Synthesis of the β anomer of the spacer-equipped tetrasaccharide side chain of the major glycoprotein of the Bacillus anthracis exosporium

Article abstract of DOI:10.1016/j.carres.2005.09.015

The β glycoside of the tetrasaccharide sequence β-Ant-(1→3)- α-l-Rhap-(1→3)-α-l-Rhap-(1→2)-l-Rhap, whose aglycon allows conjugation to proteins, was synthesized for the first time. A stepwise synthetic approach was applied with thioglycosides as glycosyl

Full text of DOI:10.1016/j.carres.2005.09.015

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 2579–2582
Rapid Communication
Synthesis of the b anomer of the spacer-equipped
tetrasaccharide side chain of the major glycoprotein of the
Bacillus anthracis exosporiumI
´ˇ*
Roberto Adamo, Rina Saksena and Pavol Kovac
NIDDK, LMC, National Institutes of Health, Bethesda, MD 20892-0815, USA
Received 10 August 2005; accepted 6 September 2005
Available online 10 October 2005
Abstract—The b glycoside of the tetrasaccharide sequence b-Ant-(1!3)-a-L-Rhap-(1!3)-a-L-Rhap-(1!2)-L-Rhap, whose aglycon
allows conjugation to proteins, was synthesized for the first time. A stepwise synthetic approach was applied with thioglycosides as
glycosyl donors, and the b anomer of the compound was obtained equipped with a spacer group whose further transformation
allows conjugation to suitable carriers. To synthesize the b-anthrosyl linkage with high stereoselectivity, a linker-equipped rhamno-
triose derivative was glycosylated with ethyl 4-azido-3-O-benzyl-2-O-bromoacetyl-4,6-dideoxy-1-thio-b-D-glucopyranoside. Further
functionalization of the tetrasaccharide thus obtained, followed by deprotection, gave the target substance.
Ó 2005 Elsevier Ltd. All rights reserved.
Keywords: Anthrax; Anthrose; Exosporium glycoprotein; Thioglycoside; Oligosaccharide
Bacillus anthracis is the etiological cause of anthrax.1 In
view of current concerns regarding the use of some form
of the bacterium as a biological weapon, development of
a potent vaccine for anthrax is a pressing issue world-
wide. B. anthracis is a spore-forming pathogen, and a
vaccine for anthrax could be based on targeting spores
with neutralizing antibodies that are specific for one of
their surface components. The tetrasaccharide b-Ant-
(1!3)-a-L-Rhap-(1!3)-a-L-Rhap-(1!2)-L-Rhap was
recently established2 as the oligosaccharide side chain
of the collagen-like region of the major glycoprotein of
the B. anthracis exosporium (Fig. 1). In connection with
developing a conjugate vaccine for anthrax following
the above-outlined strategy, we have recently synthe-
sized anthrose, as well as intermediates that could be
used to prepare its glycosyl donors.3 With the plan to
generate immunogens for antibodies specific for the
carbohydrate component of the anthrax exosporium
HO
HO
CH3
CH3
HO
CH3
HO
HO
O
O
H3C
O
O
C
NH
HO
OH
O
O
O
O
CH3
CH3
HO
OMe
HO
β–Ant–(1→3)–α–L–Rhap–(1→3)–α–L–Rhap–(1→2)–L–Rhap–
Figure 1. Structure of the tetrasaccharide side chain of the collagen-
like glycoprotein of Bacillus anthrasis exosporium.
q This work was presented at the 13th Eurocarb, August 22–26, 2005,
Bratislava, Slovakia.
*
Scheme 1. Reagents and conditions: (a) Bu2SnO, toluene; (b) Tf2O,
Corresponding author. Tel.: +1 301 496 3569; fax: +1 301 402
K2CO3, CH2Cl2; (c) CsF, MeCN.
0589; e-mail: kpn@helix.nih.gov
0008-6215/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.09.015

2580
R. Adamo et al. / Carbohydrate Research 340 (2005) 2579–2582
Me
BnO
Me
BnO
O
O
COOMe
c
O
COOBn
b
O
SMe
BnO
BnO
Me
BnO
O
O
O
a
Me
Me
BnO
O
O
BnO
AcO
OBn
OH
AcO
OBn
OBn
6
7
8
Me
BnO
O
O
COOMe
Me
BnO
O
O
COOMe
OBn
O
Me
BnO
O
O
COOMe
BnO
O
Me
BnO
O
b
Me
BnO
O
O
BnO
O
Me
BnO
OBn
O
O
OBn
Me
BnO
e
O
Me
BnO
O
O
OBn
AcO
OBn
Me
BnO
9
O
HO
d
OBn
Me
O
O
OBn
N3
12
10
N3
BnO
Me
Me
BnO
O
O
N3
BnO
BrAcO
SEt
SEt
HO
BrAcO
11
Scheme 2. Reagents and conditions: (a) 5, NIS, AgOTf, CH2Cl2; (b) NaOMe, MeOH; (c) 6, NIS, AgOTf, CH2Cl2; (d) BrCH2COBr, TMU, CH2Cl2;
(e) NIS, AgOTf, CH2Cl2.
glycoprotein, we have now prepared and here report the
first synthesis of tetrasaccharide glycoside 17, whose
aglycone can be transformed to allow conjugation to
suitable carriers.
light. The formation of the b-rhamnopyranosyl link-
age6,7 in the formation of 5 manifested8 itself in the pro-
ton-coupled 13C NMR spectrum (JC-1,H-1 156.4 Hz).
Stepwise extension of the oligosaccharide chain was
effected (Scheme 2) by glycosylation with methyl 3-O-
acetyl-2,4-di-O-benzyl-1-thio-a-L-rhamnopyranoside 6,9
first of 5 [!7, 82%, [a]D +38.5 (c 0.8, CHCl3)], and then
of the product of deacetylation of 7, alcohol 8 [94%, [a]D
+13 (c 0.6 CHCl3)], to give rhamnotrioside 9 [92%, [a]D
+22 (c 1.2, CHCl3)]. Positioning anthrose at the upstream
terminus of the desired tetrasaccharide 17 required for-
mation of a b-glucosidic linkage. Because of the pres-
ence of a 2-O-methyl group in 17, formation of such a
linkage in a highly stereoselective manner could be prob-
lematic using a glycosyl donor derived from a synthon
having a nonparticipating group at O-2, such as the
OMe group in anthrose. Therefore, glycosyl acceptor
10, obtained [96%, [a]D +14 (c 0.4, CHCl3)] by deacetyl-
ation of 9, was glycosylated with the versatile glucosyl
donor 11 [mp 82–82.5 °C, [a]D +60 (c 0.5, CHCl3)], ob-
tained (ꢀ70%) by bromoacetylation10 of ethyl 4-azido-
3-O-benzyl-4,6-dideoxy-1-thio-b-D-glucopyranoside,3 to
give the fully protected tetrasaccharide 12 [66%, [a]D
It has been proposed2 that the tetrasaccharide may be
attached to the exosporium glycoprotein through an N-
acetyl-D-galactosamine linker, but the type of glycosidic
linkage providing that attachment to the GalNAc linker
is unknown. To insure production of antibodies homo-
logous to the naturally occurring tetrasaccharide, conju-
gates from both a- and b-linked tetrasaccharides will
have to be prepared and tested for their immunogenic-
ity. Results of such studies may provide clues regarding
the mode of linkage of the tetrasaccharide in the natural
exosporium, as well as information about specificity and
cross-reactivity of antibodies formed. Described below
is the stepwise construction of tetrasaccharide glycoside
17 in which the spacer is attached through a b-rhamnos-
yl linkage. The initial glycosyl acceptor 5 [52% from 1,
[a]D +11 (c 0.6, CHCl3)] was synthesized from triflate 4
{m/z 354 ([M]+)} of benzyl 6-hydroxyhexanoate4 3 and
the stannylidene acetal 2 of 3,4-di-O-benzyl rhamnose5
(Scheme 1). Using the benzyl ester allowed monitoring
the formation and purification of the triflate by UV
;H-1II
+18 (c 1, CHCl3)]; JC-1I;H-1I 152.3 Hz, JC-1II
;H-1III
;H-1IV
160.9 Hz.
169.8 Hz, JC-1III
171.2 Hz, JC-1IV
Transformation of 12 into the target tetrasaccharide
17 was accomplished as shown in Scheme 3. Accord-
ingly, after debromoacetylation [!13, 91%, [a]D +40
(c 0.4, CHCl3)], successive methylation with MeI and
Ag2O in the presence11 of Me2S [!14, 70%, [a]D +29
All new compounds produced correct analytical figures by combus-
tion analysis, except 4, 12, 15, 16, and 17. Copies of NMR spectra of
the foregoing five compounds are available as supplementary
material.

R. Adamo et al. / Carbohydrate Research 340 (2005) 2579–2582
2581
Me
O
Me
BnO
Me
BnO
O
O
COOMe
O
O
COOMe
O
COOMe
BnO
BnO
BnO
BnO
O
O
O
Me
BnO
Me
BnO
Me
BnO
O
O
O
a
b
c
d
12
O
O
O
OBn
OBn
OBn
Me
BnO
Me
BnO
Me
BnO
O
O
O
Me
N3
BnO
Me
N3
BnO
Me
H2N
BnO
O
O
O
O
O
O
OBn
OBn
OBn
MeO
HO
13
MeO
14
15
Me
BnO
O
Me
HO
O
COOMe
O
O
COOMe
BnO
HO
O
O
Me
BnO
O
Me
HO
O
e
O
O
OBn
OH
Me
BnO
O
Me
HO
O
O
Me
O
Me
O
O
O
OBn
O
C NH
OH
C NH
BnO
HO
Me
OH
Me
Me
OH
Me
MeO
MeO
16
17
Scheme 3. Reagents and conditions: (a) NaOMe, MeOH; (b) MeI, Me2S, Ag2O, CH2Cl2; (c) H2S, Py, H2O; (d) 3-hydroxy-3-methylbutyric acid,
HATU, EDA, CH2Cl2; (e) H2, Pd/C, MeOH/AcOH.
(c 0.4, CHCl3)], and selective reduction of the azido func-
tion with H2S12 gave amine 15 (70%), whose treatment
with 3-hydroxy-3-methylbutyric acid in the presence of
HATU {N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]-
pyridin-1-ylmethylene]-N-methylmethanaminium hexa-
fluorophosphate N-oxide} gave butanamido derivative
16 (73%). Finally, hydrogenolytic debenzylation over
5% palladium-on-charcoal catalyst gave the target tetra-
saccharide 17 [82%, [a]D ꢁ38 (c 0.7, MeOH)], equipped
with a spacer that makes it amenable to conjugation to
proteins or other suitable carriers. NMR data observed
for 17 (in D2O with acetone as internal standard13) are
in Table 1. Work toward the a-linked analog of 17
and to neoglycoconjugates from 17 is in progress.
Table 1. 1H and 13C chemical shifts (d) for tetrasaccharide 17a
Residueb,c
H-1
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6
C-6
I
4.641
102.336
5.005
4.002
79.615
4.186
3.691
76.053
3.902
3.40
3.40
ꢀ1.301
19.455e
1.249
75.150d
3.508
74.979d
4.213
II
103.995
5.028
104.929
4.470
72.781
4.285
72.606
3.135
80.864
4.002
82.392
3.543
74.099
3.620
73.912
3.636
71.547
3.876
72.047
3.561
19.492
ꢀ1.315
19.424e
1.223
III
IV
106.431
86.030
75.620
59.345
73.536
19.086
a Measured at 600 MHz (1H) and 150 MHz (13C) for solutions in D2O at 20 °C.
b Sugar residues in the tetrasaccharide are serially numbered, beginning with the rhamnose residue bearing the aglycone, and are identified by a
Roman numeral superscript. Nuclei associated with the N-acyl substitutent and those with the linker aglycone are denoted with a single and double
prime, respectively.
c
00
00
0
00
00 00
00
0
dH-1 a ꢀ3.84; dCOOMe 3.680; dH-1 b ꢀ3.67; dOMe-2 3.632; dCH 2.459; dH-5 2.400; dH-2 ;4 1.612; dH-3 1.422; dMe ꢀ1.309, ꢀ1.300. dCO 180.399,
2
0
00
0
00
00
00
00
176.830; dC-3 ðquartÞ 72.990; dC-1 72.486; dOMe-2 62.811; dCOOMe 54.855; dC-2 51.687; dC-5 36.393; dC-4 31.451; dMe 31.040, 30.855; dC-3 27.728;
00
dC-2 26.858.
d The assignment can be reversed.
e The assignment can be reversed.

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R. Adamo et al. / Carbohydrate Research 340 (2005) 2579–2582
´ˇ
3. Saksena, R.; Adamo, R.; Kovac, P. Carbohydr. Res. 2005,
Acknowledgments
340, 1591–1600.
4. Nederberg, F.; Bowden, T.; Hilborn, J. Macromolecules
2004, 37, 954–965.
5. Srivastava, V. K.; Schuerch, C. J. Org. Chem. 1981, 46,
1121–1126.
This research was supported by the Intramural Research
Program of the NIH, NIDDK.
´ˇ
6. Hodosi, G.; Kovac, P. J. Am. Chem. Soc. 1997, 119, 2335–
Supplementary data
2336.
´ˇ
7. Hodosi, G.; Kovac, P. Carbohydr. Res. 1998, 308, 63–
75.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2005.09.015.
8. Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2
1974, 293–297.
9. Liptak, A.; Szabo, L.; Harangi, J. J. Carbohydr. Chem.
1988, 7, 687–699.
´ˇ
10. Kovac, P. Carbohydr. Res. 1986, 153, 237–251.
References
´ˇ
11. Kovac, P. In Alkylation, 2nd ed.; Blau, K., Halket, J. M.,
Eds.; John Wiley & Sons: Chichester, 1993; pp 109–129.
12. Adachi, T.; Yamada, Y.; Inoue, I.; Saneyoshi, M.
Synthesis 1977, 45–46.
13. Wishart, D. S.; Bigam, C. G.; Yao, J.; Abildgaard, F.;
Dyson, H. J.; Oldfield, E.; Markley, J. L.; Sykes, B. D.
J. Biolmol. NMR 1995, 6, 135–140.
1. Cote, C. K.; Rossi, C. A.; Kang, A. S.; Morrow, P. R.; Lee,
J. S.; Welkos, S. L. Microb. Pathogen. 2005, 38, 209–225.
2. Daubenspeck, J. M.; Zeng, H.; Chen, P.; Dong, S.;
Steichen, C. T.; Krishna, N. R.; Pritchard, D. G.;
Turnbough, C. L. J. Biol. Chem. 2004, 279, 30945–30953.