Synthesis of a spore surface pentasaccharide of Bacillus anthracis

Article abstract of DOI:10.1002/ejoc.200601082

An analogue 2 of the pentasaccharide 1 found on the spore surface protein BclA of Bacillus anthracis was synthesized by a (3+2) glycosylation approach. A robust building block for 3-linked α-rhamnose is presented. Benzoate groups were used to ensure α-sel

Full text of DOI:10.1002/ejoc.200601082

FULL PAPER
DOI: 10.1002/ejoc.200601082
Synthesis of a Spore Surface Pentasaccharide of Bacillus anthracis
Daniel B. Werz,[a][‡] Alexander Adibekian,[a] and Peter H. Seeberger*[a]
Keywords: Anthrose / Bacillus anthracis / Carbohydrates / Oligosaccharides / Total synthesis
An analogue 2 of the pentasaccharide 1 found on the spore
surface protein BclA of Bacillus anthracis was synthesized by
a (3+2) glycosylation approach. A robust building block for
3-linked α-rhamnose is presented. Benzoate groups were
used to ensure α-selectivity for the rhamnose units. A radical-
initiated reduction with tributyltin hydride was shown to be
able to convert the NHTCA group into an acetylated amine
as well as an azido group into a free amine on one strike.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
thracis spores and to develop a vaccine candidate. We syn-
thesized the tetrasaccharide[5] and used the antigen to create
anti-carbohydrate antibodies.[6] These antibodies are able to
detect B. anthracis endospores in a highly selective man-
ner.[6] Following our initial report, two other groups
achieved the synthesis of this tetrasaccharide[7] and corre-
sponding sequences.[8]
Introduction
Recent bioterrorism attacks have emphasized the need
for fast and efficient methods to detect and reliably identify
biowarfare agents. Anthrax is one of these highly infectious
agents and is caused by the Gram-positive spore-forming
soil bacterium Bacillus anthracis.[1,2] The spores, the durable
form of the pathogen, are remarkably resistant to physical
stress such as extreme temperatures, radiation, harsh chemi-
cals, desiccation and physical damage. These properties en-
able the spores to persist in the soil for many years.[3] Inha-
lation of specially prepared spores will kill most victims if
they are not treated within 24–48 h. In autumn 2001 several
cases of anthrax infections were the result of the intentional
release of spores by placement of contaminated letters in
the mail. The death of five people resulted in a widespread
panic and brought the US postal system to the brink of
collapse. These cases demonstrated the danger of anthrax
as a biowarfare agent to terrorize civilian populations. As a
result of these attacks a more thorough examination of the
mechanisms underlying the pathogenicity, the detection and
treatment of organisms such as B. anthracis began.
In 2004, a unique tetrasaccharide portion of the major
glycoprotein BclA from the surface of B. anthracis spores
was discovered.[4] The non-reducing end of this carbo-
hydrate is capped with a highly specific monosaccharide
that was named anthrose. This monosaccharide moiety has
not been found on the spores of other Bacillus strains in-
cluding the closest relatives such as B. cereus and B. thuring-
iensis.[4] Therefore, the tetrasaccharide served as an attract-
ive target to create antibodies for the detection of B. an-
Results and Discussion
The tetrasaccharide that has been the focus of several
syntheses to date is part of pentasaccharide 1 containing
an additional galactosamine residue, presumably by an α-
linkage.[4,9] This N-acetylated galactosamine serves to con-
nect the oligosaccharide to the surface protein BclA. Here,
we describe the first synthesis of the pentasaccharide 2 via
a convergent (3+2) approach. The key challenge associated
with the synthesis of 2 is the differentiation of the two
amino groups in the target molecule. One amine bears an
acetyl moiety while the amine on the terminal anthrose is
attached to a β-hydroxy carboxylic acid. Trichloroacetyl
(TCA) was chosen to mask the amine of galactosamine
whereas an azide was taken to mask the amino group on
anthrose (Figure 1). This protecting group pattern allows
for the assembly of the pentameric structure prior to amine
functionalization.
To improve on the previous synthesis,[5] we modified the
Fmoc-protected rhamnose building block. The sterically
more demanding benzoate (Bz) group replaced the acetate
(Ac) as participating group to ensure α-selectivity while at
the same time decreases unwanted rearrangements,[10] and
thus separation problems. The transformation of known
acetal 3[5] via diol 4 and the corresponding orthoester fol-
lowed by subsequent ring-opening resulted in the kinetically
favored axial benzoate 5. Placement of the base-labile fluor-
enylmethoxycarbonyl (Fmoc) group,[11] cleavage of the an-
omeric p-methoxyphenyl (MP) glycoside and reaction of
[a] Laboratory for Organic Chemistry, Swiss Federal Institute of
Technology (ETH) Zürich, ETH-Zürich, HCI F 315,
Wolfgang-Pauli-Str. 10, 8093 Zürich, Switzerland
E-mail: seeberger@org.chem.ethz.ch
[‡] Present address: Institut für Organische und Biomolekulare
Chemie der Georg-August-Universität Göttingen
Tammannstr. 2, 37077 Göttingen, Germany
1976
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 1976–1982

Synthesis of a Spore Surface Pentasaccharide of Bacillus anthracis
FULL PAPER
at C2. The commonly used maneuver[12] to convert the
methoxyphenyl glycoside into the corresponding trichlo-
roacetimidate yielded disaccharide unit 12.
Figure 1. Structure of pentasaccharide 1 attached to the spore sur-
face protein BclA of B. anthracis and analogue 2 that was synthe-
sized.
the formed hemiacetal with trichloroacetonitrile in the pres-
ence of traces of sodium hydride afforded building block 7
(Scheme 1).[12]
Scheme 2. Synthesis of disaccharide building block 12. Reagents
and conditions: a) 8, TMSOTf, CH2Cl2 , 0 °C, 1 h, 98 %;
b) N2H4·AcOH, CH2Cl2/MeOH (5:1), 25 °C, 12 h, 83%; c) NaH,
MeI, DMF, 1 h, 25 °C, 81%; d) 1) CAN, H3CCN/H2O (1:1), 25 °C,
85%, 2) Cl3CCN, CH2Cl2, NaH (cat.), 25 °C, 90 %.
For the trisaccharide part galactosamine building block
13[14] was treated with 4-penten-1-ol (Scheme 3). The reac-
tion with the highly reactive pentenol did not proceed with
complete β-selectivity as approximately 20% of the corre-
sponding α-anomer was obtained as well. This mixture of
anomers was subjected to the subsequent reactions since
separation could not easily be achieved at this stage. Re-
moval of Fmoc, glycosidation with rhamnose building
block 14[15] and subsequent removal of acetate by sodium
methoxide yielded disaccharide 15.
Scheme 1. Synthesis of rhamnose building block 7. Reagents and
conditions: a) HCl (pH 3), MeOH/H2O (10:1), 45 °C, 89 %;
b) PhC(OMe)3, CSA (cat.), CH2Cl2, 25 °C, 2 h; c) AcOH/H2O (4:1,
v/v), 30 min, 90% (2 steps); d) FmocCl, pyridine, 25 °C, 2 h, 88 %;
e) CAN, H2O/CH3CN (1:1), 25 °C, 2 h, 86%; f) Cl3CCN, CH2Cl2,
NaH (cat.), 25 °C, 2 h, 85 %.
Union of 15 and 7 furnished trisaccharide 16, before
Fmoc cleavage exposed the hydroxyl in 17. Even the use of
benzoate groups could not suppress completely the transes-
terification[10,16] of benzoate to position 3 during Fmoc de-
The assembly of the pentasaccharide was performed via protection. Best conditions for Fmoc removal[17] involved
a (3+2) approach. The terminal disaccharide unit was as- the use of triethylamine in an apolar solvent such as dichlo-
sembled by glycosidation of rhamnose 5, an intermediate in romethane. The commonly used protocol relying on piperi-
the synthesis of building block 7, with the known anthrose dine (20% in DMF) afforded less of the desired trisaccha-
8 (Scheme 2). The levulinoyl (Lev) group,[13] which ensured ride 17. Fully protected pentasaccharide 18 was obtained
β-selectivity, was replaced by the final methoxy substituent by coupling trisaccharide 17 and disaccharide 12.
Eur. J. Org. Chem. 2007, 1976–1982
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1977

D. B. Werz, A. Adibekian, P. H. Seeberger
FULL PAPER
Scheme 3. Assembly of pentasaccharide 18. Reagents and conditions: a) 4-penten-1-ol, TMSOTf, CH2Cl2, 4-Å mol. sieves, –30 °C, 90 min,
70%; b) 20% piperidine in DMF, 25 °C, 1 h, quant.; c) 14, TMSOTf, CH2Cl2, 0 °C, 90 min, 77%; d) NaOMe, MeOH, 25 °C, 12 h, 91%;
e) 7, TMSOTf, CH2Cl2, 0 °C, 90 min, 61%; f) NEt3, CH2Cl2, 25 °C, 4 h, 89%; g) 12, TMSOTf, CH2Cl2, –2 °C, 90 min, 73%.
Prior to the removal of all permanent protecting groups, multaneous reduction of the azide to a free amine. Excess
the N-trichloroacetyl group on galactosamine had to be tributyltin hydride under radical-forming conditions
transformed into an N-acetyl. The azide on the terminal achieved this goal.
anthrose unit had to be reduced to a free amine, followed
by acylation with 3-hydroxy-3-methylbutanoic acid. Both
Experimental Section
reductions were achieved in one step (Scheme 4). The radi-
cal-initiated reaction of excess tributyltin hydride in toluene
at 100 °C converted the NHTCA group of 18 into the de-
sired NHAc group and reduced the azide to a free amine
moiety.[18] Potential problems with the β-hydroxy carboxylic
acid due to sensitivity to strong bases caused us to remove
the benzoate groups in the next step affording 19. The ad-
dition of excess butylamine is required to trap the cleaved
benzoate groups that would react otherwise with the free
amine on anthrose. Without further purification of 19,
amide bond formation was induced using HATU and
Hünig’s base to attach the 3-hydroxy-3-methylbutanoic acid
to the amine.[19] Purification by reversed-phase HPLC
yielded 20. Even at this stage the separation of the α/β mix-
ture at the anomeric carbon carrying the linker was not
possible.[20] Hydrogenolysis of 20 furnished the pentasac-
charide target compound 2.
General Methods: All chemicals used were reagent grade and used
as supplied except where noted. Dichloromethane (CH2Cl2) was
purchased from JT Baker and purified by a Cycle-Tainer Solvent
Delivery System. Pyridine and acetonitrile were refluxed over cal-
cium hydride and distilled prior to use. Analytical thin-layer
chromatography was performed on E. Merck silica gel 60 F254
plates (0.25 mm). Compounds were visualized by dipping the plates
in a cerium sulfate ammonium molybdate solution or a sulfuric
acid/methanol solution (for fully deprotected compounds) followed
by heating. Liquid chromatography was performed using forced
flow of the indicated solvent on Sigma H-type silica (10–40 mm).
HPLC purifications were performed by a Waters system 2420,
using a reversed-phase C18 column. 1H NMR spectra were ob-
tained on a Varian VXR-300 (300 MHz), Bruker-600 (600 MHz),
and are reported in parts per million (δ) relative to CHCl3 (δ =
7.26 ppm) or in the case of CD3OD as solvent relative to TMS (δ
= 0.00 ppm). Coupling constants (J) are reported in Hertz (Hz).
13C NMR spectra were obtained on a Varian VXR-300 (75 MHz),
Bruker-600 (150 MHz) and are reported in δ relative to CDCl3 (δ
= 77.0 ppm) as an internal reference or to TMS (δ = 0.00 ppm).
For α/β mixtures we abstained from measuring [α]D values.
Conclusions
We have reported the first total synthesis of the pentasac-
charide attached to BclA spore surface protein of Bacillus
anthracis by utilizing a convergent (3+2) approach. Key to
differentiating the two amino groups is the one-pot conver-
4-Methoxyphenyl 4-O-Benzyl-α-L-rhamnopyranoside (4): Rhamno-
side 3 (9.38 g, 23.45 mmol, 1.0 equiv.) was dissolved in MeOH
(100 mL) and H2O (10 mL). A few drops of an aqueous solution
of hydrochloric acid (0.1 ) were added until pH 3 is reached. The
sion of an N-trichloroacetyl into an N-acetyl group and si- mixture was heated to 45 °C and stirred for 2 d until TLC indicated
1978
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Eur. J. Org. Chem. 2007, 1976–1982

Synthesis of a Spore Surface Pentasaccharide of Bacillus anthracis
FULL PAPER
(hexane/EtOAc, 4:1 Ǟ 2:1) yielded 5.25 g (90% over 2 steps) of 5
as a yellow oil; Rf (SiO2, cyclohexane/EtOAc, 3:1) = 0.30. [α]D
=
–85.3 (c = 0.48, CHCl3). 1H NMR (300 MHz, CDCl3): δ = 1.38 (d,
J = 6.3 Hz, 3 H), 2.18 (m, 1 H), 3.55 (dt, J = 2.4, 9.4 Hz, 1 H),
3.77 (s, 3 H), 3.98 (m, 1 H), 4.43 (m, 1 H), 4.78 (d, J = 10.8 Hz, 1
H), 4.87 (d, J = 10.8 Hz, 1 H), 5.49 (s, 1 H), 5.53 (m, 1 H), 6.82
(d, J = 9.0 Hz, 2 H), 6.99 (d, J = 9.0 Hz, 2 H), 7.31–7.38 (m, 5 H),
7.46–7.52 (m, 2 H), 7.59–7.64 (m, 2 H), 8.07 (m, 1 H) ppm.
13C NMR (75 MHz, CDCl3): δ = 18.1, 55.5, 68.1, 70.2, 73.0, 75.1,
81.5, 96.4, 114.5, 117.6, 127.9, 127.9, 128.4, 128.5, 129.5, 129.8,
133.4, 138.0, 150.0, 154.9, 166.1 ppm. MALDI-HRMS: m/z [M +
Na]+ calcd. 487.1727, obsd. 487.1719.
4-Methoxyphenyl 2-O-Benzoyl-4-O-benzyl-3-O-fluorenylmethoxy-
carbonyl-α-
L-rhamnopyranoside (6): Rhamnoside
5
(5.25 g,
11.3 mmol, 1.0 equiv.) was dissolved in pyridine (60 mL). FmocCl
(5.87 g, 22.7 mmol, 2.0 equiv.) was added in one portion and the
mixture stirred for 2 h at room temperature. Pyridine was removed
in vacuo and the residue was adsorbed on silica gel. Column
chromatography on silica gel (hexane/EtOAc, 5:1 Ǟ 4:1 Ǟ 3:1)
yielded 6.21 g (88%) of 6 as a colorless solid; Rf (SiO2, cyclohexane/
EtOAc, 3:1) = 0.53. [α]D = –31.2 (c = 0.77, CHCl3). 1H NMR
(300 MHz, CDCl3): δ = 1.40 (d, J = 6.3 Hz, 3 H), 3.48 (d, J =
4.2 Hz, 1 H), 3.78 (s, 3 H), 3.78 (t, J = 9.6 Hz, 1 H), 4.11 (m, 1 H),
4.57 (m, 1 H), 4.72 (d, J = 12.0 Hz, 1 H), 4.89 (d, J = 11.1 Hz, 1
H), 5.53 (m, 2 H), 5.83 (m, 1 H), 6.84 (d, J = 9.0 Hz, 2 H), 7.04
(d, J = 9.0 Hz, 2 H), 7.18–7.40 (m, 10 H), 7.50–7.60 (m, 4 H), 7.66
(m, 1 H), 7.75 (d, J = 7.5 Hz, 2 H), 8.12 (d, J = 7.2 Hz, 2 H) ppm.
13C NMR (75 MHz, CDCl3): δ = 18.2, 46.7, 55.7, 68.3, 70.3, 70.4,
75.3, 76.2, 78.5, 96.4, 114.5, 117.7, 119.9, 119.9, 125.0, 125.3, 127.0,
127.1, 127.7, 127.8, 127.8, 128.3, 128.5, 129.4, 130.0, 133.4, 137.7,
141.1, 141.2, 143.0, 143.5, 149.9, 154.1, 155.0, 165.4 ppm. MALDI-
HRMS: m/z [M + Na]+ calcd. 709.2408, obsd. 709.2436.
Scheme 4. Completion of the total synthesis of 2. Reagents and
conditions: a) Bu3SnH (excess), AIBN (cat.), toluene, 100 °C, 2 h,
54%; b) NaOMe, BuNH2 (excess), MeOH, 25 °C, 18 h, 73%; c) 3-
hydroxy-3-methylbutanoic acid, HATU, DIPEA, DMF, 25 °C, 4 h,
44% (after HPLC); d) Pd/C, MeOH/THF/H2O (4:4:1), H2, 25 °C,
18 h, 63%.
2-O-Benzoyl-4-O-benzyl-3-O-fluorenylmethoxycarbonyl-α-L-rham-
nopyranosyl Trichloroacetimidate (7): Rhamnoside 6 (1.91 g,
2.8 mmol, 1.0 equiv.) was dissolved in a mixture of acetonitrile
(35 mL) and water (35 mL). Cerium ammonium nitrate (4.58 g,
8.3 mmol, 3.0 equiv.) was added and the mixture was vigorously
stirred for 2 h at room temperature. After TLC indicated complete
removal of the MP group, water (50 mL) and EtOAc (100 mL) were
added. The phases were separated, the organic phase was washed
twice with water (50 mL each), then with brine (50 mL), and again
with water (50 mL). The organic phase was dried with Na2SO4,
and concentrated. The resulting crude product (deeply yellow) was
purified by column chromatography on silica gel (hexane/EtOAc,
3:1 Ǟ 2:1) to afford 1.38 g (86%) of the corresponding hemiacetal
as an orange foam. This compound (1.34 g, 2.3 mmol, 1.0 equiv.)
was dissolved in CH2Cl2 (15 mL) and trichloroacetonitrile (6.70 g,
46.2 mmol, 20.0 equiv.). A catalytic amount of sodium hydride (ca.
10 mg) was added and the mixture was stirred for 2 h at room tem-
perature. The solvent was removed in vacuo and the residue puri-
fied by flash column chromatography on silica gel (hexane/EtOAc,
2:1) to afford 1.42 g (85%) of 7 as a slightly yellow oil; Rf (SiO2,
cyclohexane/EtOAc, 3:1) = 0.64. [α]D = –24.6 (c = 1.00, CHCl3).
1H NMR (300 MHz, CDCl3): δ = 1.49 (d, J = 6.3 Hz, 3 H), 3.83
(t, J = 9.6 Hz, 1 H), 4.20 (m, 1 H), 4.26 (m, 2 H), 4.61 (m, 1 H),
4.74 (d, J = 10.8 Hz, 1 H), 4.91 (d, J = 11.1 Hz, 1 H), 5.41 (dd, J
= 3.0, 9.6 Hz, 1 H), 5.91 (m, 1 H), 6.42 (d, J = 1.8 Hz, 1 H), 7.16–
7.43 (m, 9 H), 7.52–7.60 (m, 4 H), 7.68 (m, 1 H), 7.76 (d, J =
7.5 Hz, 2 H), 8.13 (d, J = 6.9 Hz, 2 H), 8.77 (s, 1 H) ppm. 13C NMR
(CDCl3, 75 MHz): δ = 18.3, 29.8, 46.7, 68.9, 70.3, 70.7, 75.5, 76.0,
77.8, 94.9, 119.9, 119.9, 125.0, 125.3, 127.0, 127.1, 127.7, 127.8,
128.0, 128.1, 128.4, 128.5, 129.1, 130.0, 133.6, 137.4, 141.1, 141.2,
complete conversion. The mixture was extracted with EtOAc
(250 mL), washed twice with NaHCO3 solution (100 mL each),
dried with Na2SO4, and concentrated. The resulting crude product
was purified by flash column chromatography on silica gel (hexane/
EtOAc, 2:1 Ǟ 1:1) to afford 7.52 g (89%) of 4 as a colorless pow-
der: Rf (SiO2, cyclohexane/EtOAc, 2:1) = 0.11. [α]D = –109.5 (c =
0.86, CHCl3). 1H NMR (300 MHz, CDCl3): δ = 1.33 (d, J =
6.3 Hz, 3 H), 3.10 (br. s, 1 H), 3.44–3.47 (s, 2 H), 3.75 (s, 3 H), 3.88
(m, 1 H), 4.13 (br. s, 2 H), 4.71–4.84 (m, 2 H), 5.39 (d, J = 6.6 Hz,
1 H), 6.81 (d, J = 9.0 Hz, 2 H), 6.96 (d, J = 9.0 Hz, 2 H), 7.28–
7.37 (m, 5 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 18.2, 55.7,
68.0, 71.1, 71.4, 75.1, 81.5, 98.2, 114.6, 117.5, 127.8, 128.5, 138.1,
150.1, 154.7 ppm. MALDI-HRMS: m/z [M
+
Na]+ calcd.
383.1465, obsd. 383.1459.
4-Methoxyphenyl 2-O-Benzoyl-4-O-benzyl-α-
L-rhamnopyranoside
(5): Rhamnoside 4 (4.51 g, 12.5 mmol, 1.0 equiv.) was dissolved in
CH2Cl2 (100 mL). (Trimethoxymethyl)benzene (4.62 g, 25.4 mmol,
2.0 equiv.) was added in one portion and a catalytic amount of
CSA was added as well. The reaction mixture was stirred for 2 h
at room temperature until the TLC indicated complete conversion
[Rf (SiO2, cyclohexane/EtOAc, 3:1) = 0.64]. The solvent was re-
moved in vacuo and the residue was dissolved in 80% AcOH
(150 mL). The reaction mixture was stirred for 30 min at room tem-
perature. The solvent was removed in vacuo and the residue was
coevaporated with toluene. Column chromatography on silica gel
Eur. J. Org. Chem. 2007, 1976–1982
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1979

D. B. Werz, A. Adibekian, P. H. Seeberger
CHCl ). IR (thin film, CHCl ): ν = 3008, 2110, 1719, 1603, 1505,
FULL PAPER
142.9, 143.5, 154.0, 160.0, 165.2 ppm. MALDI-HRMS: m/z [M +
˜
3
3
Na]+ calcd. 746.1086, obsd. 746.1073.
1458, 1364 cm–1. 1H NMR (CDCl3, 300 MHz): δ = 1.29 (d, J =
5.7 Hz, 3 H), 1.40 (d, J = 6.0 Hz, 3 H), 3.05–3.24 (m, 3 H), 3.39
(t, J = 9.3 Hz, 1 H), 3.57 (s, 3 H), 3.76 (m, 3 H), 3.79 (s, 1 H), 4.02
(m, 1 H), 4.51 (dd, J = 3.3, 9.6 Hz, 1 H), 4.68–4.93 (m, 4 H), 5.07
(d, J = 10.5 Hz, 1 H), 5.53 (m, 1 H), 5.59 (m, 1 H), 6.85 (d, J =
9.0 Hz, 2 H), 7.03 (d, J = 9.0 Hz, 2 H), 7.26–7.47 (m, 12 H), 7.62
(m, 1 H), 8.13 (m, 2 H) ppm. 13C NMR (CDCl3, 75 MHz): δ =
18.0, 18.2, 55.5, 60.5, 67.4, 68.2, 70.4, 72.9, 75.0, 75.2, 76.2, 80.7,
82.7, 84.3, 96.2, 103.0, 114.5, 117.7, 127.8, 127.9, 128.1, 128.2,
128.3, 128.4, 129.7, 130.1, 133.1, 137.9, 138.0, 150.0, 155.0, 165.7
ppm. MALDI-HRMS: m/z [M + Na]+ calcd. 762.2997, obsd.
762.2982.
4-Methoxyphenyl 4-Azido-3-O-benzyl-4,6-dideoxy-2-O-levulinoyl-β-
D-glucopyranosyl-(1Ǟ3)-2-O-benzoyl-4-O-benzyl-α-L-rhamnopyra-
n
o
-
side (9): Rhamnose 5 (360 mg, 0.775 mmol, 1.0 equiv.) and
anthrose trichloroacetimidate 8 (485 mg, 0.280 mmol, 1.2 equiv.)
were codistilled three times with toluene and dried for 30 min in
vacuo. The mixture was dissolved in CH2Cl2 (10 mL) and cooled
to 0 °C. TMSOTf (26 mg, 21 µL, 0.116 mmol, 0.15 equiv.) was
added, the solution stirred for 1 h and quenched by addition of
some drops of pyridine. The solvent was removed and the resulting
crude product was purified by column chromatography (hexane/
EtOAc, 3:1) to afford 630 mg (98 %) of 9 as a colorless oil.
1H NMR (CDCl3, 300 MHz): δ = 1.21 (d, J = 6.0 Hz, 3 H), 1.29
(d, J = 6.3 Hz, 3 H), 2.00 (s, 3 H), 2.17–2.35 (m, 4 H), 3.12 (t, J =
9.6 Hz, 1 H), 3.27 (m, 1 H), 3.49 (t, J = 9.3 Hz, 1 H), 3.67 (t, J =
9.6 Hz, 1 H), 3.78 (s, 3 H), 3.94 (m, 1 H), 4.37 (dd, J = 3.3, 9.3 Hz,
1 H), 4.64–4.76 (m, 4 H), 4.92 (d, J = 11.7 Hz, 1 H), 5.04 (t, J =
8.4 Hz, 1 H), 5.50 (m, 2 H), 6.83 (d, J = 9.3 Hz, 2 H), 7.01 (d, J =
9.3 Hz, 2 H), 7.25–7.37 (m, 10 H), 7.48 (t, J = 7.5 Hz, 2 H), 7.60
(t, J = 7.5 Hz, 1 H), 8.05 (d, J = 8.7 Hz, 2 H) ppm. 13C NMR
(CDCl3, 75 MHz): δ = 17.8, 18.0, 27.6, 29.5, 37.3, 55.5, 67.3, 68.1,
70.7, 71.9, 73.5, 74.5, 74.8, 78.2, 79.7, 81.1, 96.1, 100.4, 114.5,
117.7, 127.2, 127.5, 127.8, 128.0, 128.3, 129.8, 130.0, 133.0, 137.4,
138.4, 150.0, 155.0, 166.0, 171.2, 206.0 ppm. MALDI-HRMS: m/z
[M + Na]+ calcd. 846.3209, obsd. 846.3193.
4-Azido-3-O-benzyl-4,6-dideoxy-2-O-methyl-β-
D-glucopyranosyl-
(1Ǟ3)-2-O-benzoyl-4-O-benzyl-α- -rhamnopyranosyl Trichloroacet-
L
imidate (12): Compound 11 (324 mg, 0.438 mmol, 1.0 equiv.) was
dissolved in a mixture of acetonitrile and water (14 mL/14 mL),
cerium ammonium nitrate (841 mg, 1.534 mmol, 3.5 equiv.) was
added in one portion and the mixture was stirred for 2 h until TLC
indicated complete conversion into the hemiacetal. The solution
was poured into brine and the aqueous phase was extracted twice
with EtOAc. The combined organic phases were washed twice with
water, dried with Na2SO4 and concentrated. The resulting crude
product was purified by column chromatography (hexane/EtOAc,
2:1) to yield 235 mg (85%) of the hemiacetal. The hemiacetal was
dissolved in CH2Cl2 (3 mL) and trichloroacetonitrile (3 mL). A
catalytic amount of sodium hydride (8 mg) was added and the mix-
ture was stirred for 60 min. The solvent was removed in vacuo and
the residue was purified by column chromatography on silica gel
(hexane/EtOAc, 3:1) to afford 259 mg (90%) of 11 as a colorless
4-Methoxyphenyl 4-Azido-3-O-benzyl-4,6-dideoxy-β-
syl-(1Ǟ3)-2-O-benzoyl-4-O-benzyl-α- -rhamnopyranoside (10):
D-glucopyrano-
L
Compound 9 (630 mg, 0.764 mmol, 1.0 equiv.) was dissolved in
CH2Cl2 (25 mL) and hydrazinium acetate (137 mg, 1.492 mmol,
1.95 equiv.) in 5 mL of MeOH was added at room temperature and
stirred for 12 h until mass spectrometry indicated complete conver-
sion. The solvent was evaporated and the crude product was puri-
fied by column chromatography (3:1 hexane/EtOAc) to afford
501 mg (83%) of 10 as a colorless oil; [α]D = 13.5 (c = 0.29, CHCl3).
oil; [α]D = 30.4 (c = 0.25, CHCl ). IR (thin film, CHCl ): ν = 3026,
˜
3
3
2923, 2103, 1723, 1672, 1600, 1451, 1267, 1092 cm–1 1H NMR
.
(CDCl3, 300 MHz): δ = 1.22 (d, J = 5.7 Hz, 3 H), 1.43 (d, J =
6.0 Hz, 3 H), 3.04–3.16 (m, 3 H), 3.37 (t, J = 9.0 Hz, 1 H), 3.52 (s,
3 H), 3.74 (t, J = 9.6 Hz, 1 H), 4.02 (m, 1 H), 4.38 (dd, J = 3.3,
9.6 Hz, 1 H), 4.63 (d, J = 8.1 Hz, 1 H), 4.71 (d, J = 10.2 Hz, 1 H),
4.80 (d, J = 10.5 Hz, 1 H), 4.88 (d, J = 10.8 Hz, 1 H), 5.02 (d, J =
9.9 Hz, 1 H), 5.57 (m, 1 H), 6.35 (m, 1 H), 7.28–7.51 (m, 12 H),
7.62 (m, 1 H), 8.09 (m, 2 H), 8.71 (s, 1 H) ppm. 13C NMR (CDCl3,
75 MHz): δ = 17.9, 18.1, 60.4, 67.3, 70.4, 70.6, 71.9, 75.2, 75.3,
75.5, 80.0, 82.5, 84.3, 90.8, 94.7, 103.1, 127.8, 128.1, 128.2, 128.3,
128.4, 128.5, 129.7, 133.2, 137.7, 137.8, 160.0, 165.4 ppm. MALDI-
HRMS: m/z [M + Na]+ calcd. 799.1675, obsd. 799.1661.
IR (thin film, CHCl ): ν = 3035, 2113, 1718, 1595, 1508, 1451,
˜
3
1
1262, 1097, 1036 cm–1. H NMR (CDCl3, 300 MHz): δ = 1.26 (d,
J = 5.7 Hz, 3 H), 1.38 (d, J = 6.3 Hz, 3 H), 2.47 (s, 1 H), 3.07 (t,
J = 9.6 Hz, 1 H), 3.25 (s, 1 H), 3.37 (t, J = 9.2 Hz, 1 H), 3.50 (m,
1 H), 3.73 (m, 1 H), 3.78 (s, 3 H), 4.00 (m, 1 H), 4.45 (dd, J = 3.3,
9.3 Hz, 1 H), 4.52 (d, J = 7.5 Hz, 1 H), 4.74–4.98 (m, 4 H), 5.50
(m, 1 H), 5.58 (m, 1 H), 6.84 (d, J = 9.3 Hz, 2 H), 7.01 (d, J =
9.3 Hz, 2 H), 7.33–7.46 (m, 10 H), 5.50 (m, 2 H), 7.61 (m, 1 H),
8.09 (d, J = 7.2 Hz, 2 H) ppm. 13C NMR (CDCl3, 75 MHz): δ =
18.3, 18.4, 55.7, 67.2, 68.4, 70.9, 72.5, 74.9, 75.1, 75.5, 77.8, 80.4,
82.5, 96.3, 103.0, 114.5, 117.7, 127.8, 128.0, 128.0, 128.1, 128.3,
128.3, 128.5, 129.8, 130.0, 133.1, 137.8, 137.9, 149.9, 154.9, 165.7
ppm. MALDI-HRMS: m/z [M + Na]+ calcd. 748.2841, obsd.
748.2828.
Pent-4-enyl 3,4-Di-O-benzyl-α-
benzyl-2-N-trichloroacetyl-α/β-
L
-rhamnopyranosyl-(1Ǟ3)-4,6-di-O-
D-galactosaminopyranoside (15): Ga-
lactosamine building block 13 (367 mg, 0.399 mmol, 1.0 equiv.) was
codistilled three times with toluene and dried in vacuo for 30 min.
CH2Cl2 (8 mL) was added as well as 4-pentenol (69 mg, 82 µL,
0.798 mmol, 2.0 equiv.) and 4 Å molecular sieves. The suspension
was cooled to –30 °C. TMSOTf (86 mg, 72 µL, 0.399 mmol,
1.0 equiv.) was added and the reaction mixture stirred for 90 min.
Then, the reaction was quenched by addition of pyridine (0.1 mL).
Column chromatography on silica gel (hexane/EtOAc, 3:1) yielded
222 mg (70%, α/β = 1:4) of the pentenyl glycoside as a colorless oil
that was subsequently submitted to the next step. The Fmoc-pro-
4-Methoxyphenyl 4-Azido-3-O-benzyl-4,6-dideoxy-2-O-methyl-β-
D-
glucopyranosyl-(1Ǟ3)-2-O-benzoyl-4-O-benzyl-α- -rhamnopyrano-
L
side (11): Compound 10 (407 mg, 0.561 mmol, 1.0 equiv.) was dis-
solved in DMF (5 mL) and cooled to 0 °C. Sodium hydride (29 mg,
0.725 mmol, 1.2 equiv.) was added, then methyl iodide (161 mg, tected pentenyl glycoside (191 mg, 0.240 mmol, 1.0 equiv.) was dis-
1.134 mmol, 2.0 equiv.) was added and the mixture was stirred for
1 h. After the TLC indicated complete conversion, the reaction
mixture was poured into an acidified aqueous solution (pH 3) and
extracted twice with EtOAc. The combined organic phases were
washed with brine, dried with Na2SO4 and concentrated. Column
chromatography on silica gel (hexane/EtOAc, 3:1) was performed
to yield 335 mg (81%) of 11 as a colorless oil; [α]D = 1.8 (c = 0.38,
solved in DMF (8 mL) and piperidine (2 mL) was added. The reac-
tion mixture was stirred for 1 h. The volatiles were removed in
vacuo, the residue dissolved in CH2Cl2 and concentrated. Column
chromatography on silica gel (hexane/EtOAc, 5:1 Ǟ 2:1) yielded
137 mg (quant.) of the Fmoc-deprotected product as colorless so-
lid. This material (137 mg, 0.239 mmol, 1.0 equiv.) and rhamnosyl
phosphate 14 (221 mg, 0.383 mmol, 1.6 equiv.) were codistilled
1980
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 1976–1982

Synthesis of a Spore Surface Pentasaccharide of Bacillus anthracis
FULL PAPER
three times with toluene and dried in vacuo. CH2Cl2 (3.5 mL) was
added and the solution cooled to 0 °C. TMSOTf (85 mg, 69 µL,
0.383 mmol, 1.6 equiv.) was added and the reaction mixture was
stirred for 90 min. TLC analysis indicated one major product. The
reaction was quenched by addition of a few drops of pyridine and
the solvents were evaporated in vacuo. Column chromatography on
silica gel (hexane/EtOAc, 2:1) yielded 172 mg (77%) of the disac-
charide as a colorless oil. This oil was subsequently submitted to
the next step and was dissolved in MeOH (6 mL) and NaOMe
solution (0.5  in MeOH) was added until pH 12 was reached. The
reaction mixture was stirred overnight. The solvents were evapo-
rated and the crude product was purified by column chromatog-
raphy on silica gel (hexane/EtOAc, 3:1 Ǟ 2:1) yielding 149 mg
(91%) of 15 as a colorless oil. 1H NMR (CDCl3, 300 MHz): δ =
1.33 (d, J = 6.3 Hz, 3 H), 1.68 (m, 2 H), 2.12 (m, 2 H), 3.48 (m, 2
was removed and the resulting crude product was purified by col-
umn chromatography (hexane/EtOAc, 5:1 Ǟ 4:1 Ǟ 3:1) to afford
79 mg (73 %) of 18 as a slightly yellow oil. 1H NMR (CDCl3,
300 MHz): δ = 0.93 (d, J = 5.9 Hz, 3 H), 1.31 (m, 12 H), 1.61 (m,
2 H), 2.04 (m, 2 H), 3.01 (m, 5 H), 3.46 (s, 3 H), 3.82 (m, 12 H),
4.60 (m, 22 H), 5.12 (m, 1 H), 5.27 (m, 1 H), 5.52 (m, 1 H), 5.58
(m, 1 H), 5.76 (m, 1 H), 7.26–7.36 (m, 41 H), 8.08 (m, 4 H) ppm.
13C NMR (CDCl3, 75 MHz): δ = 17.9, 18.1, 18.2, 18.3, 18.4, 28.7,
30.1, 56.1, 60.4, 60.5, 67.3, 67.4, 68.3, 68.4, 68.6, 69.3, 70.2, 70.3,
70.8, 72.1, 72.3, 72.5, 73.0, 73.1, 73.2, 73.3, 73.6, 74.2, 74.7, 74.9,
75.0, 75.1, 75.2, 75.3, 75.5, 75.6, 75.7, 75.8, 75.9, 76.0, 76.2, 76.3,
76.6, 76.8, 76.9, 77.0, 77.3, 77.5, 80.0, 80.3, 80.5, 82.4, 84.3, 92.3,
98.1, 98.9, 99.2, 100.1, 102.8, 114.7, 127.0, 127.1, 127.2, 127.3,
127.4, 127.6, 127.7, 127.8, 127.9, 128.0, 128.1, 128.2, 128.3, 128.4,
128.5, 128.6, 128.7, 128.8, 128.9, 129.6, 129.7, 129.8, 129.9, 130.0,
H), 3.53–3.69 (m, 4 H), 3.91 (m, 4 H), 4.05 (m, 2 H), 4.32 (m, 1 130.1, 132.9, 133.0, 133.1, 137.5, 137.6, 137.7, 137.8, 137.9, 138.0,
H), 4.48 (m, 2 H), 4.63 (m, 4 H), 4.79–5.05 (m, 6 H), 5.79 (m, 1 138.1, 138.4, 138.5, 161.7, 165.2, 165.3 ppm. MALDI-HRMS: m/z
H), 6.89 (d, J = 7.8 Hz, 1 H), 7.26–7.37 (m, 20 H) ppm. 13C NMR
(CDCl3, 75 MHz): δ = 18.1, 28.8, 30.1, 56.0, 68.5, 68.7, 69.2, 72.1,
73.5, 73.6, 74.9, 75.1, 75.7, 79.6, 92.6, 99.6, 100.8, 114.9, 127.5,
127.6, 127.7, 127.9, 128.1, 128.2, 128.2, 128.4, 128.4, 137.6, 137.7,
137.8, 138.3, 161.8 ppm. MALDI-HRMS: m/z [M + Na]+ calcd.
920.2705, obsd. 920.2752.
[M + Na]+ calcd. 1875.6597, obsd. 1875.6556.
Pent-4-enyl 3-O-Benzyl-4,6-dideoxy-4-(3-hydroxy-3-methylbutana-
mido)-2-O-methyl-β-
nopyranosyl-(1Ǟ3)-4-O-benzyl-α-
benzyl-α- -rhamnopyranosyl-(1Ǟ3)-4,6-di-O-benzyl-2-N-acetyl-α/β-
-galactosaminopyranoside (20): Pentasaccharide 18 (48 mg,
D-glucopyranosyl-(1Ǟ3)-4-O-benzyl-α-
L-rham-
L-rhamnopyranosyl-(1Ǟ2)-3,4-O-
L
D
Pent-4-enyl 4-O-Benzyl-2-O-benzoyl-α-
3,4-di-O-benzyl-α- -rhamnopyranosyl-(1Ǟ3)-4,6-di-O-benzyl-2-N-
trichloroacetyl-β- -galactosaminopyranoside (17): Disaccharide 15
L-rhamnopyranosyl-(1Ǟ2)-
0.026 mmol, 1.0 equiv.), tributyltin hydride (113 mg, 103 µL,
0.388 mmol, 15.0 equiv.) and a catalytic amount of AIBN were dis-
solved in toluene (2 mL). Argon was bubbled through the solution
for 30 min. The reaction mixture was put in a preheated oil bath
of 100 °C and stirred for 2 h. Afterwards, the reaction mixture was
cooled to room temperature. The solvent was removed and the resi-
due passed through a plug of silica gel (hexane/EtOAc, 1:1 Ǟ 0:1).
MS (ESI+) shows a strong signal for 1726 [M + H]+ and 1748 [M
+ Na]+. After removal of the solvent 24 mg (54%) of crude product
were obtained. This material (22 mg, 0.013 mmol, 1.0 equiv.) was
dissolved in MeOH (4 mL), butylamine (19 mg, 25 µL,
0.255 mmol, 20.0 equiv.) was added, then a solution of NaOMe
(0.5 , 0.4 mL). The reaction mixture was stirred for 18 h. Mass
spectrometric (ESI+) analysis indicated the removal of two benzo-
ate groups. The solution was neutralized with Amberlite IR-120
acidic resin, concentrated and passed through a plug of silica gel
(CH2Cl2/MeOH, 20:1 Ǟ 10:1). The product 19 (14 mg, 73%) was
not purified further and submitted directly to amide formation.
The starting material (14 mg, 0.0092 mmol, 1.0 equiv.) was dis-
solved in CH2Cl2 (0.5 mL). HATU (5.3 mg, 0.014 mmol, 1.5 equiv.)
and ethyldiisopropylamine (2.3 mg, 3 µL, 0.0175 mmol, 1.9 equiv.)
were dissolved in a separate flask in CH2Cl2 (0.3 mL) and added
after 2 min to the solution of the pentasaccharide. The unified solu-
tion was stirred for 1 h. Mass spectrometric (ESI+) analysis showed
still starting material after 1 h. Therefore, again 3 µL of ethyldi-
isopropylamine were added and stirred for further 3 h. The solvent
was removed in vacuo. HPLC purification by reversed-phase C18
column (H3CCN/H2O, 6:4 Ǟ 0:1, 20 % iPrOH, gradient over
L
D
(120 mg, 0.134 mmol, 1.0 equiv.) and rhamnosyl trichloroacetimid-
ate 7 (146 mg, 0.201 mmol, 1.5 equiv.) were codistilled three times
with toluene and dried in vacuo. CH2Cl2 (2 mL) was added and the
solution was cooled to 0 °C. TMSOTf (85 mg, 69 µL, 0.383 mmol,
1.6 equiv.) was added and the reaction mixture was stirred for
90 min. The reaction was quenched by addition of a few drops
of pyridine and the solvents were evaporated in vacuo. Column
chromatography on silica gel (hexane/EtOAc, 5:1 Ǟ 4:1) yielded
119 mg (61%) of trisaccharide 16 as a slightly yellow oil. This oil
was subsequently submitted the to next step and dissolved in
CH2Cl2 (3 mL). NEt3 (0.2 mL) was added and the solution stirred
for 4 h at room temperature. The solvents were removed and the
crude product was purified by column chromatography on silica
gel (hexane/EtOAc, 3:1 Ǟ 2:1) yielding 74 mg (89%) of 17 as a
colorless oil. 1H NMR (CDCl3, 300 MHz): δ = 1.26–1.45 (m, 6 H),
1.66 (m, 2 H), 2.23 (d, J = 4.2 Hz, 1 H), 3.42–3.54 (m, 3 H), 3.63–
3.73 (m, 4 H), 3.83–4.03 (m, 7 H), 4.29 (m, 2 H), 4.39 (m, 2 H),
4.49–5.09 (m, 14 H), 5.50 (s, 1 H), 5.76 (m, 1 H), 7.16 (d, J =
8.1 Hz, 2 H), 7.22–7.38 (m, 25 H), 7.50 (t, J = 7.5 Hz, 2 H), 8.06
(d, J = 7.2 Hz, 2 H) ppm. 13C NMR (CDCl3, 75 MHz): δ = 18.3,
18.4, 28.8, 30.1, 56.1, 68.2, 68.4, 69.3, 70.4, 72.3, 73.2, 73.4, 73.6,
74.8, 75.0, 75.3, 75.7, 76.3, 79.0, 79.9, 81.8, 92.4, 98.4, 99.2, 100.2,
114.8, 127.5, 127.5, 127.6, 127.7, 127.7, 127.8, 128.0, 128.1, 128.2,
128.2, 128.3, 128.4, 129.6, 129.7, 129.8, 133.2, 137.7, 137.9, 138.1,
138.2, 138.4, 161.8, 166.0 ppm. MALDI-HRMS: m/z [M + Na]+
calcd. 1260.4022, obsd. 1260.4048.
1
30 min) yielded 6.5 mg (44%) of 20 as a colorless foam: H NMR
Pent-4-enyl 4-Azido-3-O-benzyl-4,6-dideoxy-2-O-methyl-β-
pyranosyl-(1Ǟ3)-2-O-benzoyl-4-O-benzyl-α- -rhamnopyranosyl-
(1Ǟ3)-2-O-benzoyl-4-O-benzyl-α- -rhamnopyranosyl-(1Ǟ2)-3,4-O-
benzyl-α- -rhamnopyranosyl-(1Ǟ3)-4,6-di-O-benzyl-2-N-trichloro-
acetyl-α/β- -galactosaminopyranoside (18): Trisaccharide acceptor
17 (72 mg, 0.058 mmol, 1.0 equiv.) and disaccharide donor 12
(77 mg, 0.099 mmol, 1.7 equiv.) were codistilled three times with
toluene and dried for 30 min in vacuo. The mixture was dissolved
D
-gluco-
(CDCl3, 300 MHz): δ = 1.23 (m, 3 H), 1.25 (m, 3 H), 1.27 (m, 3
H), 1.28 (m, 3 H), 1.29 (m, 3 H), 1.30 (m, 3 H), 1.31 (m, 2 H), 1.55
(m, 2 H), 1.86 (s, 2 H), 1.92 (s, 3 H), 2.09 (m, 2 H), 2.52 (m, 1 H),
3.16 (m, 3 H), 3.57 (s, 3 H), 3.69 (m, 16 H), 4.05 (m, 3 H), 4.46
(m, 9 H), 4.82 (m, 8 H), 5.17 (d, J = 1.3 Hz, 1 H), 5.29 (d, J =
3.4 Hz, 1 H), 5.54 (m, 1 H), 5.78 (m, 1 H), 7.15–7.32 (m, 35 H)
ppm. 13C NMR (CDCl3, 75 MHz): δ = 14.1, 17.8, 17.9, 18.0, 18.2,
18.3, 22.7, 23.1, 23.6, 28.7, 29.3, 29.4, 29.5, 29.6, 29.7, 30.0, 31.9,
L
L
L
D
in CH2Cl2 (1.5 mL) and cooled to –2 °C. TMSOTf (3.2 mg, 3 µL, 33.4, 47.9, 55.4, 55.8, 60.6, 68.0, 68.3, 68.7, 68.9, 69.4, 70.6, 70.8,
0.015 mmol, 0.25 equiv.) was added, the solution stirred for 90 min
and quenched by addition of some drops of pyridine. The solvent
70.9, 72.1, 72.2, 72.3, 73.3, 73.4, 73.6, 73.9, 74.8, 74.9, 75.3, 76.2,
76.8, 77.0, 77.2, 78.8, 79.4, 79.8, 80.0, 80.2, 80.9, 84.4, 84.6, 98.9,
Eur. J. Org. Chem. 2007, 1976–1982
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1981

D. B. Werz, A. Adibekian, P. H. Seeberger
FULL PAPER
[3] W. L. Nicholson, N. Munakata, G. Horneck, H. J. Melosh, P.
Setlow, Microbiol. Mol. Biol. Rev. 2000, 64, 548–572.
[4] J. M. Daubenspeck, H. Zeng, P. Chen, S. Dong, C. T. Steichen,
N. R. Krishna, D. G. Pritchard, C. L. Turnbough Jr, J. Biol.
Chem. 2004, 279, 30945–30953.
99.5, 100.2, 100.9, 103.1, 114.8, 127.4, 127.5, 127.6, 127.7, 127.8,
127.9, 128.9, 128.1, 128.2, 128.3, 128.4, 128.5, 128.6, 138.0, 138.1,
138.2, 138.4, 138.5, 138.6, 170.9, 172.3 ppm. MALDI-HRMS: m/z
[M + Na]+ calcd. 1639.7861, obsd. 1639.7904.
[5] D. B. Werz, P. H. Seeberger, Angew. Chem. 2005, 117, 6474–
6476; Angew. Chem. Int. Ed. 2005, 44, 6315–6318.
[6] M. Tamborrini, D. B. Werz, J. Frey, G. Pluschke, P. H. Seeb-
erger, Angew. Chem. 2006, 118, 6731–6732; Angew. Chem. Int.
Ed. 2006, 45, 6581–6582.
[7] a) R. Saksena, R. Adamo, P. Kovácˇ, Carbohydr. Res. 2005, 340,
1591–1600; b) R. Adamo, R. Saksena, P. Kovácˇ, Carbohydr.
Res. 2005, 340, 2579–3582; c) R. Saksena, R. Adamo, P. Kovácˇ,
Bioorg. Med. Chem. Lett. 2006, 16, 615–617; d) R. Adamo, R.
Saksena, P. Kovácˇ, Helv. Chim. Acta 2006, 89, 1075–1089.
[8] A. S. Mehta, E. Saile, W. Zhong, T. Buskas, R. Carlson, E.
Kannenberg, Y. Reed, C. P. Quinn, G.-J. Boons, Chem. Eur. J.
2006, 12, 9136–9149.
[9] C. L. Turnbough Jr, personal communication.
[10] a) A. H. Haines, Adv. Carbohydr. Chem. Biochem. 1976, 33, 11–
109; b) T. Nukada, A. Berces, D. M. Whitfield, J. Org. Chem.
1999, 64, 9030–9045; c) M. Chandrasekhar, K. L. Chandra,
V. K. Singh, J. Org. Chem. 2003, 68, 4039–4045.
[11] F. Roussel, L. Knerr, M. Grathwohl, R. R. Schmidt, Org. Lett.
2000, 2, 3043–3046.
[12] a) R. R. Schmidt, J. Michel, Angew. Chem. 1980, 92, 763–764;
Angew. Chem. Int. Ed. Engl. 1980, 19, 731–732; b) R. R.
Schmidt, J. Michel, M. Roos, Liebigs Ann. Chem. 1984, 1343–
1357.
Pentyl 4,6-Dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-
β-
D
-glucopyranosyl-(1Ǟ3)-α-
L-rhamnopyranosyl-(1Ǟ3)-α-L-rham-
nopyranosyl-(1Ǟ2)-α-
L
-rhamnopyranosyl-(1Ǟ3)-2-N-acetyl-α/β-D-
galactosaminopyranoside (2): Pentasaccharide 16 (6.5 mg,
0.004 mmol, 1.0 equiv.) was dissolved in MeOH/THF/H2O (4:4:1,
2 mL). Pd on charcoal (20 mg) was added and the argon atmo-
sphere was substituted by an H2 atmosphere. The reaction mixture
was stirred for 18 h at room temperature. MS (ESI+) experiments
showed complete conversion. The mixture was filtered through a
pad of celite. The filtrate was concentrated and the residue was
purified by a reversed-phase (C18) column chromatography (H2O/
MeOH, 1:0 Ǟ 4:1 Ǟ 3:1 Ǟ 2:1 Ǟ 1:1 Ǟ 1:2 Ǟ 1:3 Ǟ 0:1). The
product-containing fractions were concentrated and the residue
dried by lyophilization. 2.5 mg (63%) of 2 were obtained as a white
solid. 1H NMR (CD3OD, 600 MHz): δ = 0.91 (t, J = 7.2 Hz, 3 H),
1.20 (m, 3 H), 1.21 (m, 3 H), 1.23 (m, 3 H), 1.24 (m, 3 H), 1.27
(m, 3 H), 1.29 (m, 3 H), 1.41 (m, 4 H), 1.55 (m, 2 H), 1.88 (s, 2
H), 1.99 (s, 3 H), 2.34 (d, J = 13.2 Hz, 1 H), 2.38 (d, J = 13.1 Hz,
1 H), 3.01 (dd, J = 7.8, 9.0 Hz, 1 H), 3.44 (m, 11 H), 3.64 (d, J =
4.4 Hz, 1 H), 3.65 (s, 3 H), 3.73 (d, J = 7.2 Hz, 1 H), 3.76 (d, J =
3.3 Hz, 1 H), 3.85 (m, 5 H), 3.91 (d, J = 2.3 Hz, 1 H), 4.04 (dd, J
= 2.0, 3.2 Hz, 1 H), 4.17 (dd, J = 1.7, 3.2 Hz, 1 H), 4.61 (d, J =
7.8 Hz, 1 H), 4.85 (d, J = 7.8 Hz, 1 H), 4.98 (d, J = 2.1 Hz, 1 H),
5.05 (d, J = 1.6 Hz, 1 H) ppm. MALDI-HRMS: m/z [M + Na]+
calcd. 1011.4731, obsd. 1011.4712.
[13] H. J. Koeners, J. Verhoeven, J. H. van Boom, Tetrahedron Lett.
1980, 21, 381–382.
[14] B. Castagner, D. B. Werz, P. H. Seeberger, manuscript in prepa-
ration.
[15] A. Ravidà, X. Liu, L. Kovacs, P. H. Seeberger, Org. Lett. 2006,
8, 1815–1818.
[16] R. M. Rowell, Carbohydr. Res. 1972, 23, 417–424.
[17] L. A. Carpino, Acc. Chem. Res. 1987, 20, 401–407.
[18] H. H. Wasserman, R. K. Brunner, J. D. Buynak, C. G. Carter,
T. Oku, R. P. Robinson, J. Am. Chem. Soc. 1985, 107, 519–521.
[19] L. A. Carpino, A. El-Faham, J. Org. Chem. 1995, 60, 3561–
3564.
[20] The non-reducing terminal sugars are commonly most relevant
to induce a specific immune response. The mixture of anomers
at the carbon carrying the linker is not expected to influence
subsequent immunological studies: a) R. Roy, Drug Discov. To-
day: Technol. 2004, 1, 327–336; b) B. Kuberan, R. J. Linhardt,
Curr. Org. Chem. 2000, 4, 653–677.
Acknowledgments
This research was supported by Swiss Federal Institute of Technol-
ogy (ETH), Zürich, by a Feodor Lynen Research Fellowship of
the Alexander von Humboldt Foundation, by an Emmy Noether
Fellowship of the Deutsche Forschungsgemeinschaft (DFG) (both
to D. B. W.) and the Fonds der Chemischen Industrie (Kekulé Fel-
lowship to A. A.).
[1] M. Mock, A. Fouet, Annu. Rev. Microbiol. 2001, 55, 647–671.
[2] P. Sylvestre, E. Couture-Tosi, M. Mock, Mol. Microbiol. 2002,
45, 169–178.
Received: December 13, 2006
Published Online: March 2, 2007
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