Synthesis of the tetrasaccharide side chain of the major glycoprotein of the Bacillus anthracis exosporium

Article abstract of DOI:10.1016/j.bmcl.2005.10.056

An α-glycoside of the tetrasaccharide sequence β-Ant-(1 → 3)-α-L-Rhap-(1 → 3)-α-L-Rhap-(1 → 2)-α-L-Rhap whose aglycon allows conjugation to suitable carriers was synthesized. The NMR characteristics of the compound are virtually identical with those of th

Full text of DOI:10.1016/j.bmcl.2005.10.056

Bioorganic & Medicinal Chemistry Letters 16 (2006) 615–617
Synthesis of the tetrasaccharide side chain of the major
glycoprotein of the Bacillus anthracis exosporiumq
*
´
Rina Saksena, Roberto Adamo and Pavol Kovacˇ
NIDDK, LMC, National Institutes of Health, Bethesda, MD 20892-0815, USA
Received 15 September 2005; revised 13 October 2005; accepted 14 October 2005
Available online 4 November 2005
Abstract—An a-glycoside of the tetrasaccharide sequence b-Ant-(1 ! 3)-a-L-Rhap-(1 ! 3)-a-L-Rhap-(1 ! 2)-a-L-Rhap whose agly-
con allows conjugation to suitable carriers was synthesized. The NMR characteristics of the compound are virtually identical with
those of the a-anomer of the tetrasaccharide isolated from the major glycoprotein of the Bacillus anthracis exosporium. Thus, the
correct structure of the natural product has been proven by chemical synthesis.
Ó 2005 Elsevier Ltd. All rights reserved.
Bacillus anthracis is the causative agent of anthrax. Be-
cause of concerns regarding the use of some of its forms
as a biological weapon, there is a worldwide effort to
develop a potent vaccine for the disease. Since B. anthra-
cis is a spore-forming pathogen, a strategy toward a
vaccine for anthrax could be based on targeting spores
with antibodies that are specific for the tetrasaccharide
b-Ant-(1 ! 3)-a-L-Rhap-(1 ! 3)-a-L-Rhap-(1 ! 2)-L-
Rhap (1), which was recently discovered2 as the oligo-
saccharide side chain of the collagen-like region of the
major glycoprotein of the pathogenꢀs exosporium. With-
in our work toward a conjugate vaccine for anthrax we
have prepared and here report a synthesis (Scheme 1) of
sequence 1 in the form of the a-glycoside 20 whose agly-
cone can be further transformed to allow conjugation to
suitable carriers.
Subsequent deacetylation of 9 gave the trisaccharide gly-
cosyl acceptor 10.
Synthesis of 20 required formation of 1,2-trans glycosidic
linkage, which could be problematic with a glycosyl donor
bearing a non-participating group at O-2, such as the 2-O-
methyl group in anthrose. Therefore, the versatile 2-O-
bromoacetylated glycosyl donor 14 (Scheme 2) was
prepared. In addition to being a participating group, the
bromoacetyl function7,8 can be selectively removed in
the presence of other acyl groups. Thus, donor 14 can
be used in alternative syntheses of 1 involving acyl-
protected intermediates. Compound 14 was obtained
as shown in Scheme 2. Accordingly, methyl 4-azido-
3-O-benzyl-4,6-dideoxy-a-D-glucopyranoside9 (11) was
bromoacetylated,10 and the formed 12 was acetolyzed to
give 13. The anomeric mixture of acetates thus obtained
was treated with EtSH under Lewis acid catalysis to give
14. Condensation of the trirhamnoside glycosyl acceptor
10 with 14 gave the fully protected tetrasaccharide 15,
which was sequentially de-O-bromoacetylated10 (!16),
methylated11 (!17), and treated with H2S,12 to selectively
reduce the azido group to amino function, to afford 18.
Treatment of 18 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-methylmethana-
minium hexafluorophosphate N-oxide} gave butanamido
derivative 19, which was fully deprotected by hydrogeno-
lytic debenzylation, to give the target tetrasaccharide 20
equipped with a spacer that makes it amenable for conju-
gation to proteins or other suitable carriers. NMR data
observed for 20 in DMSO-d6 (Tables 1 and 2) are virtually
identical to those reported2 for the solution of a-anomer
The initial glycosyl acceptor 413 was synthesized by reac-
tion of the known3 thioglycoside 2 with methyl 6-
hydroxyhexanoate4 (!3) followed by deacetylation.
Extension of the oligosaccharide chain to give rhamno-
trioside 9 was effected by glycosylation of 4 with the
disaccharide building block 8. To prepare 8, the known5
thioglycoside 6 was treated with glycosyl chloride 7,
which was obtained by treatment of 55 with chlorine.6
Keywords: Anthrax; Anthrose; Exosporium glycoprotein; Thioglyco-
side; Oligosaccharide.
q
See Ref. 1.
Corresponding author. Tel.: +1 301 496 3569; fax: +1 301 402
0859; e-mail: kpn@helix.nih.gov
*
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.056

616
R. Saksena et al. / Bioorg. Med. Chem. Lett. 16 (2006) 615–617
Scheme 1. Reagents and conditions: (a) NIS, AgOTf, methyl 6-hydroxyhexanoate, CH2Cl2, rt, 1 h; (b) NaOMe, MeOH, rt, 2 h; (c) NIS, AgOTf,
CH2Cl2, rt, 1 h; (d) AgOTf, 2,6-di-t-Bu-Me-pyridine, CH2Cl2, rt, 1 h; (e) Cl2, CH2Cl2, rt, 5 min; (f) NIS, AgOTf, CH2Cl2, rt, 1 h; (g) 3-hydroxy-3-
methylbutyric acid, HATU, CH2Cl2, rt, 1 h; (h) MeI, Ag2O, Me2S, 1,2-dimethoxyethane, rt, 24 h; (i) H2S, 3:1 pyridine–water, rt, 24 h; (j) H2, Pd/C,
MeOH, rt, 24 h.
Me
Me
Me
Me
O
O
O
O
a
N3
b
N3
N3
BnO
c
N3
SEt
BnO
BrCH2COO
BnO
BrCH2COO
BnO
BrCH2COO
OAc
HO
OMe
OMe
14
11
13
12
Scheme 2. Reagents and conditions: (a) BrCH2COBr, TMU, CH2Cl2, 0 °C ! rt, 24 h; (b) Ac2O, H2SO4, AcOH, rt, 1 h; (c) EtSH, BF3ÆEt2O, CH2Cl2,
rt, 2 h.
of 1 in the same solvent. The minute differences in chem-
ical shifts are due to the presence of the linker substituent
at O-1 of the D-rhamnose residue. This confirms, by chem-
ical synthesis, the structure assigned to the material isolat-
ed from the natural source.
Table 1. Comparison of 1H chemical shiftsa for tetrasaccharide 20 with
data reported in Ref. 1 for 1
Ring
A
H-1
H-2
H-3
H-4
H-5
H-6
4.577 2.845 3.281
4.582 2.850 3.301
4.857 3.872 3.745
4.876 3.880 3.760
4.842 3.775 3.609
4.848 3.783 3.592
4.566 3.634 3.530
4.870 3.601 3.615
3.402
3.401
3.402
3.404
3.325
3.339
3.155
3.144
3.275 1.074
3.278 1.082
3.636 1.132
3.655 1.142
3.487 1.101
3.504 1.103
3.373 1.134
3.592 1.114
B
Acknowledgment
C
D
This research was supported by the Intramural Research
Program of the NIH, NIDDK.
NH
CH2 CH3
OCH3
Otherb 7.706 2.204 1.155, 1.143 3.519
7.754 2.211 1.160, 1.148 3.526
Supplementary data
a Spectra taken at 600 MHz for a solution in DMSO-d6 at 25 °C. Data
in the second row for each ring were taken from Ref. 1.
b dOH (disappear on deuteration): 4.995, 4.945, 4.885, 4.875, 4.831,
4.779, 4.718, 4.534. Not reported in Ref. 1.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2005.10.056.

R. Saksena et al. / Bioorg. Med. Chem. Lett. 16 (2006) 615–617
617
Table 2. Comparison of 13C chemical shiftsa for tetrasaccharide 20 with data reported in Ref. 1 of 1
Ring
A
C-1
C-2
C-3
C-4
C-5
C-6
103.50; JC,H 160.7
103.4; JC,H 161
101.71; JC,H 171.3
101.6; JC,H 171
101.76; JC,H 170.6
101.6; JC,H 170
98.62; JC,H 167.6
92.8; JC,H 166
84.36
84.3
72.77
72.7
56.42
56.4
70.34
70.3
18.18
18.1
B
69.67
69.6
79.48
79.4
71.65
71.6
68.31
68.2
17.73
17.6
C
D
69.97
69.9
76.28
76.2
71.37
71.4
69.04
68.8
17.81
17.9
76.66
77.7
70.56
70.1
72.22
72.6
68.58
67.7
18.02
17.9
CO
CH2
CH3
Cquat
OCH3
Other
171.45
171.4
48.67
48.6
29.58, 29.48
29.5, 29.4
68.71
Not reported
60.16
59.9
a Spectra taken at 150 MHz for a solution in DMSO-d6 at 25 °C. Data in the second row for each ring were taken from Ref. 1.
Compound 9: 94%; [a]D ꢀ4.6° (c 0.9, CHCl3); dH-1B 5.16,
J1,2 1.7 Hz; dH-1C 5.11, J1,2 1.9 Hz, dH-1D 4.68, J1,2 1.8 Hz,
dC-1B 99.25, dC-1C 98.98, dC-1D 98.87.
Compound 10: 85%; [a]D ꢀ6.6° (c 0.6, CHCl3); dH-1B 5.21
(s), dHꢀ1C 5.11, J1,2 1.9 Hz, dH-1D 4.70, J1,2 1.8 Hz; dC-1B
98.34,dC-1C 98.93, dC-1D 98.82.
References and notes
1. This work was presented at the 13th Eurocarb, August
22–26, 2005, Bratislava, Slovakia.;
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.
3. Pozsgay, V.; Jennings, H. J. J. Org. Chem. 1988, 53, 4042.
4. Solladie, G.; Ziani-Cherif, C. J. Org. Chem. 1993, 58, 2181.
5. Liptak, A.; Szabo, L.; Harangi, J. J. Carbohydr. Chem.
1988, 7, 687.
6. Kovac, P.; Lerner, L. Carbohydr. Res. 1988, 184, 87.
7. Bertolini, M.; Glaudemans, C. P. J. Carbohydr. Res. 1970,
15, 263.
8. Kovac, P.; Glaudemans, C. P. J. Carbohydr. Res. 1985,
140, 313.
´ˇ
9. Saksena, R.; Adamo, R.; Kovac, P. Carbohydr. Res. 2005,
340, 1591.
´ˇ
10. Kovac, P. Carbohydr. Res. 1986, 153, 237.
Compound 12: 90%; [a]D +204° (c 0.5, CHCl3); dH-1 4.86,
J1,2 ꢁ3.7 Hz; dC-1 96.41.
Compound 13: 93%; dH-1a 6.24, J1,2 3.7 Hz, dH-1b 5.58, J1,2
8.4 Hz; dC-1a 89.04, dC-1b 91.41.
Compound 14: b anomer, 62%; mp 82.0–82.5 °C (from
EtOH); [a]D +60° (c 0.5, CHCl3); dH-1 4.34, J1,2 10.0 Hz; dC-1
82.82; the corresponding a-anomer, mp 31–32 °C (from
hexane), [a]D +263° (c 0.9, CHCl3); (dH-1 5.57, J1,2 5.4 Hz;
dC-1 81.27) was formed in 23%, total yield of thioglycosi-
dation, 85%.
Compound 15: 84%; [a]D +2.3° (c 0.7, CHCl3); dH-1A
ꢁ4.69, J1,2 ꢁ8.0 Hz, dH-1B 5.09 (s); dH-1C 5.10, J1,2 1.9 Hz;
dH-1D ꢁ4.66, J1,2 not determined due to overlap; dC-1A
100.62, dC-1B 100.22, dC-1C 98.75, dC-1D 98.87.
´ˇ
´ˇ
´ˇ
11. Kovac, P. In Alkylation; Blau, K., Halket, J. M., Eds., 2nd
Compound 16:93%; [a]D +10.4°(c 0.5, CHCl3);d
A
4.39,
ed.; John Wiley and Sons, Ltd.: Chichester, 1993; p 109.
12. Adachi, T.; Yamada, Y.; Inoue, I.; Saneyoshi, M. Synthesis
1977, 45.
J1,2 7.7 Hz, dH-1B 5.08 (s), dH-1C 5.11, J1,2 2.0 Hz,dH-1 4.67,
H-1D
J1,2 1.7 Hz; dC-1A 103.70, dC-1B 99.17, dC-1C 98.70, dC-1D
98.89.
13. All reactions were monitored by TLC on silica gel coated
glass slides, and the structure of products was verified by MS
and NMR spectra. All new compounds produced correct
analytical figures by combustion analysis, except for com-
pounds 7, 13, 17, and 20. Copies of NMR spectra of the four
compounds are available as Supplementary data.
Compound 3: 83%; [a]D ꢀ6° (c 1, CHCl3); dH-1 5.35, J1,2
1.8 Hz; dC-1 97.60.
Compound 17: 96%; [a]D +2° (c 1, CHCl3); dH-1A 4.61,
J1,2 8.0 Hz, dH-1B 5.11 (s); dH-1C 5.08, J1,2 1.8 Hz; dH-1D
4.66, J1,2 1.8 Hz; dC-1A 103.52, dC-1B 100.26, dC-1C 98.96,
dC-1D 98.90.
Compound 18: 88%; dH-1A 4.70, J1,2 7.7 Hz, dH-1B 5.12
(s), dH-1C 5.08, J1,2 1.9 Hz, dH-1D ꢁ4.66, J1,2 not
determined due to overlap; dC-1A 103.82, dC-1B 100.37,
dC-1C 99.03, dC-1D 98.89.
Compound 4: 96%; [a]D ꢀ39° (c 0.9, CHCl3); dH-1 4.78, J1,2
Compound 19: 97%; [a]D ꢀ26° (c 0.5, CHCl3); dH-1A 4.62,
J1,2 8.0 Hz, dH-1B 5.09 (s), dH-1C 5.07, J1,2 2.0 Hz, dH-1D
ꢁ4.66, ꢁ1.9 Hz;dC-1A 103.69, dC-1B 100.34, dC-1C 98.94,
dC-1D 98.86.
1.6 Hz; dC-1 98.83.
Compound 7: 88%; dH-1 5.98, J1,2 1.6 Hz; dC-1 90.97.
Compound 8: 63%; [a]D ꢀ42° (c 0.4, CHCl3); d
5.10, J1,2
H-1C
1.6 Hz, dH-1D 4.67, J1,2 not determined due to overlap of
signals; dC-1C 99.16, dC-1D 98.79.
Compound 20: 83%; [a]D ꢀ52.6° (c 0.5, H2O).