnose (13.62%), fucose (3.6%)
and small amounts (B1.35%) of other sugars.
Additional studies indicated that the O-
methylated mannose was the predominant
component of the polysaccharidic fraction iso-
lated by gel-filtration chromatography, sug-
gesting it was not covalently linked to the
proteins.
Fig. 1. Monosaccharide profiles of PS4A. The monosaccha-
ride peaks separated by high-performance anion-exchange
HPLC were identified using internal standards.
The structure of the isolated oligomer was
characterized in detail by one- and two-dimen-
sional NMR spectroscopy. The simplicity of
1
the H (Fig. 2) and 13C NMR spectra of
PS4A3 suggested a material with a high de-
gree of structural homogeneity. In particular,
the 13C NMR spectrum (125 MHz, D2O) com-
prised only six sharp resonances at l: 58.8,
63.6, 68.8, 75.0, 83.4, and 103.8 with minor
peaks of much lower intensity (Fig. 3). The
signal at l 75.0 was considerably broader than
the others, suggesting that it contained the
unresolved resonances of two or more car-
bons. The gated decoupled 13C NMR spec-
1
trum revealed both the anomeric JC,H
Fig. 2. 1H NMR spectrum of the oligomer. The 1H NMR
spectrum was obtained in D2O with a Bruker Avance 500
spectrometer.
coupling to be 173 Hz, consisting of an a-gly-
cosidic linkage [25] and the signal at l 58.8 to

42
X.-X. Tian et al. / Carbohydrate Research 324 (2000) 38–44
a broad singlet, now resonated at 5.40 and the
two H-6s at l 4.25 and 4.35, whereas H-3 and
H-4 were part of a multiplet at l 3.60 to 3.65.
H-5 of the major component of this acetylated
mixture resonated at l 3.85. This pattern of
chemical shift changes suggested that O-2 and
O-6 had undergone acetylation and that the
positions of the linkage and the methyl ether
were, interchangeably, at O-3 and O-4. Fi-
nally, the broad singlet nature of H-1 and
H-2, together with the axial nature of the
glycosidic bond, strongly suggested the axial
orientation for O-2, as in mannose.
Fig. 3. 13C NMR spectrum of the oligomer.
1
13
The H and C NMR spectra of the crude
hydrolysate were also simple and indicated the
presence of one major component, consistent
with the hypothesis of a highly homogenous
polymer, present as a major and a minor
1
be a quartet indicating a methyl ether. The H
NMR spectrum (500 MHz, D2O) was corre-
spondingly simple (Fig. 2). It consisted of a
broad, anomeric singlet at l 5.10, a further
broader singlet at l 4.10, a series of multiplets
centered on l 3.75, 3.60, and 3.55 and a sharp
singlet at l 3.35, confirming the existence of
the methyl ether moiety. A COSY spectrum
revealed the coupling between the broad sin-
glets at l 5.10 and 4.10, leading to the assign-
ment of the latter as H-2 (data not shown).
In order to determine the site of linkage and
methylation, the polymer was next exposed to
acetic anhydride and a catalytic amount of
4-(dimethylamino)pyridine in pyridine at
room temperature overnight, followed by con-
1
anomer. The H NMR spectrum (500 MHz,
D2O), aside from the broad singlets l 5.09 and
4.05 assigned, respectively to H-1 and H-2 of
the major anomer, was poorly resolved, which
prevented interpretation of the coupling pat-
tern and assignment of stereochemistry.
A sample of the hydrolysate, containing
NaHCO3, was recovered from the NMR ex-
periment, lyophilized and then treated with
benzoyl chloride and pyridine for 24 h at
room temperature. Concentration and prepar-
ative TLC over silica gel enabled isolation of
the major UV-active product, whose 1H NMR
spectrum (300 MHz, CDCl3) was fully re-
solved (Fig. 4) and permitted its unambiguous
identification as 1,2,4,6-tetra-O-benzoyl-3-O-
1
centration to dryness under vacuum. The H
NMR spectrum of the reaction was consider-
ably more complex than that of the polymer
itself, which indicated that some decomposi-
tion had accompanied acetylation. Neverthe-
less, it still consisted of one major component.
A COSY spectrum (500 MHz, CDCl3) re-
vealed that H-2 of the major component, still
methyl-b-
D
-mannopyranose
(5).
Conse-
quently, the hydrolysate is 3-O-methyl-
D-
mannopyranose (4) [23,24], and the oligomer
poly-a-(1“4)-linked 3-O-methylmannopyr-
anose (Fig. 5). We note that a similar
1
Fig. 4. Partial H NMR spectrum of 1,2,4,6-tetra-O-benzoyl-3-O-methyl-b-
D
-mannose (5) in CDCl3.

X.-X. Tian et al. / Carbohydrate Research 324 (2000) 38–44
43
portant regioselective 3-O-monomethylation
was achieved by sequential treatment of
known
1
[18,19] with dibutyltin and
iodomethane [20]. The regioselectivity of
closely related alkylations has previously been
adequately demonstrated in one of our labora-
tories [21,22]. The regioselectivity of this alky-
lation is further adequately supported by the
spectral data for the perbenzoylated 3-O-
methylmannose derivatives 5 and 6. The au-
thentic sample (4) had spectral data identical
to those of the major component of the hy-
drolysate. The synthesized monomer (4) also
had an identical retention time to that of the
monosaccharide in the hydrolysate of the
PS4A sample in HPLC analysis (Fig. 6). With
the structure of the oligomer fully elucidated,
it was possible to return to the spectral data
Fig. 5. Structure of the oligomer.
13
and assign the C NMR spectrum as follows
1
using standard H–13C correlation methods: l
(D2O) 58.8 (Me), 63.6 (C-6), 68.8 (C-2), 75.0
(C-4 and C-5), 83.4 (C-3) and 103.8 (C-1).
Scheme 1. Synthesis of the authentic monomer and its tetra-
benzoates.
4. Discussion
Ultrastructural studies have demonstrated
that the mycobacterial cell wall is a triple-lay-
ered structure [27]. An inner layer of moderate
electron density probably contains peptidogly-
cans. A wider (9–10 nm) electron-transparent
middle layer appears to consist of the hydro-
phobic lipids of the cell wall. The outermost
electron-opaque layer varies in thickness, elec-
tron density and appearance among species,
and under different growth conditions. Ko-
batake et al. [3] first reported that the exocel-
lular materials extracted by boiling M.
tuberculosis Aoyama B strains were polysac-
charide-rich mixtures composed of glucan
(Mw\75 kDa), arabinomannan (Mw 13 kDa)
and mannan (Mw 4 kDa). Previous studies by
our group have demonstrated that much of
the antineoplastic activity of M. bo6is BCG
Tice substrain is associated with a glucan hav-
ing a molecular weight of ꢀ65 kDa [7]. The
Connaught substrain, also clinically employed
in the treatment of superficial bladder cancer,
has a glycan component that probably differs
in structure, but, on a weight-in-weight basis,
is equivalent in biological activity [10]. These
Fig. 6. HPLC analysis of synthetic 3-O-methyl-D-mannose.
This chromatogram showed that the authentic sample had an
identical retention time with the major sugar identified in
PS4A (Fig. 1).
oligomer has been previously reported from
1
M. smegmatis and that its H NMR spectrum
bears a very strong resemblance to that of the
present sample [26].
Finally, authentic samples of the monomer
(4) and its corresponding benzoylated deriva-
tives (5) were prepared (Scheme 1). The all-im-

44
X.-X. Tian et al. / Carbohydrate Research 324 (2000) 38–44
studies confirmed the existence of high-molec-
ular-weight glycans on the exocellular surface
of slow-growing mycobacteria. Moreover, in
the case of M. bo6is, the biologically active
components are associated with the cellular
integument surrounding the bacterial cells
[10].
References
[1] A. Lemassu, M. Daffe, Biochem. J., 297 (1994) 351–357.
[2] A. Lemassu, A. Ortalo-Magne, F. Bardou, G. Silve,
M-A Laneelle, M. Daffe, Microbiology, 142 (1996)
1513–1520.
[3] H. Kobatake, T. Suekane, Y. Murakami, S-L. Niwa, A.
Okahira, H. Kushida, Yakugaku Zasshi, 101 (1981) 713–
722.
Although extracts of the rapid growing M.
6accae had similar biological activity to mate-
rials obtained from M. bo6is, it was evident
that the components responsible for the im-
munostimulating activity in this case were
structurally different. Hot-water extracts of
intact cells of M. 6accae contained polysac-
charides that were associated with a proteina-
[4] F. Suzuki, R.R. Brutkiewicz, R.B. Pollard, J. Natl. Can-
cer Inst., 77 (1986) 441–447.
[5] H. Sano, H. Nagae, M. Suzuki, E. Tagashira, X. Pakas-
saku, S. Kimishima, H. Yugi, S. Baba, J. Labelled
Compd. Radiopharm., 29 (1990) 131–142.
[6] Y. Lou, M.E. Klegerman, A. Muhammad, X. Dai, M.J.
Groves, Anticancer Res., 14 (1994) 1469–1476.
[7] R. Wang, M.E. Klegerman, I. Marsden, M. Sinnott,
M.J. Groves, Biochem. J., 311 (1995) 867–872.
[8] P.O. Devadoss, M.J. Groves, M.E. Klegerman, Biomed.
Lett., 46 (1991) 81–90.
[9] M.E. Klegerman, P.O. Devadoss, J.L. Garrido, H.R.
Reyes, M.J. Groves, FEMS Immunol. Med. Microbiol.,
15 (1996) 213–222.
[10] J.L. Garrido, M.E. Klegerman, H.R. Reyes, M.J.
Groves, Cytobios, 90 (1997) 47–65.
[11] G.M. Bahr, M.A. Shaaban, M. Garbriel, B. Al-Shimali,
Z. Siddioui, T.D. Chugh, F.M. Denath, A. Shahin, K.
Behbehan, L. Chedid, G.A.W. Rook, J.L. Stanford, Tu-
bercle, 71 (1990) 259–266.
ceous
component
[14].
The
major
polysaccharide recovered from PS4A was
identified as poly-a-(1“4)-3-O-methyl- -
D
mannose with a molecular weight of around
3.6 kDa [28]. O-Methyl sugars are not widely
distributed in nature but have been frequently
isolated from mycobacteria. Poly-a-(1“4)-3-
O-methyl- -mannose, composed of 10 to 13
D
monomers, has previously been isolated from
M. smegmatis and was considered as a biosyn-
thetic precusor of the larger methylmannose
polysaccharide [26]. Similar sugars have also
been reported in the lipopolysaccharide of
Klebsiella and Escherichia coli, which seems to
be of immunological importance even though
it is present only in small amount [29].
[12] J.L. Stanford, G.M. Bahr, G.A.W. Rook, M.A. Shaa-
ban, T.D. Chugh, M. Gabriel, B. Al-Shimali, F.
Ghardani, A. Shahin, K. Behbehan, Vaccine, 8 (1990)
525–530.
[13] B.J. Marsh, C.F. Vonreyn, R.D. Arbelt, P. Morin, Am.
J. Med. Sci., 313 (1997) 377–383.
[14] M.J. Groves, M.E. Klegerman, K. Ciftci, X.X. Tian, J.
Pharm. Pharmacol., 47 (1996) 1082.
[15] X.X. Tian, A. Li, W. Zhou, I.V. Farrugia, M.J. Groves,
Anticancer Res., 19 (1999) 237–244.
[16] R. Wang, M.E. Klegerman, M.J. Groves, J. Liq. Chro-
matogr. Relat. Technol., 19 (1995) 2691–2698.
[17] J.E. Hodge, B.T. Hofreiter, Methods Carbohydr. Chem.,
1 (1962) 388–389.
[18] H. Franzyk, M. Meldal, H. Paulsen, K. Bock, J. Chem.
Soc., Perkin Trans. 1, (1995) 2883–2898.
[19] T. Oshitari, M. Shibasaki, T. Yoshizawa, M. Tomita, K.
Takao, S. Kobayashi, Tetrahedron, 53 (1997) 10993–
11006.
[20] S. David, S. Hanessian, Tetrahedron, 41 (1985) 643–663.
[21] D. Crich, S. Sun, Tetrahedron, 54 (1998) 8321–8348.
[22] D. Crich, Z. Dai, Tetrahedron, 55 (1999) 1569–1580.
[23] M.M. Ponpipom, Carbohydr. Res., 59 (1977) 311–317.
[24] G.O. Aspinall, G. Zweifel, J. Chem. Soc., (1957) 2271–
2278.
Although O-methyl sugars are frequently
found in mycobacteria, the distribution and
biological activity of such sugars, especially
3-O-methyl- -mannose, is still not clear. This
D
study is the first report of the isolation of
homogeneous poly-3-O-methylmannose in M.
6accae. Our study also found that this sugar is
associated with water-soluble glycoproteins
that have immunostimulating activity [15].
This information is of great importance for
the further study and use of M. 6accae, which
is currently being tested as a nonspecific
immunostimulant.
[25] K. Bock, C. Pederson, J. Chem. Soc., Perkin Trans. 2,
(1974) 293–297.
[26] H. Yamada, R.E. Cohen, C.E. Ballou, J. Biol. Chem.,
254 (1979) 1972–1979.
[27] N. Rastogi, C. Frehel, H.L. David, Curr. Microbiol., 13
(1986) 237–242.
Acknowledgements
[28] X.X. Tian, Ph.D. Thesis, University of Illinois at
Chicago (1998).
[29] W. Nimmich, Biochim. Biophys. Acta, 215 (1970) 189–
191.
We thank Dr Robert A. Kleps of the Research
Resources Center of the University of Illinois at
Chicago for valuable assistance in this study.