Exploration of the use of an acylsulfonamide safety-catch linker for the polymer-supported synthesis of hyaluronic acid oligosaccharides

Article abstract of DOI:10.1016/j.carres.2009.12.021

The synthesis of hyaluronic acid oligosaccharides on polyethylene glycol (PEG) using an acylsulfonamide linker has been explored. Hyaluronic acid is a challenging synthetic target that usually involves the condensation of highly disarmed glucuronic acid building blocks. Amine-ended PEG monomethyl ether was efficiently functionalized with a hydroxyl-terminated acylsulfonamide linker. Suitably protected d-glucosamine (GlcN) and d-glucuronic acid (GlcA) monosaccharide building blocks were coupled to the polymer acceptor using the trichloroacetimidate glycosylation method. The sulfonamide safety-catch linker enables simultaneous cleavage of the monosaccharide from the polymer and orthogonal functionalization for further (bio)-conjugation of the sugar sample. Subsequent glycosylation of PEG-bound glycosyl acceptor to generate hyaluronic acid oligosaccharide chain failed. Model glycosylation experiments in solution and on soluble support using the same unreactive acceptors and donors allows for the synthesis of an orthogonally protected hyaluronic acid disaccharide and suggest that the encountered difficulties could be attributed to the presence of the N-acylsulfonamide.

Full text of DOI:10.1016/j.carres.2009.12.021

Carbohydrate Research 345 (2010) 565–571
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Exploration of the use of an acylsulfonamide safety-catch linker for the
polymer-supported synthesis of hyaluronic acid oligosaccharides
*
José L. de Paz , M. Mar Kayser, Giuseppe Macchione, Pedro M. Nieto*
Instituto de Investigaciones Químicas, Centro de Investigaciones Científicas Isla de La Cartuja, CSIC –US, Americo Vespucio, 49, 41092 Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 October 2009
Received in revised form 17 December 2009
Accepted 21 December 2009
Available online 4 January 2010
The synthesis of hyaluronic acid oligosaccharides on polyethylene glycol (PEG) using an acylsulfonamide
linker has been explored. Hyaluronic acid is a challenging synthetic target that usually involves the con-
densation of highly disarmed glucuronic acid building blocks. Amine-ended PEG monomethyl ether was
efficiently functionalized with a hydroxyl-terminated acylsulfonamide linker. Suitably protected
D-gluco-
samine (GlcN) and -glucuronic acid (GlcA) monosaccharide building blocks were coupled to the polymer
D
acceptor using the trichloroacetimidate glycosylation method. The sulfonamide safety-catch linker
enables simultaneous cleavage of the monosaccharide from the polymer and orthogonal functionaliza-
tion for further (bio)-conjugation of the sugar sample. Subsequent glycosylation of PEG-bound glycosyl
acceptor to generate hyaluronic acid oligosaccharide chain failed. Model glycosylation experiments in
solution and on soluble support using the same unreactive acceptors and donors allows for the synthesis
of an orthogonally protected hyaluronic acid disaccharide and suggest that the encountered difficulties
could be attributed to the presence of the N-acylsulfonamide.
Keywords:
Hyaluronic acid
Glycosylation
Oligosaccharide synthesis
Polymer-supported synthesis
Sulfonamide linker
Polyethylene glycol
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
synthesis.2 Its chemical nature also determines the cleavage condi-
tions required to release the final compound from the polymer
The synthesis of complex oligosaccharides is still a very challeng-
ing task and there is no general procedure for the stereoselective
synthesis of glycosides and complex glycoconjugates.1 In recent
years, solid phase synthesis has become a powerful tool for the effec-
tive preparation of targeted carbohydrates.2,3 Despite the impres-
sive advances in the solid phase synthesis of oligosaccharides,
which include the formation of the challenging b-mannosidic4 and
support. Additionally, it is highly convenient if the chosen linker
enables liberation of the synthesized saccharide with an orthogo-
nal tag for further bioconjugation.6 For example, Seeberger and
co-workers have successfully employed an octenediol linker22 for
the automated solid phase synthesis of a wide variety of complex
oligosaccharides. The final product is liberated by cross metathesis
using Grubbs catalyst and ethylene to afford n-pentenyl glycosides
where the double bond can be chemically manipulated for linkage
to carrier proteins and surfaces, creating carbohydrate vaccines or
microarrays,23 respectively.
Still, alternative anchors for the polymer-supported synthesis of
oligosaccharides should be explored, particularly concerning the
synthesis of complex glycosaminoglycans (GAGs).24–26 Ideally,
new linker designs should allow for simple and fast release from
the support with simultaneous and versatile incorporation of a un-
ique functional group that serves to attach the oligosaccharide to a
chip surface, protein or other carriers without additional function-
alization steps.3,6 Importantly, in the GAG field, linker functionali-
zation after release from the support and deprotection/sulfation
steps may require more effort than the assembly of the protected
oligosaccharide on the polymer. Moreover, these additional off-re-
sin steps on valuable and elaborated compounds usually lead to a
significant decrease in overall yield.
a
-galactosidic linkages,5 several important questions still need to
be addressed to make this approach usable for non-specialists.6
Solution-phase methodologies are an attractive alternative to solid
phase carbohydrate synthesis. Among others,7–14 these methods in-
clude oligosaccharide assembly on soluble polyethylene glycol
(PEG) polymers.15–21 PEG-supported oligosaccharide synthesis has
important advantages over solid phase synthesis such as higher
reactivity of the soluble polymer-bound sugar and the direct moni-
toring of the reaction process using routine tools, for example, stan-
dard NMR spectroscopy and thin layer chromatography (TLC), while
maintaining facilitated purification steps.
The linker that attaches the first monosaccharide to the poly-
mer support is of utmost importance for the oligosaccharide syn-
thetic process. The linker should allow for the high-yielding
attachment of the first sugar unit on the support and should be sta-
ble to the reaction conditions employed for the oligosaccharide
In this context, we consider an acylsulfonamide safety-catch
linker27–30 as an attractive alternative for oligosaccharide synthe-
sis. It is completely stable under both strongly acidic and basic
* Corresponding authors. Fax: +34 954 460565 (J.L.d.P.).
E-mail address: jlpaz@iiq.csic.es (J.L. de Paz).
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.12.021

566
J. L. de Paz et al. / Carbohydrate Research 345 (2010) 565–571
reaction conditions while the sulfamyl group can be activated at
the end of the synthesis to afford an N-alkyl-N-acylsulfonamide
intermediate that can be then displaced by a wide range of nucle-
ophiles to yield tailored glycoconjugates (Scheme 1). In fact, an
acylsulfonamide linker has been successfully employed in the solid
phase synthesis of oligosaccharides using thioglycosides as glyco-
syl donors.31,32 However, to the best of our knowledge, trichloro-
acetimidate glycosyl donors have not been previously used with
acylsulfonamide linkers.
In the context of a programme involving the development of
polymer-supported synthesis of GAG oligosaccharides, we report
herein an exploratory study on the use of an acylsulfonamide
safety-catch linker for the synthesis of hyaluronic acid oligosaccha-
rides33–35 using trichloroacetimidates as glycosyl donors. We chose
soluble PEG as support because it allowed us to monitor the reac-
tion progress and identify reaction products by using standard ana-
lytical techniques. As the only unsulfated GAG, hyaluronic acid is
Boc- and Fmoc-amino acids was achieved using benzotriazole-1-
yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP)
as coupling agent.42 In our initial studies, a carboxylic acid-termi-
nated monosaccharide 2, prepared by glycosylation of the corre-
sponding trichloroacetimidate with t-butyl 3-hydroxypropionate
followed by TFA treatment, failed to react with 1 under a host of
reaction conditions, including PyBOP activation (Scheme 2). There-
fore, an alternative approach was designed to introduce the first
monosaccharide on PEG support involving condensation of glyco-
syl donors with hydroxyl-terminated polymer 4.
Acylsulfonamide 3 was prepared by treating polymer 1 with DI-
PEA, catalytic DMAP, and the symmetrical anhydride of 2-O-acetyl-
glycolic acid prepared in situ. Subsequent deacetylation of 3
afforded acceptor 4 quantitatively as estimated by 1H NMR spec-
troscopy (Scheme 2). The first glycosylation attempts of 4 with tri-
chloroacetimidate 5 in the presence of TMSOTf failed to produce
the desired polymer 6 in good yield with multiple PEG-bound
products formed. This complex mixture of compounds probably re-
sulted from the coupling between oxocarbenium ion intermediate
derived from 5 and deprotonated acylsulfonamide.43 We envisaged
that treatment of polymer acceptor 4 with acidic resin IR-120-H+
prior glycosylation would ensure the protonation of the acylsulf-
onamide linker. Under these modified conditions, coupling of 4
with donor 5 gave PEG-bound monosaccharide 6 (Scheme 2). Inter-
estingly, no trichloroacetimidate activation was detected in the ab-
sence of Lewis-acid catalysis, although the acylsulfonamide
functional group has been recently proposed as a catalyst for gly-
cosylation of perbenzylated trichloroacetimidate donors.44 This
fact highlights the low reactivity of donor 5 compared to perbenzy-
lated ‘armed’ building blocks. Purification by selective precipita-
tion using diethyl ether was carried out several times until TLC
analysis and DOSY NMR experiments45 showed the absence of
any co-precipitated side product derived from trichloroacetimidate
5. Integration of key 1H NMR signals indicated that the coupling
proceeded to completion.
comprised of disaccharide repeating units of b-(1?4)-
D-glucuronic
acid-b-(1?3)- -N-acetylglucosamine. Hyaluronic acid is involved
D
in many biological processes, including cell adhesion, cell migra-
tion and wound healing.36–38 Due to the chemical complexity
and heterogeneity of GAGs, the preparation of well-defined syn-
thetic oligosaccharides is crucial for the establishment of struc-
ture–function relationships and the understanding of GAG–
protein interactions.39,40 GAG synthesis is a challenging task as it
generally involves coupling between electron-poor building blocks.
In particular, the synthesis of hyaluronic acid oligosaccharides usu-
ally requires the condensation of highly disarmed glucuronic acid
building blocks due to the presence of both the electron-withdraw-
ing carboxylic acid derivative and an acyl group at position 2 to
control the 1,2-trans stereochemistry of the glycosidic bonds.33–35
2. Results and discussion
First, we prepared PEG functionalized with an acylsulfonamide
linker. Commercially available amine-ended PEG monomethyl
ether was treated with 4-sulfamoyl-benzoic acid to afford sulfon-
amide 1 (Scheme 2). The 1H NMR spectrum showed signals for
amide proton at 7.51 ppm, aromatic protons at 7.98 ppm and
NH2SO2 protons at 6.29 ppm, as well as the complete disappear-
ance of the NH2CH2 methylene signal, indicating clean formation
of 1. Additionally, we employed a modified version of bromophe-
nol blue test41 to monitor coupling completion. Amine-terminated
PEG is stained blue after the addition of 0.5% bromophenol blue
solution in diethyl ether, while sulfonamide 1 is stained yellow.
Coupling conditions were sought to load the first monosaccha-
ride to polymer 1 through an acylsulfonamide safety-catch linker.
N-Protected amino acids have been efficiently coupled to
sulfonamide resins and this approach has been successfully
applied to solid phase peptide synthesis.42 Optimal loading of
To demonstrate that the acylsulfonamide linker can be selec-
tively activated and then displaced with an appropriate nucleo-
phile, polymer 6 was treated with iodoacetonitrile and then with
amine
7 to give the azido-functionalized glycoconjugate 8
(Scheme 3). Thereby, this linker allows for cleavage from the sup-
port and simultaneous orthogonal functionalization for further use
of the sugar derivative. Next, oligosaccharide assembly on polymer
6 was explored. The levulinoyl (Lev) group of 6 was easily removed
by treatment with either hydrazine acetate in CH2Cl2 or NaOMe in
MeOH to give polymer-supported acceptor 9 (Scheme 3). At-
tempted glycosylation of 9, pretreated with acidic resin as de-
scribed above, with GlcA trichloroacetimidate 10 was performed
under a range of temperature conditions (from room temperature
to ꢀ20 °C) and catalysts (TMSOTf, BF3ꢁEt2O, 4 Å acid-washed
molecular sieves). In all cases, efficient activation of glycosyl donor
O
HO
R
O
n
n
O
O
O
O
O
O
O
activation
S
NaOH
S
N
R
R
N
H
R
OH
CN
nucleophile
O
H N
N
3
2
N
R
N
H
3
n
n
Scheme 1. General strategy for activation and nucleophilic displacement of N-acylsulfonamide safety-catch linkers.

J. L. de Paz et al. / Carbohydrate Research 345 (2010) 565–571
567
PivO
PivO
AcO
O
O
COOH
NPhth
O
C
O
S
2
O
OMe
a
O
OMe
N
H
O
H N
2
H N
O
n
2
n
O
b
1
b
NH
Ph
O
O
O
LevO
O
CCl
3
O
O
Ph
O
O
S
O
S
O
NPhth
O
5
RO
O
LevO
N
H
N
H
NPhth
d
O
O
6
3: R = Ac
4: R = H
c
Scheme 2. Reagents: (a) 4-sulfamoyl-benzoic acid, DIC, HOBt, CH2Cl2; (b) 2-O-acetyl-glycolic anhydride, DIPEA, DMAP, CH2Cl2; (c) NaOMe, MeOH; (d) TMSOTf, CH2Cl2.
O
N
3
O
O
H N
O
2
Ph
2
O
O
7
O
N
3
O
LevO
O
6
N
H
2
NPhth
a
8
b or c
Ph
NH
MeO C
LevO
PivO
2
O
O
CCl
3
O
O
OPiv
O
10
O
O
O
HO
N
H
S
NPhth
O
9
Scheme 3. Reagents: (a) (i) iodoacetonitrile, DIPEA, DMF; (ii) 7, THF–DMF; (b) NaOMe, MeOH; (c) hydrazine acetate, CH2Cl2.
was monitored by TLC. NMR analysis showed a complex mixture of
completion as the first cycle resulted only in partial glycosylation
as indicated by integration of 1H NMR signals. Compound 11 was
treated with hydrazine acetate to yield polymer-bound acceptor
12. Glycosylation with 5 again did not generate the desired
disaccharide.
PEG-bound compounds and NMR signals indicative of coupling
were observed. However, no pure disaccharide could be isolated
after cleavage from the support. These results suggest that acidic
resin treatment, prior glycosylation, does not ensure, in this case,
protonation of the acylsulfonamide. It is reasonable to suppose that
the presence of charged species, such as the acylsulfonamide-de-
rived conjugate base, hinder glycosylation reactions via oxocarbe-
nium cations. It is noteworthy that linker interference was not
observed using thioglycosides as glycosylating agents and dim-
ethylthiomethylsulfonium triflate as activator.31,32
To demonstrate that encountered problems can be attributed to
the acylsulfonamide linker, model glycosylations were carried out
in solution and on PEG support without the N-acylsulfonamide lin-
ker. Monosaccharide 13 was converted into acceptor 14 that was
cleanly transformed into disaccharide 15 by condensation with tri-
chloroacetimidate 10 using either TMSOTf in CH2Cl2 or 4 Å acid-
washed molecular sieves46 in toluene (Scheme 5). Compound 15
is an orthogonally protected disaccharide building block for the
assembly of hyaluronic acid oligosaccharides. Alternatively, glyco-
syl donor 5 was coupled with hydroxyl-terminated PEG mono-
methyl ether to give bound acceptor 17 after removal of Lev
group. Polymer 17 was efficiently glycosylated with trichloroace-
timidate 10 to afford bound disaccharide 18 (Scheme 5) that
showed an NMR spectrum with key signals in accordance with
disaccharide 15 NMR data.
Considering the low reactivity of 10, the alternative GlcN-GlcA
glycosidic bond formation was also examined. Glycosylation of
acceptor 4 with donor 10 afforded polymer-bound GlcA derivative
11 (Scheme 4). In this case, the reaction was repeated to drive it to
O
MeO C
2
O
S
O
a
RO
O
10
4
PivO
+
N
H
OPiv
In conclusion, we have explored the use of an acylsulfonamide
safety-catch linker for the synthesis of hyaluronic acid oligosaccha-
rides involving the coupling of disarmed building blocks. A syn-
thetic procedure for the preparation of an acylsulfonamide PEG
support was established. GlcN and GlcA building blocks were
O
11: R = Lev
12: R = H
b
Scheme 4. Reagents: (a) TMSOTf, CH2Cl2; (b) hydrazine acetate, CH2Cl2.

568
J. L. de Paz et al. / Carbohydrate Research 345 (2010) 565–571
MeO C
Ph
O
2
Ph
O
Ph
O
b or c
O
O
a
O
LevO
O
O
O
O
O
10
OTDS
+
OTDS
PivO
OTDS
HO
LevO
NPhth
OPiv
NPhth
NPhth
13
14
15
Ph
O
O
OMe
O
d
O
O
OMe
O
O
HO
O
RO
n
n
NPhth
16: R = Lev
17: R = H
e
MeO C
O
Ph
O
2
O
OMe
O
O
O
LevO
f
O
O
PivO
n
NPhth
OPiv
18
Scheme 5. Reagents and conditions: (a) hydrazine acetate, CH2Cl2; (b) TMSOTf, CH2Cl2, 0 °C; (c) 4 Å acid-washed molecular sieves, toluene; (d) 5, TMSOTf, CH2Cl2; (e)
hydrazine acetate, CH2Cl2; (f) 10, TMSOTf, CH2Cl2.
loaded on this PEG support by glycosylation of the corresponding
trichloroacetimidates with a primary polymer-supported hydroxyl
group. The safety-catch linker enables cleavage of the saccharide
from the support and versatile functionalization for further conju-
gation of the sugar probe. However, this approach was not suitable
for oligosaccharide assembly involving glycosylation of low nucle-
ophilic acceptors with electron-poor donors. Glycosylations with
the same unreactive building blocks were successfully performed
both in solution and on non-modified PEG support to give an
orthogonally protected disaccharide for the synthesis of hyaluronic
acid sequences. These results unambiguously indicate that gly-
cosylations of secondary hydroxyl groups failed due to the pres-
ence of the N-acylsulfonamide linker. It is reasonable to suppose
that the chemical nature of the linker, in particular the high acidity
of the NH proton, can explain the presence of charged species that
hinder coupling reactions mediated by oxocarbenium ions. This
investigation also highlights the advantage of using soluble poly-
mers to optimize polymer-supported synthetic strategies because
the course of the reaction can be directly monitored by solution-
phase analytical methods.
were carried out by the Mass Spectrometry Service, Citius, Univer-
sity of Seville.
3.2. Polymer 1
1-Hydroxybenzotriazole (HOBt, 91 mg, 0.670 mmol) and N,N0-
diisopropylcarbodiimide (DIC, 104 lL, 0.670 mmol) were added
at room temperature to a solution of 4-sulfamoylbenzoic acid
(135 mg, 0.670 mmol) and amine-terminated PEG monomethyl
ether (3.04 g, 0.609 mmol) in CH2Cl2 (15 mL). After stirring for
16 h, the reaction mixture was concentrated. The residue was
redissolved in pyridine (15 mL) and diethyl ether (150 mL) was
added at 0 °C. The precipitated solid was collected by filtration,
washed twice with cold diethyl ether (2 ꢂ 150 mL) and dried under
high vacuum to give polymer 1 (3.05 g) as a white solid. The pre-
cipitation and washings with diethyl ether were repeated, if neces-
sary, to remove completely urea. Selected 1H NMR data (300 MHz,
CDCl3) d 7.98 (m, 4H, Ph), 7.51 (br s, 1H, CONH), 6.29 (br s, 2H,
SO2NH2).
3.3. Polymer 3
3. Experimental section
3.1. General procedures
DIC (0.54 mL, 3.48 mmol) was added at room temperature to a
solution of 2-O-acetylglycolic acid (821 mg, 6.9 mmol) in CH2Cl2
(10 mL). After stirring for 12 h, the solution was cooled with an
ice bath and the urea precipitate was filtered. The filtrate was
added to a round-bottomed flask containing sulfonamide polymer
Amine-ended PEG monomethyl ether (average molecular
weight 5000 Da) and PEG monomethyl ether (average molecular
weight 5000 Da) were purchased from Sigma Aldrich. TLC analyses
were performed on Silica Gel 60 F254 precoated on aluminium
plates (Merck) and the compounds were detected by staining with
anisaldehyde solution (anisaldehyde (25 mL) with sulfuric acid
(25 mL), ethanol (450 mL) and acetic acid (1 mL)) followed by heat-
ing at over 200 °C. Column chromatography was carried out on Sil-
ica 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 polarimeter. 1H and 13C NMR spectra were acquired on Bruker
DPX-300, and DRX-500 spectrometers and chemical shifts are gi-
ven in ppm (d) relative to tetramethylsilane. Electrospray mass
spectra (ES-MS) were carried out with an Esquire 6000 ESI-Ion Trap
from Bruker Daltonics. High resolution FAB mass spectra (HRMS)
1 (3.6 g, 0.69 mmol), DMAP (9 mg, 70 lmol), DIPEA (0.36 mL,
2.09 mmol) and CH2Cl2 (20 mL). After stirring at room temperature
for 24 h, the reaction mixture was concentrated. The residue was
redissolved in pyridine (20 mL) and diethyl ether (120 mL) was
added at 0 °C. The precipitated solid was collected by filtration,
washed twice with cold diethyl ether (2 ꢂ 120 mL) and dried under
high vacuum. The residue was redissolved in methanol (20 mL)
and diethyl ether (120 mL) was added at 0 °C. The precipitated so-
lid was collected by filtration, washed twice with cold diethyl ether
(2 ꢂ 120 mL) and dried under high vacuum to give polymer 3
(3.6 g) as a white solid. Selected 1H NMR data (500 MHz, CDCl3) d
8.05 (d, 2H, Ph), 7.92 (d, 2H, Ph), 7.45 (br s, 1H, CONH), 4.56 (s,
2H, CH2OCOCH3), 2.09 (s, 3H, OCOCH3).

J. L. de Paz et al. / Carbohydrate Research 345 (2010) 565–571
569
3.4. Polymer 4
added. After stirring at room temperature for 12 h, 0.2 M acetic
acid in diethyl ether (15 mL) was added at 0 °C. The precipitated
solid was collected by filtration, washed twice with 0.2 M acetic
acid in diethyl ether (2 ꢂ 15 mL) and dried under high vacuum to
give polymer 9 (135 mg) as a white solid.
Compound 3 (3.6 g, 0.68 mmol) was dissolved in methanol
(50 mL) and NaOMe (0.63 mL of a 2.17 M solution in MeOH) was
added. After stirring at room temperature for 12 h, the reaction
volume was reduced to 15 mL and 0.2 M acetic acid in diethyl ether
(120 mL) was added at 0 °C. The precipitated solid was collected by
filtration, washed twice with 0.2 M acetic acid in diethyl ether
(2 ꢂ 120 mL) and dried under high vacuum to give polymer 4
(3.5 g) as a white solid. Selected 1H NMR data (500 MHz, CDCl3) d
8.02 (d, 2H, Ph), 7.85 (d, 2H, Ph), 7.33 (br s, 1H, CONH), 3.93 (br
s, 2H, CH2OH).
3.7.2. Method B (delevulination with hydrazine acetate)
Compound 6 (144 mg, 25
lmol) was dissolved in CH2Cl2 (2 mL)
and hydrazine acetate (100
lL of a 0.5 M solution in MeOH) was
added. After stirring at room temperature for 1 h, diethyl ether
(20 mL) was added at 0 °C. The precipitated solid was collected
by filtration, washed with diethyl ether (20 mL) and dried under
high vacuum to give polymer 9 (136 mg) as a white solid. Selected
1H NMR data (500 MHz, CDCl3) d 8.05–7.94 (2d, 4H, Ph linker),
7.89–7.77 (m, 4H, NPhth), 7.50–7.33 (m, 5H, Ph), 5.58 (s, 1H,
PhCHO), 5.20 (d, J1,2 = 8.5 Hz, 1H, H-1), 4.70 (m, 1H, H-3).
3.5. Polymer 6
Acceptor 4 (568 mg, 0.11 mmol) was dissolved in CH2Cl2 (5 mL).
Amberlite IR-120-H+ (2 g) was added and the mixture was stirred
at room temperature for 30 min. The acidic resin was filtered off
and the solvent was removed in vacuo. The residue was coevapo-
rated with toluene, dried under high vacuum and then dissolved
in CH2Cl2 (3 mL). A solution of trichloroacetimidate 5 (277 mg,
3.8. Polymer 11
Acceptor 4 (550 mg, 0.11 mmol) was dissolved in CH2Cl2 (5 mL).
Amberlite IR-120-H+ (2 g) was added and the mixture was stirred
at room temperature for 30 min. The acidic resin was filtered off
and the solvent was removed in vacuo. The residue was coevapo-
rated with toluene, dried under high vacuum and then dissolved
in CH2Cl2 (3 mL). A solution of trichloroacetimidate 10 (203 mg,
0.43 mmol) in CH2Cl2 (2 mL) and then TMSOTf (99 lL of a 0.22 M
solution in CH2Cl2) were added at room temperature. After stirring
for 30 min, diethyl ether (40 mL) was added at 0 °C. The precipi-
tated solid was collected by filtration, washed with cold diethyl
ether (40 mL) and dried under high vacuum. Selective precipitation
using diethyl ether was repeated twice to give polymer 6 (618 mg)
as a white solid. Selected 1H NMR data (500 MHz, CDCl3) d 9.28 (br
s, 1H, CONHSO2), 8.00–7.91 (2d, 4H, Ph linker), 7.85–7.73 (m, 4H,
NPhth), 7.41–7.31 (m, 5H, Ph), 5.93 (dd, J2,3 = J3,4 = 9.8 Hz, 1H, H-
3), 5.52 (s, 1H, PhCHO), 5.33 (d, J1,2 = 8.5 Hz, 1H, H-1), 4.33 (m,
2H, H-2, H-6), 4.12 (br s, 2H, COCH2Osugar), 1.85 (s, 3H, COCH3).
0.33 mmol) in CH2Cl2 (2 mL) and then TMSOTf (200 lL of a
0.16 M solution in CH2Cl2) were added at room temperature. After
stirring for 1.5 h, diethyl ether (40 mL) was added at 0 °C. The pre-
cipitated solid was collected by filtration, washed with cold diethyl
ether (40 mL) and dried under high vacuum. The glycosylation and
selective precipitation procedure was repeated once to give poly-
mer 11 (545 mg) as
a
white solid. Selected 1H NMR data
(500 MHz, CDCl3) d 9.33 (br s, 1H, CONHSO2), 8.14–8.00 (2d, 4H,
Ph linker), 7.43 (br s, 1H, CONH), 5.33–5.23 (m, 2H, H-3, H-4),
5.08 (dd, 1H, H-2), 4.62 (d, J1,2 = 8 Hz, 1H, H-1), 4.24–4.13 (2d,
2H, COCH2Osugar), 4.09 (d, J4,5 = 10 Hz, 1H, H-5), 2.16 (s, 3H,
COCH3), 1.16–1.13 (2s, 18H, OCOC(CH3)3).
3.6. Azide-functionalized glycoconjugate 8
Iodoacetonitrile (73
0.25 mmol) were added to a solution of polymer 6 (289 mg,
51 mol) in DMF (3 mL). After stirring in the dark overnight,
lL, 1.01 mmol) and DIPEA (44 lL,
l
diethyl ether (40 mL) was added at 0 °C. The precipitated solid
was collected by filtration, washed with cold diethyl ether
(20 mL) and dried under high vacuum to give N-alkylated acylsulf-
3.9. Polymer 12
Compound 11 (146 mg, 26
lmol) was dissolved in CH2Cl2
onamide polymer as a brown solid. Amine 7 (101
l
L, 0.51 mmol)
(2 mL) and hydrazine acetate (100 l
L of a 0.51 M solution in
was added to a solution of PEG-bound N-alkylated acylsulfonamide
in DMF–THF (2 mL/6 mL). After stirring at room temperature over-
night, THF was removed in vacuo and diethyl ether (40 mL) was
added at 0 °C. The precipitated solid was filtered off and washed
with cold diethyl ether (40 mL). The filtrate and the washes were
combined and concentrated, and the residue was redissolved in
EtOAc (40 mL), washed with 1 N HCl, dried over MgSO4, filtered
and concentrated in vacuo. Purification by flash chromatography
(48:1 CH2Cl2–MeOH) afforded compound 8 (15 mg, 39%, not opti-
mized). TLC (8:1 CH2Cl2–MeOH) Rf = 0.50; 1H NMR (500 MHz,
CDCl3) d 7.92–7.77 (m, 4H, NPhth), 7.49–7.38 (m, 5H, Ph), 6.70
(br s, 1H, CONH), 5.99 (dd, J2,3 = J3,4 = 9.5 Hz, 1H, H-3), 5.58 (s, 1H,
PhCHO), 5.45 (d, J1,2 = 8.5 Hz, 1H, H-1), 4.45 (dd, J5,6 = 4.3 Hz,
MeOH) was added. After stirring at room temperature for 1 h,
diethyl ether (20 mL) was added at 0 °C. The precipitated solid
was collected by filtration, washed with diethyl ether (20 mL)
and dried under high vacuum to give polymer 12 (130 mg) as a
white solid. Selected 1H NMR data (500 MHz, CDCl3) d 8.12–8.00
(2d, 4H, Ph linker), 5.17 (dd, 1H, H-3), 5.01 (dd, 1H, H-2), 4.60 (d,
J1,2 = 8 Hz, 1H, H-1), 1.17–1.15 (2s, 18H, OCOC(CH3)3).
3.10. Dimethylthexylsilyl 4,6-O-benzylidene-2-deoxy-2-phtha-
limido-b-D-glucopyranoside (14)
A solution of hydrazine acetate (407 mg, 4.52 mmol) in metha-
nol (2 mL) was added to a solution of 13 (1.44 g, 2.26 mmol) in
CH2Cl2 (15 mL). After stirring for 30 min, acetone (10 mL) was
added and the reaction mixture was diluted with CH2Cl2 and
washed with H2O. The organic phase was dried over Na2SO4, fil-
tered and concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (3:1 hexane–EtOAc) to give 14
(1.11 g, 91%) as a white foam. TLC (2:1 hexane–EtOAc) Rf = 0.45;
0
J6,6 = 10.3 Hz, 1H, H-6), 4.39 (dd, 1H, H-2), 4.25 (d, Jgem = 15.3 Hz,
1H, OCH2CONH), 4.07 (d, Jgem = 15.3 Hz, 1H, OCH2CONH), 3.88–
3.34 (m, 19H, H-4, H-5, H-60, CH2CH2), 2.57–2.45 (m, 4H,
OCO(CH2)2), 1.90 (s, 3H, COCH3). ES-MS: m/z: calcd for
C36H43N5O13Na: 776.3; found: 775.6 [M+Na]+.
3.7. Polymer 9
½
a 2D0
ꢃ
ꢀ34.6 (c 0.9, CHCl3); 1H NMR (500 MHz, CDCl3) d 7.89–7.73
(m, 4H, NPhth), 7.54–7.39 (m, 5H, Ph), 5.59 (s, 1H, PhCHO), 5.53
3.7.1. Method A (delevulination with NaOMe)
(d, J1,2 = 8 Hz, 1H, H-1), 4.68 (ddd, 1H, H-3), 4.37 (dd, J5,6 = 4.5 Hz,
0
Compound 6 (140 mg, 25
lmol) was dissolved in methanol
J6,6 = 10.3 Hz, 1H, H-6), 4.24 (dd, J2,3 = 10.5 Hz, 1H, H-2), 3.87 (dd,
(2 mL) and NaOMe (17 L of a 2.17 M solution in MeOH) was
l
1H, H-60), 3.68 (m, 2H, H-4, H-5), 2.46 (d, J3,OH = 3.5 Hz, 1H, OH),

570
J. L. de Paz et al. / Carbohydrate Research 345 (2010) 565–571
1.43 (m, 1H, CH(CH3)2), 0.68–0.65 (m, 12H, C(CH3)2 and CH(CH3)2),
0.14–0.01 (2s, 6H, Si(CH3)2). 13C NMR (75 MHz, CDCl3) d 137.1–
123.3 (Ph, NPhth), 102.0, 93.9, 82.4, 68.8, 68.5, 66.2, 58.7, 33.8,
24.5, 19.8, 19.6, 18.3, 18.2, ꢀ1.8, ꢀ3.8. HRMS: m/z: calcd for
C29H37O7NSiNa: 562.2237; found: 562.2261 [M+Na]+.
7.34 (m, 5H, Ph), 5.93 (dd, J2,3 = J3,4 = 9.8 Hz, 1H, H-3), 5.53 (s, 1H,
PhCHO), 5.45 (d, J1,2 = 8.5 Hz, 1H, H-1), 4.40 (dd, J5,6 = 4.5 Hz,
0
J6,6 = 10.5 Hz, 1H, H-6), 4.30 (dd, 1H, H-2), 1.89 (s, 3H, COCH3).
3.13. Polymer 17
3.11. Dimethylthexylsilyl 3-O-(Methyl 4-O-levulinoyl-2,3-di-
Compound 16 (225 mg, 41
(2 mL) and hydrazine acetate (100 l
l
mol) was dissolved in CH2Cl2
L of a 0.82 M solution in
O-pivaloyl-b-
D-glucopyranosyluronate)-4,6-O-benzylidene-2-
deoxy-2-phthalimido-b-
D
-glucopyranoside (15)
MeOH) was added. After stirring at room temperature for 1 h,
diethyl ether (30 mL) was added at 0 °C. The precipitated solid
was collected by filtration, washed with cold diethyl ether
(30 mL) and dried under high vacuum to give polymer 17
(215 mg) as a white solid. Selected 1H NMR data (500 MHz, CDCl3)
d 7.88–7.72 (m, 4H, NPhth), 7.49–7.38 (m, 5H, Ph), 5.58 (s, 1H,
PhCHO), 5.33 (d, J1,2 = 8.5 Hz, 1H, H-1), 4.65 (m, 1H, H-3), 4.40
3.11.1. Method A (glycosylation with TMSOTf)
Acceptor 14 (100 mg, 0.19 mmol) and GlcA trichloroacetimidate
10 (152 mg, 0.24 mmol) were combined in a flask, coevaporated
with toluene and dried under vacuum. The starting materials were
0
dissolved in CH2Cl2 (2 mL) and TMSOTf (100
lL of a 0.12 M solu-
(dd, J5,6 = 4.4 Hz, J6,6 = 10 Hz, 1H, H-6), 4.25 (dd, 1H, H-2).
tion in CH2Cl2) was added at 0 °C. After 1 h, the reaction was
quenched with triethylamine (0.3 mL) and the solvent was re-
moved under reduced pressure. Purification by flash chromatogra-
phy (hexane–EtOAc 3:1) yielded 15 (136 mg, 74%) and recovered
acceptor (24 mg, 24%).
3.14. Polymer 18
Acceptor 17 (215 mg, 40
0.20 mmol), previously coevaporated with toluene and dried under
high vacuum, were dissolved in CH2Cl2 (2 mL). TMSOTf (100 L of a
lmol) and glycosyl donor 10 (124 mg,
l
3.11.2. Method B (glycosylation with 4 Å acid-washed
molecular sieves)
0.10 M solution in CH2Cl2) was added at room temperature. After
stirring for 30 min, diethyl ether (35 mL) was added at 0 °C. The
precipitated solid was collected by filtration, washed with cold
diethyl ether (35 mL) and dried under high vacuum. The glycosyl-
ation and selective precipitation procedure was repeated to drive
reaction to completion, affording polymer 18 (225 mg) as a white
solid. Selected 1H NMR data (500 MHz, CDCl3) d 7.84–7.73 (m,
4H, NPhth), 7.44–7.35 (m, 5H, Ph), 5.49 (s, 1H, PhCHO), 5.24 (d,
1H, H-1), 5.06 (m, 2H, H-30, H-40), 4.81 (dd, 1H, H-20), 4.72 (d, 1H,
H-10), 4.68 (dd, 1H, H-3), 4.31 (m, 2H, H-2, H-6a), 2.11 (s, 3H,
COCH3), 0.99–0.69 (2s, 18H, OCOC(CH3)3).
Donor 10 (116 mg, 0.19 mmol) and acceptor 14 (78 mg,
0.15 mmol) were dissolved in toluene (2 mL) in the presence of
freshly activated 4 Å acid-washed molecular sieves (1.9 g, from
Fluka, 1/8 in. rods). After stirring for 48 h at room temperature, tri-
ethylamine (0.1 mL) was added and the mixture was filtered and
concentrated. The residue was purified by flash chromatography
on silica gel (3:1 hexane–EtOAc) to give 15 (102 mg, 71%) and
recovered acceptor (23 mg, 29%). TLC (2:1 hexane–EtOAc)
Rf = 0.37; ½a 2D0
ꢃ
ꢀ9.2 (c 0.8, CHCl3); 1H NMR (500 MHz, CDCl3) d
7.85–7.72 (m, 4H, NPhth), 7.48–7.42 (m, 5H, Ph), 5.51 (s, 1H,
PhCHO), 5.45 (d, J1,2 = 8 Hz, 1H, H-1), 5.09 (m, 2H, H-30, H-40),
4.83 (dd, J2,3 = 8.3 Hz, 1H, H-20), 4.75 (d, J1,2 = 7.5 Hz, 1H, H-10),
4.69 (dd, J2,3 = J3,4 = 9.5 Hz, 1H, H-3), 4.29 (m, 2H, H-2, H-6a), 4.03
(dd, 1H, H-4), 3.87 (dd, J5,6b = J6a,6b = 10.3 Hz, 1H, H-6b), 3.70 (s,
3H, COOCH3), 3.66 (m, 1H, H-5), 3.55 (d, J4,5 = 9.5 Hz, 1H, H-50),
2.61–2.36 (m, 4H, OCO(CH2)2), 2.14 (s, 3H, COCH3), 1.38 (m, 1H,
CH(CH3)2), 1.01–0.73 (2s, 18H, OCOC(CH3)3), 0.63–0.60 (m, 12H,
C(CH3)2 and CH(CH3)2), 0.09–(ꢀ0.06) (2s, 6H, Si(CH3)2). 13C NMR
(125 MHz, CDCl3) d 177.1, 175.7, 171.0, 167.1, 137.2–123.4 (Ph,
NPhth), 101.8 (PhCHO), 98.9 (C-10), 93.4 (C-1), 81.0, 75.1, 71.8,
71.6, 71.1, 69.4, 68.9, 66.3, 57.5, 38.6, 38.2, 37.4, 33.8, 29.8, 29.7,
28.5, 27.5, 26.9, 26.6, 24.7, 24.4, 23.9, 19.8, 19.6, 18.3, 18.2, 7.9,
ꢀ1.9, ꢀ3.8. HRMS: m/z: calcd for C51H69O17NSiNa: 1018.4232;
found: 1018.4234 [M+Na]+.
Acknowledgements
We thank the Spanish Research Council (CSIC, Grant
200880I041), the Spanish Ministry of Science and Innovation
(Grant CTQ2006-01123), Junta de Andalucía (Grant P07-FQM-
02969, ‘Incentivo a Proyecto Internacional’ and fellowships for
M.M.K. and G.M.) and the European Commission (Marie Curie Rein-
tegration Grant) for financial support and Dr. Javier López-Prados
and Ms. Sara López-Galán for technical assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2009.12.021.
3.12. Polymer 16
References
Hydroxyl-terminated PEG monomethyl ether (215 mg,
1. Zhu, X. M.; Schmidt, R. R. Angew. Chem., Int. Ed. 2009, 48, 1900–1934. and
references cited therein.
2. Seeberger, P. H.; Haase, W. C. Chem. Rev. 2000, 100, 4349–4393. and references
cited therein.
3. Seeberger, P. H. Chem. Soc. Rev. 2008, 37, 19–28. and references cited therein.
4. Codee, J. D. C.; Krock, L.; Castagner, B.; Seeberger, P. H. Chem. Eur. J. 2008, 14,
3987–3994.
5. Werz, D. B.; Castagner, B.; Seeberger, P. H. J. Am. Chem. Soc. 2007, 129, 2770–
2771.
6. Seeberger, P. H. Carbohydr. Res. 2008, 343, 1889–1896.
7. Bauer, J.; Rademann, J. J. Am. Chem. Soc. 2005, 127, 7296–7297.
8. Codee, J. D. C.; Litjens, R. E. J. N.; van Den Bos, L. J.; Overkleeft, H. S.; van der
Marel, G. A. Chem. Soc. Rev. 2005, 34, 769–782.
9. Kim, S.; Tsuruyama, A.; Ohmori, A.; Chiba, K. Chem. Commun. 2008, 1816–1818.
10. Mydock, L. K.; Demchenko, A. V. Org. Lett. 2008, 10, 2107–2110.
11. Pathak, A. K.; Yerneni, C. K.; Young, Z.; Pathak, V. Org. Lett. 2008, 10, 145–148.
12. Polat, T.; Wong, C. H. J. Am. Chem. Soc. 2007, 129, 12795–12800.
13. Miura, T.; Goto, K.; Hosaka, D.; Inazu, T. Angew. Chem., Int. Ed. 2003, 42, 2047–
2051.
43
coevaporated with toluene and dried under high vacuum, were
dissolved in CH2Cl2 (2 mL). TMSOTf (101 L of a 0.11 M solution
lmol) and glycosyl donor 5 (138 mg, 0.21 mmol), previously
l
in CH2Cl2) was added at room temperature. After stirring for
15 min, diethyl ether (40 mL) was added at 0 °C. The precipitated
solid was collected by filtration, washed with cold diethyl ether
(40 mL) and dried under high vacuum. Selective precipitation
using diethyl ether was repeated twice. The solid was dissolved
in pyridine (2 mL) and acetic anhydride (1 mL) was added. After
stirring at room temperature for 3 h, diethyl ether (40 mL) was
added at 0 °C. The precipitated solid was collected by filtration,
washed with cold diethyl ether (40 mL) and dried under high vac-
uum to give polymer 16 (225 mg) as a white solid. Selected 1H
NMR data (500 MHz, CDCl3) d 7.85–7.72 (m, 4H, NPhth), 7.45–

J. L. de Paz et al. / Carbohydrate Research 345 (2010) 565–571
571
14. Encinas, L.; Chiara, J. L. Eur. J. Org. Chem. 2009, 2163–2173. and references cited
therein.
30. de Araujo, A. D.; Palomo, J. M.; Cramer, J.; Seitz, O.; Alexandrov, K.; Waldmann,
H. Chem. Eur. J. 2006, 12, 6095–6109.
15. Ando, H.; Manabe, S.; Nakahara, Y.; Ito, Y. J. Am. Chem. Soc. 2001, 123, 3848–
3849.
16. Douglas, S. P.; Whitfield, D. M.; Krepinsky, J. J. J. Am. Chem. Soc. 1995, 117,
2116–2117.
31. Kanemitsu, T.; Daikoku, S.; Kanie, O. J. Carbohydr. Chem. 2006, 25, 361–376.
32. Kanemitsu, T.; Wong, C. H.; Kanie, O. J. Am. Chem. Soc. 2002, 124, 3591–3599.
33. Huang, L. J.; Huang, X. F. Chem. Eur. J. 2007, 13, 529–540. and references cited
therein.
17. Majumdar, D.; Zhu, T.; Boons, G. J. Org. Lett. 2003, 5, 3591–3594.
18. Zhu, T.; Boons, G. J. Chem. Eur. J. 2001, 7, 2382–2389.
19. DreefTromp, C. M.; Willems, H. A. M.; Westerduin, P.; van Veelen, P.; van
Boeckel, C. A. A. Bioorg. Med. Chem. Lett. 1997, 7, 1175–1180.
20. Ojeda, R.; de Paz, J. L.; Martin-Lomas, M. Chem. Commun. 2003, 2486–2487.
21. Ojeda, R.; Terenti, O.; de Paz, J. L.; Martin-Lomas, M. Glycoconjugate J. 2004, 21,
179–195. and references cited therein.
22. Plante, O. J.; Palmacci, E. R.; Seeberger, P. H. Science 2001, 291, 1523–1527.
23. de Paz, J. L.; Noti, C.; Seeberger, P. H. J. Am. Chem. Soc. 2006, 128, 2766–
2767.
34. 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.
35. Dinkelaar, J.; Gold, H.; Overkleeft, H. S.; Codee, J. D. C.; van der Marel, G. A. J.
Org. Chem. 2009, 74, 4208–4216. and references cited therein.
36. Lapcik, L.; De Smedt, S.; Demeester, J.; Chabrecek, P. Chem. Rev. 1998, 98, 2663–
2684.
37. Raman, R.; Sasisekharan, V.; Sasisekharan, R. Chem. Biol. 2005, 12, 267–277.
38. Linhardt, R. J.; Toida, T. Acc. Chem. Res. 2004, 37, 431–438.
39. Noti, C.; de Paz, J. L.; Polito, L.; Seeberger, P. H. Chem. Eur. J. 2006, 12, 8664–
8686. and references cited therein.
24. de Paz, J. L.; Angulo, J.; Lassaletta, J. M.; Nieto, P. M.; Redondo-Horcajo, M.;
Lozano, R. M.; Gimenez-Gallego, G.; Martin-Lomas, M. ChemBioChem 2001, 2,
673–685. and references cited therein.
40. Angulo, J.; Ojeda, R.; de Paz, J. L.; Lucas, R.; Nieto, P. M.; Lozano, R. M.; Redondo-
Horcajo, M.; Gimenez-Gallego, G.; Martin-Lomas, M. ChemBioChem 2004, 5,
55–61. and references cited therein.
25. de Paz, J. L.; Martin-Lomas, M. Eur. J. Org. Chem. 2005, 1849–1858. and
references cited therein.
41. Bunin, B. A. The Combinatorial Index; Academic Press: San Diego, 1998. pp 213–
236.
26. de Paz, J. L.; Ojeda, R.; Reichardt, N.; Martin-Lomas, M. Eur. J. Org. Chem. 2003,
3308–3324. and references cited therein.
27. Backes, B. J.; Virgilio, A. A.; Ellman, J. A. J. Am. Chem. Soc. 1996, 118, 3055–3056.
28. Kenner, G. W.; McDermott, J. R.; Sheppard, R. C. J. Chem. Soc., Chem. Commun.
1971, 636–637.
42. Backes, B. J.; Ellman, J. A. J. Org. Chem. 1999, 64, 2322–2330.
43. Kim, K. S.; Fulse, D. B.; Baek, J. Y.; Lee, B. Y.; Jeon, H. B. J. Am. Chem. Soc. 2008,
130, 8537–8547.
44. Griswold, K. S.; Horstmann, T. E.; Miller, S. J. Synlett 2003, 1923–1926.
45. Johnson, C. S. Prog. NMR 1999, 34, 203–256.
29. Diaz-Moscoso, A.; Benito, J. M.; Ortiz Mellet, C.; García Fernández, J. M. J. Comb.
Chem. 2007, 9, 339–342.
46. Adinolfi, M.; Barone, G.; Iadonisi, A.; Schiattarella, M. Org. Lett. 2003, 5, 987–
989.