Synthesis of hyaluronic acid oligosaccharides and exploration of a fluorous-assisted approach

Article abstract of DOI:10.1016/j.carres.2014.05.007

The synthesis of hyaluronic acid oligomers (tri- and tetrasaccharide) is described. We have followed a pre-glycosylation oxidation strategy. Glucuronic acid units were directly employed in coupling reactions with suitably protected glucosamine derivatives. In order to simplify the purification of synthetic intermediates, a fluorous-assisted strategy has been also explored. Using this approach, a hyaluronic acid trisaccharide was prepared.

Full text of DOI:10.1016/j.carres.2014.05.007

Accepted Manuscript
Synthesis of hyaluronic acid oligosaccharides and exploration of a fluorous-
assisted approach
Giuseppe Macchione, José L. de Paz, Pedro M. Nieto
PII:
S0008-6215(14)00199-2
DOI:
http://dx.doi.org/10.1016/j.carres.2014.05.007
CAR 6746
Reference:
Carbohydrate Research
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 April 2014
6 May 2014
7 May 2014
Please cite this article as: Macchione, G., de Paz, J.L., Nieto, P.M., Synthesis of hyaluronic acid oligosaccharides
and exploration of a fluorous-assisted approach, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/
j.carres.2014.05.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of hyaluronic acid oligosaccharides and exploration of a
fluorous-assisted approach
Giuseppe Macchione, José L. de Paz,* Pedro M. Nieto*
Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de
Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla,
Americo Vespucio, 49, 41092 Sevilla, Spain.
* Corresponding author. Fax: +34 954 460165; phone: +34 954 489500 ext. 909317; e-
mail: jlpaz@iiq.csic.es
Abstract
The synthesis of hyaluronic acid oligomers (tri- and tetrasaccharide) is described. We
have followed a pre-glycosylation oxidation strategy. Glucuronic acid units were
directly employed in coupling reactions with suitably protected glucosamine
derivatives. In order to simplify the purification of synthetic intermediates, a fluorous-
assisted strategy has been also explored. Using this approach, a hyaluronic acid
trisaccharide was prepared.
Keywords
Hyaluronic acid/glycosylation/fluorous tag/oligosaccharide synthesis/glucuronic
acid/carbohydrates
Introduction
Hyaluronic acid is a negatively charged linear polysaccharide that belongs to the
glycosaminoglycan family. It is constituted by the repetition of disaccharide units of D-
glucuronic acid (GlcA)-β(1→3)-N-acetyl-D-glucosamine (GlcNAc)-β(1→4).
Hyaluronic acid is the only glycosaminoglycan that does not contain sulfate groups.
1

Despite its simple chemical structure, hyaluronic acid is involved in a wide range of
biological processes, such as cell-migration, recognition and tumor invasion.1 The
specific biological activities of hyaluronic acid strongly depend on the length and
molecular weight of the carbohydrate chain.2 For example, high molecular weight
polymers are anti-angiogenic while smaller oligosaccharide fragments induce
angiogenesis. In this context, well-defined synthetic oligosaccharides are useful to
elucidate the role of hyaluronic acid chains in nature. In particular, synthetic
oligosaccharides, with defined sequence and length, are required to study, at the
molecular level, the interactions between hyaluronic acid and certain protein receptors
that trigger the biological functions of this natural product. These studies can potentially
lead to the interference and control of a particular biological process.
Several approaches have been reported for the synthesis of hyaluronic acid
oligosaccharides.3-5 Two main strategies have been employed for the preparation of
these molecules.6 In the pre-glycosylation oxidation strategy, glucuronic acid building
blocks are directly used in coupling reactions. 7-14 The low reactivity of these units can
afford low to moderate yields in the glycosylation reactions.15-17 For this reason, an
alternative post-glycosylation oxidation approach has been also considered. In this
second method, glucose moieties are used for the assembly of the oligosaccharide
chain.6, 18-23 Then, glucose units are oxidized to the corresponding glucuronic acids to
give the final hyaluronic acid molecules. The oxidation of highly elaborated
intermediates is, however, a complex and challenging synthetic step.
Recently, several methods have been reported to accelerate the preparation of
hyaluronic acid oligomers, facilitating the purification of synthetic intermediates. Huang
and coworkers6 developed a pre-activation based iterative one-pot strategy in which
anomeric reactivity adjustment and intermediate oligosaccharide purifications are not
2

required. Using a fully automated solid-phase approach, Codée, van der Marel and
coworkers24-25 described the synthesis of hyaluronic acid fragments up to the
pentadecamer level.
Here, we describe the total synthesis of one tri- and one tetrasaccharide that correspond
to the structure of hyaluronic acid, using a stepwise strategy. Glucuronic acid building
blocks were directly employed for the glycosylation reactions. Additionally, we have
explored the use of a fluorous tag to facilitate the preparation of this type of compounds.
Results and discussion
For the preparation of hyaluronic acid oligosaccharides, we have followed the
retrosynthetic analysis shown in Scheme 1. We envisaged that final molecules, such as
tetrasaccharide 1, could be obtained from the corresponding fully protected precursors,
such as 2, by a 3-step sequence of deprotection reactions (basic hydrolysis, selective N-
acetylation and hydrogenolysis). The oligosaccharide backbone was assembled by
linking monosaccharide building blocks 3, 4 and 5. The levulinoyl (Lev) groups at
position 4 of GlcA unit 3 and position 3 of glucosamine derivative 4 were chosen as
temporary protecting groups to allow the elongation of the carbohydrate chain. The
benzoyl and N-trichloroacetyl26 groups led to the selective formation of the 1,2-trans
glycosidic linkages. A 4-methoxyphenyl group was used to protect the anomeric
position of the reducing end. The trichloroacetimidate method was chosen to form the
glycosidic linkages.27
GlcA units 3 and 528 were prepared from commercially available 1,2;5,6-di-O-
isopropylidene-α-D-glucofuranose. Glucosamine trichloroacetimidate 4 was prepared
from monosaccharide 610,26 as shown in Scheme 2. The anomeric acetate of 6 was
→
exchanged for an anomeric silyl ether ( 7). Removal of the remaining acetates
3

followed by the formation of the 4,6-benzylidene acetal and subsequent levulinoylation
at position 3 gave monosaccharide 8 in 76% yield over three steps. The regioselective
reductive opening of the 4,6-di-O-benzylidene acetal of compound 8 gave the 4-OH
→
monosaccharide 9 in high yield. Compound 9 was then acetylated ( 10), desilylated
→
→
( 11) and activated as trichloroacetimidate ( 4).
With the required building blocks at hand, we first accomplished the preparation of
trisaccharide 16 (Scheme 3). The glycosylation reaction between monomers 4 and 5
gave the desired disaccharide 12 in good yield. It is important to note that the yield of
this coupling reaction strongly depended on the protecting group distribution of the
GlcA building block. In preliminary experiments, we also employed highly disarmed
glucuronic acid derivatives as glycosyl acceptors to build the GlcNAc-GlcA linkage
(see Supporting Information). However, using GlcA units that contain acyl groups at
positions 2 and 3, the glycosylation yields were significantly lower (18-41%, see
Supporting Information). Therefore, the use of more reactive, partially benzylated unit 5
was crucial to increase the efficiency of the glycosidic bond formation. Disaccharide 12
was selectively delevulinoylated to obtain glycosyl acceptor 13. Glycosylation with
GlcA trichloroacetimidate 3 gave trisaccharide 14 in moderate yield. A considerable
amount (18%) of unreacted acceptor 13 was also recovered from the reaction mixture.
Treatment with H2O2/LiOH and then with NaOH hydrolyzed all the ester and amide
groups. Selective N-acetylation afforded precursor 15 that was hydrogenated in the
presence of Pd(OH)2 to give the fully deprotected hyaluronic acid trisaccharide 16 in
high yield.
Next, we carried out the synthesis of tetrasaccharide 1 (Scheme 4). Compound 14 was
treated with hydrazine monohydrate in pyridine/acetic acid buffer to give trisaccharide
acceptor 17. Coupling with glucosamine trichloroacetimidate 4 gave the fully protected
4

tetrasaccharide 2 in good yield. Finally, saponification followed by N-acetylation and
hydrogenolysis afforded hyaluronic acid tetramer 1.
The classical stepwise synthesis of oligosaccharides requires multiple chromatographic
purifications that hamper this process. We decided to evaluate the utility of a fluorinated
tag29-30 to facilitate the purification of the synthetic intermediates31 and, therefore, the
preparation of the hyaluronic acid oligomers. As a first goal, we managed the fluorous-
assisted preparation of trisaccharide 15 (Scheme 5). Fluorous compounds can be easily
separated from nonfluorinated molecules by a simple fluorous solid-phase extraction (F-
SPE) on FluoroFlash silica gel.32-33 Importantly, the reactions involving fluorous
compounds can be monitored by standard TLC, NMR and mass spectrometry.34
Compared to solid-phase approaches, fluorous-assisted oligosaccharide synthesis often
consumes lower amounts of building blocks since the reactions are run in solution.35
Our approach involved the attachment of the fluorous tag to the reducing end GlcA
monomer through the carboxylate group (Scheme 5). Thus, starting from known diol
19,36 oxidation with calcium hypochlorite and catalytic TEMPO followed by treatment
with heptadecafluoroundecyl iodide in DMF at 60ºC directly gave fluorinated glycosyl
acceptor 20. Using this strategy, the introduction of the fluorous tag did not involve
additional synthetic steps because the analogous acceptor 5, required for the “classical”
synthesis of oligomers 1 and 16, was also prepared from diol 19 by the same two-step
procedure (oxidation and esterification). Fluorous acceptor 20 was then glycosylated
with trichloroacetimidate 4, using the same reaction conditions employed for the
preparation of comparable disaccharide 12. The crude mixture was quickly purified by
F-SPE. The non-fluorous compounds, mainly derived from the glycosyl donor, were
first eluted with MeOH/H2O 80:20 while the desired fluorous-tagged product was eluted
with 100% methanol. After two glycosylation cycles, NMR and mass spectrometry
5

analysis of the fluorous fraction indicated the presence of the desired disaccharide 21
along with a small quantity of unreacted monosaccharide acceptor 20 (6:1 ratio,
1
according to the H-NMR data). As in solid-phase approaches, fluorous-assisted
reactions can be driven to completion by carrying out multiple cycles. Disappointingly,
we observed a significant loss (approximately 10%) of fluorous material after each F-
SPE purification step. Following a typical protocol for F-SPE (100 mg of reaction
mixture purified with 3 g. of fluorous silica gel; see Experimental Section), we detected
fluorous sample breakthrough,29 that is to say, some of the fluorous compound was
eluted with the fluorophobic solvent (MeOH/H2O 80:20). We solved these breakthrough
problems by using a higher amount of fluorous silica gel for the purification (up to 13 g
of silica per 100 mg of reaction mixture). Using this modified F-SPE procedure, we did
not detect fluorous compounds in the fluorophobic elution but we still observed a loss
of fluorous-tagged material. To explain this result, we hypothesized that some fluorous
product could be retained on the F-SPE column when using the high amount of silica
required to prevent breakthrough. This experimental limitation precluded the possibility
of performing more glycosylation cycles to completely consume acceptor 20.
Therefore, compound 21 was directly submitted to delevulinoylation to give
disaccharide 22. Fluorous trisaccharide 23 was obtained by coupling of 22 with GlcA
glycosyl donor 3, after purification by F-SPE and normal silica gel chromatography.
The overall yield for the synthesis of 23 (12%) was lower than the one obtained in the
classical non-fluorous route (27%). This fact can be explained by the observed loss of
material during F-SPE purifications, using our set of building blocks. Finally, the
fluorous tag was easily removed during basic hydrolysis. Selective N-acetylation
afforded compound 15 that can provide hyaluronic acid trisaccharide 16, as shown
before.
6

Conclusions
We have described a strategy for the preparation of hyaluronic acid oligosaccharides.
Suitably protected GlcA and glucosamine monosaccharide building blocks were used
for the assembly of the hyaluronic acid chain. Participating groups at position 2 of these
units ensured the selective formation of the desired β glycosidic bonds. A sequence of
deprotection reactions efficiently afforded the final tri- and tetrasacharide fragments. An
alternative fluorous-assisted approach was also explored. F-SPE facilitated the
purification of fluorous-tagged synthetic intermediates. We attached the fluorous tag to
the carboxylic acid of the reducing-end GlcA moiety by an esterification reaction. Thus,
the introduction and removal of the fluorous tail did not add any extra steps to the
preparation of these molecules in comparison with the classical stepwise approach. The
overall yield of the fluorous-assisted synthesis of a hyaluronic acid trisaccharide was,
nevertheless, quite low. The moderate yield of the glycosylation involving GlcA donor
3 and the observed loss of fluorous material after each F-SPE purification step can
explain this result.
Scheme 1. Retrosynthetic analysis for tetrasaccharide 1.
7

Scheme 2. Reagents and conditions: a) BnNH2, THF; TDSCl, imidazole, CH2Cl2, 80%;
b) NaOMe, MeOH; PhCH(OMe)2, p-TsOH, CH3CN; LevOH, DCC, DMAP, CH2Cl2,
76%; c) Et3SiH, CF3COOH, CH2Cl2, 85%; d) Ac2O, Py; e) (HF)n·Py, THF, 0ºC,
quantitative (two steps, from 9); f) Cl3CCN, DBU, CH2Cl2, 70%.
Scheme 3. Reagents and conditions: a) TMSOTf, CH2Cl2, 0ºC, 72%; b) NH2NH2·H2O,
Py/AcOH, CH2Cl2, 93%; c) 3, TMSOTf, CH2Cl2, 41%; d) LiOH, H2O2, THF; NaOH,
MeOH; Ac2O, MeOH, Et3N, 87%; e) H2, Pd(OH)2, H2O/MeOH, quantitative.
8

Scheme 4. Reagents and conditions: a) NH2NH2·H2O, Py/AcOH, CH2Cl2, 71%; b) 4,
TMSOTf, CH2Cl2, 0ºC, 64%; c) LiOH, H2O2, THF; NaOH, MeOH; Ac2O, MeOH,
Et3N, 64%; d) H2, Pd(OH)2, H2O/MeOH, quantitative.
Scheme 5. Reagents and conditions: a) TEMPO, Ca(ClO)2, Bu4NBr, KBr, NaHCO3,
CH2Cl2/H2O, 0ºC; C8F17-(CH2)3-I, DMF, 60ºC, 55%; b) 4, TMSOTf, CH2Cl2, 0ºC; c)
NH2NH2·H2O, Py/AcOH, CH2Cl2; d) 3, TMSOTf, CH2Cl2, 12% (three steps, from 20);
e) LiOH, H2O2, THF; NaOH, MeOH; Ac2O, MeOH, Et3N, 90%.
Experimental
General procedures: Thin layer chromatography (TLC) analyses were performed on
silica gel 60 F254 precoated on aluminium plates (Merck) and the compounds were
detected by staining with sulfuric acid/ethanol (1:9), with cerium (IV) sulfate
9

(10 g)/phosphomolybdic acid (13 g)/sulfuric acid (60 mL) solution in water (1 L), or
with anisaldehyde solution [anisaldehyde (25 mL) with sulfuric acid (25 mL), ethanol
(450 mL) and acetic acid (1 mL)], followed by heating at over 200ºC. Column
chromatography was carried out on silica gel 60 (0.2-0.5 mm, 0.2-0.063 mm or 0.040-
0.015 mm; Merck). Optical rotations were determined with a Perkin-Elmer 341
1
13
polarimeter. H- and C-NMR spectra were acquired on Bruker DPX-300, Avance III-
400 and DRX-500 spectrometers. Unit A refers to the reducing end monosaccharide in
the NMR data. Electrospray mass spectra (ESI MS) were carried out with an Esquire
6000 ESI-Ion Trap from Bruker Daltonics. High resolution mass spectra (HR MS) were
carried out by CITIUS (Universidad de Sevilla) and SIdI (Universidad Autónoma de
Madrid). F-SPE was performed using FluoroFlash silica gel.
General procedure for F-SPE: FluoroFlash silica gel (3 g.) was placed in a 10 mL
plastic tube. The F-SPE column was washed with DMF (1 mL) and then preconditioned
with MeOH/H2O 80:20 (10 mL). Next, the crude sample (100 mg) was dissolved in
DMF (0.8 mL) and loaded on the column. The fluorophobic elution was carried out
with 10 mL of MeOH/H2O 80:20. The fluorous compounds were then eluted using
100% methanol (16 mL). To regenerate the F-SPE column, we washed with acetone (10
mL).
Dimethylthexylsilyl 3,4,6-tri-O-acetyl-2-deoxy-2-trichloroacetamido-β-D-
glucopyranoside (7): Benzylamine (0.267 mL, 2.44 mmol) was added to a solution of 6
(1.00 g, 2.03 mmol) in THF (10 mL). After 3 h, an additional aliquot of benzylamine
(0.267 mL, 2.44 mmol) was added. After a further 3 h, the mixture was diluted with
CH2Cl2, and washed with 1N HCl. The organic phase was dried over Na2SO4, filtered
and the solvent was removed under reduced pressure. The residue [TLC (3:2
hexane/EtOAc) Rf 0.34] was dissolved in CH2Cl2 (5 mL). Imidazole (379 mg, 5.57
10

mmol) and thexyldimethylsilyl chloride (0.481 mL, 2.45 mmol) were added. After 24 h,
the mixture was diluted with CH2Cl2 and washed with H2O. The organic layer was dried
over MgSO4, filtered, and the solvent was removed in vacuo. Flash chromatography on
silica gel (3:1 hexane/EtOAc) afforded 7 (964 mg, 80%). TLC (3:2 hexane/EtOAc) Rf
0.66; [α]20D -6.8º (c 1.17, CHCl3); 1H-NMR (500 MHz, CDCl3): δ 6.89 (d, J 2,NH = 9
Hz, 1H, NH), 5.34 (dd, 1H, H-3), 5.10 (dd, J 3,4 = J 4,5 = 9.8 Hz, 1H, H-4), 4.90 (d, J 1,2
= 8 Hz, 1H, H-1), 4.25-4.13 (m, 2H, H-6), 4.00 (ddd, 1H, H-2), 3.76 (ddd, 1H, H-5),
2.10-2.04 (3s, 9H, COCH3), 1.62 (m, 1H, CH(CH3)2), 0.93-0.84 (m, 12H, C(CH3)2 and
CH(CH3)2), 0.18-0.15 (2s, 6H, Si(CH3)2); 13C-NMR (125 MHz, CDCl3): δ 171.1, 170.6,
169.3, 161.8, 95.7, 92.4, 72.0, 71.8, 68.9, 62.5, 57.9, 33.9, 24.8, 20.7, 20.63, 20.60,
20.0, 19.9, 18.53, 18.49, -1.9, -3.4; HR MS: m/z: calcd for C22H36O9NCl3SiNa:
616.1279; found: 616.1302 [M+Na]+.
Dimethylthexylsilyl
4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-2-
trichloroacetamido-β-D-glucopyranoside (8): Compound 7 (11.5 g, 19.4 mmol) was
dissolved in methanol (160 mL) and NaOMe (1.5 mL of a 2.17 M solution in MeOH)
was added. After 12 h, Amberlite acidic resin was added and the mixture was stirred
until the pH reached 7. The Amberlite resin was filtered off and the solvent was
removed in vacuo. The residue was coevaporated with dichloromethane and toluene and
dissolved in acetonitrile (90 mL). Benzaldehyde dimethyl acetal (3.5 mL, 23.2 mmol)
and p-toluenesulfonic acid (367 mg, 1.9 mmol) were added. After stirring at room
temperature for 3 h, EtOAc was added and the mixture was extracted with saturated
NaHCO3 solution. The organic phase was dried over Na2SO4, filtered and concentrated
in vacuo. The residue [TLC (3:2 hexane/AcOEt) Rf 0.73] was dissolved in CH2Cl2 (113
mL) and levulinic acid (9.9 mL, 97 mmol), 1,3-dicyclohexylcarbodiimide (6.0 g, 29
mmol) and DMAP (500 mg) were added. After 3 h, the mixture was diluted with
11

CH2Cl2 and washed with saturated NaHCO3 solution. The organic phase was dried over
Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (3:1 hexane/EtOAc) to give 8 (9.7 g, 76%). TLC (3:1
1
hexane/EtOAc) Rf 0.5; [α]20D -47.2º (c 3.17, CHCl3); H-NMR (300 MHz, CDCl3): δ
7.43-7.26 (m, 6H, Ph, NH), 5.40 (m, 2H, H-3, PhCHO), 4.70 (d, J 1,2 = 7.8 Hz, 1H, H-
1), 3.94 (ddd, 1H, H-2), 3.86 (dd, J 5,6a = 5.0 Hz, J 6a,6b = 10.4 Hz, 1H, H-6a), 3.61 (m,
2H, H-4, H-6b), 3.30 (ddd, 1H, H-5), 2.68-2.48 (m, 4H, OCO(CH2)2), 2.06 (s, 3H,
COCH3), 1.51 (m, 1H, CH(CH3)2), 0.80-0.73 (m, 12H, C(CH3)2 and CH(CH3)2), 0.00
(2s, 6H, Si(CH3)2); 13C-NMR (75 MHz, CDCl3): δ 205.8, 173.3, 162.2, 137.2-126.1
(Ph), 101.1, 96.2, 92.7, 79.0, 71.9, 68.5, 66.2, 58.4, 38.1, 34.0, 29.9, 28.4, 24.8, 20.2,
20.0, 18.7, 18.6, -1.7, -3.2; HR MS: m/z: calcd for C28H40O8NCl3SiNa: 674.1486;
found: 674.1448 [M+Na]+.
Dimethylthexylsilyl 6-O-benzyl-2-deoxy-3-O-levulinoyl-2-trichloroacetamido-β-D-
glucopyranoside (9): The starting material 8 (3 g, 4.6 mmol) was co-evaporated with
toluene, dissolved in dry CH2Cl2 (30 mL) and further dried by stirring over activated 4Å
molecular sieves (3 g) for 30 min. Triethylsilane (8.8 mL, 55.2 mmol) was added at
room temperature, followed by the slow addition of trifluoroacetic acid (4.5 mL, 60.7
mmol). After 10 min the reaction was filtered and diluted with CH2Cl2. The reaction
mixture was washed with saturated NaHCO3 and water, dried over Mg2SO4, filtered and
→ 1:1 hexane:
concentrated. The residue was purified by column cromatography (2:1
EtOAc) to afford 9 (2.55 g, 85%). TLC (2:1 hexane/EtOAc) Rf 0.24; [α] 20 -22.9º (c 1,
D
CHCl3); 1H-NMR (300 MHz, CDCl3): δ 7.39-7.27 (m, 5H, Ar), 6.78 (br d, 1H, J NH,2
=
9.3 Hz, NH), 5.13 (dd, 1H, J 2,3 = J 3,4 = 10.6 Hz, H-3), 4.81 (d, 1H, J 1,2 = 7.9 Hz, H-1),
4.60 (m, 2H, CH2(Bn)), 3.92 (m, 1H, H-2), 3.81-3.75 (m, 3H, H-4, H-6a and H-6b),
3.58 (m, 1H, H-5), 2.81-2.44 (m, 4H, OCO(CH2)2), 2.17 (s, 3H, COCH3), 1.60 (m, 1H,
12

CH(CH3)2), 0.86-0.82 (m, 12H, C(CH3)2 and CH(CH3)2), 0.18-0.13 (2s, 6H, Si(CH3)2);
13C-NMR (75MHz, CDCl3): δ 207.8, 173.4, 162.0 (C=O), 138.1-127.7 (Ar-CH and Ar-
C), 95.8 (C-1), 92.7 (CCl3), 75.7, 74.5 (C-3 and C-5), 73.7 (CH2(Bn)), 70.2, 69.9 (C-4
and C-6), 57.6 (C-2), 38.4 (OCO(CH2)2), 34.0 (CH(CH3)2), 29.9 (COCH3), 28.4
(OCO(CH2)2), 24.8, 20.1, 20.0, 18.7, 18.6 (CH(CH3)2), C(CH3)2) and C(CH3)2)), -1.7, -
3.2 (Si(CH3)2)); HR MS: m/z: calcd for C28H42Cl3NO8SiNa: 676.1643; found: 676.1675
[M+Na]+.
O-(4-O-acetyl-6-O-benzyl-2-deoxy-3-O-levulinoyl-2-trichloroacetamido-α,β-D-
glucopyranosyl) trichloroacetimidate (4): Ac2O (6 mL) was added to a solution of 9
(1.2 g, 1.83 mmol) in pyridine (12 mL). After stirring for 10 min at 0°C and 1 hour at
room temperature, the mixture was diluted with EtOAc and washed with 1N HCl,
saturated NaHCO3 solution and H2O. The organic layer was dried over Na2SO4, filtered
and concentrated in vacuo to give 10. TLC (3:2 hexane/EtOAc) Rf 0.53; 1H-NMR (300
MHz, CDCl3): δ 7.26 (m, 5H, Ar), 6.74 (br d, 1H, J NH,2 = 9.0 Hz, NH), 5.24 (t, 1H, H-
3), 5.04 (t, 1H, J 3,4 = J 4,5 = 9.7 Hz, H-4), 4.84 (d, 1H, J 1,2 = 7.9 Hz, H-1), 4.48 (2d, 2H,
CH2(Bn)), 3.85 (m, 1H, H-2), 3.64 (m, 1H, H-5), 3.50 (m, 2H, H-6a and H-6b), 2.66-
2.39 (m, 4H, OCO(CH2)2), 2.09 (s, 3H, COCH3), 1.95 (COCH3), 1.56 (m, 1H,
CH(CH3)2), 0.79 (m, 12H, C(CH3)2 and CH(CH3)2), 0.14,0.09 (2s, 6H, Si(CH3)2) ppm.
.
Compound 10 (1.83 mmol) was dissolved in dry THF (25 mL) and (HF)n Py complex
(5.2 mL) was added at 0ºC. After stirring for 5 days at 0ºC, the reaction mixture was
diluted with CH2Cl2 and extracted with H2O and saturated NaHCO3 solution. The
organic layer was dried over Na2SO4, filtered, and the solvents were removed in vacuo
to yield 11 (1.0 g, quantitative, 2 steps, from 9). TLC (1:1 hexane/EtOAc) Rf α 0.50, Rf
1
β 0.29; Data for the α anomer: H-NMR (300 MHz, CDCl3): δ 7.26 (m, 5H, Ar), 6.92
(br d, 1H, J NH,2 = 9.0 Hz, NH), 5.36-5.27 (m, 2H, H-1 and H-3), 5.03 (pt, 1H, H-4),
13

4.67 (2d, 2H, CH2(Bn)), 4.13 (m, 2H, H-2 and H-5), 3.47 (m, 2H, H-6a and H-6b), 2.72-
2.32 (m, 4H, OCO(CH2)2), 2.08 (s, 3H, COCH3), 1.95 (COCH3) ppm; ESI-MS: m/z:
calcd for C22H26Cl3NNaO9: 576.1; found: 576.1 [M+Na]+.
Compound 11 (527 mg, 0.95 mmol) was dissolved in dry CH2Cl2 (10 mL) and Cl3CCN
(0.95 mL, 9.5 mmol) and DBU (1.4 µL, 9.5 µmol) were added. After stirring at room
temperature for 1 h, the mixture was concentrated in vacuo. Flash chromatography on
silica gel (hexane/EtOAc 3:1 + 1% Et3N) afforded 4 (463 mg, 70%) as an α/β mixture.
TLC (2:1 hexane/EtOAc) Rf 0.45; Data for the α anomer: 1H-NMR (300 MHz, CDCl3):
δ 8.74 (s, 1H, NH), 7.26 (m, 5H, Ar), 6.97 (br d, 1H, J NH,2 = 8.2 Hz, NH), 6.46 (d, 1H, J
1,2 = 3.5 Hz, H-1), 5.44-5.32 (m, 2H, H-3 and H-4), 4.48 (2d, 2H, CH2(Bn)), 4.36 (m,
1H, H-2), 4.07 (m, 1H, H-5), 3.53 (m, 2H, H-6a and H-6b), 2.78-2.36 (m, 4H,
OCO(CH2)2), 2.11 (s, 3H, COCH3), 1.98 (s, 3H, COCH3) ppm; HR MS: m/z: calcd for
C24H26Cl6N2NaO9: 718.9667; found: 718.9669 [M+Na]+.
Benzyl [4-Methoxyphenyl 2-O-benzoyl-3-O-benzyl-4-O-(4-O-acetyl-6-O-benzyl-2-
deoxy-3-O-levulinoyl-2-trichloroacetamido-β-D-glucopyranosyl)-β-D-
glucopyranoside]uronate (12): Donor 4 (3.73 g, 5.34 mmol) and acceptor 5 (2.4 g, 4.1
mmol) were coevaporated with toluene, dried under vacuum, and dissolved in dry
CH2Cl2 (90 mL) in the presence of freshly activated 4Å molecular sieves. After stirring
for 10 min, TMSOTf (199 µL, 1.1 mmol) was added under an argon atmosphere at 0 ºC.
After stirring for 10 min at 0 ºC, the reaction mixture was neutralized with Et3N,
filtered, and concentrated to dryness. The residue was purified by column
chromatography (toluene/acetone 14:1) to afford 12 (3.31 g, 72%). TLC (hexane/EtOAc
3:2) Rf 0.38; [α] 20 +14.1º (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3): δ 7.93 (m, 2H,
D
Ar), 7.56 (m, 1H, Ar), 7.45-7.37 (m, 6H, Ar), 7.33 (m, 2H, Ar), 7.26 (m, 4H, Ar), 7.16
(m, 2H, Ar), 7.05 (m, 3H, Ar), 6.87 (m, 3H, NH and Ar), 6.69 (m, 2H, Ar), 5.44 (dd,
14

1H, J1,2 = 7.1 Hz, J2,3 = 9.0 Hz, H-2A), 5.27, 5.10 (2d, 2H, Jgem = 12.0 Hz, CH2(Bn)),
5.06 (t, 1H, J3,4 = J4,5 = 9.5 Hz, H-4B), 4.99 (d, 1H, J1,2 = 7.1 Hz, H-1A), 4.93 (t, 1H,
H-3B), 4.90, 4.63 (2d, 2H, Jgem = 11.7 Hz, CH2(Bn)), 4.41 (d, 1H, J1,2 = 8.4 Hz, H-1B),
4.32 (2d, 2H, CH2(Bn)), 4.21 (t, 1H, J3,4 = J4,5 = 9.1 Hz, H-4A), 4.06-4.00 (m, 2H, H-
5A and H-2B), 3.82 (pt, 1H, H-3A), 3.72 (s, 3H, OCH3), 3.47 (m, 1H, H-6B), 3.33-3.26
(m, 2H, H-5B and H-6B), 2.71-2.42 (m, 4H, OCO(CH2)2), 2.15 (s, 3H, COCH3), 1.97
(s, 3H, COCH3) ppm; 13C-NMR (125 MHz, CDCl3): δ 206.1 (CO(Lev)), 172.3, 169.8,
168.9, 165.1, 162.1 (CO), 155.8, 151.0 (Ar-C), 138.1-114.5 (Ar-C, Ar-CH), 101.0 (C-
1A), 100.1 (C-1B), 92.5 (CCl3), 79.6 (C-3A), 78.1 (C-4A), 75.3 (CH2(Bn)), 74.5 (C-
5A), 73.7 (C-5B), 73.6 (CH2(Bn)), 73.0 (C-2A), 72.8 (C-3B), 69.1 (C-6B), 68.8 (C-4B),
68.0 (CH2(Bn)), 56.0 (C-2B), 55.7 (OCH3), 37.8 (OCO(CH2)2), 29.7 (COCH3), 28.1
(OCO(CH2)2), 20.8 (COCH3); HR MS: m/z: calcd for C56H56NO17NaCl3: 1142.2512;
found: 1142.2543 [M+Na]+.
Benzyl [4-Methoxyphenyl 2-O-benzoyl-3-O-benzyl-4-O-(4-O-acetyl-6-O-benzyl-2-
deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-β-D-glucopyranoside]uronate
(13): Compound 12 (200 mg, 0.18 mmol) was dissolved in CH2Cl2 (2.5 mL) and
hydrazine monohydrate (340 µL of a 1 M solution in Py/AcOH 3:2) was added. After
stirring at room temperature for 2 h, the reaction mixture was quenched with acetone
(0.1 mL), diluted with EtOAc and washed with 1N HCl (aq.), saturated NaHCO3 sol.,
and H2O. The organic layer was dried over MgSO4, filtered and concentrated in vacuo.
The residue was purified by column chromatography (toluene/acetone 7:1) to yield 13
(170 mg, 93%). TLC (3:2 hexane/EtOAc) Rf 0.30; [α] 20 +16.6 º (c 1.0, CHCl3); H-
1
D
NMR (500 MHz, CDCl3): δ 7.94 (m, 2H, Ar), 7.57 (m, 1H, Ar), 7.43-7.37 (m, 7H, Ar
and NH), 7.33 (m, 2H, Ar), 7.26 (m, 4H, Ar), 7.15 (m, 2H, Ar), 7.05 (m, 3H, Ar), 6.88
(m, 2H, Ar), 6.70 (m, 2H, Ar), 5.45 (dd, 1H, J1,2 = 7.2 Hz, J2,3 = 9.1 Hz, H-2A), 5.28,
15

5.15 (2d, 2H, Jgem = 12.2 Hz, CH2(Bn)), 5.01 (d, 1H, J
= 7.2 Hz, H-1A), 4.93-4.89
1,2
(m, 2H, H-4B and CH2(Bn)), 4.62 (d, 1H, Jgem = 11.8 Hz, CH2(Bn)), 4.38 (d, 1H, J1,2
=
8.3 Hz, H-1B), 4.31 (s, 2H, CH2(Bn)), 4.17 (pt, 1H, H-4A), 4.08 (d, 1H, J4,5 = 9.5 Hz,
H-5A), 3.85-3.80 (m, 2H, H-3A and H-2B), 3.73 (s, 3H, OCH3), 3.50-3.46 (m, 2H, H-
3B and H-6B), 3.33 (m, 1H, H-6B), 3.30 (m, 1H, H-5B), 2.00 (s, 3H, COCH3) ppm;
13C-NMR (125 MHz, CDCl3): δ 170.7, 169.3, 165.1, 163.6 (CO), 155.8, 151.0 (Ar-C),
138.0-114.5 (Ar-C, Ar-CH), 100.9 (C-1A), 99.7 (C-1B), 92.6 (CCl3), 79.7 (C-3A), 78.1
(C-4A), 75.2 (CH2(Bn)), 74.4 (C-5A), 74.3 (C-3B), 73.7 (C-5B), 73.6 (CH2(Bn)), 72.9
(C-2A), 72.2 (C-4B), 69.1 (C-6B), 68.0 (CH2(Bn)), 58.7 (C-2B), 55.6 (OCH3), 20.9
(COCH3); HR MS: m/z: calcd for C51H50NO15NaCl3: 1044.2144; found: 1044.2161
[M+Na]+.
4-Methoxyphenyl
O-(Benzyl
2-O-benzoyl-3-O-benzyl-4-O-levulinoyl-β-D-
glucopyranosyluronate)-(1→3)-O-(4-O-acetyl-6-O-benzyl-2-deoxy-2-
trichloroacetamido-β-D-glucopyranosyl)-(1→4)-benzyl 2-O-benzoyl-3-O-benzyl-β-
D-glucopyranosiduronate (14): Donor 3 (275 mg, 0.38 mmol) and aceptor 13 (300 mg,
0.29 mmol) were coevaporated with toluene, dried under vacuum, and dissolved in dry
CH2Cl2 (4 mL) in the presence of activated 4Å molecular sieves. The reaction mixture
was stirred, under an argon atmosphere, for 10 min and TMSOTf (14 µL, 76 µmol) was
then added. After stirring for 15 min, the reaction mixture was neutralized with Et3N,
filtered, and concentrated to dryness. The residue was purified by column
chromatography (toluene/acetone 10:1) to yield 14 (192 mg, 41%) and starting acceptor
(56 mg, 18%). TLC (toluene/acetone 7:1) Rf 0.28; [α] 20 +15.6º (c 1.0, CHCl3); H-
1
D
NMR (500 MHz, CDCl3): δ 8.04 (m, 2H, Ar), 7.93 (m, 2H, Ar), 7.54 (m, 2H, Ar), 7.44-
7.24 (m, 17H, Ar), 7.14-7.02 (m, 12H, Ar), 6.90 (br d, 1H, J2,NH = 7.8 Hz, NH), 6.86 (m,
2H, Ar), 6.68 (m, 2H, Ar), 5.41 (dd, 1H, J1,2 = 6.9 Hz, J2,3 = 8.4 Hz, H-2A), 5.28-5-22
16

(m, 2H, H-2C and H-4C), 5.20-5.08 (m, 4H, 2CH2(Bn)), 5.00 (d, 1H, J1,2 = 6.8 Hz, H-
1A), 4.90 (t, 1H, J3,4 = J4,5 = 9.4 Hz, H-4B), 4.84-4.80 (m, 2H, H-1B and H-1C), 4.75
(d, 1H, Jgem = 11.6 Hz, CH2(Bn)), 4.59-4.53 (m, 3H, CH2(Bn)), 4.36-4.26 (m, 3H, H-4A
and CH2(Bn)), 4.18 (pt, 1H, H-3B), 4.02 (d, 1H, J4,5 = 8.8 Hz, H-5A), 4.00 (d, 1H, J4,5
=
10.0 Hz, H-5C), 3.80-3.73 (m, 2H, H-3A and H-3C), 3.72 (s, 3H, OCH3), 3.55 (m, 1H,
H-2B), 3.44 (dd, 1H, J5,6b= 3.0 Hz, J6a,6b= 10.7 Hz, H-6bB), 3.37 (m, 1H, H-5B), 3.26
(dd, 1H, J5,6a= 5.4 Hz, J6a,6b= 10.7 Hz, H-6aB), 2.55-2.19 (m, 4H, OCO(CH2)2), 2.10 (s,
3H, COCH3), 1.78 (s, 3H, COCH3) ppm; 13C-NMR (125 MHz, CDCl3): δ
206.0(CO(Lev)), 171.4 (CO(Lev)), 169.9, 168.5, 167.1, 165.2, 164.8, 161.5 (CO),
155.7, 151.1 (Ar-C), 138.1-114.5 (Ar-C, Ar-CH), 100.9 (C-1A), 99.1 (C-1C), 98.6 (C-
1B), 92.8 (CCl3), 79.4 (C-3A), 79.1 (C-3C), 76.6 (C-4A), 75.9 (C-3B), 74.8, 74.6
(CH2(Bn) and C-5A), 74.1, 73.9, 73.6 (2CH2(Bn) and C-5B), 73.1 (C-2A), 72.8, 72.7
(C-2C and C-5C), 71.3 (C-4C), 69.3 (C-6B), 68.7 ((C-4B), 68.1 (CH2(Bn)), 67.8
(CH2(Bn)), 58.5 (C-2B), 55.7 (OCH3), 37.7 (OCO(CH2)2), 29.9 (COCH3), 27.8
(OCO(CH2)2), 20.6 (COCH3); HR MS: m/z: calcd for C83H80NO24NaCl3: 1602.4034;
found: 1602.4025 [M+Na]+.
→3)
-O-(2-acetamido-2-
4-Methoxyphenyl O-(β-D-glucopyranosyluronic acid)-(1
→4)
deoxy-β-D-glucopyranosyl)-(1
-β-D-glucopyranosiduronic acid (16): H2O2 (30%,
0.31 mL) and a solution of LiOH (0.7 M, 0.19 mL) were added at -5 ºC to a solution of
14 (11.6 mg, 7.3 µmol) in THF (0.83 mL). After stirring for 24 h at room temperature,
MeOH (0.83 mL) and a solution of NaOH (4 M, 0.18 mL) were added. After stirring for
72 h at room temperature, the reaction was neutralized with Amberlite IR-120, filtered
and concentrated. The residue was dissolved in MeOH (1 mL) and Et3N (4 µL) and
Ac2O (5µL) were added at 0 ºC. After stirring for 1 h at room temperature, the reaction
was coevaporated with toluene and methanol. The residue was dissolved in MeOH (1
17

mL) and NaOH (0.25 M, 0.2 mL) was added. After stirring for 12 h the reaction was
neutralized with Amberlite IR-120 and filtered. Et3N (0.3 mL) was added and the
reaction mixture was purified by Sephadex LH-20 chromatography (CH2Cl2-MeOH
1:1). The residue was converted into the sodium salt by elution from a column of
Dowex 50WX4-Na+ with MeOH-H2O 9:1 to give 15 (6.5 mg, 87%).
Alternative synthesis of intermediate 15 from fluorous-tagged 23: H2O2 (30%, 0.3 mL)
and a solution of LiOH (0.7 M, 0.19 mL) were added at -5 ºC to a solution of 23 (15
mg, 7.7 µmol) in THF (1.2 mL). After stirring for 24 h at room temperature, MeOH (1
mL) and a solution of NaOH (4 M, 385 µL) were added. After stirring for 4 days at
room temperature, the reaction was neutralized with Amberlite IR-120, filtered and
concentrated. The residue was dissolved in MeOH (2.1 mL) and Et3N (14 µL) and Ac2O
(14 µL) were added at 0 ºC. After stirring for 1 h at room temperature, the reaction was
coevaporated with toluene and methanol. Et3N (0.3 mL) was added and the reaction
mixture was purified by Sephadex LH-20 chromatography (CH2Cl2-MeOH 1:1). The
residue was converted into the sodium salt by elution from a column of Dowex 50WX4-
Na+ with MeOH-H2O 4:1 to give 15 (6.9 mg, 90%). TLC (EtOAc:MeOH:H2O 20:5:3)
Rf 0.35; 1H-NMR (500 MHz, MeOD): δ 7.44 (m, 4H, Ar), 7.32-7.17 (m, 11H, Ar), 7.03
(m, 2H, Ar), 6.81 (m, 2H, Ar), 5.07 (d, Jgem = 11.2 Hz, 1H, CH2(Bn)), 4.89 (m, 2H,
CH2(Bn)), 4.81 (d, J1,2 = 7.8 Hz, 1H, H-1A), 4.76 (d, Jgem = 11.2 Hz, 1H, CH2(Bn)),
4.69 (d, J1,2 = 8.5 Hz, 1H, H-1B), 4.52 (d, Jgem = 11.9 Hz, 1H, CH2(Bn)), 4.43 (d, J1,2
=
7.4 Hz, 1H, H-1C), 4.36 (d, Jgem = 11.9 Hz, 1H, CH2(Bn)), 4.05 (pt, 1H, H-4A), 3.89
(pt, 1H, H-2B), 3.85 (m, 1H, H-6B), 3.80 (d, J4,5 = 9.7 Hz, 1H, H-5A), 3.74 (s, 3H,
OCH3), 3.69-3.52 (m, 6H, H-2A, H-3B, H-6B, H-5C, H-4C and H-3A), 3.48 (m, 2H, H-
5B and H-4B), 3.44-3-38 (m, 2H, H-2C and H-3C), 2.07 (s, 3H, NHCOCH3) ppm;
13C-NMR (125 MHz, MeOD) (selected data from HSQC experiment): δ 151.5-115.0
18

(Ar), 104.6 (C-1C), 102.9 (C-1A), 100.8 (C-1B), 85.1 (C-2C or C-3C), 84.8 (C-3B or
C-5C), 83.7 (C-3A), 79.7 (C-4A), 78.0 (C-5A), 77.0 (C-4B or C-5B), 75.1 (CH2(Bn)),
75.0 (CH2(Bn)), 74.9 (C-3B or C-5C), 74.2 (CH2(Bn)), 74.2 (C-2C or C-3C), 73.6 (C-
2A), 73.0 (C-4C), 70.7 (C-4B or C-5B), 70.5 (C-6B), 56.2 (C-2B), 55.7 (OCH3), 23.7
(NHCOCH3); HR MS: m/z: calcd for C48H53NO19: 473.6611; found: 473.6622 [M]2-.
A solution of 15 (6.5 mg, 6.5 µmol, as sodium salt) in H2O/MeOH (3.6 mL/0.4 mL) was
hydrogenated at 1 bar pressure in the presence of Pd(OH)2 (5 mg). After 24 h, the
suspension was filtered over Celite and concentrated. The residue was purified by
Sephadex G-10 chromatography (H2O-MeOH 9:1) to give 16 after lyophilisation (4.6
1
mg, quantitative). H-NMR (500 MHz, D2O): δ 7.10 (m, 2H, Ar), 6.98 (m, 2H, Ar),
5.00 (d, 1H, J1,2 = 7.8 Hz, H-1A), 4.60 (d, 1H, J1,2 = 8.5 Hz, H-1B), 4.47 (d, 1H, J1,2
=
7.9 Hz, H-1C), 3.94 (dd, 1H, J5,6a = 2.0 Hz, J6a,6b = 12.7 Hz, H-6B), 3.89-3.78 (m, 7H,
H-2B, H-4A, H-5A, OCH3 and H-6B), 3.75-3.68 (m, 3H, H-3B, H-5C and H-3A), 3.61
(dd, J1,2 = 7.9 Hz, J2,3 = 9.5 Hz, 1H, H-2A), 3.56 (pt, 1H, H-4B), 3.50 (m, 3H, H-3C, H-
13
4C and H-5B), 3.33 (m, 1H, H-2C), 2.04 (s, 3H, NHCOCH3) ppm; C-NMR (125
MHz, D2O) (selected data from HSQC experiment): δ 118.1, 115.0 (Ar), 102.9 (C-1C),
101.3 (C-1A), 100.5 (C-1B),82.9 (C-3B or C5C), 79.8 (C-4A or C-5A), 76.6 (C-4A or
C-5A), 75.6 (C-3B or C5C), 75.3 (C-5B or C-3C or C-4C), 73.6 (C-3A), 72.7 (C-2C),
72.5 (C-2A), 71.6 (C-5B or C-3C or C-4C), 68.5 (C-4B), 60.5 (C-6B), 55.7 (OCH3),
54.2 (C-2B), 22.4 (NHCOCH3); HR MS: m/z: calcd for C27H35NO19: 338.5907; found:
338.5904 [M]2-.
4-Methoxyphenyl O-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate)-
(1→3)-O-(4-O-acetyl-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-
→4)
glucopyranosyl)-(1
-benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosiduronate
(17): Compound 14 (124 mg, 0.08 mmol) was dissolved in CH2Cl2 (2.0 mL) and
19

hydrazine monohydrate (152 µL of a 1 M solution in Py/AcOH 3:2) was added. After
stirring at room temperature for 2 h, the reaction mixture was quenched with acetone
(0.1 mL). The mixture was diluted with EtOAc and washed with 1N HCl (aq.),
saturated NaHCO3 sol., and H2O. The organic layer was dried (MgSO4), filtered and
concentrated in vacuo. The residue was purified by column chromatography
(toluene/acetone 7:1) to yield 17 (82 mg, 71%). TLC (toluene/acetone 6:1) Rf 0.28;
[α] 2D0 +6.9º (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3): δ 8.04 (m, 2H, Ar), 7.94 (m,
2H, Ar), 7.56 (m, 2H, Ar), 7.44-7.24 (m, 17H, Ar), 7.19-7.01 (m, 12H, Ar), 6.86 (m,
2H, Ar), 6.81 (d, 1H, J2,NH = 8.0 Hz, NH), 6.68 (m, 2H, Ar), 5.41 (dd, 1H, J1,2 = 7.0 Hz,
J2,3 = 8.4 Hz, H-2A), 5.27 (2d, 2H, CH2(Bn)), 5.17 (m, 2H, H-2C and CH2(Bn)), 5.10
(d, 1H, Jgem = 12.1 Hz, CH2(Bn)), 5.00 (d, 1H, J1,2 = 6.9 Hz, H-1A), 4.88 (pt, 1H, H-
4B), 4.81 (d, 1H, H-1B), 4.77-4.74 (m, 2H, H-1C and CH2(Bn)), 4.68 (s, 2H, CH2(Bn)),
4.53 (d, 1H, Jgem = 11.7 Hz, CH2(Bn)), 4.36-4.26 (m, 3H, H-4A and CH2(Bn)), 4.13 (pt,
1H, H-3B), 4.03-4.00 (m, 2H, H-5A and H-4C), 3.92 (d, 1H, J4,5 = 9.9 Hz, H-5C), 3.76
(pt, 1H, H-3A), 3.72 (s, 3H, OCH3), 3.64-3.55 (m, 2H, H-2B and H-3C), 3.45 (dd, 1H,
J5,6b= 3.0 Hz, J6a,6b= 10.6 Hz, H-6bB), 3.40 (m, 1H, H-5B), 3.27 (dd, 1H, J5,6a= 5.2 Hz,
J6a,6b= 10.6 Hz, H-6aB), 1.75 (s, 3H, COCH3) ppm; 13C-NMR (125 MHz, CDCl3): δ
169.7, 168.6, 168.4, 165.2, 165.1, 161.4 (CO), 155.7, 151.0 (Ar-C), 138.1-114.5 (Ar-C,
Ar-CH), 100.9 (C-1A), 99.8 (C-1C), 98.7 (C-1B), 92.8 (CCl3), 80.9 (C-3C), 79.4 (C-
3A), 76.7 (C-4A), 76.2 (C-3B), 74.8, 74.7, 74.7, 74.6 (2CH2(Bn), C-5C, C-5A), 73.9
(C-5B), 73.6 (CH2(Bn)), 73.1 (C-2A), 72.9 (C-2C), 72.1 (C-4C), 69.4 (C-6B), 69.0 (C-
4B), 67.8 (2 CH2(Bn)), 58.3 (C-2B), 55.7 (OCH3), 20.6 (COCH3); HR MS: m/z: calcd
for C78H74NO22NaCl3: 1504.3666; found: 1504.3644 [M+Na]+.
4-Methoxyphenyl
O-(4-O-acetyl-6-O-benzyl-2-deoxy-3-O-levulinoyl-2-
trichloroacetamido-β-D-glucopyranosyl)-(1→4)-O-(Benzyl
2-O-benzoyl-3-O-
20

benzyl-β-D-glucopyranosyluronate)-(1→3)-O-(4-O-acetyl-6-O-benzyl-2-deoxy-2-
trichloroacetamido-β-D-glucopyranosyl)-(1→4)-benzyl 2-O-benzoyl-3-O-benzyl-β-
D-glucopyranosyluronate (2): Donor 4 (56 mg, 0.08 mmol) and aceptor 17 (80 mg,
0.05 mmol) were coevaporated with toluene, dried under vucuum, and dissolved in dry
CH2Cl2 (2 mL) in the presence of freshly activated 4Å molecular sieves. After stirring
for 10 min at 0 ºC, TMSOTf (100 µL of a 0.16 M solution in dry CH2Cl2) was added
under an argon atmosphere. After stirring for 10 min at 0 ºC, the reaction mixture was
neutralized with Et3N, filtered, and concentrated to dryness. The residue was purified by
column chromatography (toluene/EtOAc 4:1) to afford 2 (69 mg, 64%). TLC (4:1
1
toluene/EtOAc) Rf 0.30; [α] 20 +6.5º (c 1.0, CHCl3); H-NMR (500 MHz, CDCl3): δ
D
7.94 (m, 4H, Ar), 7.54 (m, 2H, Ar), 7.48-7.22 (m, 25H, Ar), 7.12 (m, 2H, Ar), 7.08-7.02
(m, 7H, Ar), 6.85 (m, 2H, Ar), 6.83 (br d, 1H, JNH,2 = 9.2 Hz, NHD), 6.74 (br d, 1H,
JNH,2 = 8.1 Hz, NHB), 6.67 (m, 2H, Ar), 5.42-5.37 (m, 2H, H-2A and CH2(Bn)), 5.16-
5.08 (m, 4H, H-2C and CH2(Bn)), 5.02-4.98 (m, 2H, H-1A and H-4D), 4.89-4.82 (m,
3H, H-1B, H-4B and H-3D), 4.79-4.72 (m, 3H, H-1C and CH2(Bn)), 4.54 (d, 1H, Jgem
=
11.5 Hz, CH2(Bn)), 4-35-4.25 (m, 5H, H-4A and 2CH2(Bn)), 4.18 (d, 1H, J1,2 = 8.5 Hz,
H-1D), 4.10-4.03 (m, 2H, H-3B and H-4C), 4.01 (d, 1H, J4,5 = 8.9 Hz, H-5A), 3.98-3.92
(m, 2H, H-2D and H-5C), 3.76 (t, 1H, J2,3 = J3,4 = 8.5 Hz, H-3A), 3.71 (s, 3H, OCH3),
3.64-3.57 (m, 2H, H-3C and H-2B), 3.46-3.38 (m, H-5B and 2H6), 3.26 (m, 2H, 2H6),
3.15 (m, 1H, H-5D), 2-76-2.42 (m, 4H, OCO(CH2)2), 2.15 (s, 3H, COCH3), 1.98 (s, 3H,
COCH3), 1.78 (s, 3H, COCH3) ppm; 13C-NMR (125 MHz, CDCl3): δ 206.2 (CO(Lev)),
172.3 (CO(Lev)), 169.8, 169.5, 168.7, 168.5, 165.2, 165.1, 162.3, 161.6 (CO), 155.7,
151.0 (Ar-C), 138.1-114.5 (Ar-C and Ar-CH), 100.9 (C-1A), 99.9, 99.8 (C-1C and C-
1D), 99.0 (C-1B), 92.7, 92.5 (2CCl3), 79.5 (C-3A), 79.4 (C-3C), 77.8 (C-4C), 77.4, 76.4
(C-3B and C-4A), 75.0, 74.7 (x3) (2CH2(Bn), C-5A and C-5C), 73.9, 73.7, 73.6 (x2)
21

(C-5D, C-5B and 2 CH2(Bn)), 73.1 (C-2A), 72.8 (C-2C), 72.6 (C-3D), 69.6, 69.4, 69.0,
68.8 (C-6B, C-6D, C-4B and C4D), 68.0 (CH2(Bn)), 67.8 (CH2(Bn)), 58.1 (C-2B), 56.0
(C-2D), 55.7 (OCH3), 37.8 (OCO(CH2)2), 29.8 (COCH3), 28.1 (OCO(CH2)2), 20.8, 20.6
(COCH3); HR MS: m/z: calcd for C100H106N4O30Cl6: 1028.2501; found: 1028.2558
[M+2NH4]2+.
4-Methoxyphenyl
O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-O-(β-D-
glucopyranosyluronic acid)-(1→3)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-
(1→4)-β-D-glucopyranosiduronic acid (1): H2O2 (30%, 0.42 mL) and a solution of
LiOH (0.7 M, 0.25 mL) were added at -5 ºC to a solution of 2 (20 mg, 10 µmol) in THF
(1 mL). After stirring for 24 h at room temperature, MeOH (1 mL) and a solution of
NaOH (4 M, 0.25 mL) were added. After stirring for 5 days at room temperature, the
reaction was neutralized with Amberlite IR-120, filtered and concentrated. The residue
was dissolved in 1 mL of MeOH and Et3N (5 µL) and Ac2O (6 µL) were added at 0 ºC.
After stirring for 2 h at room temperature, the reaction was coevaporated with toluene
and methanol. The residue was dissolved in MeOH (1 mL) and NaOH (0.3 M, 0.2 mL)
was added. After stirring for 12 h the reaction was neutralized with Amberlite IR-120
and filtered. Et3N (0.3 mL) was added and the reaction mixture was purified by
Sephadex LH-20 chromatography (CH2Cl2-MeOH 1:1). The residue was converted into
the sodium salt by elution from a column of Dowex 50WX4-Na+ with MeOH-H2O 9:1
to give 18 (8 mg, 64%). TLC (EtOAc:MeOH:H2O 20:5:3) Rf 0.25; 1H-NMR (500 MHz,
MeOD): δ 7.45 (m, 4H, Ar), 7.27-7.17 (m, 16H, Ar), 7.03 (m, 2H, Ar), 6.81 (m, 2H,
Ar), 5.04 (m, 2H, CH2(Bn)), 4.80 (d, J1,2 = 7.9 Hz, 1H, H-1A), 4.74 (2d, Jgem = 10.6 Hz,
2H, CH2(Bn)), 4.68 (d, J1,2 = 8.6 Hz, 1H, H-1B), 4.62 (d, J1,2 = 8.6 Hz, 1H, H-1D), 4.50
(2d, Jgem = 10.6 Hz, 2H, CH2(Bn)), 4.43 (d, J1,2 = 7.2 Hz, 1H, H-1C), 4.34 (2d, Jgem
=
12.0 Hz, 2H, CH2(Bn)), 4.05 (pt, 1H, H-4A), 3.93 (pt, 1H, H-4C), 3.87 (dd, J2,3= 10.1
22

Hz, J1,2 = 8.6 Hz, 1H, H-2B), 3.84-3.72 (m, 7H, H-6B, H-6D, H-5A, H-5C and OCH3),
3.67-3.51(m, 6H, H-6B, H-6D, H-2A, H-2D, H-3B and H-3A), 3.49-3.43 (m, 4H, H-4B,
H-5B, H-2C and H-3C), 3.39-3.36 (m, 3H, H-3D, H-4D and H-5D), 2.06 (2s, 6H,
NHCOCH3) ppm; 13C-NMR (125 MHz, MeOD) (selected data from HSQC
experiment): δ 152.8-115.1 (Ar), 104.3 (C-1C), 103.1 (C-1A), 100.8 (C-1B and C-1D),
85.2 (C-3B), 83.7 (C-3A), 83.3 (C-4B or C-5B or C-2C or C-3C), 79.7 (C-4A), 79.4 (C-
4C), 78.2 (C-5A or C-5C), 78.0 (C-3D or C-4D or C-5D), 77.5 (C-3D or C-4D or C-
5D), 76.9 (C-4B or C-5B or C-2C or C-3C), 76.9 (C-5A or C-5C), 75.2 (2xCH2(Bn)),
74.5 (CH2(Bn)), 74.3 (CH2(Bn)), 73.9 (C-2A), 73.8 (C-4B or C-5B or C-2C or C-3C),
72.1 (C-3D or C-4D or C-5D), 70.6 (C-6B and C-6D), 70.5 (C-4B or C-5B or C-2C or
C-3C), 57.8 (C-2D), 56.2 (C-2B), 55.8 (OCH3), 23.1 (2xNHCOCH3); HR MS: m/z:
calcd for C63H72N2O24: 620.2243; found: 620.2280 [M]2-.
A solution of 18 (8.0 mg, 6.2 µmol, as sodium salt) in H2O/MeOH (4.5 mL/0.5 mL) was
hydrogenated at 2 bar pressure in the presence of Pd(OH)2 (17 mg). After 48h, the
suspension was filtered over Celite and concentrated. The residue was purified by
Sephadex G-10 chromatography (H2O-MeOH 9:1) to give 1 after lyophilisation (5.6
1
mg, quantitative). H-NMR (500 MHz, D2O): δ 7.09 (m, 2H, Ar), 6.97 (m, 2H, Ar),
4.99 (d, J1,2 = 7.9 Hz, 1H, H-1A), 4.57 (d, J1,2 = 8.4 Hz, 1H, H-1B), 4.52 (d, J1,2 = 8.3
Hz, 1H, H-1D), 4.46 (d, J1,2 = 7.9 Hz, 1H, H-1C), 3.92 (m, 2H, H-6B and H-6D), 3.87-
3.65 (m, 13H, H-2B, H-4A, H-5A, H-6B, H-6D, OCH3, H-4C, H-5C, H-3B, H-2D and
H-3A), 3.61-3.56 (m, 2H, H-2A and H-3C), 3.55-3.44 (m, 5H, H-4B, H-3D, H-4D, H-
5D and H-5B), 3.34 (pt, 1H, H-2C), 2.04 (s, 3H, NHCOCH3), 2.02 (s, 3H, NHCOCH3)
ppm; 13C-NMR (125 MHz, D2O) (selected data from HSQC experiment): δ 118.0, 114.9
(Ar), 103.0 (C-1C), 101.2 (C-1A), 100.6 (C-1D), 100.4 (C-1B), 82.4 (C-3B or C-4C or
C-5C), 79.8 (C-4A or C-5A), 79.6 (C-3B or C-4C or C-5C), 76.5 (C-4A or C-5A), 76.1
23

(C-3B or C-4C or C-5C), 75.4 (C-4B or C-5B or C-3D or C-4D or C-5D), 75.2 (C-4B or
C-5B or C-3D or C-4D or C-5D), 73.8 (C-4B or C-5B or C-3D or C-4D or C-5D), 73.5
(C-3C), 73.6 (C-3A), 72.5 (C-2A), 72.4 (C-2C), 69.4 (C-4B or C-5B or C-3D or C-4D
or C-5D), 68.4 (C-4B or C-5B or C-3D or C-4D or C-5D), 60.4 (C-6B and C-6D), 55.6
(OCH3), 55.2 (C-2D), 54.2 (C-2B), 22.5 (2xNHCOCH3); HR MS: m/z: calcd for
C35H48N2O24: 440.1304; found: 440.1323 [M]2-.
4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-Heptadecafluoroundecyl
(4-methoxyphenyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranoside) uronate (20):
TEMPO (0.39 mL of a 0.016 M solution in CH2Cl2), Bu4NBr (0.13 mL of a 0.08 M
solution in CH2Cl2) and KBr (40 µL of a 0.5 M solution in H2O) were added dropwise
at 0ºC to a solution of diol 19 (100 mg, 0.21 mmol) in CH2Cl2 (2.1 mL). A solution of
Ca(ClO)2 (74 mg, 0.52 mmol) and NaHCO3 (75 mg, 0.9 mmol) in H2O (2.0 mL) was
then added dropwise at 0ºC. After stirring for 45 min at 0ºC, the reaction was quenched
by adding Na2SO3 (1.3 mL of a 0.8 M solution in H2O). After stirring for 15 min at 0ºC,
the reaction mixture was diluted with additional CH2Cl2 and H2O, and the organic layer
was then separated, washed with a solution of Na2SO3 (0.8 M) and brine, dried
(MgSO4), filtered and concentrated. The residue was dissolved in DMF (3 mL) and
4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl iodide (245 mg, 0.42
mmol) was added. The mixture was stirred for 3 h at 60 ºC, diluted with EtOAc, washed
with H2O, dried (MgSO4) and concentrated. Flash chromatography (toluene/EtOAc,
12:1) gave 20 (110 mg, 55%). TLC (6:1 toluene/EtOAc) Rf 0.39; [α] 20 –1.7º (c 1.0,
D
CH2Cl2); 1H-NMR (300 MHz, CDCl3): δ 8.03 (m, 2H, Ar), 7.60 (m, 1H, Ar), 7.46 (m,
2H, Ar), 7.19 (m, 5H, Ar), 6.89 (m, 2H, Ar), 6.73 (m, 2H, Ar), 5.49 (dd, J 1,2 = 7.6 Hz, J
2,3 = 9.2 Hz, 1H, H-2), 5.02 (d, 1H, J 1,2 = 7.6 Hz, H-1), 4.78 (s, 2H, CH2(Bn)), 4.30 (m,
2H, CH2(CH2)2C8F17), 4.17 (pt, 1H, H-4), 4.00 (d, J 4,5 = 9.7 Hz, 1H, H-5), 3.79 (pt, 1H,
24

H-3), 3.72 (s, 3H, OCH3), 2.29-1.97 (m, 4H, CH2(CH2)2C8F17) ppm; 13C-NMR (75
MHz, CDCl3): δ 169.0, 165.3 (CO), 156.0-114.5 (Ar), 101.5 (C-1), 80.8 (C-3), 74.7
(CH2(Bn), 74.4 (C-5), 73.1 (C-2), 71.8 (C-4), 64.4 (CH2(CH2)2C8F17), 55.5 (OCH3),
28.1-20.0 (CH2(CH2)2C8F17); HR MS: m/z: calcd for C38H31F17NaO9: 977.1594; found:
977.1627 [M+Na]+.
4-Methoxyphenyl
O-(benzyl
2-O-benzoyl-3-O-benzyl-4-O-levulinoyl-β-D-
glucopyranosyluronate)-(1→3)-O-(4-O-acetyl-6-O-benzyl-2-deoxy-2-
trichloroacetamido-β-D-glucopyranosyl)-(1→4)-
4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl
2-O-benzoyl-3-O-
benzyl-β-D-glucopyranosiduronate (23): Donor 4 (62 mg, 0.09 mmol) and aceptor 20
(65 mg, 0.07 mmol) were coevaporated with toluene and dissolved in dry CH2Cl2 (1
mL) in the presence of activated 4Å MS. The reaction mixture was stirred, under an
argon atmosphere, for 10 min at 0ºC and TMSOTf (3.2 µL, 18 µmol) was added. After
stirring for 15 min at 0ºC, the reaction mixture was quenched with triethylamine,
filtered, and then concentrated under reduced pressure. The crude product was purified
using a fluorous solid-phase extraction (FSPE) cartridge. Nonfluorous compounds were
eluted with 80:20 MeOH/water and the desired fluorous product was eluted by 100%
MeOH. This MeOH fraction was concentrated and submitted to a second coupling cycle
with additional donor 4 (16 mg, 0.022 mmol). The mixture was dissolved in dry CH2Cl2
(1 mL) in the presence of activated 4Å MS and then stirred, under an argon atmosphere,
for 10 min at 0 ºC. TMSOTf (0.82 µL, 4.5 µmol) was added. After stirring for 15 min at
0ºC, the reaction mixture was neutralized with Et3N, filtered, and concentrated to
dryness. The reaction mixture was purified by using a FSPE column as described above.
The methanolic fraction was concentrated to give disaccharide 21 and a small amount of
unreacted aceptor 20. Data for disaccharide 21: TLC (6:1 toluene/EtOAc) Rf 0.29;
25

1
Selected H NMR data (500 MHz, CDCl3): δ 5.41 (dd, 1H, H-2A), 5.18 (pt, 1H, H-3B),
5.12 (pt, 1H, H-4B), 5.09 (d, 1H, H-1A), 5.03 (d, 1H, H-1B), 4.93, 4.67 (2d, 2H,
CH2(Bn)), 4.34-4.27 (m, 3H, H-4A and CH2(Bn)), 4.18-4.07 (m, 4H, H-2B, H-5A and
COOCH2), 3.88 (pt, 1H, H-3A), 3.71 (s, 3H, OCH3), 3.67 (m, 1H, H-5B), 3.51 (dd, 1H,
H6B), 3.35 (dd, 1H, H6B), 2.76-2-43 (m, 4H, OCO(CH2)2) ppm; ESI MS: m/z: calcd for
C60H55Cl3F17NNaO17: 1514.1; found: 1514.4 [M+Na]+.
The residue containing 21 (75 mg) was dissolved in CH2Cl2 (1.5 mL) and hydrazine
monohydrate (67 µL of a 1 M solution in Py/AcOH 3:2) was added. After stirring at
room temperature for 2 h, the reaction mixture was quenched with acetone. The mixture
was concentrated in vacuo, coevaporated with toluene and loaded on a FSPE column.
1
The methanolic fraction contained acceptor 22. Selected H NMR data for 22 (500
MHz, CDCl3): δ 5.43 (dd, 1H, H-2A), 5.09 (d, 1H, H-1A), 4.95 (m, 3H, H-1B, H-4B
and CH2(Bn)), 4.66 (d, 1H, CH2(Bn)), 4.30-4.11 (m, 6H, H-4A, H-5A, COOCH2 and
CH2(Bn)), 3.87 (m, 2H, H-3A and H-2B), 3.79 (m, 1H, H-3B), 3.71 (s, 3H, OCH3), 3.61
(m, 1H, H-5B), 3.53 (dd, 1H, H-6B), 3.37 (dd, 1H, H-6B) ppm; ESI MS: m/z: calcd for
C55H49Cl3F17NO15: 1416.2; found: 1416.2 [M+Na]+.
The residue containing acceptor 22 (70 mg) and donor 3 (45 mg, 0.062 mmol) were
coevaporated with toluene and dissolved in dry CH2Cl2 (1 mL) in the presence of
activated 4Å MS. The reaction mixture was stirred, under an argon atmosphere, for 10
min at r.t. and TMSOTf (1.1 µL, 6.2 µmol) was added. After stirring for 15 min, the
reaction mixture was neutralized with Et3N, filtered, and concentrated to dryness. The
reaction mixture was loaded on a fluorous solid phase extraction (FSPE) column.
Nonfluorous compounds were eluted with 80% MeOH/water and the desired product
was eluted by 100% MeOH. This MeOH fraction was concentrated and submitted to a
second coupling cycle. The methanolic residue and donor 3 (45 mg, 0.062 mmol) were
26

dissolved in dry CH2Cl2 (1 mL) in the presence of activated 4Å MS. The reaction
mixture was stirred, under an argon atmosphere, for 10 min at r.t. and TMSOTf (1.1 µL,
6.2 µmol) was added. After stirring for 15 min at r.t., the reaction mixture was
neutralized with Et3N, filtered, and concentrated to dryness. The crude mixture was
loaded on a FSPE column. The methanolic eluent was further purified by PLC on silica
gel (6:1 toluene/EtOAc) to give trisaccharide 23 (16 mg, 12% from 20). TLC (4:1
toluene/EtOAc) Rf 0.32; [α] 20 +6.8º (c 1.0, CH2Cl2); 1H-NMR (400 MHz, CDCl3): δ
D
8.03 (m, 2H, Ar), 7.94 (m, 2H, Ar), 7.56 (m, 2H, Ar), 7.46-7.06 (m, 25H, Ar and NH),
6.86 (m, 2H, Ar), 6.72 (m, 2H, Ar), 5.40 (dd, 1H, J1,2 = 6.5 Hz, J2,3 = 8.1 Hz, H-2A),
5.27-5-22 (m, 2H, H-2C and H-4C), 5.16-5.13 (m, 2H, H-1B and CH2(Bn)), 5.10-5.06
(m, 2H, H-1A and CH2(Bn)), 4.97 (pt, 1H, H-4B), 4.85 (d, 1H, J1,2 = 7.5 Hz, H-1C),
4.76 (d, 1H, Jgem = 11.8 Hz, CH2(Bn)), 4.60-4.52 (m, 3H, CH2(Bn)), 4.44-4.29 (m, 4H,
H-4A, H-3B and CH2(Bn)), 4.16 (m, 1H, CH2(CH2)2C8F17), 4.09 (m, 1H,
CH2(CH2)2C8F17), 4.05 (d, 1H, J4,5 = 8.1 Hz, H-5A), 3.99 (d, 1H, J4,5 = 10.0 Hz, H-5C),
3.83 (pt, 1H, H-3A), 3.76 (pt, 1H, H-3C), 3.71 (s, 3H, OCH3), 3.62 (m, 1H, H-5B), 3.49
(m, 2H, H-2B and H-6aB), 3.31 (dd, 1H, J5,6b= 5.4 Hz, J6a,6b= 10.7 Hz, H-6bB), 2.54-
2.28 (m, 4H, OCO(CH2)2), 2.21-2.02 (m, 7H, CH2 (CH2)2C8F17 and COCH3), 1.71
(COCH3) ppm; 13C-NMR (100 MHz, CDCl3): δ 205.9, 171.3, 169.8, 168.6, 167.1,
165.1 164.6, 161.5 (CO), 155.6-114.4 (Ar), 100.5 (C-1A), 98.6 (C-1C), 98.4 (C-1B),
92.6 (CCl3), 79.1 (C-3A or C-3C), 78.9 (C-3A or C-3C), 76.3 (C-4A or C-3B), 75.7 (C-
4A or C-3B), 74.6, 74.3, 74.0, 73.8, 73.6, 73.1, 72.6, 72.5, 71.1 (3 x CH2(Bn), C-5A, C-
5B, C-2A, C-4C, C-5C and C-2C), 69.2 (C-6B), 68.2 (C-4B), 68.0 (CH2(Bn)), 64.5
(CH2(CH2)2C8F17), 58.8 (C-2B), 55.4 (OCH3), 37.5 (OCO(CH2)2), 29.7 (COCH3), 27.6
(OCO(CH2)2), 28.0-27.2 (CH2(CH2)2C8F17), 20.4 (COCH3); HR MS: m/z: calcd for
C87H79Cl3F17NNaO24: 1972.3684; found: 1972.3987 [M+Na]+.
27

Acknowledgements
We thank the Spanish Ministry of Economy and Competitiveness (Grants CTQ2009-
07168 and CTQ2012-32605) and the European Union (FEDER support) for financial
support.
References
1.
Sugahara, K. N.; Murai, T.; Nishinakamura, H.; Kawashima, H.; Saya, H.;
Miyasaka, M. J. Biol. Chem. 2003, 278, 32259-32265.
Stern, R.; Asari, A. A.; Sugahara, K. N. Eur. J. Cell Biol. 2006, 85, 699-715.
Palmacci, E. R.; Seeberger, P. H. Tetrahedron 2004, 60, 7755-7766.
Lu, X. W.; Kamat, M. N.; Huang, L. J.; Huang, X. F. J. Org. Chem. 2009, 74,
7608-7617.
2.
3.
4.
5.
Virlouvet, M.; Gartner, M.; Koroniak, K.; Sleeman, J. P.; Braese, S. Adv. Synth.
Catal. 2010, 352, 2657-2662.
6.
7.
8.
Huang, L. J.; Huang, X. F. Chem. –Eur. J. 2007, 13, 529-540.
Blatter, G.; Jacquinet, J. C. Carbohydr. Res. 1996, 288, 109-125.
Dinkelaar, J.; Codee, J. D. C.; van den Bos, L. J.; Overkleeft, H. S.; van der
Marel, G. A. J. Org. Chem. 2007, 72, 5737-5742.
9.
10.
Rele, S. M.; Iyer, S. S.; Chaikof, E. L. Tetrahedron Lett. 2007, 48, 5055-5060.
Dinkelaar, J.; Gold, H.; Overkleeft, H. S.; Codee, J. D. C.; van der Marel, G. A.
J. Org. Chem. 2009, 74, 4208-4216.
11.
de Paz, J. L.; Mar Kayser, M.; Macchione, G.; Nieto, P. M. Carbohydr. Res.
2010, 345, 565-571.
12.
13.
Mar Kayser, M.; de Paz, J. L.; Nieto, P. M. Eur. J. Org. Chem. 2010, 2138-2147.
Gold, H.; Munneke, S.; Dinkelaar, J.; Overkleeft, H. S.; Aerts, J. M. F. G.;
Codee, J. D. C.; van der Marel, G. A. Carbohydr. Res. 2011, 346, 1467-1478.
Lopez, A. F.; Jacquinet, J.-C.; Lopin-Bon, C. Eur. J. Org. Chem. 2013, 6934-
6947.
Zeng, Y.; Wang, Z.; Whitfield, D.; Huang, X. J. Org. Chem. 2008, 73, 7952-
7962.
de Jong, A.-R.; Hagen, B.; van der Ark, V.; Overkleeft, H. S.; Codee, J. D. C.;
van der Marel, G. A. J. Org. Chem. 2012, 77, 108-125 and references cited
therein.
14.
15.
16.
17.
18.
19.
20.
Dhamale, O. P.; Zong, C.; Al-Mafraji, K.; Boons, G.-J. Org. Biomol. Chem.
2014, 12, 2087-98.
Slaghek, T. M.; Hypponen, T. K.; Ogawa, T.; Kamerling, J. P.; Vliegenthart, J.
F. G. Tetrahedron Lett. 1993, 34, 7939-7942.
Slaghek, T. M.; Nakahara, Y.; Ogawa, T.; Kamerling, J. P.; Vliegenthart, J. F. G.
Carbohydr. Res. 1994, 255, 61-85.
Slaghek, T. M.; Hypponen, T. K.; Ogawa, T.; Kamerling, J. P.; Vliegenthart, J.
F. G. Tetrahedron: Asymmetry 1994, 5, 2291-2301.
28

21.
22.
23.
24.
Halkes, K. M.; Slaghek, T. M.; Hypponen, T. K.; Kruiskamp, P. H.; Ogawa, T.;
Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr. Res. 1998, 309, 161-174.
Yeung, B. K. S.; Hill, D. C.; Janicka, M.; Petillo, P. A. Org. Lett. 2000, 2, 1279-
1282.
Gu, G.; Adabala, P. J. P.; Szczepina, M. G.; Borrelli, S.; Pinto, B. M. J. Org.
Chem. 2013, 78, 8004-8019.
Walvoort, M. T. C.; Volbeda, A. G.; Reintjens, N. R. M.; van den Elst, H.;
Plante, O. J.; Overkleeft, H. S.; van der Marel, G. A.; Codee, J. D. C. Org. Lett.
2012, 14, 3776-3779.
25.
de Jong, A. R.; Volbeda, A. G.; Hagen, B.; van den Elst, H.; Overkleeft, H. S.;
van der Marel, G. A.; Codee, J. D. C. Eur. J. Org. Chem. 2013, 2013, 6644-
6655.
26.
27.
28.
Blatter, G.; Beau, J. M.; Jacquinet, J. C. Carbohydr. Res. 1994, 260, 189-202.
Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem. 1994, 50, 21-123.
Maza, S.; Mar Kayser, M.; Macchione, G.; Lopez-Prados, J.; Angulo, J.; de Paz,
J. L.; Nieto, P. M. Org. Biomol. Chem. 2013, 11, 3510-3525.
Zhang, F.; Zhang, W.; Zhang, Y.; Curran, D. P.; Liu, G. J. Org. Chem. 2009, 74,
2594-2597 and references cited therein.
Cai, C.; Dickinson, D. M.; Li, L.; Masuko, S.; Suflita, M.; Schultz, V.; Nelson,
S. D.; Bhaskar, U.; Liu, J.; Linhardt, R. J. Org. Lett. 2014, 16, 2240-2243.
Galan, M. C.; Benito-Alfonso, D.; Watt, G. M. Org. Biomol. Chem. 2011, 9,
3598-3610 and references cited therein.
29.
30.
31.
32.
33.
34.
35.
Zhang, W.; Curran, D. P. Tetrahedron 2006, 62, 11837-11865.
Jaipuri, F. A.; Pohl, N. L. Org. Biomol. Chem. 2008, 6, 2686-2691.
Manzoni, L.; Castelli, R. Org. Lett. 2004, 6, 4195-4198.
Zong, C.; Venot, A.; Dhamale, O.; Boons, G.-J. Org. Lett. 2013, 15, 342-345
and references cited therein.
36.
Karst, N.; Jacquinet, J. C. Eur. J. Org. Chem. 2002, 815-825.
29