Conjugation-Amenable tetrasaccharide of the side chain of the major glycoprotein of the Bacillus anthracis exosporium: A large-scale preparation

Article abstract of DOI:10.1055/s-0028-1083338

A new strategy towards the synthesis of the title tetrasac-charide is described. The novelty within the common (2+2) assembly lies in the use of a disaccharide glycosyl donor having the fully assembled anthrose as one of the constituent sugar residues. Al

Full text of DOI:10.1055/s-0028-1083338

PAPER
545
Conjugation-Amenable Tetrasaccharide of the Side Chain of the Major
Glycoprotein of the Bacillus anthracis Exosporium: A Large-Scale
Preparation
Large-Scale
Ph
u
a
Tetrasacch
j
aride ie Hou, Pavol Kováč*
NIDDK, LBC, National Institutes of Health, Bethesda, MD 20892-0815, USA
Fax +1(301)4805703; E-mail: kpn@helix.nih.gov
Received 1 September 2008; revised 27 October 2008
sugars. This minimizes the number of chemical manipula-
tions with the constructed tetrasaccharide and, together
with the fewer synthetic steps involved, renders the over-
all synthesis more efficient.
Abstract: A new strategy towards the synthesis of the title tetrasac-
charide is described. The novelty within the common (2+2) assem-
bly lies in the use of a disaccharide glycosyl donor having the fully
assembled anthrose as one of the constituent sugar residues. Also,
the final deprotection and transformation of the spacer arm into an
amine, to form a structure amenable to conjugation by different con-
jugation techniques, is a one-pot conversion. Compared to other
synthetic approaches, the present synthesis involves fewer chemical
manipulations with the assembled tetrasaccharide as well as fewer
overall numbers of synthetic steps towards this important antigenic
component of a potential conjugate vaccine for anthrax.
The assembly from smaller oligosaccharide building
blocks is often a method of choice to construct higher oli-
gosaccharides. Such blockwise strategy (2+2) was also
used in the previous synthesis4 of the tetrasaccharide se-
quence described here. There,4 the glycosyl donor for the
upstream10 disaccharide terminus contained the 4-azido-
3-O-benzyl-4,6-dideoxy-2-O-methyl-b-D-glucopyranosyl
residue, a precursor of anthrose, which was made from D-
fucose. Following the assembly of the tetrasaccharide, its
upstream terminal monosaccharide residue was trans-
formed into anthrose by a two-step conversion. Conjuga-
tion by reductive amination,11 required further chemical
manipulation with the pentenyl glycoside of the tetrasac-
charide. The synthesis presented here (Schemes 1– 3)
uses donor 6, which can be made7 from inexpensive
methyl a- or b-galactopyranoside. It is more economical
and makes tetrasaccharide 15 more readily available, as it
uses a building block 12 containing the fully assembled
anthrose. In addition, the final, one-pot deprotection ef-
fects deacetylation in the N-acyl side chain and conver-
sion of the ester group in the spacer into an amine, making
the substance ready for attachment to carriers by many
conjugation techniques.12–14
Key words: carbohydrates, oligosaccharides, glycosylations, con-
jugation, amines
The structure of the tetrasaccharide of the side chain of the
major glycoprotein of the Bacillus anthracis exosporium,
which was proposed by Daubenspeck et al.1 to be the se-
quence b-Ant-(1→3)-a-L-Rha-(1→3)-a-L-Rha-(1→2)-a-
L-Rha, was confirmed in this laboratory by chemical syn-
thesis.2 We have previously described the syntheses of the
a and b 5-methoxycarbonylpentyl glycosides of the
tetrasaccharide2,3 as well as of all structural fragments2 of
the two glycosides. A shorter (2+2)4 and a (3+1)5 synthe-
sis, as well as an asymmetric synthesis6 of the tetrasaccha-
ride sequence have also been described. Preparation of the
sequence a-L-Rha-(1→3)-a-L-Rha-(1→2)-a-L-Rha is not
a very difficult task and, thus, the chemical synthesis of
the tetrasaccharide depends largely on availability of a
suitable glycosyl donor for anthrose or its precursor. We
have recently reported7 a synthesis of a glycosyl donor for
latent anthrose that is more convenient than either our
original preparation8 or the later-developed shorter ap-
proaches.4–6,9 The availability of an anthrose precursor7
from inexpensive, commercially available starting materi-
als and the need for the tetrasaccharide in connection with
extensive studies towards a conjugate vaccine for anthrax
prompted us to develop a large-scale preparation of the se-
quence b-Ant-(1→3)-a-L-Rha-(1→3)-a-L-Rha-(1→2)-a-
L-Rha amenable to conjugation. The new (2+2) construc-
tion involves a feature that has not been attempted before,
namely the use of a disaccharide glycosyl donor contain-
ing fully assembled anthrose as one of its constituent
The spacer-equipped tetrasaccharide 13 was built up from
disaccharides 4 and 12, which were synthesized from the
common intermediate 1.15 Thus, coupling of thioglyco-
side 1 with the linker-equipped rhamnoside 22 gave disac-
charide 3, which was deacetylated to afford the
disaccharide glycosyl acceptor 4 (Scheme 1).
To obtain glycosyl donor 12, alcohol 5 was prepared from
1 as described15 and treated with the known7 imidate 6 in
the presence of trimethylsilyl trifluoromethanesulfonate
(TMSOTf), to give disaccharide 7 (89%) and a small
amount of by-product 7A (3%) (Figure 1), a product of
aglycone transfer.16
Me
O
N3
SMe
BnO
OBz
7A
SYNTHESIS 2009, No. 4, pp 0545–0550
Advanced online publication: 27.01.2009
x
x
.
x
x
.2
0
0
9
Figure 1 Molecular structure of the by-product 7A
DOI: 10.1055/s-0028-1083338; Art ID: M04708SS
© Georg Thieme Verlag Stuttgart · New York

546
S. Hou, P. Kováč
PAPER
SMe
O(CH2)5COOMe
O(CH2)5COOMe
Me
O
Me
Me
O
O
BnO
BnO
BnO
AcO
BnO
i
BnO
ii
BnO
O
O
1
91%
94%
Me
BnO
Me
BnO
O
O
AcO
O(CH2)5COOMe
HO
OBn
OBn
3
Me
O
4
BnO
BnO
HO
2
Scheme 1 Synthesis of disaccharide 4. Reagents and conditions: (i) NIS, AgOTf, 4 Å MS, CH2Cl2; (ii) NaOMe, MeOH.
SMe
Me
O
BnO
SMe
SMe
SMe
HO
OBn
O
Me
BnO
O
Me
BnO
O
Me
BnO
O
O
5
ii
O
i
iii
iv
83%
O
Me
Me
Me
94%
89%
83%
O
N3
BnO
O
OBn
O
N3
OBn
N3
BnO
OBn
BnO
Me
OBz
OMe
7
OH
8
9
N3
BnO
OCCCl3
NH
OBz
6
SMe
SMe
SMe
Me
Me
O
O
Me
BnO
O
O
O
O
BnO
BnO
Me
Me
H
N
BnO
v
85%
H
vi
93%
O
Me
O
N
O
O
OBn
O
OBn
BnO
H2N
BnO
OBn
Me
Me
Me
OAc
Me
OMe
OH
OMe
OMe
10
11
12
Scheme 2 Synthesis of glycosyl donor 12. Reagents and conditions: (i) 4 Å MS, TMSOTf, CH2Cl2, –78 °C; (ii) NaOMe, MeOH–CH2Cl2,
50 °C; (iii) KOH, MeI, DMSO; (iv) H2S, H2O–pyridine; (v) HATU, Hünig’s base, 3-hydroxy-3-methylbutanoic acid, CH2Cl2; (vi) Ac2O,
DMAP, CH2Cl2.
O(CH2)5COOMe
O(CH2)5COOMe
Me
BnO
Me
HO
O
O
BnO
O
HO
O
O
Me
BnO
Me
O
O
ii
iii
i
HO
4 + 12
85%
85%
71%
O
OBn
OH
Me
Me
O
O
BnO
Me
HO
O
Me
HO
O
H
N
H
N
O
O
O
OBn
O
OH
BnO
Me
Me
OAc
Me
Me
OAc
OMe
OMe
13
14
O(CH2)5CONH(CH2)2NH2
Me
HO
O
HO
O
Me
HO
O
O
OH
Me
O
O
HO
O
Me
H
N
O
OH
HO
Me
Me
OMe
OH
15
Scheme 3 Synthesis of tetrasaccharide 15. Reagents and conditions: (i) 4 Å MS, NIS, AgOTf, CH2Cl2, –50 °C; (ii) H2, Pd/C, MeOH–EtOAc;
(iii) H2N(CH2)2NH2, 50 °C overnight, then NaOMe, MeOH.
Synthesis 2009, No. 4, 545–550 © Thieme Stuttgart · New York

PAPER
Anthrax Tetrasaccharide: Large-Scale Preparation
547
acid was purchased from Alfa Aesar Chemical Company. Solutions
in organic solvents were dried with anhyd Na2SO4, and concentrat-
ed at 40 °C/2 kPa.
When the reaction was promoted by BF3·OEt2, a much
higher proportion of 7A was formed (this conversion is
not described in the experimental section). Compound 7
was functionalized, namely debenzoylated (Zemplén,
→ 8), methylated17 (→ 9), treated successively with H2S
(→ 10) and 3-hydroxy-3-methylbutanoic acid in the pres-
ence of HATU {N-[(dimethylamino)-1H-1,2,3-triazolo-
[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium
hexafluorophosphate N-oxide} (→ 11). The product was
acetylated to give the disaccharide glycosyl donor 12 hav-
ing the fully assembled anthrose moiety at the upstream
end (Scheme 2).
5-Methoxycarbonylpentyl 2-O-(3-O-Acetyl-2,4-di-O-benzyl-a-
L-rhamnopyranosyl)-3,4-di-O-benzyl-a-L-rhamnopyranoside
(3)
A mixture of 115 (10.81 g, 26.0 mmol), 22 (10.10 g, 21.4 mmol), and
4 Å MS (4.20 g) in CH2Cl2 (210 mL) was stirred under N2 for 15
min at r.t. After cooling to 0 °C, NIS (6.73 g, 29.9 mmol) was added,
followed by solid AgOTf (2.75 g, 10.8 mmol). Red color developed
within 5 min and, after 30 min, TLC (3:1 hexane–EtOAc) showed
that the reaction was complete. The mixture was filtered through a
Celite pad into a separatory funnel containing 10% aq Na2S2O3 (30
mL), the mixture was shaken and, after the organic phase had been
drained, the aqueous solution was extracted with CH2Cl2 (3 × 3
mL). The combined organic phase was dried, concentrated, and the
residue was chromatographed (hexane–acetone, 6:1) to afford 3;
yield: 16.08 g (91%); colorless oil; [a]D +5.65 (c 1.7, CHCl3).
Couplings of the two building blocks 4 and 12 (Scheme 3)
in the presence of N-iodosuccinimide (NIS) and AgOTf
under various conditions (these reactions are not de-
scribed in the Experimental) were accompanied by side
reactions and afforded tetrasaccharide 13 in poor yields.
1H NMR (600 MHz, CDCl3): d = 5.23 (dd, J2,3 = 3.4 Hz, J3,4 = 9.4
Hz, 1 H, H-3II), 5.08 (d, J1,2 = 2.0 Hz, 1 H, H-1II), 4.88 (d, J = 10.8
Hz, 1 H, CH2Ph), 4.70 (dd, J = 3.4, 11.2 Hz, 2 H, CH2Ph), 4.66 (d,
J1,2 = 1.8 Hz, 1 H, H-1I), 4.64–4.61 (m, 3 H, CH2Ph), 4.43–4.21
(ABq, J = 12.3 Hz, 2 H, CH2Ph), 4.02 (t, J = 2.0 Hz, 1 H, H-1I),
3.93 (dd, J2,3 = 3.4 Hz, J1,2 = 2.1 Hz, 1 H, H-2II), 3.87 ( m, 1 H, H-
5II), 3.84 (dd, J2,3 = 4.0 Hz, J3,4 = 9.4 Hz, 1 H, H-3I), 3.65 (s, 3 H,
OCH3), 3.64–3.57 (m, 3 H, H-5I, H-4II, H-1a), 3.47 (t, J = 9.5 Hz, 1
H, H-4I), 3.36–3.32 (m, 1 H, H-1b), 2.31 (t, J = 7.5 Hz, 2 H, H-5),
This situation improved considerably when the reaction
was conducted at –50 °C using AgOTf as the promoter, to
give 13 consistently in ~70% yield. It was interesting to
note that 13 was obtained in much lower yield when the
reaction was performed at –40 °C, –60 °C, –78 °C or at
ambient temperature. Debenzylation of 13 by hydrogeno-
lysis in the presence of Pd/C (→ 14) was uneventful. Sub-
sequent, overnight treatment with ethylenediamine at 50 1.93 (s, 3 H, COCH3), 1.64 (m, 2 H, H-4), 1.54 (m, 2 H, H-2), 1.37
(m, 2 H, H-3), 1.35 (d, J5,6 = 3.1 Hz, H-6II), 1.34 (d, J5,6 = 3.0 Hz,
°C effected complete conversion of the methyl ester group
in 14 to give the corresponding amino amide, but the de-
H-6I).
13C NMR (150 MHz, CDCl3): d = 174.0 (C=O), 170.0 (C=O), 99.1
(C-1II, JC-H = 171 Hz), 98.8 (C-1I, JC-H = 169 Hz), 80.4 (C-4I), 80.2
(C-3I), 79.1 (C-4II), 75.8 (C-2II), 75.4 (CH2Ph), 74.9 (C-2I), 74.8
(CH2Ph), 73.3 (C-3II), 72.7 (CH2Ph), 72.3 (CH2Ph), 68.0 (2 × C, C-
5I, C-5II), 67.1 (C-1), 51.4 (OCH3), 33.9 (C-5), 29.1 (C-2), 25.7 (C-
3), 24.7 (C-4), 21.0 (COCH3), 18.0 (C-6I), 17.8 (C-6II).
MS (TOF): m/z = 858.4 [M + NH4]+, 863.3 [M + Na]+.
HRMS (TOF): m/z [M + Na]+ calcd for C49H60O12 + Na: 863.3982;
sired, concomitant deacetylation to form 15 was incom-
plete (TLC, NMR). The latter reaction could be readily
completed under Zemplén18,19 conditions. In this way, the
two-step conversion 14 → 15 could be accomplished as a
one-pot process. The isolated product 15, obtained in 85%
yield over two steps, was in all respects identical with pre-
viously described substance.2
found: 863.3996.
Optical rotations were measured at ambient temperature with a
Jasco automatic polarimeter, Model P-2000. All reactions were
monitored by TLC on silica gel 60 gel coated glass slides. Column
chromatography was performed by elution from columns of silica
gel with CombiFlash Companion Chromatograph (Isco., Inc.). Un-
less stated otherwise, solvent mixtures less polar than those used for
TLC were used at the onset of separations. NMR spectra were mea-
sured at 300 MHz (1H) and 75 MHz (13C) with a Varian Gemini or
Varian Mercury spectrometers, or at 600 MHz (1H) and 150 MHz
(13C) with a Bruker Avance 600 spectrometer. Assignments of
NMR signals were made by hmonuclear and heteronuclear 2-di-
mensional correlation spectroscopy, run with the software supplied
with the spectrometers. When reporting assignment of NMR sig-
nals, sugar residues in oligosaccharides are serially numbered, be-
ginning with the one bearing the linker, and individual nuclei are
identified by a Roman numeral superscript. Nuclei associated with
the N-butanamido side chain and the linker are identified by Arabic
numerals, and those belonging to the aglycone linker arm are denot-
ed with a prime. Attempts were made to obtain correct combustion
analysis data for all new compounds. However, some compounds
tenaciously retained traces of solvents, despite exhaustive drying,
and analytical figures for carbon could not be obtained within
0.4%. Structures of these compounds follow unequivocally from
the mode of synthesis and NMR and MS data. Pd/C catalyst (5%,
ESCAT 103) was a product of Engelhard Industries. HATU was
purchased from Applied Biosystems. 3-Hydroxy-3-methylbutanoic
Anal. Calcd for C49H60O12: C, 69.98; H, 7.19. Found: C, 69.85; H,
7.22.
5-Methoxycarbonylpentyl 2-O-(2,4-Di-O-benzyl-a-L-rhamno-
pyranosyl)-3,4-di-O-benzyl-a-L-rhamnopyranoside (4)
NaOMe in MeOH (1 M) was added to a solution of 3 (16.08 g, 19.1
mmol) in MeOH (500 mL) until the mixture became strongly basic
to litmus. The mixture was stirred overnight at r.t., when TLC (3:1
hexane–EtOAc) showed that all starting material was converted
into a more polar product. After neutralization with Amberlite IR-
120 (H+), the solution was filtered, the filtrate was concentrated, and
the residue was chromatographed to give 4; yield: 14.5 g (94%);
colorless oil; [a]D –8.76 (c 1.2, CHCl3).
1H NMR (600 MHz, CDCl3): d = 5.15 (br s, 1 H, H-1II), 4.89 (dd,
J = 2.9, 10.7 Hz, 2 H, CH2Ph), 4.72–4.61 (m, 5 H, CH2Ph, H-1I),
4.46–4.23 (ABq, J = 12.0 Hz, 2 H, CH2Ph), 4.02 (br s, 1 H, H-2II),
3.98 (ddd, J = 3.8, 9.3 Hz, H-3II), 3.84 (dd, J = 2.9, 9.4 Hz, 1 H, H-
3I), 3.78–3.74 (m, 2 H, H-2II and H-5II), 3.65 (s, 3 H, OCH3), 3.63–
3.58 (m, 2 H, H-5I and H-1a), 3.40 (t, J = 9.4 Hz, 1 H, H-4I), 3.34
(m, 1 H, H-1b), 3.28 (t, J = 9.3 Hz, 1 H, H-4II), 2.31 (t, J = 7.5 Hz,
2 H, H-5), 2.26 (d, J = 9.4 Hz, OH-3II), 1,63 (m, 2 H, H-4), 1.54 (m,
2 H, H-2), 1.37 (m, 2 H, H-3), 1.32 (d, J5,6 = 6.2 Hz, 3 H, H-6II), 1.25
(d, J5,6 = 6.3 Hz, 3 H, H-6I).
13C NMR (150 MHz, CDCl3): d = 174.0 (C=O), 98.8 (C-1I), 98.2
(C-1II), 82.2 (C-4II), 80.5 (C-4I), 80.1 (C-3I), 78.1 (C-2II), 75.4
Synthesis 2009, No. 4, 545–550 © Thieme Stuttgart · New York

548
S. Hou, P. Kováč
PAPER
(CH2Ph), 74.9 (CH2Ph), 74.4 (C-2I), 72.6 (CH2Ph), 72.2 (CH2Ph),
71.2 (C-3II), 67.8 (C-5I), 67.5 (C-5II), 67.1 (C-1), 51.4 (OCH3), 33.9
(C-5), 29.1 (C-2), 25.7 (C-3), 24.7 (C-4), 18.0 (C-6I), 17.9 (C-6II).
MS (TOF): m/z = 816.4 [M + NH4]+, 821.3 [M + Na]+.
HRMS (TOF): m/z [M + Na]+ calcd for C47H58O11 + Na: 821.3877;
became strongly basic to litmus. The mixture was stirred overnight
at 50 °C, when TLC (4:1 hexane–EtOAc) showed that the reaction
was complete. After neutralization with Amberlite IR-120 (H+), fil-
tration, and concentration of the filtrate, chromatography gave 8;
yield: 14.5 g (94%); colorless oil.
1H NMR (300 MHz, CDCl3): d = 5.03 (d, J1,2 = 1.3 Hz, 1 H, H-1I),
4.92–4.60 (m, 6 H, CH2Ph), 4.43 (d, J1,2 = 7.7 Hz, 1 H, H-1II), 4.01–
3.95 (m, 2 H, H-5I and H-3I), 3.92 (dd, J1,2 = 1.3 Hz, J2,3 = 3.0 Hz,
1 H, H-2I), 3.65 (t, J = 9.4 Hz, 1 H, H-4I), 3.55 (ddd, J2,OH = 2.2 Hz,
1 H, H-2II), 3.38 (t, J = 9.0 Hz, 1 H, H-3II), 3.21 (m, 1 H, H-5II), 3.10
(t, J = 9.4 Hz, 1 H, H-4II), 2.54 (d, J = 2.2 Hz, 1 H, 2II-OH), 2.05 (s,
3 H, SCH3), 1.35 (d, J5,6 = 6.3 Hz, 3 H, H-6I), 1.29 (d, J5,6 = 6.1 Hz,
3 H, H-6II).
13C NMR (75 MHz, CDCl3): d = 104.0 (C-1II), 84.0 (C-1I), 82.5 (C-
3II), 80.9 (C-4I), 80.6 (C-3I), 79.5 (C-2I), 75.7 (C-2II), 75.6 (CH2Ph),
75.1 (CH2Ph), 73.1 (CH2Ph), 70.9 (C-5II), 68.7 (C-5I), 67.4 (C-4II),
18.7 (C-6II), 18.2 (C-6I), 13.9 (SCH3).
MS (TOF): m/z = 653.3 [M + NH4]+, 658.2 [M + Na]+.
HRMS (TOF): m/z [M + Na]+ calcd for C34H41N3O7S + Na:
found: 821.3860.
Anal. Calcd for C47H58O11: C, 70.65; H, 7.32. Found: C, 70.45; H,
7.25.
Methyl 3-O-(4-Azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-b-D-
glucopyranosyl)-2,4-di-O-benzyl-1-thio-a-L-rhamnopyrano-
side (7) and Methyl 4-Azido-2-O-benzoyl-3-O-benzyl-4,6-
dideoxy-1-thio-b-D-glucopyranoside (7A)
A mixture of methyl 2,4-di-O-benzyl-1-thio-a-L-rhamnopyranoside
(5;15 12.83 g, 34.3 mmol) and 4 Å MS (12.3 g) in CH2Cl2 (420 mL)
was stirred under N2 at r.t. for 15 min. The mixture was cooled to
–78 °C and a solution of TMSOTf (283 mL, 1.56 mmol) in CH2Cl2
(3 mL) was added, followed by a solution of 4-azido-2-O-benzoyl-
3-O-benzyl-4,6-dideoxy-a-D-glucopyranose 1-O-trichloroacetimi-
date (6;7 16.38 g, 31.08 mmol) in CH2Cl2 (10 mL). The stirring at
the same temperature was continued for 1 h, when TLC (7:1 hex-
ane–EtOAc) showed that the imidate was consumed. Et3N (1.0 mL)
was added to quench the reaction, the mixture was filtered through
a Celite pad, the filtrate was concentrated, and chromatography
gave aglycone transfer 7A in the first fraction; yield: 400 mg (3%);
colorless oil; [a]D +115.6 (c 1.1, CHCl3).
1H NMR (600 MHz, CDCl3): d = 5.31 (dd, J1,2 = 9.9 Hz, J2,3 = 9.4
Hz, 1 H, H-2), 4.70 (ABq, J = 10.8 Hz, 2 H, CH2Ph), 4.40 (d,
J1,2 = 9.9 Hz, 1 H, H-1), 3.71 (t, J = 9.1 Hz, 1 H, H-3), 3.40–3.25
(m, 2 H, H-4 and H-5), 2.16 (s, 3 H, SCH3), 1.41 (d, J5,6 = 6.0 Hz, 3
H, H-6).
658.2563; found: 658.2559.
Methyl 3-O-(4-Azido-3-O-benzyl-4,6-dideoxy-2-O-methyl-b-D-
glucopyranosyl)-2,4-di-O-benzyl-1-thio-b-L-rhamnopyrano-
side (9)
Powdered KOH (750 mg, 12.5 mmol) was added with stirring to a
solution of 8 (1.6 g, 2.52 mmol) in DMSO (10 mL), followed by
MeI (0.8 mL, 12.5 mmol). The mixture was stirred at r.t. under N2
for 1 h, when TLC (6:1 hexane–EtOAc) showed that the reaction
was complete. EtOAc (100 mL) was added and, after washing with
brine (3 × 30 mL), the organic phase was dried (Na2SO4) and con-
centrated. The residue was chromatographed to give 9; yield: 1.36
g (83%); mp 67–69 °C (EtOH); [a]D –37.64 (c 1.4, CHCl3).
1H NMR (300 MHz, CDCl3): d = 5.07 (d, J1,2 = 1.1 Hz, 1 H, H-1I),
4.98–4.70 (m, 5 H, CH2Ph), 4.57 (m, 2 H, including d, J1,2 = 8.0 Hz,
H-1II and CH2Ph), 4.03–3.96 (m, 2 H, H-3I and H-5I), 3.92 (dd,
J2,3 = 3.3 Hz, 1 H, H-2I), 3.68–3.6 (m, 4 H, including s, 3 H, OCH3
and t, J = 7.7 Hz, H-4I), 3.38 (t, J = 9.0 Hz, H-4II), 3.20–3.04 (m, 3
H, H-2II, H-3II, and H-5II), 2.06 (s, 3 H, SCH3), 1.35 (d, J5,6 = 6.4 Hz,
3 H, H-6II), 1.25 (d, J5,6 = 5.8 Hz, 3 H, H-6I).
13C NMR (150 MHz, CDCl3): d = 82.9 (C-1), 82.6 (C-3), 75.4
(CH2Ph), 75.3 (C-5), 71.9 (C-2), 67.9 (C-4), 18.8 (C-6), 11.4
(SCH3).
MS (TOF): m/z = 431.1 [M + NH4]+, 436.1 [M + Na]+.
HRMS (TOF): m/z [M + Na]+ calcd for C21H23N3O4S + Na:
436.1307; found: 436.1299.
Anal. Calcd for C21H23N3O4S: C, 61.00; H, 5.61. Found: C, 61.17;
H, 5.63.
13C NMR (75 MHz, CDCl3): d = 103.1 (C-1II), 84.9 (C-1I), 84.3 (C-
3II), 82.9 (C-4II), 81.0 (C-4I), 80.3 (C-2I), 79.3 (C-3I), 75.6 (CH2Ph),
75.1 (CH2Ph), 73.3 (CH2Ph), 70.1 (C-5II), 68.6 (C-5I), 67.8 (C-2II),
60.9 (OCH3), 18.6 (C-6I), 18.1(C-6II), 13.8 (SCH3).
Compound 7 was eluted out in the next fraction.
Yield: 20.46 g (89%); colorless oil; [a]D –11.95 (c 1.2, CHCl3).
1H NMR (300 MHz, CDCl3): d = 5.40 (dd, J1,2 = 7.9 Hz, J2,3 = 9.6
Hz, 1 H, H-2II), 5.05 (d, J1,2 = 1.1 Hz, 1 H, H-1I), 4.84 (d, J = 8.3 Hz,
1 H, H-1II), 4.79–4.23 (m, 6 H, CH2Ph), 4.01 (dd, J3,4 = 9.3 Hz,
J4,5 = 3.0 Hz, 1 H, H-4I), 3.94 (dd, J2,3 = 3.0 Hz, 1 H, H-2I), 3.90 (m,
1 H, H-5I), 3.64 (t, J = 9.3 Hz, 1 H, H-3II), 3.48 (t, J = 9.6 Hz, 1 H,
H-3I), 3.30 (m, 1 H, H-5II), 3.25 (t, J = 9.8 Hz, 1 H, H-4II), 1.32 (d,
J5,6 = 5.7 Hz, 3 H, H-6II), 1.16 (d, J5,6 = 4.3 Hz, 3 H, H-6I).
MS (TOF): m/z = 667.3 [M + NH4]+, 672.2 [M + Na]+.
HRMS (TOF): m/z [M + Na]+ calcd for C35H43N3O7S + Na:
672.2710; found: 672.2693.
Anal. Calcd for C35H43N3O7S: C, 64.69; H, 6.67. Found: C, 64.33;
H, 6.75.
13C NMR (75 MHz, CDCl3): d = 101.7 (C-1II), 84.4 (C-1I), 81.5 (C-
3II), 80.5 (C-3I), 80.0 (C-4I), 79.7 (C-2I), 75.3 (CH2Ph), 74.9
(CH2Ph), 74.2 (C-2II), 73.3 (CH2Ph), 70.9 (C-5II), 68.6 (C-5I), 68.0
(C-4II), 18.5 (C-6II), 17.9 (C-6I), 13.7 (SCH3).
MS (TOF): m/z = 757.3 [M + NH4]+, 762.2 [M + Na]+.
HRMS (TOF): m/z [M + Na]+ calcd for C41H45N3O8S + Na:
Methyl 3-O-(4-Amino-3-O-benzyl-4,6-dideoxy-2-O-methyl-b-D-
glucopyranosyl)-2,4-di-O-benzyl-1-thio-a-L-rhamnopyrano-
side (10)
H2O (30 mL) was added to a solution of 9 (6.8 g, 10.48 mmol) in
pyridine (50 mL) until slight turbidity, followed by a few drops of
pyridine until a clear solution was formed. A slow stream of H2S gas
was passed through the solution for 1.5 h, and the mixture was
stirred overnight at r.t., when TLC (2:1:0.1 hexane–EtOAc–25%
NH4OH) showed that the conversion was complete. After concen-
tration, the residue was chromatographed to afford 10; yield: 5.4 g
(83%); colorless oil.
762.2825; found: 762.2841.
Anal. Calcd for C41H45N3O8S: C, 66.56; H, 6.13. Found: C, 66.39;
H, 6.18.
1H NMR (600 MHz, CDCl3): d = 5.07 (d, J1,2 = 1.6 Hz, 1 H, H-1I),
5.01–4.58 (m, 7 H, including d, J1,2 = 7.5 Hz at 4.64, H-1II and
CH2Ph), 4.06 (dd, J2,3 = 3.2 Hz, J3,4 = 9.6 Hz, 1 H, H-3I), 4.00 (m, 1
Methyl 3-O-(4-Azido-3-O-benzyl-4,6-dideoxy-b-D-glucopyrano-
syl)-2,4-di-O-benzyl-1-thio-a-L-rhamnopyranoside (8)
Methanolic 1 M NaOMe was added to a solution of 7 (18 g, 24.35
mmol) in MeOH (400 mL) and CH2Cl2 (10 mL) until the solution
Synthesis 2009, No. 4, 545–550 © Thieme Stuttgart · New York

PAPER
Anthrax Tetrasaccharide: Large-Scale Preparation
549
H, H-5I), 3.96 (dd, J1,2 = 1.6 Hz, J2,3 = 3.2 Hz, 1 H, H-2I), 3.66 (m,
4 H, OCH3 and H-4I), 3.18–3.12 (m, 3 H, H-2II, H-3II, and H-5II),
2.50 (t, J = 9.3 Hz, 1 H, H-4II), 2.06 (s, 3 H, SCH3), 1.35(d,
J5,6 = 6.4 Hz, 3 H, H-6I), 1.12 (d, J5,6 = 5.8 Hz, 3 H, H-6II).
13C NMR (150 MHz, CDCl3): d = 103.9 (C-1II), 85.2 (C-5II), 84.6
(C-1I), 84.7 (C-3II), 80.8 (C-4I), 80.1 (C-2I), 78.7 (C-3I), 75.0
(CH2Ph), 74.8 (CH2Ph), 73.0 (CH2Ph), 72.5 (C-2II), 68.3 (C-5I),
60.4 (OCH3), 58.1 (C-4II), 18.0 (C-6II), 17.9 (C-6I), 13.5 (SCH3).
13C NMR (150 MHz, CDCl3): d = 171.0 (C=O), 169.3 (C=O), 103.8
(C-1II), 84.5 (C-2II), 84.3 (C-1I), 80.7 (C-4I), 80.7 (C-3¢), 80.4 (C-
3II), 80.0 (C-2I), 79.2 (C-3I), 74.8 (CH2Ph), 73.4 (CH2Ph), 73.2
(CH2Ph), 71.0 (C-5II), 68.3 (C-5I), 60.6 (OCH3), 55.7 (C-4II), 47.2
(C-2¢), 26.6 and 26.5 [C(CH3)2], 22.4 (CH3CO), 18.0 (C-6I), 17.9
(C-6II), 13.5 (SCH3).
MS (TOF): m/z = 783.3 [M + NH4]+, 788.3 [M + Na]+.
HRMS (TOF): m/z [M + NH4]+ calcd for C42H59N2O10S: 783.3890;
MS (TOF): m/z = 624.3 [M + H]+, 646.2 [M + Na]+.
found: 783.3878.
HRMS (TOF): m/z [M + H]+ calcd for C35H46NO7S: 624.2995;
found: 624.3011.
5-Methoxycarbonylpentyl 4-(3-O-Acetyl-3-methylbutanami-
do)-3-O-benzyl-4,6-dideoxy-2-O-methyl-b-D-glucopyranosyl-
(1→3)-2,4-di-O-benzyl-a-L-rhamnopyranosyl-(1→3)-2,4-di-O-
benzyl-a-L-rhamnopyranosyl-(1→2)-3,4-di-O-benzyl-a-L-
rhamnopyranoside (13)
Methyl 2,4-Di-O-benzyl-3-O-[3-O-benzyl-4,6-dideoxy-4-(3-hy-
droxy-3-methylbutanamido)-2-O-methyl-b-D-glucopyranosyl]-
1-thio-a-L-rhamnopyranoside (11)
A mixture of 4 (4.41 g, 5.53 mmol), 12 (4.23 g, 5.53 mmol), and 4
Å MS (3.0 g) in CH2Cl2 (200 mL) was stirred under N2 at r.t. for 30
min, cooled to –50 °C, and NIS (1.86 g, 8.30 mmol) followed by
AgOTf (991 mg. 3.87 mmol) was added. The stirring was continued
at the same temperature for 6 h, and then overnight at r.t., when TLC
(8:1 toluene–acetone) showed that the reaction was complete. Et3N
(2.5 mL) was added to quench the reaction and, after filtration
through a Celite pad, the filtrate was washed successively with 10%
aq Na2S2O3 (2 × 50 mL) and brine (150 mL). Concentration of the
organic phase and chromatography of the residue gave 13; yield:
5.96 g (71%); colorless oil; [a]D –21.60 (c 1.19, CHCl3).
1H NMR (600 MHz, CDCl3): d = 5.15 (d, J = 9.1 Hz, 1 H, NH), 5.09
(br s, 1 H, H-1III), 5.07 (d, J1,2 = 1.8 Hz, 1 H, H-1II), 5.00–4.42 (m,
16 H, 7 × CH2Ph, H-1I, H-1IV), 4.12 (t, J = 3.0 Hz, 1 H, H-3II), 4.10
(t, J = 3.0 Hz, 1 H, H-3III), 3.99 (t, J = 2.0 Hz, 1 H, H-2I), 3.90 (dd,
J1,2 = 1.8 Hz, J2,3 = 3.0 Hz, 1 H, H-2III), 3.82–3.75 (m, 4 H, H-3I, H-
2II, H-5II, H-5III), 3.66–3.58 (m, 10 H, H-1a, H-5I, H-4III, H-4IV,
CO2CH3 and OCH3), 3.53 (m, 1 H, H-4II), 3.39 (t, J = 9.4 Hz, 1 H,
H-4I), 3.35–3.31 (m, 2 H, H-1b and H-3IV), 3.18–3.15 (m, 2 H, H-
2IV, H-5IV), 2.55 (ABq, J = 13.7 Hz, 2 H, H-2¢), 2.30 (t, J = 7.5 Hz,
H-5), 1.86 (s, 3 H, CH3CO), 1.63 (m, 2 H, H-4), 1.54 (m, 2 H, H-2),
1.49 and 1.48 [2 s, C(CH3)2], 1.34 (m, 2 H, H-3), 1.28 (d, J5,6 = 5.4
Hz, 3 H, H-6III), 1.26 (d, J5,6 = 6.2 Hz, 3 H, H-6I), 1.23 (d, J5,6 = 6.3
Hz, 3 H, H-6II), 1.02 (d, J5,6 = 6.2 Hz, 3 H, H-6IV).
HATU (4.96 g, 13.1 mmol) followed by Hünig’s base (1.68 g, 13.1
mmol) was added to a solution of 10 (5.4 g, 8.7 mmol) and 3-hy-
droxy-3-methylbutanoic acid (1.36 g, 13.1 mmol) in CH2Cl2 (100
mL). The mixture was stirred at r.t. for 1.5 h when TLC (1:1 hex-
ane–EtOAc) showed that compound 10 was completely consumed.
After partitioning between CH2Cl2 (200 mL) and a mixture of aq
NaHCO3 (50 mL) and brine (150 mL), the organic phase was dried,
concentrated, and the residue was chromatographed to give 11;
yield: 5.28 g (85%); colorless oil; [a]D –84.32 (c 2.3, CHCl3).
1H NMR (600 MHz, CDCl3): d = 5.42 (d, J = 9.0 Hz, 1 H, NH),
5.05 (d, J1,2 = 1.2 Hz, 1 H, H-1I), 5.00–4.58 (m, 7 H, including d,
J1,2 = 7.8 Hz at 4.60 H-1II and CH2Ph), 4.00 (dd, J2,3 = 3.3 Hz,
J3,4 = 9.6 Hz, 1 H, H-3I), 3.98 (m, 1 H, H-5I), 3.96 (m, 1 H, H-2I),
3.72–3.64 (m, 5 H, H-4I, H-4II and OCH3), 3.36 (dd, J2.3 = 9.1 Hz,
J3,4 = 10.2 Hz, 1 H, H-3II), 3.30 (m, 1 H, H-5II), 3.20 (dd, J1,2 = 7.8
Hz, J2,3 = 9.1 Hz, 1 H, H-2II), 2.20 (ABq, J = 15 Hz, 2 H, H-2¢), 2.06
(s, 3 H, SCH3), 1.36 (d, J5,6 = 6.3 Hz, 3 H, H-6I), 1.23 (s, 3 H, CH3),
1.20 (s, 3 H, CH3), 1.16 (d, J5,6 = 5.6 Hz, 3 H, H-6II).
13C NMR (150 MHz, CDCl3): d = 172.3 (C=O), 103.8 (C-1II), 84.8
(C-2II), 84.2 (C-1I), 80.7 (C-4I), 80.0 (C-3II), 79.9 (C-2I), 79.2 (C-
3I), 74.8 (CH2Ph), 73.7 (CH2Ph), 73.2 (CH2Ph), 70.9 (C-5II), 69.4
(C-3¢), 68.3 (C-5I), 60.5 (OCH3), 55.6 (C-4II), 47.7 (C-2¢), 29.3 [2 ×
C, C(CH3)2], 18.0 (C-6II), 17.9 (C-6I), 13.5 (SCH3).
MS (TOF): m/z = 741.3 [M + NH4]+, 746.3 [M + Na]+.
HRMS (TOF): m/z [M + NH4]+ calcd for C40H57N2O9S: 741.3785;
13C NMR (150 MHz, CDCl3): d = 103.7 (C-1IV), 100.4 (C-1III), 98.9
(C-1II), 98.8 (C-1I), 84.5 (C-2IV), 80.8 (C-4III), 80.6 (C-4I), 80.5 (C-
4II), 80.4 (C-3I), 80.3 (C-3¢), 80.0 (C-3IV), 79.2 (C-2III), 78.7 (C-3III),
78.0 (2 × C, C-2II and C-3II), 75.4 (CH2Ph), 74.8 (2 × C, C-2I and
CH2Ph), 74.5 (CH2Ph), 73.4 (CH2Ph), 73.3 (CH2Ph), 72.3 (CH2Ph),
72.0 (CH2Ph), 70.9 (C-5IV), 68.5 (C-5II), 68.3 (C-5III), 67.8 (C-5I),
67.1(C-1), 60.5 (OCH3), 55.6 (C-4IV), 51.4 (CO2CH3), 47.2 (C-2¢),
33.9 (C-5), 29.1 (C-2), 26.6 and 26.5 [C(CH3)2], 25.7 (C-3), 24.7
(C-4), 22.4 (CH3CO), 17.9 (4 × C, C-6I, C-6II, C-6III, C-6IV).
found: 741.3781.
Anal. Calcd for C40H53NO9S: C, 66.37; H, 7.38. Found: C, 66.20; H,
7.39.
Methyl 2,4-Di-O-benzyl-3-O-[3-O-benzyl-4,6-dideoxy-4-(3-O-
acetyl-3-methylbutanamido)-2-O-methyl-b-D-glucopyranosyl]-
1-thio-a-L-rhamnopyranoside (12)
Ac2O (10 mL, 109 mmol) was added at 0 °C to a mixture of 11 (5.26
g, 7.27 mmol) and 4-dimethylaminopyridine (887 mg, 7.27 mmol)
in anhyd CH2Cl2 (50 mL). The cooling bath was removed and the
mixture was stirred overnight, when the reaction was complete
(TLC, 3:2 hexane–EtOAc). MeOH (10 mL) was added to quench
the reaction, the mixture was concentrated, and the residue was
chromatographed to give 12; yield: 5.38 g (93%); colorless oil.
1H NMR (600 MHz, CDCl3): d = 5.23 (d, J = 9.0 Hz, 1 H, NH),
5.05 (d, J1,2 = 1.4 Hz, 1 H, H-1I), 4.97–4.58 (m, 7 H, H-1II and
CH2Ph), 4.01 (dd, J2,3 = 3.2 Hz, J3,4 = 9.5 Hz, 1 H, H-3I), 3.98 (m, 1
H, H-5I), 3.95 (dd, J1,2 = 1.5 Hz, J2,3 = 3.2 Hz, 1 H, H-2I), 3.70–3.63
(m, 5 H, H-4I, H-4II and OCH3), 3.39 (dd, J2,3 = 9.0 Hz, J3,4 = 10.2
Hz, 1 H, H-3II), 3.30 (m, 1 H, H-5II), 3.19 (dd, J1,2 = 7.8 Hz,
J2,3 = 9.0 Hz, 1 H, H-2II), 2.60 (ABq, J = 13.8 Hz, 2 H, H-2¢), 2.05
(s, 3 H, SCH3), 1.91 (s, 3 H, CH3CO), 1.51 (s, 3 H, CH3), 1.50 (s, 3
H, CH3), 1.36 (d, J5,6 = 6.3 Hz, 3 H, H-6I), 1.16 (d, J5,6 = 5.6 Hz, 3
H, H-6II).
MS (TOF): m/z = 1533.6 [M + NH4]+, 1538.6 [M + Na]+.
Anal. Calcd for C88H109NO21: C, 69.68; H, 7.24; N, 0.92. Found: C,
69.42; H, 7.38; N, 1.07.
5-Methoxycarbonylpentyl 4-(3-O-Acetyl-3-methylbutanami-
do)-4,6-dideoxy-2-O-methyl-b-D-glucopyranosyl-(1→3)-a-L-
rhamnopyranosyl-(1→3)-a-L-rhamnopyranosyl-(1→2)-a-L-
rhamnopyranoside (14)
A mixture of compound 13 (7.38 g, 4.91 mmol) and 5% Pd/C cata-
lyst (4.0 g) in 5:1 MeOH–EtOAc (300 mL) was stirred in a H2 at-
mosphere at r.t. for 24 h. TLC (6:4:1 CH2Cl2–acetone–MeOH)
showed that the reaction was complete. After filtration through a
Celite pad and concentration of the filtrate, the residue was chro-
matographed to give 14; yield: 3.62 g (85%); colorless oil.
1H NMR (600 MHz, CD3OD): d = 5.06 (d, J1,2 = 1.6 Hz, 1 H, H-
1III), 4.91 (d, J1,2 = 1.7 Hz, 1 H, H-1II), 4.78 (d, J1,2 = 1.5 Hz, 1 H, H-
1I), 4.62 (d, J1,2 = 7.8 Hz, 1 H, H-1IV), 4.18 (dd, J1,2 = 1.8 Hz,
Synthesis 2009, No. 4, 545–550 © Thieme Stuttgart · New York

550
S. Hou, P. Kováč
PAPER
J2,3 = 3.2 Hz, 1 H, H-2III), 4.06 (dd, J1,2 = 2.0 Hz, J2,3 = 3.1 Hz, 1 H,
H-2II), 3.90 (dd, J2,3 = 3.3 Hz, J3.4 = 9.5 Hz, 1 H, H-3III), 3.83 (ddd,
J = 4.8, 7.9, 12.5 Hz, 1 H, H-5III), 3.80–3.77 (m, 2 H, H-3II, H-2I),
(C-3III), 81.5 (C-2I), 80.7 (C-3I), 75.6 (C-5IV), 74.9 (C-3IV), 74.0 (C-
4I), 73.9 (C-4III), 73.5 (C-4II), 73.0 (C-3II), 72.8 (C-3¢), 72.6 (2 × C,
C-2II and C-2III), 72.14 (2 × C, C-5II and C-5III), 71.6 (C-5I), 70.6 (C-
1), 62.8 (OCH3), 59.3 (C-4IV), 51.7 (C-2¢), 43.8 (C-6), 42.5 (C-7),
38.5 (C-5), 31.0 (CH3), 30.9 (2 × C, C-4 and CH3), 27.8 and 27.7
(C-2 and C-3), 19.85 (C-6IV), 19.4 (3 × C, C-6I, C-6II, C-6III).
3.74 (m, 2 H, H-5II H-3I), 3.69–3.64 (m, 7 H, H-1a, 2 × OCH3),
,
3.61–3.48 (m, 4 H, H-4IV, H-4III , H-4II , H-5I), 3.44–3.34 (m, 4 H,
H-3IV, H-5IV, H-4I, and H-1b), 3.01 (dd, J1,2 = 8.0 Hz, J2,3 = 8.9 Hz,
1 H, H-2IV), 2.72 (ABq, J = 13.5 Hz, 2 H, H-2¢), 2.34 (t, J = 7.4 Hz,
2 H, H-5), 1.97 (s, 3 H, COCH3), 1.70–1.56 (m, 4 H, H-2 and H-4),
1.55 (s, 6 H, 2 × CH3), 1.46–1.37 (m, 2 H, H-3), 1.29 (d, J = 6.2 Hz,
3 H, H-6III), 1.25 (dd, J = 5.2 Hz, 6.0 Hz, 6 H, H-6II, H-6I), 1.20 (d,
J5,6 = 6.2 Hz, 3 H, H-6IV).
MS (TOF): m/z = 872.3 [M + H]+, 894.3 [M + Na]+.
HRMS (TOF): m/z [M + H]+ calcd for C38H70N3O19: 872.4604;
found: 872.4606.
13C NMR (150 MHz, CD3OD): d = 175.8 (C=O), 172.4 (2 × C, 2 ×
C=O), 105.4 (C-1IV), 103.9 (C-1II), 103.5 (C-1III), 100.3 (C-1I),
85.7 (C-2IV), 81.7 (C-3II), 81.5 (C-3¢), 80.1 (C-2II), 79.0 (C-3II),74.8
(C-3IV), 74.3 (C-4I), 73.2 (C-4II), 73.0 (C-4III), 72.2 (C-5IV), 72.1 (C-
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.
3I), 71.8 (C-2I), 71.7 (C-2III), 70.5 (C-5II), 70.1 (C-5III), 69.9 (C-5I), Acknowledgment
68.3 (C-1), 61.2 (OCH3), 58.0 (C-4IV), 52.0 (CO2CH3), 47.5 (C-2¢),
This research was supported by the Intramural Research Program of
the NIH, NIDDK.
34.7 (C-5), 30.2 (C-2), 27.0 (C-3), 26.8 [2 × C, (C¢3)(CH3)2], 25.7
(C-4), 22.4 (CH3CO), 18.4 (C-6IV), 18.1 and 17.9 (3 × C, C-6I, C-
6II, C-6III).
MS (TOF): m/z = 886.0 [M + H]+, 903.4 [M + NH4]+, 908.3 [M +
References
Na]+.
(1) 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.
HRMS (TOF): m/z [M + H]+ calcd for C39H68NO21: 886.4284;
found: 886.4236.
(2) Saksena, R.; Adamo, R.; Kováč, P. Bioorg. Med. Chem.
(2-Aminoethylamido)carbonylpentyl 4,6-Dideoxy-4-(3-hy-
droxy-3-methylbutyramido)-2-O-methyl-b-D-glucopyranosyl-
(1→3)-a-L-rhamnopyranosyl-(1→3)-a-L-rhamnopyranosyl-
(1→2)-a-L-rhamnopyranoside (15)
2007, 15, 4283.
(3) Adamo, R.; Saksena, R.; Kováč, P. Helv. Chim. Acta 2006,
89, 1075.
(4) Werz, D. B.; Seeberger, P. H. Angew. Chem. Int. Ed. 2005,
44, 6315.
(5) Crich, D.; Vinogradova, O. J. Org. Chem. 2007, 72, 6513.
(6) Guo, H.; O’Doherty, G. A. Angew. Chem. Int. Ed. 2007, 46,
5206.
(7) Hou, S.-j.; Kováč, P. Eur. J. Org. Chem. 2008, 1947.
(8) Saksena, R.; Adamo, R.; Kováč, P. Carbohydr. Res. 2005,
340, 1591.
(9) Mehta, A. S.; Saile, E.; Zhong, W.; Buskas, T.; Carlson, R.;
Kannenberg, E.; Reed, Y.; Quinn, C. P.; Boons, G.-J. Chem.
Eur. J. 2006, 12, 9136.
(10) McNaught, A. D. Carbohydr. Res. 1997, 297, 1.
(11) Tamborrini, M.; Werz, D. B.; Frey, J.; Pluschke, G.;
Seeberger, P. H. Angew. Chem. Int. Ed. 2006, 45, 6581.
(12) Hou, S.-j.; Saksena, R.; Kováč, P. Carbohydr. Res. 2008,
343, 196.
(13) Tietze, L. F.; Schröter, C.; Gabius, S.; Brinck, U.; Goerlach-
Graw, A.; Gabius, H.-J. Bioconjugate Chem. 1991, 2, 148.
(14) Bioconjugate Techniques; Hermanson, G. T., Ed.; Academic
Press: New York, 1996.
A solution of 14 (3.60 g, 4.07 mmol) in NH2(CH2)2NH2 (10 mL)
was stirred overnight at 50 °C, when TLC (CH2Cl2–MeOH–25%
NH4OH, 10:9:1) showed that all starting material was consumed
and that two poorly resolved products (one major) were present. 1H
NMR showed absence of the signal for the CO2CH3 group and pres-
ence of a disproportionately small signal for the COCH3 group. The
mixture was concentrated and the residue was dissolved in MeOH
(100 mL). Methanolic 1 M NaOMe was added to the solution until
the solution became strongly basic to litmus, and the mixture was
kept at r.t. overnight. TLC (1:1:0.1 CH2Cl2–MeOH–25% NH4OH)
showed that only one substance, more polar than the starting mate-
rial 14 was present. Concentration and chromatography of the resi-
due gave 15, whose aqueous solution was freeze-dried; yield: 3.0 g
(85%); colorless amorphous solid.
1H NMR (600 MHz, D2O): d = 5.03 (d, J1,2 = 1.7 Hz, 1 H, H-1III),
4.92 (d, J1,2 = 1.7 Hz, 1 H, H-1II), 4.87 (d, J1,2 = 1.5 Hz, 1 H, H-1I),
4.73 (d, J1,2 = 8.0 Hz, 1 H, H-1IV), 4.27 (dd, J1,2 = 1.8 Hz, J2,3 = 3.3
Hz, 1 H, H-2III), 4.14 (dd, J1,2 = 2.0 Hz, J2,3 = 3.1 Hz, 1 H, H-2II),
3.98 (dd, J2,3 = 3.3 Hz, J3,4 = 9.7 Hz, 1 H, H-3III), 3.90 (dd, J1,2 = 1.7
Hz, J2,3 = 3.3 Hz, 1 H, H-2I), 3.87–3.74 (m, 4 H, H-3I, H-3II, H-5III,
H-5II), 3.72–3.65 (m, 2 H, H-1a, H-5I), 3.63–3.58 (m, 5 H, H-4III, H-
4IV, OCH3), 3.57–3.44 (m, 5 H, H-1b, H-4I, H-4II, H-3IV, H-5IV),
3.28–3.21 (m, 2 H, H-6), 3.12 (dt, J = 5.1, 5.9 Hz, 1 H, H-2IV), 2.75
(dd, J = 3.5, 5.6 Hz, 2 H, H-7), 2.50–2.40 (m, 2 H, H-2¢), 2.25 (q,
J = 7.1 Hz, 2 H, H-5), 1.68–1.53 (m, 4 H, H-2 and H-4), 1.45–1.32
(m, 2 H, H-3), 1.30–1.24 [m, 15 H, H-6I, H-6II, H-6III, C(CH3)2],
1.21 (d, J5,6 = 6.1 Hz, 3 H, H-6IV).
(15) Lipták, A.; Szabó, L.; Harangi, J. J. Carbohydr. Chem. 1988,
7, 687.
(16) Yu, H.; Yu, B.; Wu, X.; Hui, Y.; Han, X. J. Chem. Soc.,
Perkin Trans. 1 2000, 1445.
(17) Fügedi, P.; Nánási, P. J. Carbohydr. Nucleosides,
Nucleotides 1981, 8, 547.
(18) Zemplén, G.; Geres, A.; Hadacsy, J. Ber. Dtsch. Chem. Ges.
1936, 69, 1827.
(19) Thompson, A.; Wolfrom, M. L. Methods Carbohydr. Chem.
1963, 2, 215.
13C NMR (150 MHz, D2O): d = 180.0 (C=O), 176.8 (C=O), 106.4
(C-1IV), 104.9 (C-1II), 104.6 (C-1III), 101.0 (C-1I), 86.0 (C-2IV), 82.4
Synthesis 2009, No. 4, 545–550 © Thieme Stuttgart · New York