Synthesis of asparagine-linked bacillosamine

Article abstract of DOI:10.1016/j.carres.2006.04.031

Various types of protein glycosylation have been identified from prokaryotes. Recent investigations have revealed the presence of N-linked glycoproteins in the pathogenic bacterium, Campylobacter jejuni. The structure of this glycan is unique, consisting

Full text of DOI:10.1016/j.carres.2006.04.031

Carbohydrate Research 341 (2006) 1922–1929
Note
Synthesis of asparagine-linked bacillosamine
Mohammed Nurul Amin,a,b Akihiro Ishiwataa,c and Yukishige Itoa,b,c,
*
aRIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
bGraduate School of Science and Engineering, Saitama University, Sakura-ku, Saitama 338-8570, Japan
cCREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-1102, Japan
Received 20 February 2006; received in revised form 13 April 2006; accepted 17 April 2006
Available online 15 May 2006
Abstract—Various types of protein glycosylation have been identified from prokaryotes. Recent investigations have revealed the
presence of N-linked glycoproteins in the pathogenic bacterium, Campylobacter jejuni. The structure of this glycan is unique, con-
sisting of 5 GalNAc and 1 Glc, in addition to 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose (bacillosamine; Bac), which is N-gly-
cosidically linked to the side chain of asparagine (Asn). We synthesized Bac from a 2-azido-2-deoxy-D-galactose derivative, which
was further converted to the Asn-linked form.
Ó 2006 Published by Elsevier Ltd.
Keywords: N-Linked glycoprotein; Bacillosamine; Campylobacter jejuni
Bacillosamine (Bac), 2,4-diacetamido-2,4,6-trideoxy-
glucopyranose, is the reducing terminal monosaccharide
component of the glycoproteins derived from bacterial
species.1,2 Bac was first isolated in 1960 from the
cell-wall polysaccharides of Bacillus licheniformis
ATCC9945.3 It is located in polysaccharides, including
glycoproteins and lipopolysaccharides of various pro-
karyotes.4 A number of syntheses of Bac5 and related
compounds such as 2,4-diacetamido-2,4,6-trideoxy-D-
galactopyranose derivatives6,7 have been reported.
N-Glycosylation is widespread in eukaryotic proteins,
where the attachment of glycan occurs at the Asn-Xaa-
Ser/Thr motif. It is believed that only eukaryotes are
able to produce the N-glycosylated proteins,8,9 while
several types of prokaryotic O-glycosylations have been
reported, such as O-linked pseudaminic acid and bacillos-
amine.10 Recent investigations revealed the presence of
a novel non-flagellin glycoprotein in a pathogenic
Gram-negative bacterium, Campylobacter jejuni, which
was found to be a major antigenic protein designated
PEB3 or Cj0289c.1 This glycoprotein is multiply N-gly-
cosylated with a novel glycan at sites having the eukary-
ote-like consensus amino acid sequence of Asn-Xaa-Ser/
Thr (Fig. 1).2 The structure of this N-linked glycan,
GalNAc-a-(1!4)-GalNAc-a-(1!4)-[Glc-b-(1!3)-]Gal-
NAc-a-(1!4)-GalNAc-a-(1!4)-GalNAc-a-(1!3)-Bac-
b (1), is distinct from those of eukaryotic origin.10c,d In
the latter case, glycans that share a common pentasac-
charide
[Man-a-(1!3)-]Man-a-(1!6)-Man-b-(1!4)-
GlcNAc-b-(1!4)-GlcNAc-b-(1!Asn) are decorated
by various sugar residues. However, their biosynthetic
pathways are similar to each other, in a sense that pre-
assembled lipid-linked oligosaccharide (Glc3Man9Glc-
NAc2-PP-Dol or Glc1GalNAc5Bac1-PP-Udp; Dol:
dolichyl, Udp: undecaprenyl) is transferred to a nascent
peptide either in the ER lumen (eukaryotes) or peri-
plasm (C. jejuni) by oligosaccharyl transferase (OST).9,11
The N-linked glycoprotein from C. jejuni was found
to be immunodominant, and N-glycosylation is essential
for their adhesion to host cells.10c,d Considering the
potential utility for immunochemical studies aiming at
the development of antibodies and vaccines against this
pathogenic bacterium as well as for understanding the
mechanisms of protein N-glycosylation, C. jejuni N-
glycan in Asn-linked form would be attractive as a
synthetic target.6
*
Corresponding author. Tel.: +81 48 467 9430; fax: +81 48 462
4680; e-mail: yukito@riken.jp
0008-6215/$ - see front matter Ó 2006 Published by Elsevier Ltd.
doi:10.1016/j.carres.2006.04.031

M. N. Amin et al. / Carbohydrate Research 341 (2006) 1922–1929
1923
GalNAcα1→4GalNAcα1→4
GalNAcα1→4GalNAcα1→4GalNAcα1→3Bacβ1→Asn-X-Ser/Thr
Glcβ1→3
Glc1GalNAc5
Bac-Asn
OH
HO
O
HO
AcNH
O
HO
AcNH
O
OH
OH
Ph
OH
OH
O
O
O
O
O
HO
HO
O
O
OH
AcNH
HO
HO
OTBDPS
O
N3
AcNH
O
OH
3
O
O
HO
AcNH
AcNH
O
Me
O
H
1
N
O
AcNH
NH
O
Me
O
H
Me
O
AcHN
HO
Me
N
O
N3
HO
O
O
t-Bu
AcHN
HO
OTBDPS
NHAc
O
NHFmoc
OH
N3
AcNH
2
5
4
Figure 1.
We recently completed the synthesis of the hexasac-
charide region (Glc1GalNAc5) of this glycan using the
2-azido-2-deoxy-D-galactose (GalN3) derivative 3 as
the common precursor of GalNAc components.12 In
order to target the whole structure 1, we chose the 2,4-
diazido-2,4,6-trideoxy-D-glucose derivative 5 as the Bac
equivalent. This diazide was selected as the acceptor
for the oligosaccharide construction toward 1, because
it has been reported that the hydroxyl groups of par-
tially protected N-acetylglucosamine derivatives are
poor acceptors.13 Now, we describe the synthesis of 5
and its conversion to free Bac (4) as well as to aspara-
gine-linked Bac (Bac-Asn, 2), also starting with 3
(Fig. 1).
Our synthesis commenced with tert-butyldiphenylsilyl
2-azido-4,6-O-benzylidene-2-deoxy-b-D-galactopyrano-
side 3,14 which is readily obtainable from D-galactal.
Protection of the 3-hydroxyl group with a 3-O-naph-
thylmethyl (NAP) gave 6 (Scheme 1).15 Protection with
NAP proved to be highly suitable for our purpose, be-
cause of its stability under various reaction conditions,
including acetal hydrolysis and reductive dehalogen-
ation, and chemoselective removal under oxidative con-
ditions. Removal of the benzylidene acetal of 6 in 4:1:1
AcOH–H2O–MeOH gave diol 7 in 92% yield.15c,16 Since
17
attempted iodination of 7 with Ph3P, imidazole and I2
resulted in the reduction of the azide, diol 7 was first
tosylated regioselectively to give 8, then converted to
iodide 9. Chemoselective reduction was conducted with
NaBH3CN18 in diglyme to afford 6-deoxy derivative 10
without affecting the N3 groups.19 In a large-scale prep-
aration, this compound was obtained in 67% yield
through a one-pot transformation from 8.
An additional azido group was introduced at the 4-
position with inversion of configuration. Thus, by treat-
ment with Tf2O and pyridine, compound 10 was
converted to triflate 11, which was subjected to nucleo-
philic substitution with NaN3,20 to give 4-azide 12 in
91% yield in two steps. The D-gluco configuration was
1
rigorously confirmed by the H NMR spectrum, which

1924
M. N. Amin et al. / Carbohydrate Research 341 (2006) 1922–1929
Ph
O
R2
Me
O
HO
NAPO
R
f, g
b,c.d,e
O
O
O
R1
OTBDPS
OTBDPS
OTBDPS
RO
NAPO
N3
N3
R
N3
R
R1 R2
OH
7
8
9
H
3
6
H
N3
OTf
H
11
12
a
OTs
NAP
I
H
10
Me
Me
Me
k
i, j
h
O
O
O
RHN
HO
N3
HO
AcHN
HO
OTBDPS
NHR
OH
NHAc
OTBDPS
N3
R
4
5
H
Ac
13
14
Scheme 1. Reagents and conditions: (a) 2-(bromomethyl)naphthalene, NaH, Bu4NI, THF, 95%; (b) 4:1:1 AcOH–H2O–MeOH, 80 °C, 92%; (c) TsCl,
pyridine, DMAP, 94%; (d) NaI, diglyme, 120 °C, 91%; (e) NaCNBH3, diglyme, 120 °C, 70%; (f) (CF3SO2)2O, pyridine, CH2Cl2; (g) NaN3, DMF,
91% in two steps; (h) DDQ, 10:1 CH2Cl2–H2O, quant; (i) Pd(OH)2/C, H2, MeOH; (j) Ac2O, MeOH, 98% in two steps; (k) NH4F, MeOH, 90%.
revealed a triplet (J 9.6 Hz) at 3.09 ppm, assignable as
H-4. Removal of the NAP ether was achieved by
DDQ oxidation15c to afford 5 in quantitative yield.
To complete the synthesis of Bac, the azido groups
were reduced with hydrogen–Pd(OH)2/C to afford
13, which was acetylated to give 14 in 98% yield.
Bacillosamine 4 was obtained in 90% yield through
deprotection of the anomeric TBDPS group by NH4F
in MeOH.5a,21
10 via triflate 11 to efficiently introduce nitrogen
functionality. Furthermore, N-asparagine-linked bacillos-
amine 2 was obtained through the synthesis of Alloc-
protected glycosyl amine 15, followed by one-pot
Pd(0)–PhSiH3-mediated removal of Alloc and coupling
with an aspartoyl fluoride. Synthetic studies of the novel
N-glycan (1) from C. jejuni are now in progress.12
Synthetic Bac (4) was converted to the glycosylamine,
according to Kochetkov’s procedure22 (satd ammonium
bicarbonate), which was isolated as an allyl carbamate
15 (Scheme 2). At this stage, the stereochemical homoge-
neity was confirmed by 1H NMR, which revealed the H-
1 signal at 4.73 ppm (JH1ꢀH2 10.0 Hz). It was subjected
to coupling with a protected aspartoyl fluoride in the
1. Experimental
1.1. General procedures
All reactions sensitive to air and/or moisture were car-
ried out under nitrogen or argon atmosphere with anhy-
drous solvents. Column chromatography was performed
on silica gel 60N, 100–210 mesh (Kanto Kagaku Co.,
Ltd). Preparative TLC was performed on Silica Gel 60
254, 0.5-mm plates (E. Merck). Melting points were
determined with a Buchi 510 melting point apparatus.
¨
Optical rotations were measured with a JASCO DIP
370 polarimeter. 1H NMR spectra were recorded at
400 MHz on a JEOL JNM-AL 400 spectrometer, and
chemical shifts are referred to internal CDCl3
23
presence of Pd(PPh3)4 and PhSiH3 to provide 2 in
86% yield as a pure b anomer (d 4.86 ppm, J 9.6 Hz).
In conclusion, we developed the synthesis of N-aspar-
agine-linked bacillosamine from D-galactose by employ-
ing: (1) NAP ether as a compatible protection group; (2)
regioselective formation of iodide 9 from diol 7 and
chemoselective reduction of 9 in the presence of NAP
ether and azide; and (3) inversion at the 4-position of
F
Me
O
Me
a, b
c
Me
H
H
O
O
AcHN
HO
AcHN
HO
O
N
AcHN
HO
N
O
OH
NHAc
O
t
-Bu
NHAc
NHAc
O
O
NHFmoc
2
4
15
Scheme 2. Reagents and conditions: (a) NH4HCO3, H2O, 45 °C; (b) NaHCO3, AllocCl, 10:1 dioxane–H2O, 71% in two steps; (c) NaHCO3,
FmocAsp(F)Ot-Bu, PhSiH3, Pd(PPh3)4, 10:1 dioxane–H2O, 86%.

M. N. Amin et al. / Carbohydrate Research 341 (2006) 1922–1929
1925
(7.24 ppm), D2O (4.65 ppm), or CD3OD (3.30 ppm). 13
C
80 °C for 22 h. The concentrated crude mixture was
purified by silica gel column chromatography (5:2 hex-
ane–EtOAc) to provide the title compound as a white
NMR spectra were recorded at 100 MHz on the same
instrument, and chemical shifts are referred to internal
CDCl3 (77.00 ppm), CD3OD (49.00 ppm), or dioxane
(67.19 ppm) in D2O. MALDI-TOF mass spectra were
recorded on a SHIMADZU Kompact MALDI AXI-
MA-CFR spectrometer with 2,5-dihydroxybenzoic acid
as the matrix. ESI-TOF mass spectra were recorded on
a JEOL AccuTOF JMS-T700LCK with CF3CO2Na as
the internal standard. Elemental analyses were per-
formed with a Fisons EA1108 instrument.
27
solid (12.06 g, 92%): mp 117–118 °C; ½aꢁD +9.09 (c
1
1.00, CH2Cl2); H NMR (CDCl3, 400 MHz): d 1.12 (s,
t-Bu, 9H), 3.02 (m, H-5, 1H), 3.24 (dd, J 3.2, 10.0 Hz,
H-3, 1H), 3.44 (dd, J 4.0, 12.0 Hz, H-6a, 1H), 3.66 (dd,
J 7.2, 12.0 Hz, H-6b, 1H), 3.74 (dd, J 7.6, 10.4 Hz, H-
2, 1H), 3.79 (d, J 2.8 Hz, H-4, 1H), 4.44 (d, J 7.6 Hz,
H-1, 1H), 4.82 (s, ArCH2, 2H), 7.34–7.50 (m, Ar, 9H),
7.69–7.85 (m, Ar, 8H); 13C NMR (CDCl3, 100 MHz): d
19.12, 26.82, 62.22, 65.37, 66.13, 72.27, 74.40, 78.91,
96.97, 125.56, 126.16, 126.27, 126.84, 127.37, 127.64,
127.82, 128.46, 129.80, 129.90, 132.53, 133.05, 133.56,
134.41, 135.65, 135.70; MALDI-TOF MS: [M+Na]+
calcd for C33H37N3O5SiNa, 606.24; found, 606.10;
HRMS ESI-TOF: [M+Na]+ calcd for C33H37N3O5SiNa,
606.2424; found, 606.2400. Anal. Calcd for C33H37N3O5-
Si: C, 67.90; H, 6.39; N, 7.20. Found: C, 67.62; H, 6.28;
N, 6.87.
1.2. tert-Butyldiphenylsilyl 2-azido-4,6-O-benzylidene-2-
deoxy-3-O-naphthylmethyl-b-D-galactopyranoside (6)
To a solution of tert-butyldiphenylsilyl 2-azido-4,6-O-
benzylidene-2-deoxy-b-D-galactopyranoside (3) (9.00 g,
16.9 mmol) in dry tetrahydrofuran 2-(bromo-
methyl)naphthalene (3.93 g, 17.8 mmol) was added
sodium hydride (570 mg, 23.8 mmol), followed by tetra-
butylammonium iodide (500 mg, 1.35 mmol), under Ar
atmosphere at room temperature. The reaction was stir-
red at ambient temperature for 6 h. TLC (7:3 hexane–
EtOAc) indicated a single faster moving product (Rf
0.65) than the starting materials (Rf 0.50). Ice chips were
added to the mixture, which was extracted twice with
EtOAc. The combined organic layers were washed with
brine and dried over MgSO4, concentrated in vacuo and
subjected to silica gel chromatographic purification
(using a gradient solvent system of 8:1 to 7:1 to 6:1
hexane–EtOAc) to afford the title compound as a white
1.4. tert-Butyldiphenylsilyl 2-azido-2-deoxy-3-O-
naphthylmethyl-6-O-(p-toluenesulfonyl)-b-D-galacto-
pyranoside (8)
A solution of compound 7 (5.20 g, 8.92 mmol) in dry
pyridine was treated with TsCl (1.87 g, 9.81 mmol) in
the presence of 4-dimethylaminopyridine (DMAP,
49.0 mg, 0.40 mmol) under Ar atmosphere at ambient
temperature for 72 h. Monitoring of the reaction by
TLC (5:2 hexane–EtOAc) showed a single product
(Rf 0.60). Ice chips were added in order to quench
the excess reagent. The reaction mixture was extracted
with EtOAc and the organic layer was washed with
NaHCO3, H2O, and brine, dried over Na2SO4, and
concentrated. The crude product was purified by silica
gel column chromatography (5:2 hexane–EtOAc) to
afford the title compound as a white amorphous solid
25
foamy amorphous solid (10.8 g, 95%): ½aꢁD +39.6 (c
1
1.00, CH2Cl2); H NMR (CDCl3, 400 MHz): d 1.12 (s,
t-Bu, 9H), 2.83 (br s, H-5, 1H), 3.27 (dd, J 3.6 Hz,
10.4 Hz, H-3, 1H), 3.75 (dd, J 1.6, 12.0 Hz, H-6a, 1H),
3.89 (dd, J 1.2, 12.4 Hz, H-6b, 1H), 3.91–4.11 (m, H-2,
H-4, 2H), 4.37 (d, J 8.0 Hz, H-1, 1H), 4.83 and 4.87
(2d, J 12.8 Hz, ArCH2, 1H each), 5.37 [s, PhCH(O)2,
1H], 7.29–7.54 (m, Ar, 14H), 7.69–7.82 (m, Ar, 8H);
13C NMR (CDCl3, 100 MHz): d 19.29, 26.93, 64.73,
66.19, 68.79, 71.65, 72.39, 77.73, 96.75, 101.05, 125.60,
125.93, 126.09, 126.39, 126.44, 127.16, 127.39, 127.61,
127.78, 128.14, 128.17, 128.99, 129.50, 129.64, 132.96,
135.23, 135.78, 135.93; MALDI-TOF MS: [M+Na]+
calcd for C40H41N3O5SiNa, 694.27; found, 694.72;
HRMS ESI-TOF: [M+Na]+ calcd for C40H41N3O5Si-
Na, 694.2729; found, 694.2713. Anal. Calcd for
C40H41N3O5Si: C, 71.51; H, 6.15; N, 6.25. Found: C,
71.26; H, 6.15; N, 6.10.
27
(6.18 g, 94%): ½aꢁD +17.19 (c 1.00, CH2Cl2); 1H
NMR (CDCl3, 400 MHz): d 1.11 (s, t-Bu, 9H), 2.39
(s, CH3Ar, 3H), 2.49 (br s, 4-OH, 1H), 3.17 (dd, J
2.4, 10.0 Hz, H-3, 1H), 3.23 (br t, J 6.4 Hz, H-5,
1H), 3.66 (dd, J 8.0, 9.2 Hz, H-2, 1H), 3.77 (d, J
3.2 Hz, H-4, 1H), 3.91 (dd, J 7.2, 10.0 Hz, H-6a, 1H),
4.12 (dd, J 7.2, 9.6 Hz, H-6b, 1H), 4.24 (d, J 8.0 Hz,
H-1, 1H), 4.74 (s, ArCH2, 2H), 7.33–7.47 (m, Ar,
10H), 7.65–7.83 (m, Ar, 11H); 13C NMR (CDCl3,
100 MHz): d 19.10, 21.59, 26.75, 60.33, 64.97, 67.93,
71.69, 72.23, 78.72, 96.45, 125.46, 126.13, 126.23,
126.77, 127.33, 127.47, 127.59, 127.76, 127.78, 128.39,
129.66, 129.71, 129.77, 132.18, 132.33, 132.61, 132.96,
132.97, 134.22, 135.69, 135.76, 144.75; MALDI-TOF:
[M+Na]+ calcd for C40H43N3O7SSiNa, 760.24; found,
760.41. Anal. Calcd for C40H43N3O7SSi: C, 65.10; H,
5.87; N, 5.69; S, 4.35. Found: C, 65.09; H, 5.76; N,
5.57; S, 4.59.
1.3. tert-Butyldiphenylsilyl 2-azido-2-deoxy-3-O-
naphthylmethyl-b-D-galactopyranoside (7)
Compound 6 (15.12 g, 22.53 mmol) was dissolved in
THF. A mixture of 4:1:1 AcOH–H2O–MeOH was
added to the solution, and the mixture was stirred at

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M. N. Amin et al. / Carbohydrate Research 341 (2006) 1922–1929
1.5. tert-Butyldiphenylsilyl 2-azido-2,6-dideoxy-6-iodo-3-
O-naphthylmethyl-b-D-galactopyranoside (9)
3.67 (dd, J 8.0 Hz, 10.0 Hz, H-2, 1H), 4.29 (d, J 8 Hz,
H-1, 1H), 4.82 (br s, ArCH2, 2H), 7.33–7.51 (m, Ar,
9H), 7.70–7.85 (m, Ar, 8H); 13C NMR (CDCl3,
100 MHz): d 16.03, 19.23, 26.87, 65.25, 68.21, 70.01,
72.01, 79.52, 96.55, 125.6, 126.08, 126.19, 126.77,
127.19, 127.39, 127.63, 127.82, 128.4, 129.55, 129.66,
132.82, 133.02, 133.04, 133.14, 134.58, 35.82, 135.93;
MALDI-TOF MS: [M+Na]+ calcd for C33H37N3O4Si-
Na, 590.24; found, 590.58; HRMS ESI-TOF: [M+Na]+
calcd for C33H37N3O4SiNa, 590.2437; found, 590.2451.
A mixture of compound 8 (2.50 g, 3.39 mmol) and NaI
(2.54 g, 17.0 mmol) in diethylene glycol dimethyl ether
was stirred in a brown flask under Ar atmosphere at
120 °C for 10 h until TLC (5:2 hexane–EtOAc) showed
the complete conversion of starting materials into a fas-
ter moving (Rf 0.70) product. The reaction mixture was
co-evaporated with toluene and then purified by flash
chromatography (5:1 hexane–EtOAc) to afford the title
compound as a white, foamy solid mass (2.14 g, 91%):
1.7. tert-Butyldiphenylsilyl 2,4-diazido-2,4,6-trideoxy-3-
O-naphthylmethyl-b-D-glucopyranoside (12)
24
½aꢁD +4.96 (c 0.30, CH2Cl2); 1H NMR (CDCl3,
400 MHz): d 1.10 (s, t-Bu, 9H), 2.29 (br s, 4-OH), 2.99
(dd, J 6.0, 10.0 Hz, H-6a, 1H), 3.19 (m, H-5, 1H),
3.23–3.29 (m, H-3, H-6b, 2H), 3.66 (dd, J 8.0, 10.0 Hz,
H-2, 1H), 4.04 (br s, H-4, 1H), 4.28 (d, J 8.0 Hz, H-1,
1H), 4.82 (br s, ArCH2, 2H), 7.32–7.49 (m, Ar, 9H),
7.69–7.85 (m, Ar, 8H); 13C NMR (CDCl3, 100 MHz):
d 19.27, 26.92, 53.46, 64.89, 65.69, 72.38, 74.76,
79.19, 96.4, 125.47, 126.09, 126.18, 126.8, 127.24,
127.43, 127.58, 127.76, 128.4, 129.58, 129.74, 132.35,
132.73, 132.98, 134.2, 135.75, 135.86; MALDI-TOF
MS: [M+Na]+ calcd for C33H36N3O4ISiNa, 716.14;
found, 716.65; HRMS ESI-TOF: [M+Na]+ calcd for
C33H36IN3O4SiNa, 716.1395; found, 716.1417. Anal.
Calcd for C33H36N3O4ISi: C, 57.14; H, 5.23; N,
6.06; I, 18.30. Found: C, 57.09; H, 5.00; N, 5.98; I,
18.07.
To a solution of compound 10 (1.33 g, 2.34 mmol) in dry
CH2Cl2 (15 mL) at 0 °C was added anhydrous pyridine
(470 lL, 5.86 mmol) and trifluoromethanesulfonic anhy-
dride (590 lL, 3.51 mmol). The solution was stirred at
the same temperature under Ar atmosphere for 1.5 h.
The reaction was monitored by TLC (3:1 hexane–
EtOAc), which showed a faster moving product (Rf
0.75) and the absence of starting materials. Ice chips
were added, and the mixture was extracted with CH2Cl2.
The organic layer was washed with H2O, NaHCO3, and
brine, followed by drying over Na2SO4. It was concen-
trated to give the crude triflate 11 as a light yellow oil,
which was immediately dissolved in anhydrous N,N-
dimethylformamide (10 mL) and treated with NaN3
(0.763 g, 11.7 mmol) at ambient temperature under
argon atmosphere. After 3 h, TLC (6:1 hexane–EtOAc)
revealed a single faster moving product (Rf 0.75). The
reaction mixture was diluted with EtOAc and washed
with water and brine. The organic phase was dried over
Na2SO4, concentrated, and purified by silica gel column
chromatography (8:1 hexane–EtOAc) to provide 12 as a
1.6. tert-Butyldiphenylsilyl 2-azido-2,6-dideoxy-3-O-
naphthylmethyl-b-D-galactopyranoside (10)
A mixture of compound 8 (7.50 g, 10.17 mmol) and NaI
(6.10 g, 40.7 mmol) in diethylene glycol dimethyl ether
was stirred at 120 °C in a brown flask under Ar atmo-
sphere until TLC (5:2 hexane–EtOAc) indicated the
complete conversion of starting materials (Rf 0.60) into
a faster moving product (Rf 0.70, compound 9). The
mixture was cooled to room temperature and then
24
pale-yellow semisolid product (1.26 g, 91%): ½aꢁD +30.27
1
(c 0.30, CH2Cl2); H NMR (CDCl3, 400 MHz): d 1.09
(d, J 6.0 Hz, H-6, 3H), 1.10 (s, t-Bu, 9H), 2.79 (m, H-
5, 1H), 3.09 (t, J 9.6 Hz, H-4, 1H), 3.17 (t, J 9.6 Hz,
H-3, 1H), 3.46 (dd, J 8.0, 9.2 Hz, H-2, 1H), 4.33 (d, J
7.6 Hz, H-1, 1H), 4.91 and 5.00 (2d, J 11.0 Hz, ArCH2,
each 1H), 7.32–7.52 (m, Ar, 9H), 7.66–7.69 (m, Ar, 4H),
7.80–7.84 (m, Ar, 4H); 13C NMR (CDCl3, 100 MHz):
d18.20, 19.28, 26.93, 67.83, 69.09, 70.51, 75.41, 81.39,
96.47, 125.90, 125.95, 126.01, 127.12, 127.42, 127.54,
127.89, 128.09, 129.62, 129.77, 132.47, 132.95, 132.97,
133.11, 134.65, 135.68, 135.74; MALDI-TOF MS:
[M+Na]+ calcd for C33H36N6O3SiNa, 615.25; found,
615.34; HRMS ESI-TOF: [M+Na]+ calcd for
C33H36N6O3SiNa, 615.2518; found, 615.2516.
ꢀ
NaCNBH3 (7.67 g, 0.122 mol) was added. The mixture
was stirred at 120 °C under argon atmosphere for 1 day.
Monitoring of the reaction by TLC (5:2 hexane–EtOAc)
revealed the absence of starting materials and two
slower moving products at Rf 0.50 and Rf 0.20, which
correspond to the desired product and compound 7,
respectively. The reaction mixture was diluted with
EtOAc and washed with water and brine, dried over
Na2SO4, and concentrated. The crude product was puri-
fied by silica gel column chromatography (5:2 hexane–
EtOAc) to afford the title compound as a colorless pasty
27
mass (3.86 g, 67%): ½aꢁD +11.03 (c 0.47, CH2Cl2): FTIR
1.8. tert-Butyldiphenylsilyl 2,4-diazido-2,4,6-trideoxy-b-
D-glucopyranoside (5)
1
(KBr, thin film) m (cmꢀ1): 2112 (N3); H NMR (CDCl3,
400 MHz): d 1.12 (s, t-Bu, 9H), 1.13 (d, J 6.0 Hz, H-6,
3H), 2.32 (br s, 4-OH, 1H), 3.10 (m, H-5, 1H), 3.23
(dd, J 3.6 Hz, 10.0 Hz, H-3, 1H), 3.62 (br s, H-4, 1H),
To a mixture of compound 12 (1.20 g, 2.03 mmol),
CH2Cl2 (15 mL), and H2O (1.5 mL) was added 2,3-di-

M. N. Amin et al. / Carbohydrate Research 341 (2006) 1922–1929
1927
chloro-5,6-dicyano-1,4-benzoquinone (DDQ, 0.552 g,
2.43 mmol) at room temperature under Ar atmosphere.
The mixture was stirred for 15 h. TLC (25:1 hexane–
EtOAc) showed a slower moving spot (Rf 0.20). The
reaction was quenched with ascorbic acid–citric acid
buffer (1.5 g of L-ascorbic acid, 1.8 g of citric acid mono-
hydrate, and 1.38 g of NaOH in 150 mL of H2O) and ex-
tracted with EtOAc. The combined organic layers were
washed with satd aq NaHCO3 and brine and dried over
Na2SO4. The dried organic phase was concentrated and
purified by silica gel column chromatography (25:1 hex-
ane–EtOAc) to afford compound 5 as a white solid
Calcd for C26H36N2O5Si: C, 64.43; H, 7.49; N, 5.78.
Found: C, 64.74; H, 7.22; N, 5.66.
1.10. 2,4-Diacetamido-2,4,6-trideoxy-D-glucopyranose (4)
To a solution of compound 14 (33.0 mg, 0.068 mmol) in
dry MeOH (5 mL) was added NH4F (13 mg, 0.34 mmol)
under Ar atmosphere at ambient temperature. The
resultant mixture was stirred for 10 h at room tempera-
ture and then 4 h at 40 °C. TLC (2:1 CHCl3–MeOH)
indicated the completeness of the reaction. Silica gel
(300 mg) was added to the reaction mixture, and the sol-
vent was removed under reduced pressure. The dried sil-
ica gel was transferred to a silica column and eluted with
3:1 CHCl3–MeOH. Concentration of the proper frac-
tion gave the title compound as a white solid (15.0 mg,
90%): mp 259–261 °C, dec (lit.4 mp 262–264 °C, dec);
25
(0.916 g, quant): mp 76–78 °C; ½aꢁD +12.48 (c 1.00,
CH2Cl2); 1H NMR (CDCl3, 400 MHz): d 1.08 (d, J
6.0 Hz, H-6, 3H), 1.10 (s, t-Bu, 9H), 2.53 (d, J 9.6 Hz,
3-OH, 1H), 2.81 (qd, J 6.0, 10.0 Hz, H-5), 3.03 (t, J
9.6 Hz, H-4, 1H), 3.25 (dt, J 3.6, 9.4 Hz, H-3, 1H),
3.34 (dd, J 7.6, 9.6 Hz, H-2, 1H), 4.34 (d, J 7.6 Hz, H-
1, 1H), 7.32–7.42 (m, Ar, 6H), 7.65–7.69 (m, Ar, 4H);
13C NMR (CDCl3, 100 MHz): d 18.00, 19.17, 26.86,
67.57, 69.14, 70.61, 74.13, 96.47, 127.27, 127.49,
129.71, 129.88, 132.49, 132.93, 135.74, 135.82; MAL-
DI-TOF MS: [M+Na]+ calcd for C22H28N6O3SiNa,
475.18; found, 475.36. Anal. Calcd for C22H28N6O3Si:
C, 58.38; H, 6.24; N, 18.57. Found: C, 58.22; H, 6.12;
N, 18.48.
26
½aꢁD ꢀ144.74 (c 1.00, H2O); 1H NMR (D2O,
400 MHz): (a anomer, major), d 1.03 (d, J 6.4 Hz, H-
6, 3H), 1.88 (s, CH3CO, 3H), 1.89 (s, CH3CO, 3H),
3.48 (t, J 10.0 Hz, H-4, 1H), 3.61 (t, J 10.8 Hz, H-3,
1H), 3.79 (dd, J 3.6, 10.8 Hz, H-2, 1H), 3.85 (qd, J
6.4, 10.0 Hz, H-5, 1H), 5.04 (d, J 3.6 Hz, H-1, 1H);
13C NMR (D2O, 100 MHz): d 17.65, 22.63, 22.86,
22.87, 22.90, 55.29, 57.68, 58.02, 67.25, 69.11, 71.67,
72.36, 91.28, 95.22, 174.85, 174.89, 174.97, 175.11;
MALDI-TOF MS: [M+Na]+ calcd for C10H18N2O5Na,
269.11; found, 268.88; HRMS ESI-TOF: [M+Na]+
calcd for C10H18N2O5Na, 269.1105; found, 269.1113.
1H NMR (D2O, 400 MHz): (b anomer, minor), d 1.06
(d, J 6.0 Hz, H-6, 3H), 1.96 (s, CH3CO, 3H), 1.97 (s,
CH3CO, 3H), 3.40–3.60 (m, H-2, 3, 4, 5, 4H), 4.54 (d,
J 8.8 Hz, H-1, 1H).
1.9. tert-Butyldiphenylsilyl 2,4-diacetamido-2,4,6-tri-
deoxy-b-D-glucopyranoside (14)
Compound 5 (75.0 mg, 0.166 mmol) was dissolved in
dry MeOH and Pd(OH)2/C (20 wt %) (6 mg) was added
to the solution (Caution! Extreme fire hazard!). The
mixture was stirred under a hydrogen atmosphere for
4 h. Monitoring of the reaction mixture by TLC (3:1
CHCl3–MeOH) showed a polar spot (Rf 0.05) that gave
a purple color with ninhydrin. The reaction mixture was
filtered and the filtrate was treated with AC2O (0.30 mL,
3.1 mmol). After 10 min, TLC (3:1 CHCl3–MeOH) re-
vealed a faster moving product (Rf 0.65). The reaction
mixture was concentrated and dried under vacuum over-
night to afford the title compound as a white solid
1.11. N-Allyloxycarbonyl 1-amino-2,4-diacetamido-2,4,6-
trideoxy-b-D-glucopyranose (15)
To a solution of 2,4-diacetamido-2,4,6-trideoxy-b-D-glu-
copyranose (4) (20.0 mg, 0.081 mmol) in H2O was added
enough amount of solid NH4HCO3 to make the solution
satd. It was stirred at 45 °C for 60 h. TLC (1:1 CHCl3–
MeOH) revealed a single slower moving (Rf 0.15) prod-
uct. Water was added and evaporated in vacuo twice.
The product was lyophilized, the white crude solid was
dissolved in 10:1 dioxane–water. To the solution NaH-
CO3 (50.0 mg, 0.59 mmol) was added, and the mixture
was stirred at 0 °C for 0.5 h. Allyl chloroformate
(65 lL, 0.61 mmol) was then added dropwise. The reac-
tion mixture was stirred for 13 h at ice-bath to room
temperature. TLC analysis (3:1 CHCl3–MeOH) indi-
cated the formation of a product (Rf 0.55). Small pieces
of ice were added, and the mixture was concentrated un-
der reduced pressure. Concentrated crude was purified
by silica gel column chromatography (3:1 CHCl3–
MeOH) to give compound 15 as an off-white solid
27
(79 mg, 98%): mp 204–205 °C; ½aꢁD ꢀ14.32 (c 1.00,
1
CH3OH); H NMR (CD3OD, 400 MHz): d 1.05 (d, J
6.0 Hz, H-6, 3H), 1.07 (s, t-Bu, 9H), 1.93 (s, CH3CO,
3H), 1.95 (s, CH3CO, 3H), 3.06 (qd, J 6.0, 10.0 Hz, H-
5), 3.43 (t, H-4, J 10.0 Hz, 1H), 3.50 (t, J 10.0 Hz, H-
3, 1H), 3.78 (dd, J 8.4, 10.4 Hz, H-2, 1H), 4.55 (d, J
8.4 Hz, H-1, 1H), 7.34–7.44 (m, Ar, 6H), 7.66–7.72 (m,
Ar, 4H); 13C NMR (CD3OD, 100 MHz): d 18.20,
20.04, 22.87, 23.20, 27.36, 59.09, 60.21, 72.01, 72.10,
97.23, 128.27, 128.48, 130.74, 130.78, 134.38, 134.41,
136.86, 137.05, 173.41, 173.49; MALDI-TOF MS:
[M+Na]+ calcd for C26H36N2O5SiNa, 507.23; found,
507.28; HRMS ESI-TOF: [M+Na]+ calcd for
C26H36N2O5SiNa, 507.2291; found, 507.2292. Anal.
25
(19.0 mg, 71%): mp 248–259 °C (dec); ½aꢁD ꢀ46.56 (c

1928
M. N. Amin et al. / Carbohydrate Research 341 (2006) 1922–1929
1
0.50, H2O); H NMR (CD3OD, 400 MHz): d 1.17 (d, J
6.0 Hz, H-6, 3H), 1.96 (s, CH3CO, 3H), 1.97 (s, CH3CO,
3H), 3.46–3.54 (m, H-3, H-4, H-5, 3H), 3.71 (dd, J 10.0,
11.2 Hz, H-2, 1H), 4.54 (d, J 5.2 Hz, CHAHBCHCH2–,
2H), 4.73 (d, J 10.0 Hz, H-1, 1H), 5.18 (dd, J 1.2,
6.4 Hz, CHAHB@CHCH2–, 1H), 5.28 (dd, J 1.2,
17.2 Hz, CHAHB@CHCH2–, 1H), 5.91 (m, CHAHB@
CHCH2–, 1H); 13C NMR (CD3OD, 100 MHz): d
18.45, 22.81, 22.94, 56.80, 58.88, 66.63, 73.79, 82.66,
117.55, 133.83, 157.88, 173.43, 174.10; MALDI-TOF
MS: [M+Na]+ calcd for C14H23N3O6Na, 352.14; found,
352.39; HRMS ESI-TOF: [M+Na]+ calcd for
C14H23N3O6Na, 352.1496; found, 352.1485.
and the ‘Ecomolecular Science’ and ‘Chemical Biology’
programs of RIKEN. We also thank Ms. A. Takahashi
for technical assistance.
References
1. Wacker, M.; Linton, D.; Hitchen, P. G.; Nita-Lazar, M.;
Haslam, S. M.; North, S. J.; Panico, M.; Morris, H. R.;
Dell, A.; Wren, B. W.; Aebi, M. Science 2002, 298, 1790–
1793.
2. Young, N. M.; Brisson, J.-R.; Kelly, J.; Watson, D. C.;
Tessier, L.; Lanthier, P. H.; Jarrell, H. C.; Cadotte, N.;
Michael, F. St.; Aberg, E.; Szymanski, C. M. J. Biol.
Chem. 2002, 277, 42530–42539.
1.12. Na-Fluoren-9-ylmethyloxycarbonyl-Nc-(2,4-diacet-
amido-2,4,6-trideoxy-b-D-glucopyranosyl)-L-asparagine
t-butyl ester (2)
3. Scha¨ffer, C.; Scherf, T.; Christian, R.; Kosma, P.; Zayni,
S.; Messner, P.; Nathan, S. Eur. J. Biochem. 2001, 268,
857–864.
4. Sharon, N.; Jeanloz, R. W. J. Biol. Chem. 1960, 235, 1–5.
5. (a) Liav, A.; Hildesheim, J.; Zehavi, U.; Sharon, N.
Carbohydr. Res. 1974, 33, 217–227; (b) Bundle, D.;
Josephson, S. Can. J. Chem. 1980, 58, 2679–2685.
6. Chemoenzymatic syntheses of undecaprenylpyrophos-
phate-linked glycans have been reported. (a) Weerapana,
E.; Glover, K. J.; Chen, M. M.; Imperiali, B. J. Am. Chem.
Soc. 2005, 127, 13766–13767; (b) Glover, K. J.; Weera-
pana, E.; Imperiali, B. Proc. Natl. Acad. Sci. U.S.A. 2005,
102, 14255–14259.
A mixture of compound 15 (3.0 mg, 0.009 mmol), Na-
HCO3 (8 mg, 0.1 mmol), FmocAsp(F)OtBu (6.0 mg,
0.15 mmol), and PhSiH3 (10 lL, 0.081 mmol) in 10:1
dioxane–water was treated with Pd(PPh3)4 (1 mg,
0.9 lmol) at 0 °C. The stirring was continued at ambient
temperature for 21 h. Monitoring of the reaction by TLC
(4:1 CHCl3–MeOH) indicated the formation of a UV-ac-
tive product (Rf 0.60). The mixture was filtered, and the
filtrate was concentrated. The concentrated mass was
subjected to preparative thin layer chromatography
(6:1 CHCl3–MeOH) to provide compound 2 (5.0 mg,
´
7. (a) Medgeyes, A.; Farkas, E.; Liptak, A.; Pozsgay, V.
Tetrahedron 1997, 53, 4159–4178; (b) Hermans, J. P. G.;
Elie, C. J. J.; van der Marel, G. A.; van Boom, J. H. J.
Carbohydr. Chem. 1987, 6, 451–462.
8. Dwek, R. A. Chem. Rev. 1996, 96, 683–720.
24
86%) as a white solid: mp 233–234 °C (dec); ½aꢁD 14.0
9. Essentials in Glyocbiology; Varki, A., Cummings, R.,
Esko, J., Freeze, H., Hart, G., Marth, J., Eds.; Cold
Spring Harbor Laboratory Press: New York, 1999.
10. (a) Benz, I.; Schmidt, M. A. Mol. Microbiol. 2002, 45,
267–276; (b) Kornfeld, R.; Kornfeld, S. Annu. Rev.
Biochem. 1985, 54, 631–664; (c) Szymanski, C. M.; Yao,
R.; Ewing, C. P.; Trust, T. J.; Guerry, P. Mol. Microbiol.
1999, 32, 1022–1030; (d) Linton, D.; Allan, E.; Karlyshev,
A. V.; Cronshaw, A. D.; Wren, B. W. Mol. Microbiol.
2002, 43, 497–508; (e) Schmidt, M. A.; Riley, L. W.; Benz,
I. Trends Microbiol. 2003, 11, 554–561.
11. Szymanski, C. M.; Logan, S. M.; Linton, D.; Wren, B. W.
Trends Microbiol. 2003, 11, 233–238.
12. Chemical synthesis of Glc1GalNAc5 has been completed
very recently. Ishiwata, A.; Ohta, S.; Ito, Y. Carbohydr.
Res. in press, doi:10.1016/j.carres.2006.03.011.
13. Crich, D.; Dudkin, V. J. Am. Chem. Soc. 2001, 123, 6189–
6825.
14. Nakahara, Y.; Iijima, H.; Shibayama, S.; Ogawa, T.
Carbohydr. Res. 1991, 216, 211–225.
15. (a) Gaunt, M. J.; Yu, J.; Spencer, J. B. J. Org. Chem. 1998,
63, 4172–4173; (b) Gaunt, M. J.; Boschetti, C. E.; Yu, J.;
Spencer, J. B. Tetrahedron Lett. 1999, 40, 1803–1806; (c)
Xia, J.; Abbas, S. A.; Locke, R. D.; Piskorz, C. F.;
Alderfer, J. L.; Matta, K. L. Tetrahedron Lett. 2000, 41,
169–173; (d) Liao, W.; Locke, R. D.; Matta, K. L. Chem.
(c 0.15, CHCl3–CH3OH, 1:1); 1H NMR (CDCl3–
CD3OD, 1:1, 400 MHz): d 1.17 (d, J 6.0 Hz, H-6, 3H),
1.42 (s, t-Bu, 9H), 1.92 (s, CH3CO, 3H), 1.97 (s, CH3CO,
3H), 2.70 (dd, J 6.4, 6.8 Hz, Asn-CH2, 2H), 3.40–3.47 (m,
H-3, H-5, 2H), 3.55 (dd, J 9.6, 10.0 Hz, H-4, 1H), 3.73
(dd, J 9.6, 10.0 Hz, H-2, 1H), 4.19 (br t, J 6.4 Hz,
FmocCH, 1H), 4.28 (dd, J 7.2, 10.4 Hz, FmocCHAHB,
1H), 4.41 (dd, J 7.2, 10.4 Hz, FmocCHAHB, 1H), 4.46
(t, J 5.6 Hz, Asn-aCH), 4.86 (d, J 9.6 Hz, H-1, 1H),
7.26–7.38 (m, Fmoc, 4H), 7.60 (d, J 7.2 Hz, Fmoc,
2H), 7.74 (d, J 7.6 Hz, Fmoc, 2H); 13C NMR (CDCl3–
CD3OD, 1:1, 100 MHz): d 18.29, 22.89, 22.92, 28.14,
38.16, 47.66, 51.77, 56.08, 58.00, 67.52, 73.05, 73.32,
78.21, 79.48, 82.69, 120.22, 125.37, 125.42, 127.38,
128.03, 141.59, 141.61, 141.61, 144.06, 144.15, 157.15,
170.77, 171.63, 172.84, 173.78; MALDI-TOF MS:
[M+Na]+ calcd for C33H42N4O9Na, 661.28; found,
661.21; HRMS ESI-TOF: [M+Na]+ calcd for
C33H42N4O9Na, 661.2819; found, 661.2850.
´
Commun. (Cambridge) 2000, 369–370; (e) Liptak, A.;
Acknowledgments
´
´
´
Borbas, A.; Janossy, L.; Szilagyi, L. Tetrahedron Lett.
´ ´
´
´
2000, 41, 4949–4953; (f) Csavas, M.; Borbas, A.; Szilagyi,
This work was partly supported by Grant-in-Aid for
Creative Scientific Research from the Japan Society
for the Promotion of Science (Grant No. 17GS0420)
´
L.; Liptak, A. Synlett 2002, 887–890.
16. Smith, M.; Rammler, D. H.; Goldberg, I. H.; Khorana, H.
G. J. Am. Chem. Soc. 1962, 84, 430–440.

M. N. Amin et al. / Carbohydrate Research 341 (2006) 1922–1929
1929
17. Millar, J. G.; Underhill, E. W. J. Org. Chem. 1986, 51,
4726–4728.
21. Zhang, W.; Robins, M. J. Tetrahedron Lett. 1992, 33,
1177–1180.
18. Hutchins, R. O.; Kandasamy, D.; Maryanoff, A.; Masila-
mani, D.; Maryanoff, B. E. J. Org. Chem. 1977, 42, 82–91.
19. Kuzuhara, H.; Sato, K.; Emoto, S. Carbohydr. Res. 1975,
43, 211–225.
22. (a) Likhosherstov, L. M.; Novikova, O. S.; Derevitskaja,
V. A.; Kochetkov, N. K. Carbohydr. Res. 1986, 146, c1–
´
c5; (b) Lubineau, A.; Auge, J.; Droillat, B. Carbohydr.
Res. 1995, 266, 211–219.
20. Bruce, I.; Fleet, G. W. J.; Girdhar, A.; Haraldsson, M.;
Peach, J. M.; Watkin, D. J. Tetrahedron 1990, 46, 19–32.
23. Ishiwata, A.; Takatani, M.; Nakahara, Y.; Ito, Y. Synlett
2002, 634–636.