Preparation of orthogonally protected chitosan oligosaccharides: observation of an anomalous remote substituent effect.

Article abstract of DOI:10.1016/S0008-6215(02)00124-6

Orthogonally protected chitosan tetrasaccharides were synthesized in a convergent fashion by trichloroacetimidate activation. The anomeric substituent at the reducing end of the disaccharide acceptor has a remarkably strong influence on glycosidic coupling; a thiophenyl-substituted disaccharide was observed to be an unusually poor glycosyl acceptor in comparison with the corresponding allyloxy-substituted disaccharide.

Full text of DOI:10.1016/S0008-6215(02)00124-6

Carbohydrate Research 337 (2002) 1319–1324
www.elsevier.com/locate/carres
Note
Preparation of orthogonally protected chitosan oligosaccharides:
observation of an anomalous remote substituent effect
Siong-Tern Liew, Alexander Wei*
Department of Chemistry, Purdue Uni6ersity, 1393 Brown Building, West Lafayette, IN 47907-1393, USA
Received 22 September 2001; accepted 22 May 2002
Abstract
Orthogonally protected chitosan tetrasaccharides were synthesized in a convergent fashion by trichloroacetimidate activation.
The anomeric substituent at the reducing end of the disaccharide acceptor has a remarkably strong influence on glycosidic
coupling; a thiophenyl-substituted disaccharide was observed to be an unusually poor glycosyl acceptor in comparison with the
corresponding allyloxy-substituted disaccharide. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Chitin; Protecting groups; Glycosylation
protected chitosan tetrasaccharide with an allyl group
at the reducing end as a functionalizable tether.6 In the
course of this work, we have determined that remote
substituents can have surprisingly dramatic effects on
reactivity during glycosidic coupling.
Repeating-unit oligosaccharides and their esters have
been identified as signaling elements for a broad spec-
trum of biological processes, such as elicitors in plant
defense mechanisms,1 nodulation factors of leguminous
roots,2 or regulators of fibroblast growth factor-medi-
ated cellular processes.3 In several cases there is strong
evidence that the positional distribution of acyl or
sulfate groups on the carbohydrate repeat units has a
critical role in determining the specificity of biological
action. For example, chitosan sulfated mostly at the
O-2 and O-3 positions has demonstrated potent in vitro
activity against HIV-1 infection of T-lymphocytes,
whereas chitin sulfated at the O-6 position has exhib-
ited strong anticoagulatory properties.4 However, the
intrinsic polydispersity of fractionated or semisynthetic
carbohydrates introduces ambiguity in determining the
structural basis for biological activity.
Systematic studies with repeating-unit oligosaccha-
rides of defined sizes and substitution patterns can be
useful for establishing unambiguous structure–activity
relationships. To this end we are developing protecting
group systems whose cleavage conditions are compat-
ible with sensitive functional groups such as sulfates.5
Here we present a concise synthesis of an orthogonally
Glucosamine-derived acceptor and donor 1 and 2
were synthesized using known procedures6,7 and cou-
pled in the presence of catalytic trimethylsilyl
trimethanesulfonate (TMSOTf) to yield disaccharide 3
on a multigram scale (see Scheme 1). Trichloroacetimi-
date-activated glycosylation gave consistently better
yields in our hands than direct activation of the thio-
phenyl glycoside.8 It should be mentioned that oxida-
tive cleavage of the anomeric thiophenyl group with
N-bromosuccinimide (NBS) was accompanied by par-
tial bromination of the p-methoxybenzyl (PMB) pro-
tecting group at O-6; however, this did not appear to
interfere with subsequent transformations.9 The pro-
tected disaccharide 3 was converted straightforwardly
into glycosyl acceptors 4 and 6 and into glycosyl donor
5, but attempts to couple disaccharides 4 and 5 were
unsuccessful despite numerous variations in reaction
conditions. Increasing the amount of TMSOTf to 1.3–
1.5 equiv did not improve the yield of coupling reac-
tions involving 4, indicating that coordination of the
Lewis acid by the thiophenyl group was not a rate-de-
termining factor. However, substituting the anomeric
S-thiophenyl unit with an O-allyl group greatly in-
* Corresponding author. Tel.: +1-765-4945257; fax: +1-
765-494-0239
E-mail address: alexwei@purdue.edu (A. Wei).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00124-6

1320
S.-T. Liew, A. Wei / Carbohydrate Research 337 (2002) 1319–1324
Table 1
creased the reactivity of the nucleophile, enabling tetra-
saccharide 7 to be obtained in 70% yield. Intermediate
7 was then transformed into orthogonally protected
tetrasaccharide 8 by aminolysis of phthalimide and
acetate groups with ethylenediamine,10 followed by re-
protection as a tetraazide (see Scheme 2).11 The par-
tially brominated PMB groups were cleaved to yield the
corresponding tetraol 9, whose chemical composition
was confirmed by elemental analysis.
TMSOTf-mediated coupling of mono- and disaccharide
trichloroacetimidates with different glycosyl acceptorsa
Glycosyl acceptor
Coupling yieldb (%)
Monosaccharide 2 Disaccharide 5
78
60
A systematic investigation of the glycosylation step
with different coupling partners revealed thiophenyl
disaccharide 4 to be a singularly poor glycosyl acceptor
(see Table 1), encouraging us to examine the influence
of the remote anomeric substituent in further detail.
Chromatographic recovery and analysis of the compo-
nents from the attempted coupling of acceptor 4 with
donor 5 revealed a significant amount of TMS-pro-
tected acceptor, whereas relatively little TMS ether was
15
56
0
50 (70c)
a All=allyl, Phth=phthalimido, PMB(*)=p-methoxyben-
zyl (or m-bromo-p-methoxybenzyl).
b Reaction conditions: 1.0 equiv glycosyl acceptor (0.1 M),
,
1.5 equiv glycosyl donor, 0.3 equiv TMSOTf, 4 A mol. sieves,
CH2Cl2, −20 °C to rt.
c Same as b, but with 1.2 equiv of glycosyl donor, −20 to
4 °C.
recovered from the coupling reaction between 6 and 5.
Acceptors 4 and 6 were treated with TMSOTf at
−78 °C in CH2Cl2 in the presence of tetramethylurea as
a proton scavenger to determine their relative rates of
TMS ether formation, but no significant differences
were observed. We next considered the possibility of the
thiophenyl moiety having a role as a trimethylsilyl
transfer agent; however, adding one molar equivalent
of thiophenyl disaccharide 3 to the coupling reaction
between 6 and 5 affected neither the yield of the
tetrasaccharide 7 nor the amount of TMS-protected
side product.
Scheme 1. Synthesis of chitosan disaccharide intermediates.
All=allyl, Phth=phthalimido, PMB(*)=p-methoxybenzyl
(or m-bromo-p-methoxybenzyl). Reagents and conditions: a.
(i) ClCH2COCl, pyridine, Et2O, −20 °C; (ii) NBS, 9:1
CH3COCH3–water, −20 to 0 °C; (iii) CCl3CN, DBU,
CH2Cl2, 0 °C (70% over three steps). b. 2 (1.5 equiv), TMS-
,
OTf (0.3 equiv), 4 A mol. sieves, CH2Cl2, −20 °C (78%). c.
Thiourea, 1:1 MeOH–CH2Cl2, 40 °C (95%). d. Same as a, ii
,
and iii (77% over two steps). e. (i) allyl alcohol, 4 A mol.
sieves, TMSOTf, CH2Cl2, −20 °C to rt; (ii) thiourea, 1:1
MeOH–CH2Cl2, 40 °C (95% over two steps).
Substituent effects on glycosylation rates have been
extensively investigated,12,13 but to the best of our
knowledge remote substituent effects of this nature
have not been documented despite the widespread use
of thioarylglycosides as intermediates in oligosaccharide
synthesis.8,10 Although the basis for the deactivating
influence of the remote thiophenyl group on glycosyl
acceptors remains undetermined, the anomalous effect
may have some broader implications, e.g., in the design
of solid-phase syntheses for constructing oligosaccha-
ride libraries.14,15
Scheme 2. Synthesis of orthogonally protected chitosan tetra-
saccharide. All=allyl, Phth=phthalimido, PMB(*)=p-
methoxybenzyl (or m-bromo-p-methoxybenzyl). Reagents
and conditions: a. (i) ethylenediamine, n-BuOH, 100 °C; (ii)
TfN3, CuSO4, K2CO3, 10:9:1 CH2Cl2–MeOH–water; (iii)
Ac2O, pyridine (39% yield over three steps). b. DDQ, 4:1
CH2Cl2–water, rt (60%).

S.-T. Liew, A. Wei / Carbohydrate Research 337 (2002) 1319–1324
1321
1. Experimental
CCl3CN (6.85 mL, 68.4 mmol) and DBU (153 mL, 1.03
mmol). After 2 h the reaction mixture was concentrated
in vacuo and purified by flash chromatography (10–
20% EtOAc–toluene, +1% Et3N) to yield trichloro-
acetimidate 2 (1.98 g, 70% over three steps) as a white
solid with a 4:1 mixture of PMB- and (3-bromo)-PMB-
protected ethers. [h]D +58.6° (c 1.03, CH2Cl2); IR
General methods.—All chemicals were obtained from
Aldrich Chemical Co. and used as received. All experi-
ments were conducted under an atmosphere of dry
argon unless otherwise noted. IR spectra were acquired
with a Nicolet Nexus 670 FTIR spectrometer. Optical
rotations were measured at 20–25 °C with a Rudolph
Research AUTOPOL® III polarimeter. 1H and 13C
spectra were recorded on a Varian Unity Inova NMR
spectrometer operating at 300 and 75 MHz, respec-
tively, or on a Bruker DRX 500 operating at 500 and
125 MHz, respectively. Chemical shifts are referenced
to the solvent used (7.27 and 77.23 ppm for CDCl3,
3.31 and 49.15 ppm for CD3OD, 7.16 and 128.39 ppm
for C6D6) unless otherwise stated. Electrospray-ioniza-
tion mass spectra (ESIMS) were acquired using either a
Hewlett–Packard 5989B or a Finnigan 4000 mass spec-
trometer. The chromatographic purity of the products
was assessed using TLC on Silica Gel 60 F254 (E.
Merck) with either ethanolic p-anisaldehyde or ninhy-
drin staining solutions. Chromatographic separations
were performed using silica gel (ICN SiliTech 32–63D).
Elemental analysis was performed in the Department of
Chemistry, Purdue University. In cases where com-
pounds were partially brominated, molar equivalents
and yields were determined using the relative intensities
1
(neat): 1747, 1719, 1385, 1221 cm−1; H NMR (CDCl3,
300 MHz): l 8.71 (1H, d, J 6.3 Hz), 7.89–7.74 (4H, m),
7.27 (2H, dd, J 2.4, 9.0 Hz), 6.92 (2H, d, J 8.7 Hz), 6.66
(1H, dd, J 1.8, 9.0 Hz), 5.96 (1H, dt, J 4.5, 9.9 Hz), 5.42
(1H, dt, J 2.4, 9.9 Hz), 4.71–4.42 (3H, m), 4.13–3.63
(8H, m), 1.93 (3H, d, J 1.2 Hz); 13C NMR (CDCl3, 75
MHz): l 170.49, 167.65, 166.44, 160.86, 159.67, 155.84,
134.74, 133.49, 131.46, 131.33, 130.08, 129.71, 128.80,
123.97, 114.08, 112.07, 93.85, 73.88, 73.84, 73.43, 72.72,
71.44, 71.37, 70.54, 68.26, 67.99, 56.56, 55.55, 53.82,
40.75, 20.75. ESIMS: m/z 713 [M+Na]+, 792, 794
[M+Br+Na]+.
Phenyl
p - methoxybenzyl - 2 - phthalimido - i -
(1“4)-3-O-acetyl-2-deoxy-6-O-p-methoxybenzyl-2-
phthalimido-1-thio-i- -glucopyranoside (3).—A solu-
3-O-acetyl-4-O-chloroacetyl-2-deoxy-6-O-
D
- glucopyranosyl-
D
tion of trichloroacetimidate 2 (4.05 g, 5.86 mmol, a 4:1
mixture of PMB- and (3-bromo)-PMB-protected ethers)
and thiophenyl glycoside 1 (2.2 g, 3.91 mmol) in dry
CH2Cl2 (43 mL) was stirred under argon at room
1
,
of the H NMR peaks in the aromatic region to esti-
temperature with 4 A molecular sieves (4.3 g) for 30
mate the fraction of brominated product.
min. The mixture was cooled to −20 °C, then treated
with TMSOTf (212 mL, 1.173 mmol). After 5 h, the
reaction was quenched with Et3N and concentrated to
an oil. The residue was purified by flash chromatogra-
phy (10–20% EtOAc–toluene) to yield thiophenyl dis-
accharide 3 (3.37 g, 78%) as a white solid with a 4:1
mixture of PMB- and (3-bromo)-PMB-protected ethers.
[h]D +20.9° (c 1.03, CH2Cl2); IR (neat): 1777, 1747,
1715, 1385, 1229 cm−1; 1H NMR (CDCl3, 300 MHz): l
7.87–7.74 (8H, m), 7.38–7.16 (13H, m), 5.79–5.68 (2H,
m), 5.61 (1H, d, J 10.5 Hz) 5.45 (1H, d, J 8.4 Hz), 5.29
(1H, t, J 9.3 Hz), 4.46–3.40 (21H, m), 1.88 (3H, s), 1.85
(3H, s); 13C NMR (CDCl3, 75 MHz): l 170.56, 170.40,
168.01, 167.51, 166.35, 134.62, 134.38, 133.23, 131.98,
131.72, 131.64, 131.51, 130.62, 129.95, 129.68, 129.44,
129.06, 128.34, 123.91, 114.05, 113.93, 112.17, 97.37,
83.14, 78.78, 74.28, 73.27, 72.71, 72.41, 72.31, 71.73,
70.75, 68.24, 67.77, 56.55, 55.55, 55.17, 54.20, 40.78,
20.81, 20.69. ESIMS: m/z 1115 [M+Na]+, 1194, 1196
[M+Br+Na]+.
3-O-Acetyl-4-O-chloroacetyl-2-deoxy-6-O-p-meth-
oxybenzyl-2-phthalimido-i-D-glucopyranosyl trichloro-
acetimidate (2).—Toluene (3×25 mL) was evaporated
from thiophenyl glycoside 1 (2.3 g, 4.08 mmol), and the
residue was dissolved in anhyd Et2O (38 mL). The
solution was cooled to −15 °C, and pyridine was
added (1.65 mL, 20.42 mmol) followed by dropwise
addition of chloroacetyl chloride (1.3 mL, 16.33 mmol).
The resulting mixture was stirred for 1 h from −15 to
−10 °C. Saturated aq NaHCO3 was added, and the
mixture was extracted with CH2Cl2 (3×50 mL). The
combined extracts were evaporated to dryness, and the
resulting residue was dissolved in 9:1 CH3COCH3–
water (60 mL) and cooled to −20 °C, followed by
addition of NBS (2.78 g, 15.64 mmol). After stirring for
1 h from −20 to −10 °C, the reaction was quenched
with satd aq NaHCO3 (20 mL) and diluted with water
(20 mL), followed by extraction with dichloromethane
(3×50 mL). The combined organic extract was dried
over MgSO4, filtered and concentrated in vacuo. Chro-
matography on silica gel (20–40% EtOAc–hexanes gra-
dient elution) afforded 1.87 g of the intermediate lactol
as a white solid. This product was azeotropically dried
with toluene (3×50 mL) and dissolved in dry CH2Cl2
Phenyl 3-O-acetyl-2-deoxy-6-O-p-methoxybenzyl-2-
phthalimido-i-
D
-glucopyranosyl-(1“4)-3-O-acetyl -2-
deoxy-6-O-p-methoxybenzyl-2-phthalimido-1-thio-i-
D
-
glucopyranoside (4).—A mixture of thiophenyl disac-
charide 3 (500 mg, 0.458 mmol, a 4:1 mixture of PMB-
and (3-bromo)-PMB-protected ethers) and thiourea
(175 mg, 2.29 mmol) in 1:1 MeOH–CH2Cl2 (25 mL)
was stirred at 40 °C for 14 h, then concentrated to an
,
(35 mL), followed by addition of 4 A molecular sieves
(3.5 g). The mixture was stirred at room temperature
for 30 min, then cooled to 0 °C and treated with

1322
S.-T. Liew, A. Wei / Carbohydrate Research 337 (2002) 1319–1324
67.29, 56.50, 55.48, 55.09, 54.11, 40.69, 20.78, 20.61.
ESIMS: m/z 1166 [M+Na]+, 1245, 1247 [M+Br+
Na]+, 1324, 1326, 1328 [M+2 Br+Na]+.
oil. The residue was purified by flash chromatography
(0–2% MeOH–CH2Cl2) to yield disaccharide acceptor
4 (442 mg, 95%) as a white solid with a 4:1 mixture of
PMB- and (3-bromo)-PMB-protected ethers. [h]D
+5.2° (c 1.03, CH2Cl2); IR (neat): 2941, 1777, 1746,
Allyl
phthalimido-i-
deoxy-6-O-p-methoxybenzyl-2-phthalimido-i-
3-O-acetyl-2-deoxy-6-O-p-methoxybenzyl-2-
-glucopyranosyl-(1“4)-3-O-acetyl-2-
-gluco-
D
1
1715, 1384, 1225, 1049 cm−1; H NMR (CDCl3, 300
D
MHz): l 7.86–7.74 (8H, m), 7.50–6.87 (13H, m), 5.73
(1H, t, J 0.5 Hz), 5.63–5.57 (2H, m), 5.47 (1H, d, J 8.1
Hz), 4.53–3.42 (20H, m), 3.00 (1H, s), 1.92 (3H, s),
1.86, (3H, s); 13C NMR (CDCl3, 125 MHz): l 171.23,
171.09, 170.18, 167.95, 167.44, 159.60, 159.27, 134.90,
134.54, 134.42, 134.31, 133.13, 132.99, 131.68, 131.47,
130.54, 129.64, 129.56, 129.35, 128.99, 128.42, 128.34,
128.24, 124.10, 123.85, 123.71, 117.73, 116.08, 114.13,
113.84, 113.10, 112.11, 111.76, 110.51, 109.34, 100.99,
97.46, 83.01, 78.81, 74.39, 73.89, 73.53, 73.38, 73.21,
72.65, 71.78, 71.34, 70.20, 70.04, 67.72, 65.01, 56.50,
55.49, 55.11, 54.17, 52.46, 20.81; ESIMS: m/z 1039
[M+Na]+, 1118, 1120 [M+Br+Na]+.
pyranoside (6).—A solution of disaccharide donor 5 (20
mg, 0.0175 mmol, a 4:1 mixture of PMB- and (3-
bromo)-PMB-protected ethers) and allyl alcohol (2.5
mL, 0.035 mmol) in dry CH2Cl2 (0.5 mL) was stirred
,
under argon at room temperature with 4 A molecular
sieves (50 mg) for 30 min. The mixture was then cooled
to −20 °C, followed by addition of TMSOTf (3.2 mL,
17.5 mmol). After 3 h, the reaction was quenched with
Et3N, filtered and evaporated to dryness. The remain-
ing residue was dissolved in 1:1 MeOH–CH2Cl2 (1
mL). Thiourea (6.7 mg, 87.5 mmol) was added, and the
mixture was stirred at 40 °C for 14 h, then concentrated
to an oil. The residue was purified by flash chromatog-
raphy (0–2% MeOH–CH2Cl2) to yield disaccharide
acceptor 6 (16 mg, 95%) as a white solid with a 4:1
mixture of PMB- and (3-bromo)-PMB-protected ethers.
Compound 6 was also prepared in 63% yield with a
4:1 mixture of PMB- and (3-bromo)-PMB-protected
ethers by coupling trichloroacetimidate 2 (7.31 g, 11.55
mmol, a 4:1 mixture of PMB- and (3-bromo)-PMB-pro-
tected ethers) with allyl 3-O-acetyl-4-O-chloroacetyl-2-
3-O-Acetyl-2-deoxy-6-O-p-methoxybenzyl-2-phthal-
imido-i-
acetyl-2-deoxy-6-O-p-methoxybenzyl-2-phthalimido-i-
-glucopyranosyl trichloroacetimidate (5).—To a solu-
D
-glucopyranosyl-(1“4)-3-O-acetyl-4-O-chloro-
D
tion of thiophenyl disaccharide 3 (340 mg, 0.311 mmol,
a 4:1 mixture of PMB- and (3-bromo)-PMB-protected
ethers) in 9:1 CH3COCH3–water (5 mL) at −20 °C
was added NBS (333 mg, 1.868 mmol). After stirring
for 1 h from −20 to 0 °C, the reaction was quenched
with satd aq NaHCO3 (1 mL) and diluted with water (1
mL), followed by extraction with CH2Cl2 (3×5 mL).
The combined extracts were dried over MgSO4, filtered
and concentrated to an oil. Chromatography on silica
gel (20–40% EtOAc–hexanes) afforded 280 mg of the
intermediate lactol as a white solid. This was azeotrop-
ically dried with toluene (3×5 mL), and the residue
was dissolved in dry CH2Cl2 (7 mL), followed by
deoxy-6-O-p-methoxybenzyl-2-phthalimido-b-D-glucopy
ranoside6b (6.54 g, 13.87 mmol). [h]D −21.0° (c 1.03,
CH2Cl2); IR (neat): 2867, 1773, 1746, 1715, 1610, 1509,
1384, 1240, 1042 cm−1; 1H NMR (CDCl3, 300 MHz): l
7.86–7.72 (8H, m), 7.30–6.85 (8H, m), 5.77–5.51 (3H,
m), 5.48 (1H, d, J 8.4 Hz), 5.28 (1H, d, J 8.4 Hz), 5.05
(2H, dq, J 1.2, 17.1 Hz), 4.54–3.44 (21H, m), 3.09 (1H,
dd, J 3.3, Hz), 1.91 (3H, s), 1.88 (3H, s); 13C NMR
(CDCl3, 75 MHz): l 171.30, 171.17, 168.05, 159.63,
159.32, 155.77, 134.49, 133.83, 133.01, 131.76, 131.48,
130.55, 130.06, 129.77, 129.61, 129.41, 128.43, 123.72,
117.70, 114.18, 113.98, 113.88, 112.19, 111.75, 97.39,
97.20, 77.56, 74.70, 74.63, 74.05, 73.63, 73.53, 73.46,
72.75, 72.64, 71.91, 71.69, 71.24, 70.14, 70.01, 67.63,
56.56, 55.53, 55.27, 55.21, 20.93, 20.87; ESIMS: m/z
987 [M+Na]+, 1066, 1068 [M+Br+Na]+.
,
addition of 4 A molecular sieves (700 mg). The mixture
was stirred at room temperature for 30 min, then
cooled to 0 °C. Cl3CCN (421 mL, 4.2 mmol) and DBU
(13 mL, 0.084 mmol) were added. After 5 h the reaction
mixture was concentrated and purified by flash chro-
matography (10–15% EtOAc–toluene, +1% Et3N) to
yield disaccharide donor 5 (288 mg, 75% over two
steps) as a white solid with a 4:1 mixture of PMB- and
(3-bromo)-PMB-protected ethers. [h]D +21.8° (c 1.03,
CH2Cl2); IR (neat): 1742, 1715, 1493, 1388, 1225, 1054
Allyl
methoxybenzyl - 2 - phthalimido - i -
(1“4)-3-O-acetyl-2-deoxy- 6-O-p-methoxybenzyl-2-
phthalimido-i- -glucopyranosyl-(1“4)-3-O-acetyl-2-
deoxy-6-O-p-methoxybenzyl-2-phthalimido-i- -gluco-
pyranosyl-(1“4)-3-O-acetyl-2-deoxy-6-O-p-methoxy-
benzyl-2-phthalimido-i- -glucopyranoside (7).—A so-
3-O-acetyl-4-O-chloroacetyl-2-deoxy-6-O-p-
D
- glucopyranosyl-
1
cm−1; H NMR (CDCl3, 300 MHz): l 8.59 (1H, s),
D
7.90–7.72 (8H, m), 7.52–6.88 (8H, m) 6.53 (1H, d, J
8.7 Hz), 5.86–5.74 (2H, m), 5.51 (1H, d, J 8.4 Hz), 5.32
(1H, t, J 9.3 Hz), 4.57–3.49 (21H, m), 1.93 (3H, s), 1.87
(3H, s); 13C NMR (CDCl3, 125 MHz): l 170.44, 170.21,
167.72, 166.28, 160.75, 155.77, 155.61, 134.62, 134.49,
133.21, 131.91, 131.58, 131.24, 129.88, 128.71, 128.56,
123.77, 114.02, 113.90, 112.15, 111.94, 111.64, 97.12,
93.79, 75.37, 73.66, 72.55, 71.96, 71.46, 70.97, 68.43,
D
D
lution of disaccharide donor 5 (288 mg, 0.252 mmol, a
4:1 mixture of PMB- and (3-bromo)-PMB-protected
ethers) and disaccharide acceptor 6 (202 mg, 0.210
mmol, a 4:1 mixture of non- and mono-3-brominated-

S.-T. Liew, A. Wei / Carbohydrate Research 337 (2002) 1319–1324
1323
2112, 1750, 1606, 1497, 1365, 1228, 1053 cm−1
;
1H
PMB-protected ethers) in dry CH2Cl2 (2 mL) was
,
stirred under argon at room temperature with 4 A
NMR (CDCl3, 300 MHz): l 7.51–6.87 (16H, m), 5.98
(1H, sp, J 5.4 Hz), 5.33 (2H, dq, J 1.5, 17.4 Hz),
5.06–3.07 (50H, m), 2.13 (3H, s), 2.11 (3H, s), 2.06 (3H,
s), 1.96 (3H, s), 1.64 (3H, s); 13C NMR (CDCl3, 125
MHz): l 170.55, 170.45, 170.35, 170.30, 170.23, 169.71,
159.73, 156.07, 155.83, 133.50, 133.40, 133.26, 131.11,
131.02, 130.84, 130.04, 129.98, 129.86, 129.31, 128.78,
128.68, 128.56, 120.77, 119.91, 118.23, 116.82, 114.25,
114.13, 113.94, 112.12, 112.04, 111.93, 111.76, 110.55,
107.63, 105.05, 103.07, 101.19, 101.09, 100.93, 100.87,
74.75, 74.58, 74.24, 74.11, 73.50, 73.37, 73.31, 73.09,
72.73, 72.52, 72.41, 72.25, 70.64, 68.75, 68.16, 67.98,
67.30, 64.27, 64.21, 64.15, 64.10, 64.04, 56.52, 56.47,
55.46, 21.03, 20.92. ESIMS: m/z 1574, 1576 [M+Br+
Na]+, 1675, 1677, 1679 [M+2 Br+Na]+.
molecular sieves (200 mg) for 30 min. The reaction
mixture was then cooled to −20 °C, followed by addi-
tion of TMSOTf (11.5 mL, 0.063 mmol). The reaction
was stirred from −20 °C to 4 °C over a period of 4 h,
then quenched with Et3N and concentrated to an oil.
The residue was purified by flash chromatography (10–
60% THF–hexanes) to yield tetrasaccharide 7 (289 mg,
70%) as a white solid with an approximately 4:1 mix-
ture of PMB- and (3-bromo)-PMB-protected ethers.
[h]D −7.7° (c 1.03, CH2Cl2); IR (neat): 1777, 1746,
1
1711, 1610, 1497, 1384, 1225, 1046 cm−1; H NMR
(CDCl3, 300 MHz): l 7.85–7.69 (16H, m), 7.45–6.75
(16H, m), 5.75–5.22 (10H, m), 5.02 (2H, t, J 11.7 Hz),
4.42–3.21 (43H, m), 1.85 (3H, s), 1.83 (3H, s), 1.77 (3H,
s), 1.72 (3H, s); 13C NMR (CDCl3, 75 MHz): l 170.49,
170.30, 168.34, 167.62, 166.30, 159.26, 155.78, 155.59,
155.45, 134.66, 134.37, 133.81, 133.23, 132.83, 132.55,
131.95, 131.58, 131.26, 130.42, 130.30, 130.03, 129.31,
129.06, 128.78, 128.34, 128.15, 123.66, 117.67, 113.95,
113.84, 113.79, 112.17, 112.04, 111.52, 97.03, 77.54,
74.61, 74.38, 74.25, 73.63, 72.61, 72.55, 72.41, 71.72,
71.40, 71.30, 70.84, 70.11, 68.36, 67.79, 67.49, 67.35,
56.56, 55.55, 55.50, 55.20, 40.76, 20.80, 20.66. ESIMS:
m/z 1969 [M+Na]+, 2048, 2050 [M+Br+Na]+,
2104, 2106, 2108 [M+2 Br+Na]+.
Allyl
3,4-di-O-acetyl-2-azido-2-deoxy-i-
D
-gluco-
-glu-
pyranosyl-(1“4)-3-O-acetyl-2-azido-2-deoxy-i-
copyranosyl-(1“4)-3-O-acetyl-2-azido-2-deoxy-i-
glucopyranosyl-(1“4)-3-O-acetyl-2-azido-2-deoxy-i-
D
D
-
-
D
glucopyranoside (9).—To a solution of tetraazide 8 (35
mg, 0.023 mmol, a 4:1 mixture of PMB- and (3-bromo)-
PMB-protected ethers) in 4:1 CH2Cl2–water (5 mL) at
room temperature was added DDQ (160 mg, 0.701
mmol). After stirring for 21 h, the reaction was
quenched with satd aq NaHCO3 (5 mL), followed by
extraction with CH2Cl2 (3×5 mL). The combined ex-
tracts were dried over MgSO4, filtered and concentrated
to an oil. Chromatography on silica gel (20–25%
CH3COCH3–toluene) yielded tetraol 9 (15 mg, 60%) as
a white solid: [h]D −2.2° (c 0.45, CH2Cl2); IR (neat):
Allyl 3,4-di-O-acetyl-2-azido-2-deoxy-6-O-p-methoxy-
benzyl - i -
azido-2-deoxy-6-O-p-methoxybenzyl-i-
syl - (1“4) - 3 - O - acetyl - 2 - azido - 2 - deoxy - 6 - O - p -
methoxybenzyl-i- -glucopyranosyl-(1“4)-3-O-acetyl-
2-azido-2-deoxy-6-O-p-methoxybenzyl-i- -glucopyrano-
D
- glucopyranosyl - (1“4) - 3 - O - acetyl - 2-
D
-glucopyrano-
D
3517, 2111, 1751, 1368, 1228, 1156, 1038 cm−1 1H
;
D
NMR (CDCl3, 300 MHz): l 5.98 (1H, sp, J 5.4 Hz),
5.33 (2H, dq, J 1.5, 17.4 Hz), 5.06–3.38 (34H, m),
2.28–2.05 (15H, m). 13C NMR (CDCl3, 125 MHz): l
170.90, 170.51, 170.17, 170.06, 133.27, 118.59, 101.28,
101.17, 101.07, 101.02, 75.34, 75.28, 75.23, 74.80, 74.71,
74.44, 72.84, 72.70, 72.59, 72.44, 70.98, 68.97, 64.41,
64.31, 64.26, 64.20, 61.31, 60.93, 60.84, 60.65, 21.16,
21.11, 20.99, 20.87, 20.83. Anal. calcd for
C37H52N12O22: C, 43.70; H, 5.15; Found: C, 43.70; H,
5.22%.
side (8).—Tetrasaccharide 7 (100 mg, 0.051 mmol, a 4:1
mixture of PMB- and (3-bromo)-PMB-protected ethers)
in 1:1 n-BuOH–H2N(CH2)2NH2 (16 mL) was stirred at
100 °C in a sealed vessel for 24 h. The reaction mixture
was then cooled to room temperature and transferred
to a round-bottomed flask, followed by azeotropic re-
moval of all volatiles with toluene (5×25 mL) to
afford the crude amine as a yellow solid. The solid was
redissolved with 90% MeOH (20 mL) and treated with
freshly prepared TfN3 in CH2Cl2 (20 mL), CuSO4 (1
mg), and K2CO3 (3 mg). After 24 h at room tempera-
ture, the mixture was concentrated to dryness and
passed through a short silica gel column (2:1 EtOAc–
hexanes). The partially purified tetraazide was treated
with 3:2 pyridine–Ac2O (4 mL). After 2 days at room
temperature, the reaction was poured into cold water,
followed by extraction with CH2Cl2 (3×10 mL). The
extracts were washed with satd aq NaHCO3 (2×20
mL), dried, concentrated, and purified by flash chro-
matography (10–40% EtOAc–hexanes) to afford tetra-
azide 8 (30 mg, 39% over three steps) as a white solid
with a 4:1 mixture of PMB- and (3-bromo)-PMB-pro-
tected ethers. [h]D −30.3° (c 1.03, CH2Cl2); IR (neat):
Acknowledgements
The authors gratefully acknowledge financial assis-
tance from the American Chemical Society Petroleum
Research Foundation (33341-G4, 36069-AC1), the
American Heart Association Midwest Affiliates
(30399Z), the Indiana Elks Charities, Inc., and the
American Cancer Society (IRG-58-006-41). The au-
thors also wish to acknowledge Dr. H. Daniel Lee for
providing elemental analyses, and Dr. Karl V. Wood
and Arlene Rothwell for mass spectrometry services.

1324
S.-T. Liew, A. Wei / Carbohydrate Research 337 (2002) 1319–1324
(d) Debenham, J. S.; Rodebaugh, R.; Fraser-Reid, B. J.
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