Synthesis of differentially protected glucosamine building blocks and their evaluation as glycosylating agents

Article abstract of DOI:10.1080/07328300903105108

The modular assembly of heparin oligosaccharides requires glucosamine building blocks with amine protecting groups for α-selective glycosylations that can be readily removed. The synthesis of N-4-nitrobenzensulphonamide (nosyl)- and N-2,4-dinitrophenyl (DNP)-protected glucosamine building blocks and their evaluation as glycosylating agents is described. The N-nosyl-protected glucosamine building blocks were challenging to prepare and their glycosylations resulted in inseparable mixtures of products. The N-DNP-protected glucosamines, however, were readily synthesized and resulted in α-selective couplings to protected l-iduronic acid derivatives.

Full text of DOI:10.1080/07328300903105108

Journal of Carbohydrate Chemistry, 28:395–420, 2009
ꢀ
C
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328300903105108
Synthesis of Differentially
Protected Glucosamine
Building Blocks and Their
Evaluation as Glycosylating
Agents
Pascal Bindscha¨ dler,1 Lukas O. Dialer,1 and
Peter H. Seeberger2
1Laboratory for Organic Chemistry, Swiss Federal Institute of Technology (ETH)
Zu¨rich, Wolfgang-Pauli-Strasse 10, CH-8093 Zu¨rich, Switzerland
2Max-Planck-Institute of Colloids and Interfaces, Department of Biomolecular
Systems, Am Mu¨hlenberg 1, 14476 Potsdam-Golm, Germany
The modular assembly of heparin oligosaccharides requires glucosamine building
blocks with amine protecting groups for α-selective glycosylations that can be read-
ily removed. The synthesis of N-4-nitrobenzensulphonamide (nosyl)- and N-2,4-
dinitrophenyl (DNP)-protected glucosamine building blocks and their evaluation as
glycosylating agents is described. The N-nosyl-protected glucosamine building blocks
were challenging to prepare and their glycosylations resulted in inseparable mixtures of
products. The N-DNP-protected glucosamines, however, were readily synthesized and
resulted in α-selective couplings to protected L-iduronic acid derivatives.
Keywords
Heparin;
Oligosaccharides;
Carbohydrates;
Glycosylation;
4-
Nitrobenzenesulphonamide protecting group; 2,4-Dinitrophenyl protecting group
INTRODUCTION
Glycosaminoglycans (GAGs) are a large class of polysaccharides that are found
on the cell surface and in the extracellular matrix. GAGs are attached to
proteins to form proteoglycans. Heparin and heparan sulfate are the most
complex GAGs, a family of molecules that also includes chondroitin sulfate,
Received December 5, 2008; accepted June 9, 2009.
Address correspondence to Peter H. Seeberger, Max-Planck-Institute of Colloids and
Interfaces, Department of Biomolecular Systems, Am Mu¨hlenberg 1, 14476 Potsdam-
Golm, Germany. E-mail: peter.seeberger@mpikg.mpg.de
395

P. Bindscha¨ dler et al.
396
Figure 1: General heparin target structure A with differently functionalized amine groups.
keratin sulfate, and dermatan sulfate. The various biological roles of GAGs
are dependent on their ability to bind selectively to different growth factors,
enzymes, morphogens, cell adhesion molecules, and cytokines.[1] Many aspects
of GAG chemistry,[2] biology[1,3] and structure-activity relationships (SARs)[4]
have been reviewed.
The unbranched, highly sulfated polysaccharide heparin is composed
of disaccharide units consisting of a uronic acid (UA) 1,4-linked to a D-
glucosamine (GlcN) unit. L-Iduronic acid (IdoA, 90%) predominates over its
C-5-epimer D-glucuronic acid (GlcA, 10%). Typically, a heparin disaccharide
contains several sulfate groups: O-sulfation occurs at the C-2 hydroxyl and/or
C-3 hydroxyl of the uronic acids, as well as at the C-3 hydroxyl and/or C-6 hy-
droxyl of the amino sugar. Additionally, the glucosamine unit can be N-sulfated
or N-acetylated or, in rare cases, remains unmodified.
The azido group is usually the only group used for masking the amine for
the modular assembly of heparin oligosaccharides by chemical means.[2,4a,5]
Therefore, the amines generally cannot be differentiated in the final deprotec-
tion and functionalization steps,[6] resulting in either an all N-acetylated or
an all N-sulfated oligosaccharide.[7] In order to differentiate the amines and
to introduce both N-acetate and N-sulfate groups in the same oligosaccha-
ride, leading to a structural motive such as A (Fig. 1), a second protecting
group for the amine is required. This protecting group has to fulfill several
requirements:
1. It has to be nonparticipating, in order to yield preferentially the desired
α-linked product when coupled to an L-iduronic acid acceptor.
2. It has to be orthogonal to the azido group.
3. It has to be electron withdrawing to lower the basicity of the amine group
for compatibility with TMSOTf-catalyzed couplings, and to avoid the for-
mation of an aziridinium intermediate that would lead to 1,2-trans glyco-
sylation.

Synthesis and Evaluation of Glucosamine Building Blocks
397
Figure 2: N-nosyl-protected (B, C) and N-DNP-protected (D, E), tri-O-acetylated and
tri-O-benzylated glucosamine N-phenyltrifluoroacetimidate glycosylating agents.
Two possible protecting groups that may meet these criteria were eval-
uated: N-4-nitrobenzensulphonamide (nosyl, Nos)[8] and N-2,4-dinitrophenyl
(DNP)[9] (Fig. 2).
Both protecting groups have already been applied to the synthesis of
oligosaccharides: The N-DNP group was used for the protection of glu-
cosamines and was shown to be stable to Ko¨nigs-Knorr coupling conditions.[9]
The N-DNP group resists treatment with numerous amines[10] and is compati-
ble with the conditions used for Fmoc- and Lev-cleavage used for the assembly
of oligosaccharides.[11] Cleavage of N-DNP by basic resin[9d,9f,9g] fits well into
the standard deprotection scheme for heparin oligosaccharides.[4a,5c] The N-
nosyl group was used by Fu¨gedi et al. for the protection of an aza-glycoside
and was shown to be stable to TMSOTf-catalyzed couplings.[12] Deprotection
[8b,8c,8e]
of the N-nosyl group using PhSH and K2CO3 or Cs2CO3
renders this
group orthogonal to the azido group.[13]
The aim of this study was to evaluate whether N-nosyl and N-DNP are
suitable protecting groups for the amine of glucosamine. Four glycosylating
agents B-E (Fig. 2) were to be synthesized from D-glucosamine to examine
their influence on the α/β-selectivity of glycosylation reactions. It is of par-
ticular interest to determine whether these protected glucosamine glycosylat-
ing agents yield preferentially the desired α-coupling product when glycosy-
lating L-iduronic acid, as observed for C-2 azide glucosamines.[2,5c] Glycosyl
N-phenyltrifluoroacetimidates were chosen as glycosylating agents, an attrac-
tive alternative[14] to the commonly used glycosyl trichloroacetimidates.[15] Tri-
O-acetylated (B, D) or tri-O-benzylated (C, E) glucosamines represent dis-
armed and armed glycosides, respectively.[16]
RESULTS AND DISCUSSION
The synthesis of the glucosamine building blocks started from crystalline,
glucosamine acetate 1[17] that is readily available on a 100-g scale from D-
glucosamine hydrochloride (Sch. 1). Selective cleavage of the anomeric acetyl

P. Bindscha¨ dler et al.
398
Scheme 1: Synthesis of glucosamines 3 and 5.
group, using gaseous ammonia,[18] was followed by TBDPS protection of the
resulting hemiacetal to afford imine 2. Hydrolysis of 2 was achieved by treat-
ment with hydroxylamine hydrochloride in THF and water to afford amine 3 in
92% yield. Alternatively, imine 2 was subjected to Zemple´n conditions[19] and
tri-O-benzylated to obtain imine 4 in 87% yield over two steps (Sch. 1). Hydrol-
ysis of the imino group of 4 using p-toluenesulfonyl hydrazine and acetic acid
in a mixture of diethyl ether and ethanol[20] furnished amine 5 in 90% yield.
The N-nosyl-protecting group was installed by reacting amine 3 with 4-
nitrobenzenesulfonyl chloride and triethylamine[8c] to afford N-nosyl-protected
amine 6a in 78% yield (Sch. 2). Cleavage of the anomeric silyl group us-
ing TBAF/acetic acid in THF followed by reaction of the resulting hemiac-
etal with trifluoro-N-phenylacetimidoyl chloride and Cs2CO3 in a mixture
of CH2Cl2 and THF furnished glycosylating agent B in 50% yield over two
steps.
Following the same reaction path, tri-O-benzylated amine 5 was converted
into glycosylating agent C (Sch. 2). However, the two-step manipulation of the
Scheme 2: Synthesis of N-nosyl-protected glycosylating agents B and C.

Synthesis and Evaluation of Glucosamine Building Blocks
399
Scheme 3: β-Selective glycosylation of N-nosyl-protected glycosylating agent B.
anomeric hydroxyl group was found to proceed in only 6% yield due to multi-
ple side reactions. Therefore, it was decided to evaluate solely tri-O-acetylated
derivative B in glycosylation reactions.
In a first reaction, glycosylating agent B was coupled with methanol in
CH2Cl2 using TMSOTf as activator to yield methyl glycoside 7 exclusively as
β-anomer in 63% yield (Sch. 3).
Coupling of glycosylating agent B with either linker-equipped[5c,21] L-
iduronic acid acceptor 8[5c] or methyl glycoside 9[22] resulted in an insepara-
ble mixture of products (Sch. 4). This result was attributed to side reactions
involving the N-nosyl-protecting group.
The problems observed during the preparation of N-nosyl-protected glyco-
sylating agents B and C, as well as the unselective coupling behavior of tri-O-
acetyl derivative B, may be attributed to the acidic nature of the sulfonamide
proton.
Scheme 4: Glycosylation attempts with N-nosyl-protected glycosylating agent B.

P. Bindscha¨ dler et al.
400
Scheme 5: Synthesis of N-DNP-protected glycosylating agents D, E, 11, and 12.
The N-2,4-dinitrophenyl (DNP)-protecting group was investigated next.
Amine 3 was protected by nucleophilic aromatic substitution using 1-fluoro-
2,4-dinitrobenzene (Sanger’s reagent)[23] and KHCO3 as base in THF and
H2O[9] to obtain masked amine 10a in 94% yield (Sch. 5). N-DNP-protected glu-
cosamine 10a was then converted into glycosylating agent D by cleavage of the
anomeric silyl group using TBAF/acetic acid in THF followed by reaction of the
resulting hemiacetal with trifluoro-N-phenylacetimidoyl chloride and Cs2CO3
in CH2Cl2 (62% yield, two steps). Similarly, tri-O-benzylated amine 5 reacted
with Sanger’s reagent to furnish N-DNP-protected 10b in 96% yield (Sch. 5).
Intermediate 10b was converted to three different glycosylating agents: Cleav-
age of the anomeric silyl group using TBAF/acetic acid in THF was followed by
reaction of the resulting hemiacetal either with trifluoro-N-phenylacetimidoyl
chloride and Cs2CO3 in CH2Cl2 to afford N-phenyltrifluoroacetimidate E (94%
yield, two steps) or with trichloroacetonitrile and substoichiometric amounts of
sodium hydride to obtain trichloroacetimidate 11 (81% yield, two steps). Glyco-
sylating agent 11 was subsequently converted in 99% yield into dibutyl phos-
phate 12 by reacting 11 with dibutyl phosphate in toluene.[24]
In comparison with the synthesis of N-nosyl-protected derivatives B and
C, the protection of the primary amine as N-DNP group and conversion to the
corresponding glycosylating agents proceeded well, both for tri-O-acetylated
derivative D and tri-O-benzylated glycosylating agents E, 11, and 12. In

Synthesis and Evaluation of Glucosamine Building Blocks
401
Scheme 6: Glycosylations of N-DNP-protected glycosylating agent D.
addition, all N-DNP-protected compounds displayed a bright yellow color per-
mitting easy detection after column chromatography.
First, tri-O-acetylated glucosamine building block D was evaluated. Cou-
pling with methylglycoside 9[22] using TMSOTf as activator afforded disaccha-
ride 13 as a mixture of α/β-anomers (α/β 1:2.5) in excellent yield (92%, Sch. 6).
Attempts to enhance the α-selectivity using diethyl ether as cosolvent[25] did
not meet with success, as mainly the β-product was obtained. This coupling
prompted extended investigations to couplings involving L-iduronic acid 14 as
acceptor.
Preparation of iduronic acid 14 commenced with the glycosylation of thio-
i
glycoside 16[26] with PrOH using dimethylsulfonium triflate (DMTST)[27] as
activator and tri-tert-butylpyrimidine (TTBP) as base to yield 17 in 66% yield
(Sch. 7). Despite the participating pivaloyl ester at the C2 hydroxyl, 17 was
obtained as an inseparable mixture of anomers (α/β 8:1).[26] Cleavage of the
levulinoyl ester was achieved by treatment of 17 with hydrazine monohy-
drate and a mixture of pyridine and acetic acid as buffer in CH2Cl2 to af-
ford acceptor 14 in 85% yield. Glycosylating agent D was then coupled with
14 in CH2Cl2 using TMSOTf as activator. At this stage, no diethyl ether was
Scheme 7: Synthesis of L-iduronic acid acceptor 14.

P. Bindscha¨ dler et al.
402
Table 1: TMSOTf-catalyzed couplings of glycosylating agents E, 11, and 12 to
L-iduronic acid 14.
α/β-
Yield (%) ratio
Entry Glycosylating Agent
Conditions
1
2
3
4
5
E (R = C(NPh)CF3)
E (R = C(NPh)CF3)
−30◦C to −5◦C, 45 min, CH2C12
11 (R = C(NH)CC13) −35◦C to −10◦C, 45 min, CH2C12
11 (R = C(NH)CC13) −30◦C to −10◦C, 45 min, Et2O/CH2Cl2
12 (R = P(O)(OBu)2) −78◦C to −50◦C, 30 min, CH2C12
58
87
41
57
89
5:1
2.5:1
3.5:1
3.5:1
1.5:1
−30◦C to −10◦C, 45 min, Et2O/CH2Cl2
employed, in the hope that L-iduronic acid acceptor 14 would induce complete
α-selectivity.[5b,5c,18b,28] As expected, preferably α-linked disaccharide 15 was
obtained (ratio of α/β-anomers: 5:1) in 21% yield (Sch. 6).
Moreover, the couplings of tri-O-benzylated N-DNP-protected glucosamine
building blocks E, 11, and 12 with L-iduronic acid 14 were investigated and
optimized (Table 1). When N-phenyltrifluoroacetimidate E was used in CH2Cl2
alone, disaccharide 18 was obtained in moderate yield (58%), but with good
α-selectivity (α/β 5:1, entry 1). Using diethyl ether as cosolvent distinctively
increased the yield to 87%. However, the α-selectivity dropped to α/β = 2.5:1
(entry 2). In the case of couplings with trichloroacetimidate 11, no significant
difference in α/β ratios was obtained by varying the solvent (α/β 3.5:1). Again,
the use of diethyl ether as cosolvent to CH2Cl2 significantly raised the yield
from 41% to 57% (entries 3 and 4). Couplings involving glycosyl phosphate 12
could be performed at very low temperature (−78◦C to −50◦C), resulting in
89% yield. However, the α-selectivity dropped (α/β 1.5:1, entry 5).
L-Iduronic acid acceptors are known to induce α-selectivity upon reaction
with C-2- azide glycosylating agents.[5b,5c,18b,28] Likewise, the coupling of dis-
armed tri-O-acetylated glycosylating agent D with acceptor 14 preferentially
yielded α-linked disaccharide 15. When armed glycosylating agents E, 11, and
12 were coupled with 14, corresponding disaccharide 18 was furnished with
good to very good α-selectivity.
In nature, β-linked glucosamines are prominently found in many carbo-
hydrate structures. Although synthetically less challenging than α-linkages to
glucosamine, additional orthogonal amine-protecting groups that selectively
yield β-linked glucosamines would be useful.[29] Reaction of trichloroace-
timidate 11 with methyl glycoside 9[22] using TMSOTf as activator and

Synthesis and Evaluation of Glucosamine Building Blocks
403
Scheme 8: Glycosylation attempt of N-DNP-protected glycosylating agent 11 in acetonitrile.
acetonitrile as solvent was investigated (Sch. 8). Acetonitrile is well known
to increase β-selectivity in coupling reactions.[30] In the course of the reaction,
glycosylating agent 11 was consumed; however, even after prolonged reaction
time at room temperature, no disaccharide was formed. ESI-mass analysis
of the reaction mixture hinted at the formation of acetonitrile-adduct 19
(Sch. 8).[31,32]
CONCLUSION
Reported is the synthesis of N-nosyl- and N-DNP-protected glucosamine build-
ing blocks and their evaluation in glycosylation reactions. Readily accessible
glucosamines 3 and 5 may serve as intermediates for the synthesis and subse-
quent evaluation of multiple other amine-protecting groups.
During the first investigations on N-nosyl as protecting group for a
glucosamine glycosylating agent reported here, several challenges were
encountered: The synthesis of glycosylating agents B and C turned out to be
challenging. Glycosylations involving tri-O-acetyl derivative B either yielded
undesired β-linked product 7 exclusively or resulted in inseparable mixtures of
products. On the other hand, N-DNP-protected N-phenyltrifloroacetimidates
D and E, as well as trichloroacetimidate 11 and phosphate 12, were prepared
in high yield. The glycosylations of disarmed tri-O-acetyl derivative D, as
well as armed tri-O-benzyl derivatives E, 11, and 12, showed the expected
preference for α-selective couplings to L-iduronic acid derivative 14.
Future investigations will reveal whether incorporating N-DNP-protected
glucosamine building blocks into the modular assembly of heparin oligosaccha-
rides is feasible to increase the diversity of synthetic heparin oligosaccharides.
EXPERIMENTAL
General
All chemicals used were reagent grade and used as supplied except
where noted. Dichloromethane (CH2Cl2), diethyleter (Et2O), toluene, and

P. Bindscha¨ dler et al.
404
tetrahydrofuran (THF) were purified by a J.C. Meyer Cycle-Tainer Solvent
Delivery System. Pyridine, acetonitrile, and triethylamine were distilled over
CaH2 prior to use. Trichloroacetonitrile (CCl3CN) was distilled over P2O5 un-
der Ar and stored at 0◦C. Reactions were performed under an Ar-atmosphere
except where noted. Analytical thin layer chromatography (TLC) was per-
formed on Merck silica gel 60 F254 plates (0.25 mm). Compounds were visual-
ized by UV irradiation or dipping the plate in a cerium (IV) sulfate/ammonium
molybdate/H2O/H2SO4 solution followed by heating. Flash column chromatog-
raphy was carried out using forced flow of the indicated solvent on Fluka
Kieselgel 60 (230–400 mesh). Recycling preparative HPLC (LC-9101, Japan
Analytical Industry Co., Ltd.); flow rate: 3.5 mL/min; solvent: CHCl3. 1H NMR
spectra were recorded on a Varian VXR-300 (300 MHz) or Bruker DRX400
(400 MHz) spectrometer and are reported in ppm (δ) relative to CHCl3 (7.25
ppm) as internal reference. Coupling constants (J) are reported in Hz. 13C
NMR spectra were obtained using a Varian VXR-300 (75 MHz) or Bruker
DRX400 (100 MHz) and are reported in ppm (δ) relative to CDCl3 (77 ppm).
MALDI and ESI high-resolution mass spectra were performed by the MS-
service at the Laboratory for Organic Chemistry (ETH Zu¨rich). IR spectra were
recorded on a Perkin-Elmer 1600 FTIR spectrometer (neat) or on a Perkin-
Elmer Spectrum 100 Series spectrometer (CHCl3). Optical rotations were mea-
sured using a Perkin-Elmer 241 polarimeter (10 cm, 1-mL cell); solvents and
concentrations (in g/100 mL) are indicated.
General Procedures
tert-Butyldiphenylsilyl 3,4,6-Tri-O-acetyl-2-deoxy-2-
(4-methoxy-benzylideneamino)-β-D-glucopyranose (2)
Through a solution of 1[17b] (30.4 g, 65.3 mmol) in THF (180 mL) and MeOH
(78 mL) at 0◦C was bubbled anhydrous ammonia at a modest rate. After 1.5 h,
nitrogen was bubbled through the solution to remove excess ammonia and the
solvent was removed in vacuo to afford a dark orange oil. The residue was co-
evaporated three times with toluene and once with CH2Cl2 and dried in vacuo
for 12 h to get a yellow solid that was dissolved in CH2Cl2 (130 mL). At rt, imi-
dazole (8.89 g, 131 mmol) and TBDPS-Cl (18.7 mL, 72 mmol) were added. The
mixture was stirred for 2 h at rt, diluted with EtOAc (500 mL), washed with
saturated aqueous NH4Cl solution (2 × 200 mL) and brine (200 mL), dried over
MgSO4, and concentrated. Flash column chromatography on silica gel (3:1 →
2:1 cyclohexane/EtOAc) afforded 2 (32.8 g, 76%) as a yellow foam. Rf 0.49 (cy-
clohexane/EtOAc 3:2). α r.t.: +49.4 (c = 1.0, CHCl3). IR (CHCl3): 3008, 2960,
[ ]D
2860, 1749, 1649, 1606, 1579, 1513, 1464, 1428, 1366, 1308, 1113, 1035, 909
1
t
cm−1. H NMR (CDCl3, 300 MHz) δ 0.98 (s, 9H, Bu), 1.87 (s, 3H, Ac), 1.99 (s,
3H, Ac), 2.02 (s, 3H, Ac), 3.43 (dd, J = 9.7, 7.5 Hz, 1H, H-2), 3.54 (ddd, J =

Synthesis and Evaluation of Glucosamine Building Blocks
405
10.0, 5.6, 2.5 Hz, 1H, H-5), 3.86 (s, 3H, OMe), 4.00 (dd, J = 12.1, 2.5 Hz, 1H,
H-6), 4.17 (dd, J = 12.1, 5.6 Hz, 1H, H-6ꢁ), 4.93 (d, J = 7.5 Hz, 1H, H-1), 5.08
(t, J = 9.8 Hz, 1H, H-4), 5.32 (t, J = 9.6 Hz, 1H, H-3), 6.91–6.95 (m, 2H, HAr),
7.17–7.43 (m, 6H, HAr), 7.58–7.61 (m, 2H, HAr), 7.65–7.71 (m, 4H, HAr), 8.24
(s, 1H, N = C-H). 13C NMR (CDCl3, 75 MHz) δ 19.3, 20.7, 20.8, 26.8, 55.4,
62.6, 69.0, 71.6, 73.6, 76.5, 96.7, 113.9, 127.3, 127.4, 128.7, 129.6, 130.1, 132.5,
132.9, 135.7, 135.9, 161.8, 163.7, 169.6, 169.8, 170.5. MALDI-HRMS: m/z calcd
for C36H44NO9Si [M+H]+ 662.2780, obsd 662.2775.
tert-Butyldiphenylsilyl 3,4,6-Tri-O-acetyl-2-amino-
2-deoxy-β-D-glucopyranose (3)
To a solution of 2 (250 mg, 0.378 mmol) in THF (1.8 mL) was added
NH2OH·HCl (79 mg, 1.3 mmol) in H2O (2.2 mL). The mixture was stirred for 45
min at rt, diluted with EtOAc, and washed with saturated aqueous NaHCO3
solution. The aqueous phase was re-extracted twice with EtOAc. The com-
bined organic phases were dried over MgSO4 and concentrated. The residue
was purified by flash column chromatography on silica gel (7:3 → 2:3 cyclohex-
ane/EtOAc) to afford 3 (189 mg, 92%) as a yellow, viscous oil. Rf 0.42 (cyclo-
hexane/EtOAc 3:7). α r.t.: +3.0 (c = 1.0, CHCl3). IR (CHCl3): 3008, 2962, 2860,
[ ]D
1
1748, 1428, 1367, 1113, 1042, 909 cm−1. H NMR (CDCl3, 300 MHz) δ 1.10 (s,
9H), 1.25 (br. s, 2H), 1.95 (s, 3H), 1.96 (s, 3H), 2.04 (s, 3H), 2.98 (dd, J = 10.3,
7.8 Hz, 1H), 3.37 (ddd, J = 9.8, 5.8, 2.3 Hz, 1H), 3.90 (dd, J = 12.0, 2.3 Hz,
1H), 4.06 (dd, J = 12.1, 5.6 Hz, 1H), 4.37 (d, J = 7.8 Hz, 1H), 4.82 (t, J = 9.7
Hz, 1H), 4.96 (t, J = 9.7 Hz, 1H), 7.32–7.46 (m, 6H), 7.66–7.70 (m, 4H). 13C
NMR (CDCl3, 75 MHz) δ 19.3, 20.7, 20.8, 20.9, 27.0, 58.0, 62.5, 69.1, 71.6, 75.1,
98.9, 127.3, 127.6, 129.8, 129.9, 132.7, 132.8, 135.6, 135.8, 169.6, 170.4, 170.5.
MALDI-HRMS: m/z calcd for C28H38NO8Si [M+H]+ 544.2361, obsd 544.2356.
tert-Butyldiphenylsilyl 3,4,6-Tri-O-benzyl-2-deoxy-2-
(4-methoxy-benzylideneamino)-β-D-glucopyranose (4)
At rt, a solution of 2 (5.0 g, 7.56 mmol) in MeOH (75 mL) and CH2Cl2
(38 mL) was treated with a solution of sodium (73 mg, 3.17 mmol) in MeOH
(10 mL). After 2.5 h, more sodium (36 mg, 1.59 mmol) in MeOH (5 mL) was
added. After 30 min stirring at rt, the mixture was neutralized with IR-120-
H+ Amberlite resin to pH 6, filtered, and concentrated. The residue was co-
evaporated three times with toluene and dissolved in THF (75 mL). At 0◦C,
NaH (2.12 g, 52.9 mmol, 60% dispersion in mineral oil) and benzyl bromide
(7.2 mL, 60.5 mmol, filtered over alumina) were added. After warming to rt,
TBAI (28 mg, 76 µmol) was added and the mixture was heated at reflux. Af-
ter 2 h, the reaction was allowed to cool to rt, quenched with MeOH (50 mL),
diluted with CH2Cl2, and washed with saturated aqueous NaHCO3 solution.

P. Bindscha¨ dler et al.
406
The aqueous phase was re-extracted once with CH2Cl2. The combined organic
phases were dried over MgSO4 and concentrated. Flash column chromatogra-
phy on silica gel (1:1 → 0:1 cyclohexane/CH2Cl2 and then 12:1 to 9:1 cyclo-
hexane/EtOAc) afforded 4 (5.31 g, 87%) as a colorless oil. Rf 0.18 (cyclohex-
ane/EtOAc 9:1). α r.t.: +80.7 (c = 1.0, CHCl3). IR (CHCl3): 3067, 3008, 2933,
[ ]D
2860, 1648, 1606, 1579, 1513, 1454, 1428, 1390, 1361, 1307, 1113, 1061 cm−1
.
t
1H NMR (CDCl3, 300 MHz) δ 0.99 (s, 9H, Bu), 3.25–3.29 (m, 1H, H-5), 3.40
(t, J = 8.1 Hz, 1H, H-2), 3.52 (dd, J = 10.9, 1.9 Hz, 1H, H-6), 3.67 (dd, J =
11.1, 4.2 Hz, 1H, H-6ꢁ), 3.74 (t, J = 8.9 Hz, 1H, H-4), 3.80 (t, J = 9.0 Hz, 1H,
H-3), 3.89 (s, 3H, OMe), 4.45 (d, J = 12.1 Hz, 1H,CHPh), 4.54 (d, J = 10.9
Hz, 1H, CHPh), 4.56 (d, J = 12.1 Hz, 1H, CHPh), 4.62 (d, J = 10.9 Hz, 1H,
CHPh), 4.63 (d, J = 10.6 Hz, 1H, CHPh), 4.85 (d, J = 11.2 Hz, 1H, CHPh),
4.85 (d, J = 7.5 Hz, 1H, H-1), 6.94–6.98 (m, 2H, HAr), 7.08–7.38 (m, 21H, HAr),
7.62–7.66 (m, 2H, HAr), 7.69–7.74 (m, 4H, HAr), 8.33 (s, 1H, N = C-H). 13C
NMR (CDCl3, 75 MHz) δ 19.4, 27.0, 55.5, 68.9, 73.6, 74.9, 75.1, 75.2, 77.8, 79.3,
83.9, 96.9, 113.9, 127.2, 127.3, 127.4, 127.5, 127.6, 127.8, 128.2, 128.2, 128.2,
128.3, 129.3, 129.5, 130.0, 133.0, 133.5, 136.0, 136.0, 138.2, 138.4, 138.5, 161.6,
163.1. MALDI-HRMS: m/z calcd for C51H56NO6Si [M+H]+ 806.3871, obsd
806.3888.
tert-Butyldiphenylsilyl 2-Amino-3,4,6-tri-O-benzyl-2-deoxy-
β-D-glucopyranose (5)
A solution of 4 (3.99 g, 4.95 mmol) in Et2O (15 mL) and EtOH (22 mL) was
cooled to 0◦C. AcOH (2.6 mL) and TosNHNH2 (1.88 g, 9.89 mmol) were added.
The mixture was stirred for 3.5 h at rt, diluted with EtOAc, and washed with
saturated aqueous NaHCO3 solution. The aqueous phase was re-extracted
with EtOAc. The combined organic phases were dried over MgSO4 and con-
centrated. Flash column chromatography on silica gel (4:1 → 7:3 cyclohex-
ane/EtOAc) afforded 5 (3.07 g, 90%) as a yellow, viscous oil. Rf 0.42 (cyclo-
hexane/EtOAc 3:1). α r.t.: −6.3 (c = 1.0, CHCl3). IR (CHCl3): 3385, 3067, 3008,
[ ]D
1
2933, 2860, 1589, 1496, 1454, 1428, 1362, 1110, 1062, 1028 cm−1. H NMR
(CDCl3, 300 MHz) δ 1.15 (s, 9H), 1.60 (br. s, 2H), 3.04 (dd, J = 9.8, 7.6 Hz,
1H), 3.13 (ddd, J = 9.8, 3.7, 1.7 Hz, 1H), 3.37 (t, J = 9.3 Hz, 1H), 3.45 (dd,
J = 11.1, 1.7 Hz, 1H), 3.65 (dd, J = 11.2, 4.0 Hz, 1H), 3.75 (t, J = 9.3 Hz,
1H), 4.37 (d, J = 12.1 Hz, 1H), 4.41 (d, J = 7.5 Hz, 1H), 4.51 (d, J = 12.1 Hz,
1H), 4.65 (d, J = 10.9 Hz, 1H), 4.73 (d, J = 11.2 Hz, 1H), 4.81 (d, J = 10.9
Hz, 1H), 4.96 (d, J = 11.5 Hz, 1H), 7.22–7.45 (m, 21H), 7.72–7.79 (m, 4H).
13C NMR (CDCl3, 75 MHz) δ 19.4, 27.2, 59.1, 68.6, 73.6, 74.7, 75.3, 78.5, 85.2,
98.9, 127.3, 127.4, 127.5, 127.5, 127.6, 127.7, 127.8, 128.2, 128.4, 128.4, 129.5,
129.7, 132.9, 133.6, 135.8, 135.9, 138.1, 138.4, 138.5. MALDI-HRMS: m/z calcd
for C43H49NO5SiNa [M+Na]+ 710.3272, obsd 710.3254.

Synthesis and Evaluation of Glucosamine Building Blocks
407
tert-Butyldiphenylsilyl 3,4,6-Tri-O-acetyl-2-deoxy-2-(4-
nitrobenzenesulfonylamino)-β-D-glucopyranose (6a)
A solution of 3 (2.02 g, 3.71 mmol) in CH2Cl2 (35 mL) was cooled to 0◦C and
treated with NEt3 (2.1 mL, 14.8 mmol) and 4-nitrobenzenesulphonyl chloride
(2.88 g, 13.0 mmol). The reaction was stirred for 3 h at rt. Then, more NEt3 (0.5
mL, 3.7 mmol) and 4-nitrobenzenesulphonyl chloride (0.82 g, 3.7 mmol) were
added and the mixture was stirred for additional 2 h at rt, diluted with CH2Cl2,
and washed with saturated aqueous NH4Cl solution. The aqueous phase was
re-extracted once with CH2Cl2 and the combined organic phases were dried
over MgSO4 and concentrated. Flash column chromatography on silica gel (4:1
→ 3:2 cyclohexane/EtOAc) afforded 6a (2.12 g, 78%) as a pale yellow foam. Rf
0.37 (cyclohexane/EtOAc 3:2). α r.t.: +2.0 (c = 1.0, CHCl3). IR (CHCl3): 3364,
[ ]D
3032, 2934, 2861, 1750, 1533, 1428, 1350, 1312, 1114, 1091, 1046, 909 cm−1
.
1H NMR (CDCl3, 300 MHz) δ 1.02 (s, 9H, tBu), 1.83 (s, 3H, Ac), 1.96 (s, 3H, Ac),
2.01 (s, 3H, Ac), 3.40 (ddd, J = 9.6, 5.3, 2.5 Hz, 1H, H-5), 3.76–3.85 (m, 1H,
H-2), 3.95 (dd, J = 12.1, 2.5 Hz, 1H, H-6), 4.05 (dd, J = 12.1, 5.3 Hz, 1H, H-6ꢁ),
4.61 (d, J = 7.8 Hz, 1H, H-1), 4.90 (d, J = 9.3 Hz, 1H, NH), 4.96 (t, J = 9.8 Hz,
1H, H-3), 5.07 (t, J = 9.5 Hz, 1H, H-4), 7.29–7.55 (m, 10H, HAr), 7.86–7.95 (m,
4H, HAr). 13C NMR (CDCl3, 75 MHz) δ 19.3, 20.7, 20.8, 20.8, 26.7, 60.6, 62.1,
68.7, 71.5, 73.3, 95.9, 124.0, 127.5, 127.6, 127.9, 129.9, 130.0, 132.6, 132.8,
135.3, 135.5, 146.6, 149.4, 169.1, 170.4, 170.6. MALDI-HRMS: m/z calcd for
C34H40N2O12SSiNa [M+Na]+ 751.1963, obsd 751.1978.
3,4,6-Tri-O-acetyl-2-deoxy-2-(4-nitrobenzenesulfonylamino)-β-D-
glucopyranosyl N-Phenyl trifluoroacetimidate (B)
A solution of 6a (166 mg, 0.228 mmol) in THF (4.6 mL) was treated with
AcOH (0.13 mL, 2.28 mmol) and a 1M solution of TBAF in THF (0.91 mL, 0.91
mmol). The mixture was stirred for 2 h at rt, diluted with EtOAc, washed with
saturated aqueous NaHCO3 solution and brine, dried over MgSO4, and con-
centrated. The residue was purified by flash column chromatography on silica
gel (3:2 → 1:1 toluene/EtOAc) to yield the hemiacetal (92 mg, 82%) as a pale
yellow foam. An aliquot of the resulting hemiacetal (75 mg, 0.153 mmol) was
dissolved in THF (1 mL) and CH2Cl2 (1 mL). At 0◦C, the solution was treated
with CF3C(NPh)Cl[33] (0.10 mL, 0.63 mmol) and Cs2CO3 (0.10 g, 0.31 mmol)
and stirred for 15 min at 0◦C. After stirring for 45 min at rt, the mixture was
filtered through Celite (CH2Cl2) and concentrated. The residue was purified
by column chromatography (7:3 → 3:2 cyclohexane/EtOAc) to afford B (62 mg,
61% [50% over two steps]) as a mixture of α/β-anomers (α/β = 1:>20). Color-
less, viscous oil. Rf 0.18 (cyclohexane/EtOAc 7:3). IR (neat): 3257, 2959, 1730,
1
1597, 1533, 1489, 1452, 1351, 1311, 1165, 1044 cm−1. H NMR (CDCl3, 300
MHz, only signals of β-anomer) δ 1.85 (s, 3H), 1.97 (s, 3H), 2.07 (s, 3H), 3.85
(br, 1H), 4.02–4.09 (m, 2H), 4.26 (dd, J = 12.7 Hz, 4.4 Hz, 1H), 5.15 (t, J =

P. Bindscha¨ dler et al.
408
9.9 Hz, 1H), 5.34 (t, J = 10.1 Hz, 1H), 6.25 (br. s, 1H), 6.55 (d, J = 9.1 Hz, 1H),
6.69–6.71 (m, 2H), 7.09–7.14 (m, 1H), 7.25–7.30 (m, 2H), 8.02–8.05 (m, 2H),
8.25–8.29 (m, 2H). 13C NMR (CDCl3, 75 MHz, only signals of β-anomer) δ 20.4,
20.5, 20.7, 55.6, 61.3, 67.2, 69.8, 70.0, 93.9, 119.0, 120.3, 124.4, 124.9, 126.2,
128.0, 128.8, 129.2, 142.2, 146.3, 149.9, 169.0, 170.3, 171.3. ESI-HRMS: m/z
calcd for C26H26F3N3O12SNa [M+Na]+ 684.1082, obsd 684.1077.
tert-Butyldiphenylsilyl 3,4,6-Tri-O-benzyl-2-deoxy-2-(4-
nitrobenzenesulfonylamino)-β-D-glucopyranose (6b)
At 0◦C, a solution of 5 (3.07 g, 4.46 mmol) in CH2Cl2 (60 mL) was portion-
wise treated with NEt3 (2.5 mL, 17.8 mmol) and 4-nitrobenzenesulphonyl chlo-
ride (3.46 g, 15.6 mmol). The reaction was stirred for 4 h at rt, diluted with
CH2Cl2, and washed with saturated aqueous NH4Cl solution. The aqueous
phase was re-extracted once with CH2Cl2 and the combined organic phases
were dried over MgSO4 and concentrated. Flash column chromatography on
silica gel (9:1 → 7:3 cyclohexane/EtOAc) afforded 6b (3.49 g, 90%) as a pale
yellow foam. R 0.44 (cyclohexane/EtOAc 7:3). α r.t.: −33.1 (c = 1.0, CHCl3).
[ ]D
f
IR (neat): 3330, 3031, 2932, 2859, 1607, 1530, 1454, 1348, 1162, 1092 cm−1
.
1H NMR (CDCl3, 400 MHz) δ 1.02 (s, 9H, Bu), 3.02–3.05 (m, 1H, H-5), 3.19
(t, J = 8.8 Hz, 1H, H-3), 3.37 (dd, J = 10.9, 2.6 Hz, 1H, H-6), 3.49 (dd, J =
10.9, 3.8 Hz, 1H, H-6ꢁ), 3.57–3.64 (m, 1H, H-2), 3.68 (t, J = 8.5 Hz, 1H, H-
4), 4.22 (d, J = 11.6 Hz, 1H, CHPh), 4.28 (d, J = 12.1 Hz, 1H, CHPh), 4.35
(d, J = 7.3 Hz, 1H, H-1), 4.38 (d, J = 12.8 Hz, 1H, CHPh), 4.46 (d, J = 10.8
Hz, 1H, CHPh), 4.52 (d, J = 10.8 Hz, 1H, CHPh), 4.56 (d, J = 7.7 Hz, 1H,
NH), 4.58 (d, J = 11.4 Hz, 1H, CHPh), 6.90–6.96 (m, 4H, HAr), 7.10–7.36 (m,
17H, HAr), 7.59–7.63 (m, 4H, HAr), 7.71–7.76 (m, 4H, HAr). 13C NMR (CDCl3,
100 MHz) δ 19.2, 26.8, 60.5, 68.5, 73.6, 74.1, 74.5, 74.8, 78.0, 81.9, 96.4, 123.8,
125.0, 126.5, 127.4, 127.6, 127.6, 127.7, 127.8, 128.2, 128.4, 128.4, 129.8, 130.0,
132.8, 133.2, 136.0, 137.5, 137.6, 138.2, 147.1, 149.3. MALDI-HRMS: m/z calcd
for C49H52N2O9SSiNa [M+Na]+ 895.3055, obsd 895.3040.
t
3,4,6-Tri-O-benzyl-2-deoxy-2-(4-nitrobenzenesulfonylamino)-D-glucopyranosyl
N-Phenyl trifluoroacetimidate (C)
At rt, a solution of 6b (200 mg, 0.229 mmol) in THF (5 mL) was treated
with AcOH (131 µL, 2.29 mmol) and TBAF·3H2O (0.29 g, 0.92 mmol). The
mixture was stirred at rt for 4 h. Then, TBAF·H2O (0.39 g, 1.37 mmol) and
H2O (0.05 mL) were added, and the mixture was stirred for additional 15 h at
rt, diluted with EtOAc, and washed with saturated aqueous NH4Cl solution.
The aqueous phase was re-extracted once with EtOAc. The combined organic
phases were dried over MgSO4 and concentrated. The residue was purified
by flash column chromatography on silica gel (7:3 → 3:2 cyclohexane/EtOAc)
to afford the hemiacetal (94 mg, 65%) that was dissolved in THF (1 mL) and
CH2Cl2 (2 mL). At 0◦C, the solution was treated with CF3C(NPh)Cl[33] (47 µL,

Synthesis and Evaluation of Glucosamine Building Blocks
409
0.30 mmol) and Cs2CO3 (145 mg, 0.44 mmol) and stirred for 1.5 h at 0◦C and
for 4 h at rt. Then, the mixture was filtered through Celite (CH2Cl2) and con-
centrated. The residue was purified by flash column chromatography on sil-
ica gel (1:0 → 4:1 cyclohexane/EtOAc) to afford C (11 mg, 9% [6% over two
steps.) as a mixture of α/β-anomers. Yellow solid. Rf 0.51 (cyclohexane/EtOAc
7:3). IR (neat): 3317, 3034, 2870, 1718, 1607, 1530, 1498, 1453, 1349, 1312,
1
1208, 1071 cm−1. H NMR (CDCl3, 300 MHz) δ 3.28–3.97 (m, 6H), 3.97–4.96
(m, 7H), 6.36–8.36 (m, 25H). 13C NMR (CDCl3, 75 MHz) δ 56.7, 67.7, 72.1,
73.2, 74.8, 75.3, 78.7, 94.4, 95.1, 113.7, 117.5, 119.1, 120.4, 122.8, 123.4, 123.8,
123.9, 124.4, 124.6, 126.2, 126.9, 127.6, 127.9, 129.1, 130.2, 134.9, 137.0, 137.3,
142.7, 145.4, 148.9, 149.4. MALDI-HRMS: m/z calcd for C41H38F3N3O9SNa
[M+Na]+ 828.2173, obsd 828.2175.
Methyl 3,4,6-Tri-O-acetyl-2-deoxy-2-(4-nitrobenzenesulfonylamino)-β-D-
glucopyranoside (7)
N-Phenyl trifluoroacetimidate B (58 mg, 0.088 mmol) was coevaporated
three times with toluene, dried in vacuo for 1 h, and dissolved in CH2Cl2
(2 mL). At −30◦C, MeOH (5.4 µL, 0.132 mmol) and TMSOTf (2.4 µL, 13 µmol)
were added. The reaction was warmed to 0◦C over 2 h, quenched with NEt3
(0.1 mL), and concentrated. The residue was coevaporated twice with toluene
and purified by flash column chromatography on silica gel (9:1 → 7:3 cyclohex-
ane/EtOAc) to afford 7 (28 mg, 63%) as pure β-anomer (colorless oil). Rf 0.53
(cyclohexane/EtOAc 7:3). α r.t.: −36.4 (c = 1.0, CHCl3). IR (neat): 3275, 3108,
[ ]D
1
2926, 1748, 1531, 1451, 1350, 1312, 1229, 1166, 1090 cm−1. H NMR (CDCl3,
400 MHz) δ 1.97 (s, 3H, Ac), 2.01 (s, 3H, Ac), 2.04 (s, 3H, Ac), 3.00 (s, 3H,
OMe), 3.38–3.50 (m, 1H, H-2), 3.57–3.62 (m, 1H, H-5), 4.03 (dd, J = 12.4, 2.4
Hz, 1H, H-6), 4.11 (d, J = 8.3 Hz, 1H, H-1), 4.19 (dd, J = 12.4, 4.7 Hz, 1H,
H-6ꢁ), 4.99–5.03 (m, 2H, H-3/H-4), 5.42 (d, J = 8.9 Hz, 1H, NH), 7.96–8.00
(m, 2H, HAr), 8.24–8.27 (m, 2H, HAr). 13C NMR (CDCl3, 100 MHz) δ 20.6,
20.7, 20.8, 56.8, 58.5, 61.8, 68.2, 71.8, 72.6, 102.3, 123.8, 128.6, 147.0, 149.9,
169.2, 170.7, 171.7. MALDI-HRMS: m/z calcd for C19H24N2O12SNa [M+Na]+
527.0942, obsd 527.0936.
tert-Butyldiphenylsilyl 3,4,6-Tri-O-acetyl-2-deoxy-2-(2,4-dinitrophenylamino)-
β-D-glucopyranose (10a)
A solution of 3 (2.20 g, 4.04 mmol) in THF (21 mL) and H2O (5.3 mL)
was treated with KHCO3 (405 mg, 4.04 mmol) and 1-fluoro-2,4-dinitrobenzene
(1.53 mL, 12.1 mmol) and stirred for 2 h at rt. Then, more 1-fluoro-2,4-
dinitrobenzene (0.51 mL, 4.04 mmol) was added. The mixture was stirred for
an additional 4 h at rt, diluted with EtOAc, and washed with saturated aque-
ous NH4Cl solution. The aqueous phase was re-extracted once with EtOAc,
and the combined organic phases were dried over MgSO4 and concentrated.
Flash column chromatography on silica gel (9:1 → 3:1 cyclohexane/EtOAc)

P. Bindscha¨ dler et al.
410
afforded 10a (2.69 g, 94%) as a yellow foam. Rf 0.66 (cyclohexane/EtOAc 1:1).
α
r.t.: −7.3 (c = 1.0, CHCl3). IR (neat): 3318, 3075, 2934, 1747, 1618, 1593,
[ ]D
1524, 1428, 1334, 1112, 1042 cm−1. H NMR (CDCl3, 400 MHz) δ 0.98 (s, 9H,
tBu), 1.90 (s, 3H, Ac), 2.01 (s, 3H, Ac), 2.06 (s, 3H, Ac), 3.52–3.56 (m, 1H, H-
5), 4.02–4.08 (m, 2H, H-2, H-6), 4.22 (dd, J = 12.2, 5.6 Hz, 1H, H-6ꢁ), 4.75 (d,
J = 7.7 Hz, 1H, H-1), 5.15 (t, J = 9.3 Hz, 1H, H-4), 5.19 (t, J = 9.5 Hz, 1H,
H-3), 7.20–7.42 (m, 8H, HAr), 7.56–7.58 (m, 2H, HAr), 8.15 (dd, J = 9.5, 2.5 Hz,
1H, DNP), 8.44 (d, J = 9.3 Hz, 1H, DNP), 9.01 (d, J = 2.7 Hz, 1H, DNP). 13C
NMR (CDCl3, 100 MHz) δ 18.9, 20.4, 20.5, 20.6, 26.6, 59.2, 62.1, 68.5, 71.9, 97.5,
115.5, 123.8, 127.6, 127.6, 129.7, 130.3, 130.3, 131.8, 135.3, 135.8, 136.8, 148.3,
169.4, 170.2, 170.4. MALDI-HRMS: m/z calcd for C34H39N3O12SiNa [M+Na]+
732.2195, obsd 732.2209.
1
3,4,6-Tri-O-acetyl-2-deoxy-2-(2,4-dinitrophenylamino)-D-glucopyranosyl
N-Phenyl trifluoroacetimidate (D)
A solution of 10a (200 mg, 0.282 mmol) in THF (5 mL) was treated with
AcOH (0.16 mL, 2.82 mmol) and TBAF·3H2O (360 mg, 1.13 mmol). The mix-
ture was stirred for 1.5 h at rt, diluted with EtOAc, and washed with sat-
urated aqueous NaHCO3 solution. The aqueous layer was re-extracted once
with EtOAc, and the combined organic phases were dried over MgSO4 and
concentrated. The residue was purified by flash column chromatography on
silica gel (7:3 → 1:1 cyclohexane/EtOAc) to yield the hemiacetal (94 mg, 71%)
as a yellow foam. Combined samples of the resulting hemiacetal (654 mg, 1.39
mmol) were dissolved in CH2Cl2 (26 mL). At 0◦C, the solution was treated with
CF3C(NPh)Cl[33] (0.86 mL, 5.4 mmol) and Cs2CO3 (904 mg, 2.77 mmol) and
stirred for 15 min at 0◦C and for 75 min at rt. Then, the mixture was filtered
through Celite (CH2Cl2) and concentrated. The residue was purified by flash
column chromatography on silica gel (9:1 → 3:2 cyclohexane/EtOAc) to yield D
(783 mg, 88% [62% over two steps]) as a mixture of α/β-anomers. Yellow foam.
Rf 0.50 (cyclohexane/EtOAc 1:1). IR (neat): 3333, 2964, 1748, 1617, 1594, 1524,
1337, 1153, 1114, 1043 cm−1. 1H NMR (CDCl3, 400 MHz) δ 1.89 (s, 3H), 2.09 (s,
3H), 2.13 (s, 3H), 4.11–4.19 (m, 2H), 4.35–4.43 (m, 2H), 5.31 (t, J = 9.8 Hz, 1H),
5.55 (t, J = 9.9 Hz, 1H), 6.52 (br. s, 1H), 6.72–6.74 (m, 2H), 7.09–7.14 (m, 1H),
7.17 (d, J = 9.6, 1H), 7.25–7.30 (m, 2H), 8.30 (dd, J = 9.5, 2.7 Hz, 1H), 8.74 (d,
J = 9.6 Hz, 1H), 9.14 (d, J = 2.7 Hz, 1H). 13C NMR (CDCl3, 75 MHz) δ 20.7,
20.9, 54.7, 61.4, 67.3, 70.3, 72.0, 92.8, 114.0, 118.8, 124.0, 124.8, 128.7, 130.0,
131.4, 137.0, 142.1, 147.0, 169.2, 169.4, 170.0. MALDI-HRMS: m/z calcd for
C26H25F3N4O12Na [M+Na]+ 665.1313, obsd 665.1318.
tert-Butyldiphenylsilyl 3,4,6-Tri-O-benzyl-2-deoxy-2-(2,4-dinitrophenylamino)-
β-D-glucopyranose (10b)
A solution of 5 (3.77 g, 5.48 mmol) in THF (16 mL) and H2O (4 mL)
was treated with KHCO3 (549 mg, 5.48 mmol) and 1-fluoro-2,4-dinitrobenzene

Synthesis and Evaluation of Glucosamine Building Blocks
411
(2.11 mL, 16.5 mmol) and stirred for 2 h at rt. Then, the reaction was di-
luted with EtOAc and washed with saturated aqueous NH4Cl solution. The
aqueous phase was re-extracted once with EtOAc, and the combined organic
phases were dried over MgSO4 and concentrated. Flash column chromatog-
raphy on silica gel (1:1 → 1:9 cyclohexane/CH2Cl2 and then 17:3 → 4:1 cy-
clohexane/EtOAc) afforded 10b (4.49 g, 96%) as a yellow foam. Rf 0.39 (cyclo-
hexane/EtOAc 4:1). α r.t.: +21.8 (c = 1.0, CHCl3). IR (neat): 3327, 3031, 2859,
[ ]D
1
1618, 1592, 1522, 1428, 1332, 1279, 1113, 1065 cm−1. H NMR (CDCl3, 400
t
MHz) δ 0.98 (s, 9H, Bu), 3.25–3.27 (m, 1H, H-5), 3.52–3.59 (m, 2H, H-3, H-6),
3.71 (dd, J = 10.9, 3.9 Hz, 1H, H-6ꢁ), 3.83–3.93 (m, 2H, H-2, H-4), 4.52 (d, J =
11.4 Hz, 1H, CHPh), 4.52 (d, J = 13.3 Hz, 1H, CHPh), 4.59 (d, J = 11.5 Hz,
1H, CHPh), 4.60 (d, J = 7.85 Hz, 1H, H-1), 4.68 (d, J = 11.0 Hz, 1H, CHPh),
4.81 (d, J = 12.8, 1H, CHPh), 4.84 (d, J = 11.2, 1H, CHPh), 6.97–6.99 (m, 2H,
HAr), 7.09–7.45 (m, 21H, HAr), 7.62–7.64 (m, 22H, HAr), 8.11 (dd, J = 9.6, 2.6
Hz, 1H, DNP), 8.34 (d, J = 9.4 Hz, 1H, DNP), 8.99 (d, J = 2.7 Hz, 1H, DNP).
13C NMR (CDCl3, 100 MHz) δ 18.9, 26.6, 60.8, 68.2, 73.6, 74.9, 75.0, 75.7, 83.8,
97.4, 116.0, 123.8, 127.5, 127.5, 127.7, 127.8, 127.9, 128.6, 129.4, 130.0, 130.1,
130.2, 132.3, 132.3, 135.5, 136.0, 136.3, 137.3, 137.9, 138.1, 149.0. MALDI-
HRMS: m/z calcd for C49H51N3O9SiNa [M+Na]+ 876.3287, obsd 876.3287.
3,4,6-Tri-O-benzyl-2-deoxy-2-(2,4-dinitrophenylamino)-D-glucopyranosyl
N-Phenyl trifluoroacetimidate (E)
At rt, a solution of 10b (4.32 g, 5.06 mmol) in THF (140 mL) was treated
with AcOH (2.9 mL, 50.6 mmol) and TBAF·3H2O (6.45 g, 20.2 mmol). The
mixture was stirred for 1 h at rt, diluted with EtOAc, and washed with satu-
rated aqueous NaHCO3 solution. The aqueous layer was re-extracted once with
EtOAc, and the combined organic phases were dried over MgSO4 and concen-
trated. The residue was purified by flash column chromatography on silica gel
(4:1 → 2:1 cyclohexane/EtOAc) to yield hemiacetal 20 (3.01 g, 96%) as a yellow
foam. An aliquot of hemiacetal 20 (616 mg, 1.0 mmol) was dissolved in CH2Cl2
(20 mL), treated at 0◦C with CF3C(NPh)Cl[33] (0.62 mL, 3.9 mmol) and Cs2CO3
(655 mg, 2.0 mmol), and stirred for 15 min at 0◦C. After stirring for 12 h at
rt, the mixture was filtered through Celite (CH2Cl2) and concentrated. The
residue was purified by flash column chromatography on silica gel (9:1 → 4:1
cyclohexane/EtOAc) to yield E (766 mg, 97% [94% over two steps]) as a mixture
of α/β-anomers (α/β = 1:2.5). Yellow foam. Rf 0.55 (cyclohexane/EtOAc 7:3). IR
1
(neat): 3332, 3062, 2871, 1718, 1617, 1591, 1522, 1332, 1207, 1115 cm−1. H
NMR (CDCl3, 400 MHz, only signals of β-anomer) δ 3.77–4.12 (m, 6H), 4.55
(d, J = 11.4 Hz, 1H), 4.60 (d, J = 11.1 Hz, 1H), 4.66–4.71 (m, 2H), 4.83–4.93
(m, 2H), 6.41 (br. s, 1H), 6.76–6.77 (m, 2H), 6.94–7.41 (m, 19H), 8.14 (dd, J =
9.4, 2.5 Hz, 1H), 8.61 (d, J = 9.3 Hz, 1H), 9.06 (d, J = 2.7 Hz, 1H). 13C NMR
(CDCl3, 100 MHz, only signals of β-anomer) δ 55.7, 57.5, 67.8, 68.0, 73.6, 73.7,
74.8, 75.4, 75.6, 75.9, 76.2, 81.8, 82.8, 94.2, 115.5, 115.6, 119.1, 123.8, 123.9,

P. Bindscha¨ dler et al.
412
124.7, 124.7, 127.8, 128.0, 128.1, 128.3, 128.5, 129.7, 130.9, 131.0, 136.7, 136.9,
137.0, 137.4, 137.5, 137.6, 137.7, 142.8, 148.1, 148.3. MALDI-HRMS: m/z calcd
for C41H37F3N4O9Na [M+Na]+ 809.2405, obsd 809.2420.
3,4,6-Tri-O-benzyl-2-deoxy-2-(2,4-dinitrophenylamino)-D-glucopyranosyl
trichloroacetimidate (11)
An aliquot of hemiacetal 20 (616 mg, 1.0 mmol) was dissolved in CH2Cl2
(10 mL), treated at 0◦C with Cl3CCN (1.44 mL, 10 mmol) and NaH (spatula
tip), and stirred for 1.5 h at 0◦C. After stirring for 12 h at rt, the mixture
was cooled again to 0◦C and more NaH (spatula tip) was added. After stirring
at rt for an additional 4 h, the reaction was filtered through Celite (CH2Cl2)
and concentrated. The residue was purified by flash column chromatography
on silica gel (9:1 → 7:3 cyclohexane/EtOAc) to afford 11 (642 mg, 84% [81%
over two steps]) as a mixture of α/β-anomers (α/β = 4:1). Yellow foam. Rf 0.39
(cyclohexane/EtOAc 7:3). IR (neat): 3330, 3031, 2872, 1676, 1591, 1522, 1333,
1285, 1135, 1059, 1026 cm−1. 1H NMR (CDCl3, 400 MHz) δ 3.73–4.20 (m, 6H),
4.53–4.92 (m, 6H), 5.90 (d, J = 7.5 Hz, 0.2H), 6.43 (d, J = 3.4 Hz, 0.8H),
6.90–7.38 (m, 16H), 8.16 (dd, J = 9.4, 2.5 Hz, 0.8H), 8.20 (dd, J = 9.8, 2.9
Hz, 0.2H), 8.52 (d, J = 9.5 Hz, 0.8H), 8.61 (d, J = 9.3 Hz, 0.2H), 8.69 (s, 0.2 H),
8.80 (s, 0.8H), 9.03–9.04 (m, 1H). 13C NMR (CDCl3, 100 MHz) δ 55.8, 57.3, 67.7,
68.2, 73.6, 73.7, 73.7, 74.7, 75.5, 76.0, 76.1, 81.5, 82.4, 90.7, 95.0, 97.1, 115.6,
115.8, 123.7, 123.9, 127.8, 128.0, 128.1, 128.1, 128.5, 129.7, 130.8, 136.7, 136.8,
136.9, 137.0, 137.5, 137.6, 137.6, 137.8, 148.2, 148.4, 160.5, 161.0. MALDI-
HRMS: m/z calcd for C35H33Cl3N4O9Na [M+Na]+ 781.1205, obsd 781.1217.
3,4,6-Tri-O-benzyl-2-deoxy-2-(2,4-dinitrophenylamino)-D-glucopyranosyl
dibutylphosphate (12)
Trichloroacetimidate 11 (110 mg, 145 µmol) was coevaporated three times
with toluene and taken up in toluene (7 mL). Dibutyl phosphate (38 µL,
188 µmmol) was added dropwise at rt. The reaction mixture was stirred for
3 h at rt and concentrated. The crude product was purified by flash column
chromatography on silica gel (9:1 → 3:1 cyclohexane/EtOAc) to yield 12 (116
mg, 99%) as a mixture of anomers (α/β = 0.45/0.55). Yellow solid. Rf 0.24 (cy-
clohexane/EtOAc 7:3). IR (neat): 3340, 2963, 1729, 1618, 1592, 1523, 1454,
1275, 1135, 1028, 941 cm−1. 1H NMR (CDCl3, 400 MHz) δ 0.72–1.81 (m, 14H),
3.68–4.02 (m, 10H), 4.47–4.67 (m, 4H), 4.80–4.89 (m, 2H), 5.30 (t, J = 7.8 Hz,
0.55H), 5.78 (dd, J = 5.4, 3.1 Hz, 0.45H), 6.43 (br. s, 0.45H), 6.64 (br. s, 0.55H),
6.88–7.34 (m, 15H), 8.12 (dd, J = 9.6, 2.7 Hz, 0.45H), 8.17 (dd, J = 9.6, 2.7
Hz, 0.55H), 8.51 (d, J = 9.3 Hz, 0.55H), 8.58 (d, J = 9.2 Hz, 0.45H), 9.02 (d,
J = 2.6 Hz, 0.45H), 9.03 (d, J = 2.6 Hz, 0.55H). 13C NMR (CDCl3, 100 MHz)
δ 13.4, 13.5, 18.4, 18.6, 31.9, 32.2, 56.2, 59.0, 68.1, 68.4, 72.9, 73.7, 75.0, 75.7,
75.9, 81.5, 83.2, 96.0, 97.7, 115.7, 123.7, 123.9, 127.9, 128.0, 128.2, 128.5, 129.7,

Synthesis and Evaluation of Glucosamine Building Blocks
413
130.8, 136.6, 136.9, 137.6. 31P NMR (CDCl3, 162 MHz) δ −2.51, −1.85. MALDI-
HRMS: m/z calcd for C41H50N3O12PNa [M+Na]+ 830.3024, obsd 830.3008.
Methyl 3,4,6-Tri-O-acetyl-2-deoxy-2-(2,4-dinitrophenylamino)-D-
glucopyranosyl-(1→4)-2,3,6-tri-O-benzyl-β-D-glucosaminopyranoside (13)
Methyl glycoside 9[22] (50 mg, 0.108 mmol) and N-phenyl trifluoroacetimi-
date D (90 mg, 0.140 mmol) were coevaporated three times with toluene, dried
in vacuo for 1 h, and dissolved in CH2Cl2 (0.8 mL) and Et2O (3.2 mL). Freshly
activated acid-washed 4 molecular sieves (140 mg) were added. At −30◦C, the
mixture was treated with TMSOTf (3.9 µL, 22 µmol). The reaction was warmed
to −10◦C over 1 h, quenched with pyridine (0.1 mL), filtered through Celite
(CH2Cl2), and concentrated. The residue was coevaporated twice with toluene
and purified by flash column chromatography on silica (4:1 → 1:1 cyclohex-
ane/EtOAc) and recycling preparative HPLC (CHCl3) to afford 13 (96 mg, 92%)
as a pale yellow solid (α/β-ratio: 1:2.5). Rf 0.04 (cyclohexane/EtOAc 4:1). IR
1
(neat): 3332, 3022, 2878, 1748, 1617, 1593, 1523, 1337, 1215, 1039 cm−1. H
NMR (CDCl3, 400 MHz) δ 1.65 (s, 0.9H), 1.71 (s, 0.9H), 1.90 (s, 2.1H), 2.00 (s,
0.9H), 2.03 (s, 2.1H), 2.06 (s, 2.1H), 2.87 (d, J = 9.7 Hz, 0.7H), 3.24–5.13 (m,
21.7H), 5.29 (t, J = 9.9 Hz, 0.3H), 5.88 (d, J = 4.0 Hz, 0.3H), 6.76–6.98 (m,
2.7H), 7.26–7.51 (m, 13.3H), 8.05 (dd, J = 9.6, 2.7 Hz, 1H), 8.26 (d, J = 8.9 Hz,
0.7H), 8.78 (d, J = 9.6 Hz, 0.3H), 9.01 (d, J = 2.7 Hz, 0.3H), 9.05 (d, J = 0.7H).
13C NMR (CDCl3, 100 MHz) δ 20.5, 20.5, 20.6, 20.7, 20.7, 53.4, 54.7, 57.1, 57.8,
61.3, 61.6, 67.6, 67.7, 67.9, 68.4, 68.9, 71.5, 72.5, 73.6, 74.1, 74.6, 81.5, 81.9,
82.5, 84.6, 96.4, 100.9, 104.7, 114.3, 115.8, 123.8, 124.2, 126.1, 127.1, 127.8,
128.1, 128.3, 128.5, 129.0, 130.8, 130.9, 136.5, 137.0, 137.6, 137.9, 138.1, 138.2,
138.4, 139.0, 147.3, 148.2, 169.4, 169.5, 169.6, 170.1, 170.4, 170.5. MALDI-
HRMS: m/z calcd for C46H51N3O17Na [M+Na]+ 940.3111, obsd 940.3102.
Iso-propyl (Methyl 3-O-Benzyl-4-O-levulinoyl-2-O-pivaloyl-L-
idopyranosyluronate) (17)
Thioglycoside 16[26] (500 mg, 0.95 mmol) and tri-tert-butylpyrimidine
(TTBP, 1.18 g, 4.8 mmol) were coevaporated twice with toluene and dissolved
i
in CH2Cl2 (19 mL), freshly activated 4 molecular sieves and PrOH (365 µL,
4.8 mmol) were added, and the mixture was stirred at rt for 15 min. At 0◦C,
MeSSMe (0.42 mL, 4.8 mmol) and MeOTf (0.6 mL, 4.8 mmol) were added drop-
i
wise and the reaction was stirred at rt for 10 h. Then, more PrOH (0.72 mL,
48 mmol) was added and the reaction was stirred for an additional 3 h at
rt, filtered through Celite (CH2Cl2), and washed with a 1:1 mixture of satu-
rated aqueous NaHCO3 and saturated aqueous Na2S2O3 solutions. The aque-
ous layer was re-extracted once with CH2Cl2, and the combined organic phases
were dried over MgSO4, filtered, and concentrated. Flash column chromatog-
raphy on silica gel (9:1 → 4:1, cyclohexane/EtOAc) afforded 17 (330 mg, 66%)

P. Bindscha¨ dler et al.
414
as a colorless oil (ratio of α/β-anomers: 8:1). Rf 0.54 (cyclohexane/EtOAc 1:1).
1H NMR (CDCl3, 300 MHz, only signals of α-anomer) δ 1.18–1.23 (m, 15H),
2.50–2.82 (m, 4H), 3.71 (t, J = 2.6 Hz, 1H), 3.78 (s, 3H), 3.93–4.01 (m, 1H),
4.67 (d, J = 11.7 Hz, 1H), 4.80 (d, J = 11.7 Hz, 1H), 4.87 (br., 1H), 4.93 (d,
J = 2.4 Hz, 1H), 5.08 (br. s, 1H), 5.22 (t, J = 2.6 Hz, 1H), 7.26–7.38 (m, 5H).
13C NMR (CDCl3, 100 MHz) δ 14.9, 21.4, 23.2, 26.5, 27.1, 27.1, 27.9, 29.7, 29.7,
37.7, 38.7, 38.8, 52.3, 52.4, 66.4, 66.6, 67.4, 68.2, 68.5, 70.5, 72.0, 72.6, 73.5,
77.2, 82.8, 96.7, 127.4, 127.5, 127.6, 127.7, 127.8, 128.2, 128.4, 128.5, 137.2,
137.8, 168.9, 169.2, 171.6, 171.6, 177.3, 177.3, 205.8, 205.9. MALDI-HRMS:
m/z calcd for C27H38O10Na [M+Na]+ 545.2357, obsd 545.2358.
Iso-propyl (Methyl 3-O-Benzyl-2-O-pivaloyl-α-L-idopyranosyluronate) (14)
To a solution of 17 (255 mg, 488 µmol) in CH2Cl2 (4.9 mL) at rt was added
pyridine (1.18 mL), AcOH (0.78 mL), and hydrazine monohydrate (47.4 µL,
976 µmol). The solution was stirred for 1.5 h at rt, quenched with acetone
(1 mL), and concentrated. The residue was coevaporated twice with toluene
and purified by flash column chromatography on silica gel (6:1 → 3:1, cyclo-
hexane/EtOAc) to afford 14 (176 mg, 85%) as a colorless oil. Rf 0.63 (cyclo-
hexane/EtOAc 1:1). α r.t.: −46.9 (c = 1.0, CHCl3). IR (neat): 3505, 2973, 2934,
[ ]D
1
1739, 1456, 1370, 1279, 1210, 1104, 1049, 942 cm−1. H NMR (CDCl3, 300
MHz) δ 1.18–1.24 (m, 15H), 2.69 (d, J = 11.9 Hz, 1H), 3.68 (br. s, 1H), 3.79 (s,
3H), 3.96–4.06 (m, 2H), 4.58 (d, J = 11.5 Hz, 1H), 4.80 (d, J = 11.5 Hz, 1H),
4.90 (br. s, 1H), 4.94 (br. s, 1H), 5.04 (br. s, 1H), 7.27–7.35 (m, 5H). 13C NMR
(CDCl3, 100 MHz) δ 21.4, 23.1, 23.3, 27.1, 38.8, 52.3, 53.4, 67.2, 67.8, 68.3, 68.4,
70.6, 71.6, 74.5, 97.0, 127.5, 127.7, 128.3, 137.8, 170.1, 176.7. MALDI-HRMS:
m/z calcd for C22H32O8Na [M+Na]+ 447.1989, obsd 447.1992.
Iso-propyl 3,4,6-Tri-O-acetyl-2-deoxy-2-(2,4-dinitrophenylamino)-D-
glucopyranosyl-(1→4)-(Methyl 3-O-Benzyl-2-O-pivaloyl-α-L-
idopyranosyluronate) (15)
L-Iduronic acid acceptor 14 (37 mg, 87 µmol) and N-phenyl trifluoroacetim-
idate D (114 mg, 177 µmol) were coevaporated three times with toluene and
dried in vacuo for 1 h. Freshly activated acid-washed 4 molecular sieves (150
mg) were added and the mixture was dissolved in CH2Cl2 (5 mL). At −20◦C,
TMSOTf (5 µL, 27 µmol) was added. The reaction was warmed to +5◦C over
1 h, quenched with pyridine (0.1 mL), filtered through Celite (CH2Cl2), and
concentrated. The residue was coevaporated once with toluene and purified by
flash column chromatography on silica (9:1 → 3:2 cyclohexane/EtOAc) and re-
cycling preparative HPLC (CHCl3) to afford 15 (16 mg, 21%) as a pale yellow oil
(ratio of α/β-anomers: 5:1). Rf 0.23 (cyclohexane/EtOAc 13:7). IR (neat): 3333,
1
2972, 2931, 1740, 1616, 1592, 1523, 1335, 1218, 1106, 1029 cm−1. H NMR
(CDCl3, 400 MHz, only signals of α-anomer) δ 1.07–1.16 (m, 6H, (CH3)2CH),

Synthesis and Evaluation of Glucosamine Building Blocks
415
1.14 (s, 9H, Piv), 1.67 (s, 3H, Ac), 1.96, (s, 3H, Ac), 2.05 (s, 3H, Ac), 3.82 (s, 3H,
OMe), 3.86–3.91 (m, 1H, (CH3)2CH), 3.95 (td, J = 9.9, 3.7, H-22), 4.07–4.11 (m,
2H, H-31, H-62), 4.18–4.21 (m, 1H, H-52), 4.25 (dd, J = 7.0, 5.3, 1H, H-41), 4.31
(dd, J = 12.4, 4.1, 1H, H-6ꢁ2), 4.32 (d, J = 11.8, 1H, CHHPh), 4.69 (d, J = 13.3,
1H, CHHPh), 4.71 (d, J = 5.3, 1H, H-51), 4.92 (dd, J = 6.2, 4.5, 1H, H-21), 5.07
(t, J = 9.9, 1H, H-42), 5.16 (d, J = 4.5, 1H, H-11), 5.26 (t, J = 9.9, 1H, H-32), 5.27
(d, J = 4.1, 1H, H-12), 6.89–7.02 (m, 5H, HAr), 8.08 (dd, J = 9.5, 2.6, 1H, DNP),
8.66 (d, J = 9.7, 1H, DNP), 8.93, (d, J = 2.7, 1H, DNP). 13C NMR (CDCl3, 100
MHz, only signals of α-anomer) δ 20.4, 20.5, 20.7, 21.8, 27.1, 38.8, 52.5, 54.9,
61.5, 67.7, 68.9, 70.5, 71.6, 71.8, 72.5, 72.9, 73.8, 77.8, 97.7, 97.8, 114.0, 124.2,
126.3, 127.3, 128.0, 129.8, 130.9, 136.7, 137.5, 147.3, 169.5, 169.6, 169.9, 170.5,
177.2. MALDI-HRMS: m/z calcd for C40H51N3O19Na [M+Na]+ 900.3009, obsd
900.3004.
Iso-propyl 3,4,6-Tri-O-benzyl-2-deoxy-2-(2,4-dinitrophenylamino)-D-
glucopyranosyl-(1→4)-(Methyl 3-O-Benzyl-2-O-pivaloyl-α-L-
idopyranosyluronate) (18)
L-Iduronic acid acceptor 14 (33 mg, 78 µmol) and N-phenyl trifluoroace-
timidate E (122 mg, 155 µmol) were coevaporated three times with toluene
and dried in vacuo for 1 h. Freshly activated acid-washed 4 molecular sieves
(155 mg) were added and the mixture was dissolved in CH2Cl2 (4.6 mL). At
−30◦C, TMSOTf (4 µL, 22 µmol) was added. The reaction was warmed to
−5◦C over 45 min, quenched with pyridine (0.1 mL), filtered through Celite
(CH2Cl2), and concentrated. The residue was coevaporated twice with toluene
and purified by flash column chromatography on silica gel (1:0 → 4:1 cyclohex-
ane/EtOAc) and recycling preparative HPLC (CHCl3) to afford 18 (46 mg, 58%)
as a yellow solid (ratio of α/β-anomers: 5:1). Rf 0.32 (cyclohexane/EtOAc 4:1).
IR (neat): 3332, 2931, 1738, 1618, 1590, 1523, 1454, 1334, 1131, 1029 cm−1
.
1H NMR (CDCl3, 400 MHz) δ 1.15–1.30 (m, 15H), 3.49 (s, 0.51H), 3.52–4.04
(m, 7H), 3.83 (s, 2.49H), 4.16–4.20 (m, 0.17H), 4.18 (t, J = 7.0 Hz, 0.83H), 4.33
(dd, J = 7.3, 5.6 Hz, 0.83H), 4.36–4.88 (m, 9.34H), 4.90 (br. s, 0.17H), 4.94 (br.
s, 0.17H, H-1-IdoA), 5.01 (dd, J = 6.6, 5.2 Hz, 0.83H), 5.21 (d, J = 5.1 Hz,
0.83H), 5.29 (d, J = 3.7 Hz, 0.83H), 6.80–6.82 (m, 2H), 6.94–7.38 (m, 19H),
8.00 (dd, J = 9.6, 2.6 Hz, 0.83H), 8.13 (dd, J = 9.6, 2.7 Hz, 0.17H), 8.43 (d, J
= 8.1 Hz, 0.17H), 8.74 (d, J = 9.5 Hz, 0.83H), 8.92 (d, J = 2.7 Hz, 0.17H), 9.06
(d, J = 2.7 Hz, 0.83H). 13C NMR (CDCl3, 100 MHz) δ 21.5, 21.8, 23.3, 23.3,
27.2, 27.3, 38.7, 38.8, 51.8, 52.2, 56.0, 59.3, 66.9, 67.1, 67.8, 68.5, 70.3, 71.2,
71.8, 73.0, 73.4, 74.7, 76.0, 76.2, 78.0, 78.3, 82.3, 84.0, 96.8, 97.5, 98.2, 104.4,
115.5, 116.2, 123.7, 123.9, 126.3, 126.4, 127.2, 128.1, 129.4, 129.7, 130.1, 130.6,
136.1, 136.7, 136.9, 137.0, 137.6, 137.7, 137.9, 138.0, 138.3, 148.1, 148.9, 169.5,
169.6, 177.0, 177.3. MALDI-HRMS: m/z calcd for C55H63N3O16Na [M+Na]+
1044.4101, obsd 1044.4124.

P. Bindscha¨ dler et al.
416
Entry 2: Acceptor 14 (31 mg, 73 µmol) and N-phenyl trifluoroacetimidate
E (115 mg, 146 µmol) were coevaporated three times with toluene and dried in
vacuo for 1 h. Freshly activated acid-washed 4 molecular sieves (145 mg) were
added and the mixture was dissolved in Et2O (3.5 mL) and CH2Cl2 (0.9 mL).
At −30◦C, TMSOTf (4 µL, 22 µmol) was added. The reaction was warmed to
−10◦C over 45 min, quenched with pyridine (0.1 mL), filtered through Celite
(CH2Cl2), and concentrated. The residue was coevaporated twice with toluene
and purified by flash column chromatography on silica gel (1:0 → 4:1 cyclohex-
ane/EtOAc) and recycling preparative HPLC (CHCl3) to afford 18 (65 mg, 87%)
as a yellow solid (ratio of α/β-anomers: 2.5:1).
Entry 3: Acceptor 14 (32 mg, 75 µmol) and trichloroacetimidate 11 (115 mg,
151 µmol) were coevaporated three times with toluene and dried in vacuo for 1
h. Freshly activated acid-washed 4 molecular sieves (150 mg) were added and
the mixture was dissolved in Et2O (3.7 mL) and CH2Cl2 (0.9 mL). At −29◦C,
TMSOTf (4 µL, 23 µmol) was added. The reaction was warmed to −10◦C over
45 min, quenched with pyridine (0.1 mL), filtered through Celite (CH2Cl2), and
concentrated. The residue was coevaporated twice with toluene and purified by
flash column chromatography on silica gel (1:0 → 4:1 cyclohexane/EtOAc) and
recycling preparative HPLC (CHCl3) to afford 18 (44 mg, 57%) as a yellow solid
(ratio of α/β-anomers: 3.5:1).
Entry 4: Acceptor 14 (39 mg, 92 µmol) and trichloroacetimidate 11 (91 mg,
119 µmol) were coevaporated three times with toluene and dried in vacuo for 1
h. Freshly activated acid-washed 4 molecular sieves (150 mg) were added and
the mixture was dissolved in CH2Cl2 (4.2 mL). At −35◦C, TMSOTf (3.3 µL,
18 µmol) was added. The reaction was warmed to −10◦C over 45 min, quenched
with pyridine (0.1 mL), filtered through Celite (CH2Cl2), and concentrated. The
residue was coevaporated twice with toluene and purified by flash column chro-
matography on silica gel (1:0 → 4:1 cyclohexane/EtOAc) and recycling prepar-
ative HPLC (CHCl3) to afford 18 (39 mg, 41%) as a yellow solid (ratio of α/β-
anomers: 3.5:1).
Entry 5: Acceptor 14 (29 mg, 68 µmol) and phosphate 12 (100 mg,
124 µmol) were coevaporated three times with toluene and dried in vacuo for
1 h. Freshly activated acid-washed 4 molecular sieves (130 mg) were added
and the mixture was dissolved in CH2Cl2 (3.9 mL). At −78◦C, TMSOTf (4
µL, 22 µmol) was added. The reaction was warmed to −50◦C over 30 min,
quenched with pyridine (0.1 mL), filtered through Celite (CH2Cl2), and con-
centrated. The residue was coevaporated twice with toluene and purified by
flash column chromatography on silica gel (1:0 → 4:1 cyclohexane/EtOAc) and
recycling preparative HPLC (CHCl3) to afford 18 (62 mg, 89%) as a yellow solid
(ratio of α/β-anomers: 1.5:1).

Synthesis and Evaluation of Glucosamine Building Blocks
417
ACKNOWLEDGMENTS
We are grateful to ETH Zu¨rich and the Swiss National Science Foundation
(SNF) for generous support of our work. P.B. thanks Novartis for a doctoral
fellowship.
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