Oligosaccharide synthesis on a soluble, hyperbranched polymer support via thioglycoside activation

Article abstract of DOI:10.1016/j.tet.2005.06.101

The synthesis of linear and branched di-, tri- and tetramannosides on a commercially available hyperbranched polyester as a soluble, high loading support is described. Glycosylation products were isolated in 26-63% yield as mixtures of anomers after total

Full text of DOI:10.1016/j.tet.2005.06.101

Tetrahedron 61 (2005) 8329–8338
Oligosaccharide synthesis on a soluble, hyperbranched polymer
support via thioglycoside activation
*
Eric Assen B. Kantchev, Scott J. Bader and Jon R. Parquette
Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA
Received 9 May 2005; revised 14 June 2005; accepted 27 June 2005
Available online 20 July 2005
Abstract—The synthesis of linear and branched di-, tri- and tetramannosides on a commercially available hyperbranched polyester as a
soluble, high loading support is described. Glycosylation products were isolated in 26–63% yield as mixtures of anomers after total
hydrolytic degradation of the polymer. All polymer-bound intermediates were purified through simple extraction or precipitation. Solution-
phase NMR and MALDI-TOF were used to monitor the progress of the reaction directly on the hyperbranched polymer support.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
hyperbranched polymers are highly branched polymers
that are prepared in a single synthetic step with degrees of
branching that are typically less than 50%. Therefore, these
imperfect analogs may serve as practical, low cost
alternatives to dendrimers (Fig. 1). Despite the tremendous
potential of these readily accessible materials to serve as
inexpensive, high-loading dendritic supports, they have
received only limited attention as synthetic14 or catalyst15
supports.
The development of insoluble polymeric supports1 to
facilitate the purification of synthetic intermediates has
dramatically streamlined the synthesis of natural and
unnatural molecules.2 However, the lower kinetic reactivity
of polymer-supported substrates, which depends greatly on
the swelling characteristics of the polymer–substrate
conjugate, impedes the routine application of solution-
phase synthetic procedures to the solid phase. The use of
highly-crosslinked, macroporous supports that do not
require swelling for reactivity,3 and insoluble resins grafted
with soluble linear4 or dendritic polymers,5 partially
circumvents these kinetic impediments. Similarly, the
solubility of non-crosslinked polymers, such as linear
polystyrene (PS) and poly(ethylene glycol) (PEG),6 permits
supported reactions to be performed in solution. However,
the practical utility of many of these linear supports is
limited by low loading capacities. Dendrimers7 and
hyperbranched polymers8 possess an (xK1)nC1 (nZ
degree of polymerization) number of terminal groups for
an ABx-type repeat unit. Accordingly, polyamidoamine
(PAMAM),9 carbosilane,10 polyglycerol,11 and dye-con-
jugated12 dendrimers have been employed as high-loading,
soluble supports for organic synthesis.13 The utility of
dendrimer-based supports is severely limited by the high
cost of their synthesis. However, a perfectly branched
dendrimer architecture is not absolutely required for
efficacy as a high loading support. For example,
Although the preparation of peptides16 and nucleotides17 on
solid phase has become routine, oligosaccharide synthesis
on polymer supports18 remains problematic due to the
structural complexity of oligosaccharides and difficulties
associated with glycosyl bond formation. However, recent
success in the automation of solid-phase oligosaccharide
Keywords: Carbohydrates; Glycosylations; Polymers; Dendrimers; Solid-
phase.
*
Corresponding author. Tel.: C1 614 2925886; fax: C1 614 2921685;
e-mail: parquett@chemistry.ohio-state.edu
Figure 1. The hyperbranched polymer (Boltorne).
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.06.101

8330
E. A. B. Kantchev et al. / Tetrahedron 61 (2005) 8329–8338
synthesis bodes well for the development of a routine solid-
phase approach to these molecules.19 In a preliminary
communication,20 we demonstrated that the synthesis of
disaccharides via thioglycoside activation21 could be
performed using this hyperbranched polymer as a support.
We report herein the extension of this approach to the
preparation of linear and branched di-, tri- and tetrasacchar-
ides. This paper focuses on hyperbranched polymer
supported assembly of oligomannosides22,23 because of their
important role as a structural constituent of N-glycans.24
2. Results and discussion
The hyperbranched polymer support employed in this study
is constructed via an acid-catalyzed polymerization of
dimethylolpropionic acid in the presence of pentaerythritol
as a central core (Fig. 1, commercially available as
Boltorne H-50).25 This support exhibits several properties
that facilitate purification and analysis of polymer-bound
intermediates: (1) the polymer-bound intermediates tend to
exhibit high solubility in most aprotic solvents but very low
solubility in methanol, from which they can be quantitat-
ively precipitated. Although purification by size exclusion
chromatography (SEC) remains as a potential purification
method, we found that precipitation was much more
expedient, especially on preparative scale. (2) Direct mass
spectral analysis of the polymer-bound disaccharides can be
achieved by photolytic release of the disaccharide from the
support with the MALDI-TOF laser. (3) The support
undergoes rapid hydrolytic degradation to water-soluble
materials thereby permitting product purification by
extraction. This method typically provides more efficient
cleavage from the support than photolysis of the
2-nitrobenzyl linkage. (4) The high intrinsic loading
capacity of Boltorne H-50 polymer (8.8 mmol/g OH-
groups; nominal MwZ14,500, pdiZ2.0) permits relatively
large amounts of substrates per gram of polymer support to
be immobilized.
Scheme 1. Reagents and conditions: (a) BF3$OEt2, CH2Cl2, 0 8C;
(b) K2CO3, MeOH–THF; (c) TBDMSCl, cat. DMAP, Et3N, CH2Cl2;
(d) NaOH, THF–H2O, then dil H2SO4; (e) EDCI, cat. DMAP, THF–
pyridine, then CH3COCl; (f) HF–pyridine, THF; (g) NIS, cat. TfOH,
CH2Cl2, K40 8C.
2.1.2. Synthesis of linear tri- and tetrasaccharides. Linear
tri-and tetrasaccharides were prepared from 12 by an
iterative glycosylation–deprotection sequence (Scheme 2).
Glycosylation of 12 with 5 equiv of the thioethyl glycoside
donor 1330 using N-iodosuccinimide (NIS) and cat.
trifluoromethanesulfonic acid (TfOH) in acetonitrile at
K40 8C followed by precipitation into methanol afforded
polymer-bound trisaccharide 14. Hydrolytic degradation of
the support with NaOH in H2O–THF at 65 8C and treatment
with diazomethane31 afforded the trisaccharide 15 as a
mixture of anomers in 47% yield after chromatographic
purification. Similarly, treatment of 12 with the activated
thioglycoside 10, silyl deprotection with HF–pyridine, and
further glycosylation of trisaccharide 16 with 13 afforded
the tetrasaccharide 18 in 26% isolated yield as a mixture of
anomers following hydrolytic liberation of the product from
the polymer support. The complexity of the NMR spectra of
the oligosaccharide products 15 and 18 did not allow the
ratios of the individual anomers to be determined. However,
in the case of 15 small amounts of pure trisaccharides were
obtained and their gate-decoupled 13C NMR spectra were
consistent with a,a,a (d 98.4 (1JC–HZ168.5 Hz), 100.7
(1JC–HZ171.7 Hz), 102.4 (1JC–HZ164.5 Hz)) and a,b,a (d
98.4 (1JC–HZ173.4 Hz), 98.5 (1JC–HZ156.8 Hz), 100.2
(1JC–HZ173.4 Hz)) configurations.32
2.1. O-6 Glycoside bond
2.1.1. Preparative scale synthesis of a supported
disaccharide. The 2-nitrobenzyl alcohol photolabile linker
(2)26 was glycosylated with 1,6-di-O-acetyl-2,3,4-tri-O-
benzyl-a-mannose (3)27 affording the mannoside 4 (a:bZ
6:1) in 78% yield (Scheme 1). The anomers could not be
separated and were carried through the rest of the synthesis
as a mixture. Deprotection of the 6-O-acetyl group with
potassium carbonate in methanol–THF followed by silyla-
tion with TBDMSCl/Et3N afforded the methyl ester 6.
Subsequent hydrolysis in a biphasic mixture of THF and
aqueous KOH provided the carboxylic acid 7, which was
coupled to the Boltorn H-50 polyester (1) using EDCI in
THF–pyridine. After an extractive aqueous work-up, the
polymer-mannoside conjugate 8 was desilylated with excess
HF$pyridine and glycosylated with the thioethyl donor 1028
affording polymer-supported disaccharide 11 (99% for both
steps, loading level: 0.59 mmol/g).29 Desilylation with
HF–pyridine provided multigram quantities of the poly-
mer-acceptor conjugate 12 in 88% yield (loading level:
0.64 mmol/g).29b
2.2. O-2 Glycoside bond: disaccharide synthesis
Mannose–mannose a-glycosidic bonds in N-glycans occur

E. A. B. Kantchev et al. / Tetrahedron 61 (2005) 8329–8338
8331
Scheme 2. Reagents and conditions: (a) NIS-cat. TfOH, CH3CN, K40 8C; (b) HF$pyridine, THF; (c) NaOH, THF–H2O, 65 8C, then dil H2SO4; (d) CH2N2,
ether.
1
most often at C-6, C-2 and more rarely at C-3 hydroxyl
groups.23 Accordingly, disaccharides were elaborated from
C-2 on the hyperbranched polymer support. The
2-nitrobenzyl linker-mannose conjugate (24) was prepared
from 1,2-di-O-acetyl-3,4,6-tri-O-benzyl-a-mannose33 (19)
via a BF3$OEt2-promoted glycosylation with linker alcohol
2 as shown in Scheme 3. The acetyl protected mannoside
was formed as a mixture of two anomers (20, 21) in a 6:1
ratio. It is noteworthy that hydrolysis of the acetoxy group in
b-anomer 21 proceeded much slower than for a-anomer 20,
presumably a consequence of the increased steric bulk of the
b-substituent. Therefore, selective hydrolysis of the
a-anomer 20 provided 22 in 70% isolated yield as a single
anomer, along with 7% of recovered 21. The
b-configuration of 21 was confirmed by gate-decoupled
13C NMR (d 99.4, JC–HZ155.3 Hz).32 Silylation of the
2-hydroxyl group in 22 with TBDMSOTf and lutidine34
followed by saponification of the methyl ester provided the
carboxylic acid 24, which was coupled with EDCI to the
Boltorne H-50 (1) polymer support in 86% yield (loading
level: 0.64 mmol/g).29b Deprotection of the axial
2-OTBDMS group in the product (25) proved to be
significantly more difficult in comparison with the
6-OTBDMS analog 11, most likely for steric reasons
(Scheme 4). Accordingly, exposure to a large excess of
HF–pyridine in THF at reflux was required to achieve 95%
deprotection of the TBDMS groups. Fortuitously, the
factors responsible for hindering C-2 desilylation did not
impede glycosylation of the C-2 hydroxyl group in the
polymer-bound acceptor 26. Accordingly, glycosylation of
26 with donors 13 and 2735 provided disaccharides 28 in
41% and 29 in 63% isolated yield as pure a-anomers
(Scheme 4, Table 1).
Scheme 3. Reagents and conditions: (a) BF3$OEt2, CH2Cl2, 0 8C;
(b) K2CO3, MeOH–THF (from 20; 21 was recovered in 7% isolated
yield); (c) TBDMSOTf, 2,6-lutidine, CH2Cl2; (d) NaOH, THF–H2O, then
dil H2SO4.
Scheme 4. Reagents and conditions: (a) EDCI, cat. DMAP, THF–pyridine,
then excess CH3COCl; (b) HF$pyridine, THF, 65 8C; (c) NIS-cat. TfOH,
CH3CN, K40 8C; (d) KOH, THF–H2O, 65 8C, then dil H2SO4; (e) CH2N2,
ether.

8332
E. A. B. Kantchev et al. / Tetrahedron 61 (2005) 8329–8338
Table 1. Disaccharides extended from C-2 (Scheme 4)
coupling of 3-nitro-4-(tetrahydropyran-2-yloxymethyl)ben-
zoic acid, 31, to the Boltorne H-50 polymer (loading
1.36 mmol/g)29b and acid-catalyzed THP deprotection, was
glycosylated with the glycosyl donor 27. Cleavage of the
2,3-isopropylidene protecting group in 33 with 1,3-
propanedithiol–trifluoroacetic acid (TFA) followed by
simultaneous glycosylation at O-2 and O-3 with donor
provided supported trisaccharide 35 after precipitation with
methanol. Release of the product by treatment with aqueous
KOH and methylation with diazomethane afforded
branched trisaccharide 36 as a mixture of anomers in 39%
overall yield from 32, after chromatographic purification.
The ratios of the individual anomers could not be
determined due to the complexity of the 1H NMR spectrum
of 36. However, inspection of the gate-decoupled 13C NMR
spectrum32 revealed that a,a,a-stereoisomer was the major
constituent of the mixture.
Donor
Disaccharide
Yield
(%)
63
27
28
41
13
29
3. Conclusion
2.3. Synthesis of 2,3-branched trisaccharides
This work demonstrates the potential for hyperbranched
polymers to serve as high loading, soluble supports for
multistep synthesis of large, complex molecules. We
prepared linear and branched di-, tri-, and tetrasaccharides
on the hyperbranched polyester Boltorne H-50 as a soluble
support in good yields on a preparative scale. Boltorn
polyester shares numerous advantageous characteristics
with linear polymers previously used as soluble supports.
For example, separation from soluble by-products and
excess of reagents can be accomplished by simple
precipitation from a poor solvent (methanol).36 The high
solubility of all protected intermediates in most organic
solvents ensured that synthetic protocols and analytical
techniques used for conventional chemistry (solution 1H and
13C NMR and MALDI-TOF MS) could be applied to the
supported reactions and intermediates with minimal
changes, and specialized equipment was not required. In
contrast to the traditional linear soluble supports, the high
loading capacity (theoretically 8.8 mmol/g) and low cost
(commercially available at US$ 5/kg) allows economical,
large scale preparations to be possible. As a potential
drawback, the base-labile nature of the polyester backbone
limits the range of reactions and conditions applicable to
Boltorn polymer supports. However, the preparation of new,
more chemically robust hyperbranched polymers from
readily available starting materials should remedy this
problem.
The synthesis of 2,3-branched oligosaccharides was
addressed using 2,3-isopropylidene thioethyl donor 27 as a
protected branching unit (Scheme 5). Accordingly, poly-
mer-supported linker 32, prepared by EDCI mediated
4. Experimental
4.1. General
THF was distilled from sodium/benzophenone ketyl;
CH3CN, CH2Cl2 and pyridine were distilled from calcium
˚
hydride; methanol was dried over 3 A molecular sieves.
Chromatographic separations were performed on silica gel
˚
60 (230–400 mesh, 60 A) using the flash technique in the
Scheme 5. Reagents and conditions: (a) DHP, cat. TsOH, CH2Cl2; (b)
KOH, H2O–THF, then dil H2SO4; (c) EDCI, cat. DMAP, THF–pyridine,
then excess CH3COCl; (d) cat. HCl, MeOH–CH2Cl2; (e) NIS-cat. TfOH,
CH3CN, K40 8C; (f) HS(CH2)3SH, TFA, CH2Cl2; (g) KOH, THF–H2O,
65 8C, then dil H2SO4; (h) CH2N2, ether.
indicated solvents as mobile phase. TLC was performed on
silica gel 60-F254 plates. Visualization of the compounds
was accomplished by UV-detection (254 nm) or staining
with 10% H2SO4. The solvents used for extraction and

E. A. B. Kantchev et al. / Tetrahedron 61 (2005) 8329–8338
8333
column chromatography were removed on a rotary
evaporator (40 mm Hg). All glycosylation reactions were
performed under anhydrous conditions under an argon
atmosphere. The precipitated polymers were centrifuged in
an Eppendorf centrifuge at 5000 rpm for time sufficient to
achieve complete precipitation (5–60 min). NMR spectra
were recorded at Bruker DPX-250, AC-300, DPX-400 and
DRX-500 spectrometers, referenced to the residual deuter-
ated solvent peaks and the chemical shifts expressed with
respect to tetramethylsilane (TMS). MALDI-TOF mass
spectra were recorded using 2,3-dihydrobenzoic acid as
matrix in THF. Elemental analyzes were obtained at
Atlantic Microlabs, Norcross, GA. All samples for
elemental analysis were dried for 16 h in vacuum over
P2O5 at 56 8C (refluxing acetone).
(6). To a solution of 5 (2.46 g, 3.8 mmol, 1 equiv) in dry
CH2Cl2 (10 mL) and Et3N (2.8 mL), DMAP (50 mg,
0.4 mmol, 0.11 equiv) and TBDMSCl (0.60 g, 4.0 mmol,
1.05 equiv) were added in succession and the solution
stirred for 4 h. The mixture was diluted with ethyl acetate
(50 mL), washed successively with water (30 mL), 5%
H2SO4 (3!30 mL), saturated NaHCO3 solution (2!
30 mL), brine (20 mL), dried (MgSO4) and evaporated.
After column chromatography (hexane/ethyl acetate, 7:1), 6
(2.63 g, 3.5 mmol, 91%) was obtained as a yellow foam. 1H
NMR (500 MHz, CDCl3) d 0.13 (s, 6H), 0.94 (s, 9H), 3.63–
3.67 (m, 1H), 3.91–4.01 (m, 3H), 4.02–4.06 (m, 5H), 4.70–
5.16 (m, 8H), 7.24–7.36 (m, 15H), 7.69 (d, JZ8.1 Hz, 1H),
8.29 (dd, JZ8.1, 1.3 Hz, 1H), 8.75 (d, JZ1.3 Hz, 1H). 13C
NMR (125 MHz, CDCl3) d K4.77, K4.73, 18.2, 26.3, 53.2,
63.8, 65.9, 72.8, 72.9, 73.1, 74.6, 75.1, 75.4, 80.0, 98.2,
126.3, 128.1–128.8 (multiple carbons), 129.1, 130.9, 134.5,
138.7, 138.8, 139.0, 139.2, 147.6, 165.2. Anal. Calcd for
C42H51NO10Si: C, 66.56; H, 6.78; N, 1.85. Found: C, 66.60;
H, 6.63; N, 1.74.
4.1.1. 4-Methoxycarbonyl-2-nitrobenzyl 6-O-acetyl-2,3,
4-tri-O-benzyl-a/b-D-mannopyranoside (4). Methyl
4-hydroxymethyl-2-nitrobenzoate 2 (3.51 g, 6.6 mmol,
1 equiv) and 3 (1.52 g, 7.2 mmol, 1.09 equiv) and were
dissolved in dry CH2Cl2 (12 mL). The solution was cooled in
an ice bath and BF3$OEt2 (4.1 mL, 4.68 g, 33 mmol, 5 equiv)
were added. After stirring at 0 8C for 6 h, the solution was
diluted with CH2Cl2 (50 mL), washed with water (50 mL),
saturated NaHCO3 solution (2!100 mL) and brine (20 mL).
The organic layer was dried (MgSO4) and the solvent was
evaporated. The acetate 4 (3.54 g, 5.1 mmol, 78%) was
obtained as a pale yellow foam after column chromatography
4.1.4. 4-Oxycarbonyl-2-nitrobenzyl 2,3,4-tri-O-benzyl-6-
O-tert-butyldimethylsilyl-a/b-D-mannopyranoside (7).
Solution of 6 (2.63 g, 3.47 mmol, 1 equiv) in THF
(30 mL) was mixed with freshly prepared 2 M aqueous
KOH solution (30 mL). The mixture was stirred for 6 h, then
acidified with 10% H2SO4 (pHZ3) and extracted with ethyl
acetate (3!30 mL), washed with water (5!30 mL), dried
(MgSO4) and evaporated. The carboxylic acid 7 (2.48 g,
1
(toluene/ether, 6:1). H NMR (300 MHz, CDCl3) d 2.07 (s,
1
3H), 3.78–3.85 (m, 2H), 3.92–4.05 (m, 5H), 4.14–4.39 (m,
2H), 4.60–5.19 (m, 9H), 7.54(d, JZ8.1 Hz, 1H), 8.24(dd, JZ
8.1, 1.7 Hz, 1H), 8.70 (d, JZ1.7 Hz, 1H). 13C NMR (75 MHz,
CDCl3) d 20.7, 52.6, 63.3, 65.6, 70.7, 71.8, 72.2, 74.3, 74.3,
75.1, 79.5, 97.9, 125.8, 127.5–129.2(multiple carbons), 130.5,
133.9, 137.8, 138.0, 138.2, 147.1, 164.6, 170.7. Anal. Calcd
for C38H39NO11: C, 66.56; H, 5.69; N, 2.04. Found: C,
66.82; H, 5.69; N, 2.10.
3.33 mmol, 96%) was obtained as pale yellow foam. H
NMR (500 MHz, CDCl3) d 0.13 (s, 6H), 0.94 (s, 9H), 3.63–
3.67 (m, 1H), 3.91–4.01 (m, 3H), 4.02–4.06 (m, 5H), 4.70–
5.16 (m, 8H), 7.24–7.36 (m, 15H), 7.69 (d, JZ8.1 Hz, 1H),
7.80 (broad s, 1H), 8.29 (dd, JZ8.1, 1.3 Hz, 1H), 8.75 (d,
JZ1.3 Hz, 1H). 13C NMR (125 MHz, CDCl3) d K4.83,
K4.73, 18.2, 26.3, 53.2, 63.8, 65.9, 72.8, 74.7, 73.1, 75.2,
75.3, 75.7, 89.9, 98.1, 126.3, 128.1–128.8 (multiple
carbons), 129.1, 130.9, 134.5, 138.7, 138.8, 139.0, 139.2,
147.5, 168.9. Anal. Calcd for C41H49NO10Si: C, 66.20; H,
6.64; N, 1.88. Found: C, 66.47; H, 6.64; N, 1.71.
4.1.2. 4-Methoxycarbonyl-2-nitrobenzyl 2,3,4-tri-O-ben-
zyl-a/b-D-mannopyranoside (5). The acetate 4 (7.17 g,
10.5 mmol, 1 equiv) was dissolved in a mixture of THF
(15 mL) and methanol (45 mL) and cooled in an ice bath.
Finely powdered K2CO3 (2.77 g, 20 mmol, 1.90 equiv) was
added and the mixture stirred for 1 h 15 min. The solution
was acidified with 10% aqueous H2SO4 (5 mL), the organic
solvents were removed and the organic material was
extracted in ethyl acetate (100 mL). The organic layer was
washed with saturated NaHCO3 solution (2!50 mL), brine
(30 mL), dried (MgSO4) and evaporated. The alcohol 5
(5.06 g, 7.88 mmol, 75%) was obtained as pale yellow foam
after column chromatography (hexane/ethyl acetate, 2:1).
1H NMR (300 MHz, CDCl3) d 1.96 (broad s, 1H), 3.75–3.81
(m, 2H), 3.85–4.08 (m, 8H), 4.61–5.09 (m, 8H), 7.08–7.43
(m, 15H), 7.30–7.48 (m, 15H), 7.61 (d, JZ8.1 Hz, 1H), 8.23
(dd, JZ8.1, 1.5 Hz, 1H), 8.69 (d, JZ1.5 Hz, 1H). 13C NMR
(75 MHz, CDCl3) d 52.7, 62.1, 65.8, 72.3, 72.9, 72.9, 74.5,
74.5, 75.2, 79.5, 98.4, 125.8, 127.5, 127.5–129.2 (multiple
carbons), 130.5, 134.0, 137.9, 138.1, 138.2, 138.2, 147.1,
164.6. Anal. Calcd for C36H37NO10: C, 67.17; H, 5.79; N,
2.18. Found: C, 67.44; H, 5.91; N, 1.93.
4.1.5. 4-Methoxycarbonyl-2-nitrobenzyl 6-(6-(3,4,6-tri-
O-benzyl-a-D-mannopyranosyl)-2,3,4-tri-O-benzyl-a/b-
D-mannopyranosyl)-2,3,4-tri-O-benzyl-a/b-D-manno-
pyranoside (15). A. To a solution of 7 (2.30 g, 3.09 mmol,
1 equiv), Boltorn H-50 polymer 1 (1.41 g, 12.4 mmol OH-
groups, 4 equiv) and DMAP (42 mg, 0.34 mmol,
0.11 equiv) in dry pyridine (3.7 mL) and dry THF
(4.8 mL), EDCI (0.65 g, 3.40 mmol, 1.10 equiv) was
added and the mixture stirred for 16 h. It was then cooled
in an ice bath and acetyl chloride (0.83 mL, 0.92 g,
11.7 mmol, 3.78 equiv) was added dropwise. The mixture
was warmed up to room temperature and stirred for
additional 8 h, poured in water (100 mL) and extracted
with ethyl acetate (2!30 mL). The combined organic
extracts were washed with 10% H2SO4 (2!30 mL),
saturated NaHCO3 solution (2!30 mL), brine (30 mL),
dried (MgSO4) and evaporated, affording 8 (4.20 g,
2.60 mmol, 84%, loading level: 0.62 mmol/g) as pale
yellow foam.
4.1.3. 4-Methoxycarbonyl-2-nitrobenzyl 2,3,4-tri-O-ben-
zyl-6-O-tert-butyldimethylsilyl-a/b-D-mannopyranoside
B. A solution of 8 from the previous step in THF (25 mL)
was then treated with HF$pyridine (0.83 g, 8.33 mmol,

8334
E. A. B. Kantchev et al. / Tetrahedron 61 (2005) 8329–8338
3.19 equiv) and the solution stirred for 16 h. The solution
was diluted with ethyl acetate (50 mL) and washed with
water (50 mL), 10% H2SO4 (2!20 mL) and saturated
NaHCO3 solution (2!20 mL). After drying (MgSO4) and
evaporation, 9 (3.80 g) was obtained as a pale yellow foam.
This material was used directly for the next step.
Calcd for C90H93NO20: C, 71.65; H, 6.21; N, 0.93. Found:
C, 71.25; H, 6.27; N, 0.97.
A small portion of the product was chromatographically
separated into two fractions. The (a,a,a)-trisaccharide: H
1
NMR (500 MHz, CDCl3) d 1.89 (broad s, 1H), 3.63–4.03
(m, 22H), 4.12–4.17 (m, 1H), 4.51–4.96 (m, 22H), 5.03–5.
15 (m, 3H), 7.25–7.49 (m, 45H), 7.58 (d, JZ8.1 Hz, 1H), 8.
23 (dd, JZ8.1, 1.5 Hz, 1H), 8.66 (d, JZ1.5 Hz, 1H). 13C
NMR (125 MHz, CDCl3) d 53.2, 66.2, 67.0, 68.1, 68.9, 69.
4, 71.5, 71.6, 71.8, 72.7, 72.9, 73.3, 73.8, 74.3, 74.5, 74.7,
74.7, 74.9, 75.1, 75.3, 75.5, 75.5, 75.7, 79.7, 80.2, 82.7, 98.
4 (1JC–HZ168.5 Hz), 100.7 (1JC–HZ171.7 Hz), 102.4
(1JC–HZ164.5 Hz), 126.4, 128.0–128.9 (multiple carbons),
130.9, 134.5, 138.4–139.2 (multiple carbons), 147.4, 165.2.
C. The polymer-immobilized acceptor
9 (3.80 g,
2.61 mmol, 1 equiv) and the thioethyl glycoside 10
(6.09 g, 10.40 mmol, 4 equiv) were dissolved in dry
CH2Cl2 (10 mL). After a clear solution had formed, the
flask was cooled to K40 8C. NIS (2.56 g, 11.40 mmol,
4.40 equiv) was added, followed by TfOH (90 mL, 156 mg,
1.04 mmol, 0.40 equiv). After stirring for 30 min at
K40 8C, the mixture was diluted with CH2Cl2 (50 mL),
washed with 10% NaHSO3 solution (100 mL), saturated
NaHCO3 solution (50 mL), brine (10 mL), dried (MgSO4)
and concentrated in vacuum. The crude polymer was
dissolved in CH2Cl2 (10 mL) and precipitated out of
methanol (250 mL). After decanting of the supernatant,
rinsing with methanol (3!10 mL) and drying in vacuum,
the disaccharide 11 (4.36 g, mmol, 99%, loading level:
0.59 mmol/g) was obtained as a pale yellow foam.
The (a,b,a)-trisaccharide: 1H NMR (500 MHz, CDCl3) d 1.
89 (broad s, 1H), 3.63–4.03 (m, 22H), 4.12–4.17 (m, 1H), 4.
51–4.96 (m, 22H), 5.03–5.15 (m, 3H), 7.25–7.49 (m, 45H),
7.59 (d, JZ8.0 Hz, 1H), 8.22 (dd, JZ8.0, 1.3 Hz, 1H), 8.70
(d, JZ1.3 Hz, 1H). 13C NMR (125 MHz, CDCl3) d 53.2, 60.
8, 66.1, 67.4, 67.5, 68.4, 69.3, 71.6, 71.8, 71.9, 72.0, 72.8,
73.1, 73.5, 73.8, 74.7, 74.8, 74.9, 75.1, 75.4, 75.4, 75.6, 75.
6, 80.0, 80.0, 80.2, 98.4 (1JC–HZ173.4 Hz), 98.5 (1JC–H
Z
156.8 Hz), 100.2 (1JC–HZ173.4 Hz), 126.3, 127.9–128.9
(multiple carbons), 130.1, 134.5, 138.4–138.8 (multiple
carbons), 147.8, 165.2.
D. From 11 (4.22 g, 2.57 mmol, 1 equiv), dissolved in THF
(20 mL) and HF$pyridine (0.57 g, 5.66 mmol, 2.20 equiv),
12 (3.52 g, mmol, 88%, loading level: 0.64 mmol/g) was
obtained as a yellow foam following the procedure (Part B)
for preparation of 9.
4.1.6. 4-Methoxycarbonyl-2-nitrobenzyl 6-(6-(6-(3,4,6-
tri-O-benzyl-a-D-mannopyranosyl)-2,3,4-tri-O-benzyl-
a/b-D-mannopyranosyl)-2,3,4-tri-O-benzyl-a/b-D-man-
nopyranosyl)-2,3,4-tri-O-benzyl-a/b-D-mannopyrano-
side (18). From the glycosyl acceptor 12 (321 mg,
0.2 mmol, 1 equiv) and the glycosyl donor 10 (0.68 g,
1 mmol, 5 equiv) in dry CH3CN (1 mL), following the
procedure for preparation of compound 14, polymer-
supported, 6-O-TBDMS-protected trisaccharide (432 mg)
was obtained as a pale yellow foam after precipitation from
methanol (30 mL, containing five drops Et3N), decanting of
the supernatant, washing with methanol (3!2 mL) and
drying in vacuum. The polymer was treated with HF$pyr-
idine (0.5 mL, 0.5 g, 5.0 mmol, 2.50 equiv) in THF (3 mL),
following the procedure for compound 12. From the
glycosyl acceptor 16 (obtained as described above,
0.1 mmol, 1 equiv) and the glycosyl donor 13 (0.54 g,
1 mmol, 5 equiv) in dry CH3CN (1 mL), following the
procedure for preparation of 14, the polymer-supported
tetrasaccharide 17 (534 mg) was obtained as a pale yellow
foam after precipitation into methanol (30 mL), decanting
of the supernatant, rinsing with methanol (3!2 mL) and
drying in vacuum. From 17 (536 mg), following the general
procedure for compound 15, the tetrasaccharide (mixture of
anomers) 18 (100 mg, 0.52 mmol, 26%) was obtained as a
pale yellow foam after chromatography on silicagel
E. The glycosyl acceptor 12 (156 mg, 0.1 mmol, 1 equiv)
and the glycosyl donor 13 (0.27 g, 0.5 mmol, 5 equiv) were
dissolved in dry CH3CN (0.6 mL). After stirring at room
temperature for 15 min, the mixture was cooled to K40 8C.
NIS (135 mg, 0.6 mmol, 6 equiv) and TfOH (2 mL, 3.4 mg,
0.022 mmol, 0.20 equiv) were added in succession. After
stirring for 20 min at K40 8C, the reaction mixture was
diluted with ethyl acetate (5 mL) and washed with 10%
NaHSO3 solution (5 mL), saturated NaHCO3 solution
(5 mL) and brine (2 mL). After drying (MgSO4) and
concentrating in vacuum, the polymer-immobilized trisac-
charide 14 (194 mg) was purified by dissolving in CH2Cl2
(0.5 mL) and precipitating out of methanol (15 mL),
decanting the supernatant, rinsing with methanol (3!
3 mL) and drying under high vacuum.
F. The immobilized trisaccharide 14 was heated at reflux in
a mixture of THF (2 mL) and 2 M aqueous NaOH solution
(2 mL) for 16 h. H2SO4 (10%) was added (pHZ3) and the
product was extracted into ethyl acetate (3!5 mL). The
combined organic extracts were washed with water (5!
10 mL), brine (5 mL), dried (MgSO4) and evaporated. The
crude product was dissolved in diethyl ether (2 mL) and an
excess of freshly prepared diazomethane solution in diethyl
ether was added dropwise until the evolution of N2 had
ceased. The excess of diazomethane was destroyed by
dropwise addition of glacial acetic acid. The ether solution
was washed with saturated NaHCO3 solution (2!5 mL),
brine (5 mL), dried (MgSO4) and evaporated. The
trisaccharide 15 (73 mg, 0.047 mmol, 47%) was obtained
as a pale yellow foam after chromatography on silica gel
(hexane/ethyl acetate, 2:1). MALDI-TOF MS: m/z calcd for
C90H93NNaO20 (MCNa)C: 1530.62, found: 1525.8. Anal.
1
(hexane/ethyl acetate, 2:1). H NMR (400 MHz, CDCl3) d
2.00 (broad s, 1H), 3.05–4.05 (m, 33H), 4.22–5.13 (m, 33H),
6.94–7.32 (m, 60H), 7.42 (m, 1H), 8.11 (m, 1H), 8.68 (m,
1H). 13C NMR (125 MHz, CDCl3) d 53.2, 66.8–82.0
(multiple carbons), 98.4, 98.5, 98.9, 102.2, 126.4, 127.8–
128.8 (multiple carbons), 129.3, 134.4, 138.7–139.0
(multiple carbons), 147.8, 165.2. MALDI-TOF MS: m/z
calcd for C117H121NNaO25 (MCNa)C: 1962.81, found

E. A. B. Kantchev et al. / Tetrahedron 61 (2005) 8329–8338
8335
1957.6. Anal. Calcd for C117H121NO25: C, 72.39; H, 6.28;
N, 0.72. Found: C, 72.55; H, 6.19; N, 0.82.
CH2Cl2 (45 mL), 2,6-lutidine (2.1 mL, 1.93 g, 19 mmol,
2.04 equiv) were added, followed by dropwise addition of
TBSOTf (2.6 mL, 2.99 g, 11.3 mmol, 1.22 equiv). After 5 h,
the solution was diluted with CH2Cl2 (150 mL), washed
with 10% aqueous H2SO4 (100 mL), saturated NaHCO3
solution (2!150 mL), brine (50 mL), dried (MgSO4) and
evaporated. After column chromatography (hexane/ethyl
acetate, 6:1), 23 (5.5 g, 7.25 mmol, 78%) was obtained as a
yellow foam. 1H NMR (500 MHz, CDCl3) d K0.49 (s, 3H),
K0.43 (s, 3H), 0.77 (s, 9H), 3.45–4.00 (m, 10H), 4.38–4.84
(m, 9H), 4.99 (d, JZ9.3 Hz, 1H), 7.03–7.58 (m, 15H), 7.57
(d, JZ8.1 Hz, 1H), 8.07 (dd, JZ8.1, 1.6 Hz, 1H), 8.54 (d,
JZ1.6 Hz, 1H). 13C NMR (125 MHz, CDCl3) d K1.88,
K1.28, 18.6, 26.2, 53.1, 66.3, 69.7, 70.2, 72.8, 73.5, 73.7,
74.9, 75.5, 80.2, 101.2, 126.3, 127.8–128.8 (multiple
carbons), 129.3, 131.0, 134.5, 138.8, 139.0, 139.0, 147.7,
165.2.
4.1.7. 4-Methoxycarbonyl-2-nitrobenzyl 2-O-acetyl-3,4,6-
tri-O-benzyl-a/b-D-mannopyranoside (20, 21). From 19
(4.74 g, 22.4 mmol, 1.20 equiv) and 2 (10.0 g, 18.7 mmol,
1 equiv), activated with BF3$OEt2 (6.0 mL, 6.92 g,
48.8 mmol, 2.61 equiv) in dry CH2Cl2 (30 mL), 20 and 21
(10.4 g, 15.1 mmol, 81%, a/b 6:1) were obtained as pale
yellow foam after column chromatography (toluene/ether,
5:1) as described for compound 4. The major anomer (20):
1H NMR (500 MHz, CDCl3) d 2.22 (s, 3H), 3.77 (m, 1H),
3.82–3.85 (m, 3H), 3.97–4.03 (m, 5H), 4.08 (m, 1H), 4.55–
4.82 (m, 8H), 4.92 (d, JZ10.7 Hz, 1H), 5.00–5.06 (m, 2H),
5.19 (m, 1H), 5.34 (s, 1H), 5.49 (m, 1H), 7.23–7.40 (m,
15H), 7.79 (d, JZ8.1 Hz, 1H), 8.32 (dd, JZ8.1, 1.7 Hz,
1H), 8.77 (d, JZ1.7 Hz, 1H). 13C NMR (125 MHz, CDCl3)
d 21.5, 53.2, 66.3, 67.0, 69.1, 72.4, 72.7, 74.0, 74.6, 75.7,
78.2, 98.2, 126.4, 128.1–128.9 (multiple carbons), 129.2,
131.1, 134.7, 138.2, 138.6, 138.7, 147.5, 165.2, 170.9. Anal.
Calcd for C38H39NO11: C, 66.56; H, 5.73; N, 2.04. Found:
C, 6.47; H, 5.47; N, 1.97.
B. 4-Oxycarbonyl-2-nitrobenzyl 3,4,6-tri-O-benzyl-2-O-
tert-butyldimethylsilyl-a-D-mannopyranoside (24)
Following the procedure for compound 7, from 23 (3.60 g,
4.75 mmol, 1.00 equiv) dissolved in THF (23 mL) and 2 M
aqueous KOH solution (23 mL) after stirring for 4 h, 24
(3.53 g, 4.75 mmol, 100%) was obtained as a pale yellow
4.1.8. 4-Methoxycarbonyl-2-nitrobenzyl 3,4,6-tri-O-ben-
zyl-b-D-mannopyranoside (22) and 4-methoxycarbonyl-
2-nitrobenzyl 2-O-acetyl-3,4,6-tri-O-benzyl-b-D-manno-
pyranoside (21 recovered). Following the procedure for
compound 5, acetate deprotection of the mixture of 20 and
21 (10.4 g, 15.2 mmol, 1 equiv) with K2CO3 (2.10 g,
15 mmol, 1 equiv) in dry THF (15 mL) and dry methanol
(50 mL) after 1 h 45 min afforded 22 (6.83 g, 10.6 mmol,
70%) as a pale yellow foam after column chromatography
1
foam. H NMR (400 MHz, CDCl3) d 0.14 (s, 3H), 0.19 (s,
3H), 0.97 (s, 9H), 3.80–4.20 (m, 10H), 4.57–5.02 (m, 9H),
5.18 (d, JZ9.3 Hz, 1H), 7.23–7.47 (m, 15H), 7.77 (d, JZ
8.2 Hz, 1H), 8.26 (dd, JZ8.2, 1.5 Hz, 1H), 8.69 (d, JZ
1.5 Hz, 1H). 13C NMR (100 MHz, CDCl3) d K4.25, K3.92,
18.7, 26.3, 66.5, 69.7, 70.2, 72.9, 73.5, 73.7, 75.0, 75.6,
80.1, 101.1, 126.8, 128.0–128.9 (multiple carbons), 129.3,
130.5, 134.9, 138.5, 138.7, 138.9, 139.9, 147.7, 168.5. Anal.
Calcd for C41H49NO10Si: C, 66.20; H, 6.64; N, 1.88. Found:
C, 66.00; H, 6.72; N, 1.87.
1
(hexane/ethyl acetate, 2:1). H NMR (400 MHz, CDCl3) d
2.50 (broad s, 1H), 3.60–3.72 (m, 2H), 3.85 (m, 2H), 3.92 (s,
3H), 4.04 (s, 1H), 4.46 (d, JZ9.4 Hz, 1H), 4.47 (d, JZ
12.2 Hz, 1H), 4.58 (d, JZ12.1 Hz, 1H), 4.68 (s, 2H), 4.77
(d, JZ10.7 Hz, 1H), 4.77 (d, JZ10.7 Hz, 1H), 4.90–5.06
(m, 3H), 7.13–7.32 (m, 15H), 7.65 (d, JZ8.1 Hz, 1H), 8.19
(dd, JZ8.1, 1.7 Hz, 1H), 8.63 (d, JZ1.7 Hz, 1H). 13C NMR
(125 MHz, CDCl3) d 53.2, 66.3, 68.7, 69.1, 72.2, 72.6, 74.0,
74.5, 75.7, 80.3, 99.8, 126.4, 128.0–129.0 (multiple
carbons), 129.3, 131.1, 134.5, 138.2, 138.5, 138.8, 147.7,
165.2. Anal. Calcd for C36H37NO10: C, 67.17; H, 5.79; N,
2.18. Found: C, 67.08; H, 5.93; N, 2.11.
4.1.10. 4-Methoxycarbonyl-2-nitrobenzyl 2-(4,6-di-O-
benzyl-2,3-O-isopropylidene-a-D-mannopyranosyl)-3,4,
6-tri-O-benzyl-a-D-mannopyranoside (28). Following the
procedure for compound 8, the carboxylic acid 24 (1.33 g,
1.79 mmol, 1.00 equiv) and Boltorn H-50 polymer (0.82 g,
7.16 mmol, 4.00 equiv) were coupled in dry pyridine (2 mL)
and dry THF (7 mL) using EDCI (0.38 g, 1.97 mmol,
1.10 equiv) and DMAP (0.02 g, 0.18 mmol, 0.10 equiv).
The polymer was then capped with acetyl chloride
(0.51 mL, 0.71 g, 7.16 mmol, 400 mol%). The polymer-
immobilized product 25 (2.30 g, 1.54 mmol, 86%) was
obtained as a pale yellow foam after the usual work-up and
precipitation from methanol (50 mL), decanting of the
supernatant, rinsing with methanol (3!5 mL) and drying in
high vacuum. To a solution of 25 (1.83 g, 1.24 mmol,
1.00 equiv) in THF (7 mL), HF$pyridine (0.76 g, 7.7 mmol,
6.21 equiv) was added and the solution stirred for 12 h at
reflux. After cooling to room temperature, the mixture was
diluted with ethyl acetate (75 mL) and washed with water
(50 mL), 10% aqueous H2SO4 (2!50 mL), saturated
NaHCO3 solution (2!50 mL) and brine (50 mL). After
drying (MgSO4) and evaporation, 26 was obtained as a pale
yellow foam (1.46 g, 1.15 mmol, 94%, loading level:
0.79 mmol/g). The glycosylation-cleavage procedure used
compounds 14 and 15 was applied using 2 M aqueous KOH
solution. From glycosyl acceptor 26 (126 mg, 0.1 mmol,
1.00 equiv) and glycosyl donor 27 (222 mg, 0.5 mmol,
Unreacted 21 (0.70 g, 1.1 mmol, 7%) was recovered as a
pale yellow foam. H NMR (400 MHz, CDCl3) d 2.19 (s,
1
3H), 3.46 (m, 1H), 3.64–3.74 (m, 3H), 3.84–3.92 (m, 5H),
4.43–4.85 (m, 9H), 4.92 (m, 1H), 5.06 (d, JZ19.2 Hz, 1H),
5.28 (d, JZ19.2 Hz, 1H), 5.69 (d, JZ3.4 Hz, 1H), 7.14–
7.30 (m, 15H), 7.84 (d, JZ10.2 Hz, 1H), 8.22 (dd, JZ10.2,
2.0 Hz, 1H), 8.65 (d, JZ2.0 Hz, 1H). 13C NMR (125 MHz,
CDCl3) d 21.1, 53.2, 68.3, 68.5, 69.3, 72.1, 74.0, 74.7, 75.7,
76.2, 80.6, 99.4, 126.2, 128.1–128.9 (multiple carbons),
129.3, 130.8, 134.7, 138.0, 138.6, 139.6, 147.1, 165.3,
170.1.
4.1.9. 4-Oxycarbonyl-2-nitrobenzyl 3,4,6-tri-O-benzyl-2-
O-tert-butyldimethylsilyl-a-D-mannopyranoside (24). A.
4-Methoxycarbonyl-2-nitrobenzyl 3,4,6-tri-O-benzyl-2-O-
tert-butyldimethylsilyl-a-D-mannopyranoside (23)
To a solution of 22 (6.0 g, 9.3 mmol, 1.00 equiv) in dry

8336
E. A. B. Kantchev et al. / Tetrahedron 61 (2005) 8329–8338
5.00 equiv), 28 (63 mg, 0.063 mmol, 63%) was isolated
after column chromatography (hexanes/ethyl acetate, 3:1)
mixture was stirred vigorously for 6 h at room temperature.
The mixture was acidified with 10% aqueous H2SO4 (pHZ
4), extracted with ethyl acetate (3!30 mL), washed with
water (5!50 mL), brine (30 mL), dried (MgSO4) and the
solvent was evaporated to afford 31 (3.08 g, 92%). 1H NMR
(250 MHz, CDCl3) d 1.51–1.82 (m, 7H), 3.50–3.57 (m, 1H),
3.77–3.81 (m, 1H), 4.74–4.76 (m, 1H), 4.91 (d, JZ16.4 Hz,
1H), 5.16 (d, JZ16.4 Hz, 1H), 7.93 (d, JZ8.2 Hz, 1H), 8.26
(dd, JZ8.2, 1.7 Hz, 1H), 8.69 (d, JZ1.7 Hz, 1H). 13C NMR
(63 MHz, CDCl3) d 19.8, 25.7, 30.8, 53.0, 62.9, 66.1, 99.3,
126.1, 129.4, 130.6, 134.4, 140.4, 147.6, 165.3. HR-QTOF-
MS: m/z calcd for C13H15NNaO6 (MCNa)C304.0797,
found 304.0782.
1
as a pale yellow foam. H NMR (500 MHz, CDCl3) d 1.42
(s, 3H), 1.53 (s, 3H) 3.49–3.80 (m, 7H), 3.90–3.98 (m, 6H),
4.18 (s, 1H), 4.30–4.38 (m, 2H), 4.49–4.55 (m, 5H), 4.65–
5.04 (m, 6H), 5.14 (d, JZ1.8 Hz), 5.40 (s, 1H), 7.15–7.35
(m, 25H), 7.60 (d, JZ8.1 Hz, 1H), 8.17 (dd, JZ8.1, 1.7 Hz,
1H), 8.68 (d, JZ1.7 Hz, 1H). 13C NMR (125 MHz, CDCl3)
d 26.9, 28.5, 53.1, 66.1, 69.2, 69.5, 70.1, 72.7, 73.0, 73.1,
73.8, 73.8, 74.4, 74.4, 75.0, 75.6, 76.3, 76.3, 79.0, 79.6, 99.4
(1JC–HZ173.1 Hz), 99.4 (1JC–HZ173.1 Hz), 109.7, 126.2,
127.7–128.7 (multiple carbons), 128.9, 134.5, 138.6–138.7
(multiple carbons), 147.3, 165.3. Anal. Calcd for
C59H63NO15: C, 69.06; H, 6.19; N, 1.37. Found: C, 68.66;
H, 6.31; N, 1.36.
4.1.14. 4-Methoxycarbonyl-2-nitrobenzyl 2-(3,4,6-tri-O-
benzyl-D-mannopyranosyl)-3-(3,4,6-tri-O-benzyl-D-man-
nopyranosyl)-4,6-di-O-benzyl-D-mannopyranoside (36).
The carboxylic acid 31 (2.97 g, 10.5 mmol, 1.00 equiv),
Boltorn H-50 polymer (1) (4.72 g, 42 mmol, 4.00 equiv)
and DMAP (130 mg, 1.1 mmol, 0.11 equiv) were dissolved
in dry pyridine (15 mL) and dry THF (20 mL). EDCI
(2.20 g, 11.5 mmol, 1.10 equiv) was added and the reaction
mixture was stirred for 16 h. The mixture was cooled in an
ice bath and acetyl chloride (2.85 mL, 3.14 g, 40 mmol,
3.81 equiv) was added dropwise. The reaction was warmed
to room temperature with stirring for 8 h, poured into water
(100 mL) and extracted with ethyl acetate (2!30 mL). The
combined organic extracts were washed with 10% H2SO4
(2!30 mL), saturated NaHCO3 solution (2!30 mL), brine
(30 mL), dried (MgSO4) and the solvent was evaporated to
afford the THP-protected polymer-immobilized linker,
which was used directly for the next step. THP-protected
32 was dissolved in CH2Cl2 and methanol (40 mL each) and
concd HCl (2 mL) was added. After stirring for 4 h,
saturated aqueous NaHCO3 solution was added (pHZ8).
The organic solvents were removed on rotary evaporator
and the product was extracted in ethyl acetate (3!30 mL).
The combined organic extracts were dried (MgSO4) and
concentrated in vacuo. The polymer-immobilized alcohol
32 (5.27 g, 68%, loading level: 1.36 mmol/g) was obtained
as a pale yellow foam after dissolving the crude polymer in
dichloromethane (10 mL) and precipitation into methanol
(150 mL), decanting of the supernatant, rinsing with
methanol (3!10 mL), and drying in high vacuum. From
the polymer-supported linker 32 (73.5 mg, 0.1 mmol,
1.00 equiv) and the glycosyl donor 27 (0.23 g, 0.5 mmol,
5.00 equiv) in dry CH3CN (0.6 mL), following the
procedure for preparation of 14, the monosaccharide 33
(110 mg) was obtained as a pale yellow foam after
precipitation from methanol (15 mL, containing five drops
of Et3N), decanting of the supernatant, rinsing with
methanol (3!2 mL), and drying in high vacuum. To 33
(110 mg, 0.1 mmol, 1.00 equiv) dissolved in dry CH2Cl2
(1 mL), 1,3-propanedithiol (100 mL, 108 mg, 0.5 mmol,
5.00 equiv) and TFA (30 mL, 44 mg, 0.39 mmol,
3.90 equiv) were added and the solution was stirred
overnight. Excess Et3N (60 mL) was used to quench the
acid and the solution was concentrated. The diol 34 was
obtained by precipitation into methanol (30 mL), decanting
of the supernatant, rinsing with methanol (3!2 mL) and
drying in high vacuum. The product was directly applied
into the next step. Following the procedure for preparation
of 14, the glycosylation of the diol 34 and the glycosyl donor
4.1.11. 4-Methoxycarbonyl-2-nitrobenzyl 2-(3,4,6-tri-O-
benzyl-a-D-mannopyranosyl)-3,4,6-tri-O-benzyl-a-D-
mannopyranoside (29). The glycosylation-cleavage pro-
cedure for compounds 14 and 15 was applied using 2 M
aqueous KOH. From glycosyl acceptor 26 (126 mg,
0.1 mmol, 1.00 equiv) and glycosyl donor 13 (268 mg,
0.5 mmol, 5.00 equiv), 29 (44 mg, 0.041 mmol, 41%) was
isolated after column chromatography (hexanes/ethyl
1
acetate, 2:1) as a pale yellow foam. H NMR (400 MHz,
CDCl3) d 2.40 (broad s, 1H), 3.65–3.95 (m, 15H), 4.03–4.14
(m, 2H), 4.65–4.80 (m, 14H), 5.06–5.13 (m, 3H), 7.12–7.33
(m, 30H), 7.58 (d, JZ8.1 Hz, 1H), 8.17 (dd, JZ8.1, 1.7 Hz,
1H), 8.65 (d, JZ1.7 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d 53.1, 66.2, 68.9, 69.5, 69.6, 72.1, 72.6, 72.9, 73.1, 73.8,
73.8, 74.8, 75.0, 75.3, 75.4, 75.6, 79.6, 80.4, 99.4 (1JC–H
Z
172.4 Hz), 101.6 (1JC–HZ172.0 Hz), 126.3, 127.8–128.8
(multiple carbons), 128.9, 128.9, 134.5, 138.4–138.7
(multiple carbons), 147.7, 165.3. Anal. Calcd for
C63H65NO15: C, 70.31; H, 6.09; N, 1.30. Found: C, 70.26;
H, 6.23; N, 1.31.
4.1.12. Methyl 4-((tetrahydro-2[H]-pyran-2-yl)-oxy-
methyl)-3-nitrobenzoate (30). To a suspension of methyl
4-hydroxymethyl benzoate (2) (10.56 g, 50 mmol,
1.00 equiv) in dry dichloromethane (20 mL), 2,3-dihydro-
4[H]-pyrane (9.12 mL, 8.42 g, 100 mmol, 2.00 equiv) was
added, followed by p-toluenesulfonic acid monohydrate
(0.19 g, 1 mmol, 0.02 equiv). After 15 min, the clear
solution formed was diluted with dichloromethane
(150 mL), washed with saturated NaHCO3 solution
(50 mL), dried (MgSO4) and the solvent was evaporated
in vacuum. The THP ether 30 (14.21 g, 96%) was obtained
as a pale yellow oil after chromatography on silicagel
(hexane/ethyl acetate, 5:1 containing 0.5% Et3N). 1H NMR
(250 MHz, CDCl3) d 1.48–1.85 (m, 8H), 3.44–3.50 (m, 1H),
3.73–3.79 (m, 1H), 3.82 (s, 3H), 4.68–4.72 (m, 1H), 4.87 (d,
JZ16.1 Hz, 1H), 5.10 (d, JZ16.1 Hz, 1H), 7.87 (d, JZ
8.1 Hz, 1H), 8.20 (dd, JZ8.2, 1.5 Hz, 1H), 8.60 (d, JZ
1.5 Hz, 1H). 13C NMR (63 MHz, CDCl3) d 19.8, 25.7, 30.8,
53.0, 62.9, 66.1, 99.3, 126.1, 129.4, 130.6, 134.4, 140.4,
147.6, 165.3. HR-QTOF-MS: m/z calcd for C14H17NNaO6
(MCNa)C318.0954, found 318.0952.
4.1.13. 4-((Tetrahydro-[2H]-pyran-2yl)-oxymethyl)-3-
nitrobenzoic acid (31). A solution of 30 (3.51 g,
11.9 mmol, 100 mol%) in THF (50 mL) was mixed with
2 M aqueous KOH solution (50 mL) and the two-phase

E. A. B. Kantchev et al. / Tetrahedron 61 (2005) 8329–8338
8337
(g) Barrett, A. G. M.; Cramp, S. M.; Roberts, R. S. Org. Lett.
1999, 1, 1083–1086.
13 (0.54 g, 1 mmol, 10.00 equiv) in dry CH3CN (0.7 mL)
was accomplished using NIS (0.27 g, 1.2 mmol,
12.00 equiv) and TfOH (10 mL, 17 mg, 0.1 mmol,
100 mol%). The polymer-immobilized trisaccharide 35
was obtained as a pale yellow foam after precipitation into
methanol (15 mL), decanting of the supernatant, rinsing
with methanol (3!2 mL) and drying in high vacuum.
Following the procedure for compound 15, the trisaccharide
(mixture of anomers) 36 (56 mg, 0.039 mmol, 39%) was
obtained as a pale yellow foam after chromatography on
silicagel (hexane/ethyl acetate, 2:1). The major anomer
5. Examples: (a) Basso, A.; Bradley, M. Tetrahedron Lett. 2003,
44, 2699–2702. (b) Chhabra, S. R.; Mahajan, A.; Chan, W. C.
J. Org. Chem. 2002, 67, 4017–4029. (c) Basso, A.; Bradley,
M.; Evans, B.; Pegg, N. Chem. Commun. 2001, 697–698.
(d) Basso, A.; Evans, B.; Pegg, N.; Bradley, M. Tetrahedron
Lett. 2000, 41, 3763–3767. (e) Basso, A.; Pegg, N.; Evans, B.;
Bradley, M. Eur. J. Org. Chem. 2000, 3887–3891. (f) Fromont,
C.; Bradley, M. Chem. Commun. 2000, 283–284. (g) Malenfant,
P. R. L.; Jayaraman, M.; Frechet, J. M. Chem. Mater. 1999, 11,
3420–3422. (h) Wells, N. J.; Basso, A.; Bradley, M.
Biopolymers 1998, 47, 381–396. Mahajan, A.; Chhabra, S.
R.; Chan, W. C. Tetrahedron Lett. 1999, 40, 4909–4912.
(i) Wells, N. J.; Davies, M.; Bradley, M. J. Org. Chem. 1998,
63, 6430–6431. (j) Swali, V.; Wells, N. J.; Langley, G. J.;
Bradley, M. J. Org. Chem. 1997, 62, 4902–4903.
6. For reviews, see: (a) Toy, P. H.; Janda, K. D. Acc. Chem. Res.
2000, 33, 546–554. (b) Gravert, D. J.; Janda, K. D. Chem. Rev.
1997, 97, 489–509.
1
(a,a,a): H NMR (500 MHz, CDCl3) d 2.00 (broad s, 2H),
3.75–5.26 (m, 58H), 7.28–7.37 (m, 40H), 7.75–8.00 (m,
1H), 8.07–8.23 (m, 1H), 8.72–8.78 (m, 1H). 13C NMR
(125 MHz, CDCl3) d 53.2, 66.2, 66.4, 68.3, 69.1, 69.1, 69.3,
69.6, 69.7, 72.1, 72.1, 72.4, 72.5, 72.7, 73.8, 73.9, 74.0,
74.7, 74.7, 74.9, 75.2, 75.4, 75.5, 76.3, 80.3, 80.6, 99.1
(1JC–HZ174.8 Hz), 99.2 (1JC–HZ174.8 Hz), 99.9 (1JC–H
Z
170.8 Hz), 126.3, 128.1–129.2 (multiple carbons), 130.7,
134.7, 137.9–139.3 (multiple carbons), 147.1, 165.3.
MALDI-TOF MS: m/z calcd for C80H87NNaO20 (MC
Na)C: 1440.57, found 1441.30; m/z calcd for C80H87NKO20
(MCK)C: 1456.68, found 1457.27. Anal. Calcd for
C80H87NO20: C, 70.27; H, 6.18; N, 0.99. Found: C, 70.04;
H, 6.33; N, 0.89.
7. General introduction to dendrimers: Newkome, G. R.;
Moorefield, C. N.; Vogtle, F. Dendrimers and Dendrons:
Concepts, Syntheses, Applications; Wiley-VCH: New York,
2001.
8. Kim, Y. H.; Webster, O. Hyperbranched Polymers. In Star and
Hyperbranched Polymers; Mishra, M. K., Kobayashi, S., Eds.;
Plastics Engineering; Marcel Dekkar: New York, 1999; 53,
p 201.
Acknowledgements
9. Kim, R. M.; Manna, M.; Hutchins, S. M.; Griffen, P. R.; Yates,
N. A.; Bernick, A. M.; Champman, K. T. Proc. Natl. Acad.
Aci. U.S.A. 1996, 93, 10012–10017.
This work was supported by The Ohio State University
through a University Seed Grant and a graduate assistant-
ship to E.A.K. The generous gift of large quantity of
Boltorne H-50 polymer from the manufacturer, Perstorp
polyols, is greatly appreciated.
10. (a) Le Notre, J.; Firet, J. J.; Sliedregt, L. A. J. M.; Van Steen,
B. J.; Van Koten, G.; Gebbink, R. J. M. K. Org. Lett. 2005, 7,
363–366. (b) Hovestad, N. J.; Ford, A.; Jastrzebski, J. T. B. H.;
van Koten, G. J. Org. Chem. 2000, 65, 6338–6344.
11. Roller, S.; Siegers, C.; Haag, R. Tetrahedron 2004, 60,
8711–8720.
12. Zhang, J.; Aszodi, J.; Chartier, C.; L’Hermite, N.; Weston, J.
Tetrahedron Lett. 2001, 42, 6683–6686.
References and notes
13. For a recent reviews, see: (a) Gebbink, R. J. M.; Klein, K.;
Cornelis, A.; Van Klink, G. P. M.; Van Koten, G. Rev. Mol.
Biotechnol. 2002, 90, 183–193. (b) Haag, R. Chem. Eur. J.
2001, 7, 327–335.
1. (a) Dorwald, F. Z. Organic Synthesis on Solid Phase, 2nd ed.;
Wiley-VCH: New York, 2002. (b) For a critical review of solid
resin supports, see: Vaino, A. R.; Janda, K. D. J. Comb. Chem.
2000, 2, 579–596.
14. (a) Haag, R.; Sunder, A.; Hebel, A.; Roller, S. J. Comb. Chem.
2002, 4, 112–119. (b) Hebel, A.; Haag, R. J. Org. Chem. 2002,
67, 9452–9455.
2. (a) Wessjohann, L. A. Curr. Opin. Chem. Biol. 2000, 4,
303–309. (b) Watson, C. Angew. Chem., Int. Ed. 1999, 38,
1903–1908.
15. (a) Bergbreiter, D. E.; Kippenberger, A. M.; Tao, G. Chem.
Commun. 2002, 2158–2159. (b) Schloegl, M.; Riethmueller,
S.; Troll, C.; Moeller, M.; Rieger, B. Macromolecules 2004,
37, 4004–4007. (c) Stiriba, S.-E.; Slagt, M. Q.; Kautz, H.;
Gebbink, R. J. M. K.; Thomann, R.; Frey, H.; Van Koten, G.
Chem. Eur. J. 2004, 10, 1267–1273.
3. Recent examples: (a) Egusa, K.; Kusumoto, S.; Fukase, K.
Eur. J. Org. Chem. 2003, 3435–3445. (b) Epple, R.; Kudirka,
R.; Greenberg, W. A. J. Comb. Chem. 2003, 5, 292–310. (c)
Fukase, K.; Nakai, Y.; Egusa, K.; Porco, J. A., Jr.; Kusumoto,
S. Synlett 1999, 1074–1078.
16. Atherton, E.; Sheppard, R. C. Solid-phase Peptide Synthesis: A
Practical Approach; Oxford University Press: Oxford, UK,
1989.
4. (a) Barrett, A. G. M.; Cramp, S. M.; Hennessy, A. J.;
Procopiou, P. A.; Roberts, R. S. Org. Lett. 2001, 3, 271–273.
(b) Arnauld, T.; Barrett, A. G. M.; Hopkins, B. T.; Zecri, F.
Tetrahedron Lett. 2001, 42, 8215–8222. (c) Lindsley, C. W.;
Hodges, J. C.; Filzen, G. F.; Watson, B. M.; Geyer, A. G. J.
Comb. Chem. 2000, 2, 550–559. (d) Hodges, J. C.;
Harikrishnan, L. S.; Ault-Justus, S. J. Comb. Chem. 2000, 2,
80–88. (e) Barrett, A. G. M.; Cramp, S. M.; Roberts, R. S.;
Zecri, F. J. Comb. Chem. High Throughput Screen. 2000, 3,
131–138. (f) Arnauld, T.; Barrett, A. G. M.; Cramp, S. M.;
Roberts, R. S.; Zecri, F. J. Org. Lett. 2000, 2, 2663–2666.
17. Pon, R. T. In Artificial DNA; Khudyakov, Y. E., Fields, H. A.,
Eds.; CRC: USA, 2003; pp 1–70.
18. Recent examples: (a) Bauer, J.; Rademann, J. J. Am. Chem.
Soc. 2005, 127, 7296–7297. (b) Dondoni, A.; Marra, A.;
Massi, A. Angew. Chem., Int. Ed. 2005, 44, 1672–1676. (c)
Seeberger, P. H.; Routenberg Love, K. Angew. Chem., Int. Ed.
2004, 43, 602–605. (d) Wu, X.; Schmidt, R. R. Eur. J. Org.
Chem. 2004, 2826–2832. (e) Wu, X.; Schmidt, R. R. J. Org.

8338
E. A. B. Kantchev et al. / Tetrahedron 61 (2005) 8329–8338
Chem. 2004, 69, 1853–1857. (f) Timmer, M. S. M.; Codee, J.
41, 4549–4553. (c) Unverzagt, C.; Seifert, J. Tetrahedron Lett.
1997, 38, 7857–7860. (d) Haneda, K.; Inazu, T.; Yamamoto,
K.; Nakahara, Y.; Kobata, A. Carbohydr. Res. 1996, 292,
61–70.
D. C.; Overkleeft, H. C.; van Boom, J. H.; van der Marel, G. A.
Synlett 2004, 2155–2158. (g) Mogemark, M.; Elofsson, M.;
Kihlberg, J. J. Org. Chem. 2003, 68, 7281–7288.
(h) Tennant-Eyles, R. J.; Davis, B. G.; Faibanks, A. J.
Tetrahedron: Asymmetry 2003, 14, 1201–1210. Additional
references in: (i) Diederichsen, U.; Wagner, T. In Organic
Synthesis Highlights V; Schmalz, H.-G., Wirth, T., Eds.;
Wiley-VCH: Weinheim, Germany, 2003; pp 384–394.
(j) Marcaurelle, L. A.; Seeberger, P. H. Curr. Opin. Chem.
Biol. 2002, 6, 289–296. (k) Solid Support Oligosaccharide
Synthesis and Combinatorial Carbohydrate Libraries; See-
berger, P. H., Ed.; Wiley: New York, 2001. (l) Seeberger, P.
H.; Haase, W. C. Chem. Rev. 2000, 100, 4349–4394. (m)
Haase, W. C.; Seeberger, P. H. Curr. Org. Chem. 2000, 4,
481–511. (n) Osborn, H. M. I.; Khan, T. H. Tetrahedron 1999,
55, 1807–1850.
24. Reviews on N-glycans: (a) Schachter, H. J. Clin. Invest. 2001,
108, 1579–1582. (b) Schachter, H. Glycoconj. J. 2000, 17,
465–483. (c) Marth, J. D. In Essentials of Glycobiology; Varki,
A., Ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor,
NY, USA, 1999; pp 85–100.
25. Malmstroem, E.; Johansson, M.; Hult, A. Macromolecules
1995, 28, 1698–1750.
26. For a previous application of this linker in a solid supported
oligosaccharide synthesis, see: Zehavi, U.; Sadeh, S.; Herch-
man, M. Carbohydr. Res. 1983, 124, 23–34.
27. Preparation: Zhang, G. T.; Gou, Z. W.; Hui, Y. Z. Synth.
Commun. 1997, 27, 1907–1911.
28. Preparation: Boons, G. J.; Grice, P.; Leslie, R.; Ley, S. V.
Tetrahedron Lett. 1993, 34, 8523–8526.
19. Plante, O. J.; Palmacci, E. R.; Seeberger, P. H. Science 2001,
291, 1523–1527.
29. (a) All polymer-immobilized intermediates were characterized
by 1H and, when necessary, 13C NMR; (b) The loading levels
of polymer-supported intermediates were determined by 1H
NMR in CDCl3 with DMF as an internal standard. Yields were
calculated from the loading level and the total mass of the
crude or precipitated polymer obtained after drying under high
vacuum.
20. Kantchev, A. B.; Parquette, J. R. Tetrahedron Lett. 1999, 40,
8049–8053.
21. Review: Garregg, P. J. Adv. Carbohydr. Chem. Biochem. 1997,
52, 179–266.
22. Representative publications regarding chemical synthesis of
oligomannosides related to the N-glycans: (a) Smiljanic, N.;
Halila, S.; Moreau, V.; Djedaini-Pilard, F. Tetrahedron Lett.
2003, 44, 8999–9002. (b) Ratner, D. M.; Swanson, E. R.;
Seeberger, P. H. Org. Lett. 2003, 5, 4717–4720. (c) Matsuo, I.;
Ito, Y. Carbohydr. Res. 2003, 338, 2163–2168. (d) Du, Y.;
Zhang, M.; Kong, F. Tetrahedron 2001, 57, 1757–1763.
(e) Prahl, I.; Unverzagt, C. Tetrahedron Lett. 2000, 41,
10189–10193. (f) Chiesa, M. V.; Schmidt, R. R. Eur. J. Org.
Chem. 2000, 3541–3554. (g) Duffels, A.; Ley, S. V. J. Chem.
Soc., Perkin Trans. 1 1999, 375–378. (h) Derpre, D.; Duffels,
A.; Green, L. G.; Lenz, R.; Ley, S. V.; Wong, C. H. Chem. Eur.
J. 1999, 5, 3326–3340. (i) Wang, W.; Kong, F. Angew. Chem.,
Int. Ed. 1999, 38, 1247–1250. (j) Becker, B.; Furneaux, R. H.;
Reck, F.; Zubikov, O. A. Carbohydr. Res. 1999, 315, 148–158.
(k) Rademann, J.; Geyer, A.; Schmidt, R. R. Angew. Chem.,
Int. Ed. 1998, 37, 1241–1245. (l) Weiler, S.; Schmidt, R. R.
Tetrahedron Lett. 1998, 39, 2299–2302. (m) Dan, A.;
Lergenmueller, M.; Amano, M.; Nakahara, Y.; Ogawa, T.;
Ito, Y. Chem. Eur. J. 1998, 4, 2182–2190. (n) Grice, P.; Ley,
S. V.; Pietruszka, J.; Osborn, H. M. I.; Priepke, H. W. M.;
Warriner, S. L. Chem. Eur. J. 1997, 3, 431–440.
30. Preparation: Zhang, Y. M.; Mallet, J. M.; Sinay, P. Carbohydr.
Res. 1992, 236, 73–88.
31. Diazomethane was generated by treatment of N-nitroso-N-
methyl urea (Preparation: Arndt, F. Organic Syntheses; Wiley:
New York, 1943; Collect. Vol. 2, pp 462–464) with 50%
aqueous KOH solution in diethyl ether: Hudlicky, T.; Koszyk,
F. J.; Kutchan, T. M.; Sheth, J. P. J. Org. Chem. 1980, 45,
5020–5027.
32. The configuration of the glycosyl linkages in mannosides can
be assigned using the values for 1JC–H obtained from gated 13
C
NMR spectra: Barresi, F.; Hindsgaul, O. Can. J. Chem. 1994,
72, 1447–1465. Values above 165 Hz are indicative for
a-mannosides.
33. Preparation: Ponpipom, M. M. Carbohydr. Res. 1977, 59,
311–317.
34. Corey, E. J.; Cho, H.; Ruecker, C.; Hua, D. H. Tetrahedron
Lett. 1981, 22, 3455–3458.
35. Preparation: Sarbajna, S.; Misra, A. K.; Roy, N. Synth.
Commun. 1998, 28, 2559–2570.
36. In all cases described in this work, the polymer-immobilized,
benzyl protected oligosaccharides were insoluble in methanol
and hexanes, and were freely soluble in diethyl ether,
chloroform, dichloromethane, acetonitrile, THF, and acetone.
23. Representative publications regarding chemoenzymatic syn-
thesis of N-glycan-related oligomannosides: (a) Singh, S.; Ni,
J. H.; Wang, L. X. Bioorg. Med. Chem. Lett. 2003, 13,
327–330. (b) Seifert, J.; Unverzagt, C. Tetrahedron Lett. 2000,