Stereoselective synthesis of light-activatable perfluorophenylazide- conjugated carbohydrates for glycoarray fabrication and evaluation of structural effects on protein binding by SPR imaging

Article abstract of DOI:10.1039/c1ob05040k

A series of light-activatable perfluorophenylazide (PFPA)-conjugated carbohydrate structures have been synthesized and applied to glycoarray fabrication. The glycoconjugates were structurally varied with respect to anomeric attachment, S-, and O-linked carbohydrates, respectively, as well as linker structure and length. Efficient stereoselective synthetic routes were developed, leading to the formation of the PFPA-conjugated structures in good yields over few steps. The use of glycosyl thiols as donors proved especially efficient and provided the final compounds in up to 70% total yield with high anomeric purities. PFPA-based photochemistry was subsequently used to generate carbohydrate arrays on a polymeric surface, and surface plasmon resonance imaging (SPRi) was applied for evaluation of carbohydrate-protein interactions using the plant lectin Concanavalin A (Con A) as a probe. The results indicate better performance and equal efficiency of S- and O-linked structures with intermediate linker length.

Full text of DOI:10.1039/c1ob05040k

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PAPER
Stereoselective synthesis of light-activatable perfluorophenylazide-conjugated
carbohydrates for glycoarray fabrication and evaluation of structural effects
on protein binding by SPR imaging†
Lingquan Deng,a Oscar Norberg,a Suji Uppalapati,b Mingdi Yan*a,b and Olof Ramstro¨m*a
Received 7th January 2011, Accepted 14th February 2011
DOI: 10.1039/c1ob05040k
A series of light-activatable perfluorophenylazide (PFPA)-conjugated carbohydrate structures have
been synthesized and applied to glycoarray fabrication. The glycoconjugates were structurally varied
with respect to anomeric attachment, S-, and O-linked carbohydrates, respectively, as well as linker
structure and length. Efficient stereoselective synthetic routes were developed, leading to the formation
of the PFPA-conjugated structures in good yields over few steps. The use of glycosyl thiols as donors
proved especially efficient and provided the final compounds in up to 70% total yield with high
anomeric purities. PFPA-based photochemistry was subsequently used to generate carbohydrate arrays
on a polymeric surface, and surface plasmon resonance imaging (SPRi) was applied for evaluation of
carbohydrate-protein interactions using the plant lectin Concanavalin A (Con A) as a probe. The
results indicate better performance and equal efficiency of S- and O-linked structures with intermediate
linker length.
crobalance (QCM), surface plasmon resonance (SPR), scanning
probe microscopy (SPM) and nanoobject-based systems have been
recognized.4–7
Introduction
Carbohydrates and carbohydrate-containing biomolecules are
ubiquitous in nature. These compounds form a basis in the
generation of a vast variety of biological complexities inherent
in the development, growth, and function of intact organisms,
diversifying the effects of the relatively small number of genes
in a typical genome. Due to essential roles of glycans and
glycoconjugates in biological processes, considerable efforts are
being made to decipher the many mechanisms and effects of
the glycocode.1–3 Indeed, glycans modulate or mediate various
fundamental processes in cell-cell-, cell-matrix-, and cell-molecule
interactions, and also act as regulatory switches within the nucleus
and cytoplasm. In most cases, involved glycans are attached to
proteins or lipids, and these conjugates have proven to be major
information carriers between cells and their surroundings. Thus,
in order to further understand these biological processes, detailed
elucidation of carbohydrate-protein interactions is important.
New analytical methods and techniques have more recently been
developed to meet this objective, among which a series of solid-
supported techniques, including microarrays, quartz crystal mi-
Many carbohydrate-mediated recognition events occur on cell
surfaces. The presentation of glycans at the cell surfaces with
respect to, e.g., density, distribution, orientation, and linker length
and flexibility strongly influences the recognition specificity. This
feature underscores the importance of studying carbohydrate-
protein interactions at surfaces rather than in solution. Fur-
thermore, multivalent display and cooperative recognition, which
greatly enhance the binding strength, can be achieved by surface
supported techniques,8,9 thus making them particularly well suited
for studies of carbohydrate-protein interactions. A variety of
methods have been developed to achieve the immobilization of
carbohydrates on surfaces so that subsequent binding studies were
possible. Of these, photochemical immobilization has proven to
be a very robust and versatile method in this pursuit, either by
using a pre-immobilized layer of photoreactive groups on the
surface to further bind underivatized sugars, or by introducing a
photoreactive tag onto carbohydrates to allow one-step function-
alization under UV irradiation.10–19 In the latter case, PFPA-based
photochemistry,20,21 has been utilized in our laboratories to enable
rapid, covalent and nondestructive immobilization of carbohy-
drates onto solid supports, resulting in the development of new
methodologies in microarray and QCM biosensor systems.15–17
Glycoarray technology, which permits screening of a number of
biological interactions in parallel, is a very promising technique
that has received much attention ever since its introduction into the
scientific community.22–29 In general, analysis of such arrays relies
aDepartment of Chemistry, KTH - Royal Institute of Technology, Teknikrin-
gen 30, S-10044, Stockholm, Sweden. E-mail: ramstrom@kth.se; Fax: + 46
8 7912333
bDepartment of Chemistry, Portland State University, P.O. Box 751,
Portland, Oregon, 97207-0751, USA. E-mail: yanm@pdx.edu; Fax: + 1
503 725-9525
† Electronic supplementary information (ESI) available: NMR spectra of
all new compounds, primary SPRi data and corresponding experimental
description. See DOI: 10.1039/c1ob05040k
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on various protein labeling formats in a step-wise batch mode.
However, surface plasmon resonance imaging is a technique that
can combine the advantages of glycoarray technology with the
characteristic merits of the SPR technique to evaluate biological
interactions on surfaces in a label-free manner.30–35 This also
enables the efficient analysis of multiple complex interactions in
real-time. These features are attractive, leading to a recent upsurge
of interest in exploring carbohydrate-based SPR.27 On the other
hand, since biomolecular interactions are delicately affected by the
chemistry and topography of the generated surfaces.36 Tailoring
and evaluating the surface chemistry, e.g., to enhance the specific
binding and to reduce the nonspecific binding, is therefore also of
fundamental importance in glycoarray and sensor development.
However, systematic investigations of varied surface chemical
structures and their effects on target biological interactions on
such platforms are generally lacking.
In the present study, this has been addressed by evaluating
the lectin binding performances of a number of light-activatable,
PFPA-conjugated carbohydrate structures on surfaces. The gly-
coconjugates were varied with respect to glycosidic bond type
(S-, and O-glycosides, respectively), linker length and linker
structure, in order to probe the sensitivity and specificity of the
photogenerated surface system, and to provide optimal surface
structures for protein binding studies using such a platform. To
take full advantage of the glycoarray technology for the study of
carbohydrate-protein interactions, carbohydrate immobilization
strategies are of vital importance. Since covalent immobilization
methods offer more stable arrays than non-covalent alternatives,
the former strategy has drawn greater attention. This strategy
however requires efficient and straight-forward synthetic methods,
resulting in robust functionalized surfaces. Herein, this challenge
has also been addressed, where several synthetic methods were
developed for efficient and stereo-controlled synthesis of the
final PFPA-carbohydrate conjugates. Using these probes, photo-
induced generation of glycoarrays and subsequent evaluation of
biological interactions was subsequently addressed, and surface
plasmon resonance imaging was used to combine microarray and
imaging analysis for the multiplex examination of the binding
capacities.
Results and discussion
Development of synthetic pathways
Six glycoconjugate structures (Fig. 1) were designed for the
study of carbohydrate-protein interactions on the glycoarray. Two
carbohydrate head-groups, D-mannose (Man) and D-galactose
(Gal) were chosen as model carbohydrates, linked to the photo-
activatable perfluorophenylazide moiety via different linkers and
different glycosidic bonds. Structure 1 composed a mannose head-
group linked by an O-glycosyl bond to a triethylene glycol (TEG)
based linker, which was further coupled to the PFPA moiety.
In comparison, compound 2 was linked through an S-glycosyl
bond while all other features were the same as for compound
1. Compounds 2–4 were all S-linked glycoconjugates, however
differing in linker lengths from three ethylene glycol (EG) units
to nine EG units. Compound 5 was designed to possess a triazole
ring in the linker part with a linker length comparable to that of
compound 3. Finally, compound 6 was galactose-based, different
from the other five structures, used as a reference compound in
the study.
Four different synthetic pathways for the preparation of the
target glycoconjugates were designed. Initially, the glycoconju-
gates 1 and 6 were synthesized following previously established
synthetic routes.17 Briefly, this involved anomeric coupling of
the peracetylated parent donor to an azido-alcohol acceptor
under Lewis acid activation. This product was subsequently
reduced, subjected to ion exchange, and coupled to 4-azido-
2,3,5,6-tetrafluorobenzoic acid using EDCI/triethylamine to give
the acetyl-protected PFPA-conjugate. Deprotection under basic
Fig. 1 PFPA-conjugated carbohydrate structures evaluated.
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conditions yielded the final product. This synthetic pathway being
straightforward, the total yield was, however, low. To address this,
a second route was instead designed (Scheme 1). In this case, the
acetylated carbohydrate-azide 9 was deprotected prior to amide
formation. This manipulation enabled the subsequent reduction of
azide 10 without formation of any side products. Final conjugation
using NHS-PFPA 1115 instead of EDCI coupling resulted in good
product yields.
This pathway, although considerably more efficient, still in-
volved a protection-glycosylation-deprotection route. On the other
hand, glycosylation using unprotected carbohydrates is more
desirable as it reduces the number of synthetic steps and could
enhance reactivity.37 In the second pathway, free carbohydrate-
azide 10 (Scheme 1) was employed for the final PFPA-coupling,
which prompted attempts of using unprotected carbohydrate
structures in the entire synthetic route. Taking advantage of
the fact that sulfur is less basic and more nucleophilic than
oxygen, glycosyl thiols are increasingly used in the synthesis of
thiooligosaccharides, thioglycopeptides and thioglycoproteins.38–45
The use of glycosyl thiols was thus adopted in a more efficient,
yet simpler, third synthetic route. In order to realize the third route,
however, a series of different linker structures were first synthesized
(Scheme 2). These were based on the oligoethylene glycol (OEG)
motif, widely used in a variety of biotechnical and biomedical
applications.46 These were primarily chosen because of their water
solubility, stability, and ability to reduce nonspecific binding
of proteins and other biomolecules. However, the integration
of ethylene glycols with defined length into other synthetic
molecules requires a selective functionalization and prolongation
of symmetrical diols/diol derivatives. In this study, an iterative
method to produce different bifunctional and monodispersed
OEGs with up to 9 units was developed (Scheme 2).
For the shortest TEG derivative 14, the commercially available
chloride 12 was chosen as starting material. The TEG azide 8
was prepared quantitatively from the chloride 12 by using sodium
azide in DMF at 90 ◦C overnight. Tosylation at the other end of
the azide 8 was achieved in good yield according to an established
method.47 SN2 substitution of the tosylate 13 was subsequently
achieved by use of sodium iodide in DMF at 50 ◦C for 5 h and the
desired iodide 14 was obtained in a yield of 82%. To make longer
OEG derivatives, OEG diols 15 and 19 were firstly transformed
into OEG ditosylates 16 and 20, respectively. The ditosylates
were subsequently prolonged by nucleophilic substitution with
the deprotonated azide 8 at only one end of the molecule to afford
tosylates 17 and 21 in good yields. The final iodides 18 and 22 were
obtained following similar conditions as for 14.
The glycosyl thiol pathway was next addressed (Scheme 3).
Mannose thiolate 2348 was thus used as donor and the S-
linked glycoconjugates 24–26 were conveniently obtained from
SN2 coupling of the free thiol sugar 23 and the iodide linker
molecules 14, 18 and 22. Subsequent reduction and PFPA coupling
using the methods mentioned above provided the final S-linked
glycoconjugates 2, 3 and 4 in total yields up to around 70%.
This synthetic pathway proved highly efficient, requiring few steps
Scheme 1 a) BF3·Et2O, DCM, 0 ◦C-RT, 24 h (60%); b) NaOMe, MeOH, 1 h (quant.); c) H2, Pd/C, MeOH, 1 h (quant.); d) 11, DMF, dark, 2.5 h (62%).
Scheme 2 a) NaN3, DMF, 90 ◦C, overnight (quant.); b) TsCl, TEA, DMAP, THF, 0 ◦C-RT, 12 h (78%); c) NaI, DMF, 50 ◦C, 5 h (82%); d) TsCl, TEA,
DMAP, THF, 0 ◦C–RT, overnight (81%); e) 8, NaH, DMF, 1.5 h (59%); f) NaI, DMF, 50 ◦C, 5 h (81%); g) TsCl, TEA, DMAP, DCM, 0 ◦C-RT, 2 h
(84%); h) 8, NaH, DMF, 3 h (63%); i) NaI, acetone, reflux, 1 h (86%).
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Scheme 3 a) DMF, 50 ◦C, 1 h (n = 1, 87%; n = 4, 84%; n = 7, 84%); b) H2, Pd/C, MeOH, 1 h (n = 1, 4, 7, quant.); c) 11, DMF, dark, 2.5 h (n = 1, 78%;
n = 4, 81%; n = 7, 82%).
and minimal purification, potentiating its use for glycobiological
studies. However, unexpected anomerization during the develop-
ment of this synthetic route occurred, and efforts to improve the
anomeric purities were thus undertaken (see details below).
Carbohydrate-protein interactions depend not only on the
carbohydrate structure per se, but also on the mode of presentation
with respect to linker length and linker structure. In addition
to evaluating the OEG type of linker structures with three, six
and nine ethylene glycol units, the fourth synthetic pathway was
developed in which an alternative linker structure15 was designed
and synthesized (compound 5, Fig. 1). This structure was based
on Cu-catalyzed azide-alkyne cycloaddition,49 yielding a linker-
based triazole ring. This alternative method to functionalize car-
bohydrate structures is equally attractive, owing to the efficiency
of the cycloaddition reaction, and therefore the binding properties
of this structural element were evaluated in comparison with the
OEG type of linkers.
Sulfur-based glycosyl donors are increasingly used in
synthesis,38–45 partly because of their reactivities, and partly
due to the high biocompatibility and improved resistance to-
wards hydrolytic enzymes for the products.40,51 However, the
general understanding of thioglycosylation reactions, and es-
pecially of their stereocontrol, is limited.52,53 This was fur-
ther addressed in the the present study. Initially, 1-thio-a-D-
mannopyranose 23 was used to displace a linker tosylate group
(13), leading to the intermediate carbohydrate-azide. However,
in contrast to the expectation, a mixture was produced un-
der the reaction conditions used, and NMR-analysis revealed
that both S-linked a- and b-mannose-azides were obtained
(Fig. 2).54
These results prompted further investigation of the thiogly-
cosylation process, and the origin of the observed anomeriza-
tion. Generally, 1-thiosaccharides are regarded relatively stable
due to the poor orbital overlap between the anomeric carbon
and the sulfur atom, impeding ring opening and subsequent
mutarotation. However, a recent in-depth study on the mu-
tarotation of 1-thio-aldoses in aqueous media has shown that
anomerization of certain 1-thiosaccharides readily occurs at
different pH in aqueous media.55 Ring opening and exocyclic
cleavage were found to account for the conspicuous mutarota-
tions.
Stereocontrol in the synthesis of S-linked glycoconjugates
The stereoselective formation of the glycosidic linkage is one of the
most challenging aspects in glycoconjugate and oligosaccharide
synthesis.50 This remains a challenge not only for O-linked
structures, but also for thioglycosides.
Fig. 2 1H-NMR spectrum of a- and b-anomers of compound 24 in D2O.
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The mechanistic nature of the SN2 reaction combined with the
mutarotation effect prompted the screening of the reaction con-
ditions for stereocontrolled thioglycosylation.54 Different reaction
time, temperature and solvents were screened, and the effects of
two electrophiles, tosylate and halide, were compared. Most sig-
nificantly, when the linker iodide 14 and the 1-thiomannopyranose
23 were coupled in aprotic polar solvents like DMF at 50 ◦C for
1 h, complete stereoselectivity in favor of the a-anomer product
was realized (Fig. 3).
nitrene from PFPA.56 The resulting matrix then served as the
substrate for the photoligation of the different PFPA-derivatized
carbohydrates. A range of protein-resistant polymers, including
poly(2-ethyl-2-oxazoline) (PEOX), poly(ethylene glycol) (PEG),
and poly(ethylene oxide) (PEO), were initially evaluated.57 Of
these, PEOX was chosen due to a more stable performance in
the instrumentation setup used. The different PFPA-carbohydrate
conjugates (Fig. 1) were subsequently photo-immobilized on the
surfaces, after which the sensors were ready for subsequent protein
binding studies by SPRi.
Con A,58 the plant lectin originally extracted from the jack-
bean, Canavalia ensiformis, was used as a model lectin to probe the
carbohydrate-protein interactions in this study. Firstly, the optimal
concentration of the glycoconjugate needed for the lectin binding
studies was determined. Printing solutions ranging from 0.1 mM to
100 mM, and subsequently in a more detailed study from 2 mM to
20 mM were evaluated, and the resulting sensors were interrogated
with Con A (10 mM). As a result, 10 mM of glycoconjugate was
chosen for following studies since it afforded the highest binding
with relatively low concentration of glycoconjugates used, possibly
due to the effect of surface carbohydrate ligand density on lectin
binding (cf. ESI: Figure S21†).18 The binding capacities of Con
Stereocontrol of the O-linked carbohydrate conjugates 1, 5 and 6
was conveniently achieved using conventional neighboring group
participation protocols (cf . Scheme 1).
Photo-induced glycoarray fabrication and SPRi analysis
Glycoarray fabrication was realized using the PFPA-conjugated
carbohydrates using a double surface photoligation methodology
(Scheme 4).16 Gold substrates were first covalently activated
to establish a PFPA-layer, and subsequently covered with a
polymer film followed by UV irradiation. The polymer was
covalently immobilized on the gold surface by a fast and efficient
CH insertion reaction of the photogenerated perfluorophenyl
Fig. 3 1H-NMR spectrum of compound 24 in D2O.
Scheme 4 Double surface photoligation of different glycoconjugate structures on gold surfaces for glycoarray fabrication.
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A towards the six different glycoconjugates were next examined.
SPRi data for a single sensor printed with six spots of each
glycoconjugate and interrogated with Con A is shown in Fig. 4.
3, the triazole-ring-containing counterpart 5 shows slightly higher
binding capacity when applied to lectin interrogation. This result
is in agreement with previous results, showing that biomolecules
retain their biological activities after the introduction of triazole
rings.60–62
From these results, it is clear that all the synthesized glycoconju-
gates demonstrate good selectivity for the lectin, however varying
in sensitivity. Especially, the O- and S-linked glycoconjugates 1 and
2 both show high performances, indicating that the replacement
of the glycosidic oxygen for a sulfur atom results in no significant
loss of binding. Furthermore, the results presented here show that
this photogenerated glycoarray system is capable of distinguishing
small variations in the glycoconjugate structures and conveying
their effects on binding in a convenient manner.
Conclusions
The results reported in this study demonstrate that good se-
lectivities and sensitivities can be achieved with photogenerated
glycoarrays on SPRi sensors. Furthermore, the effects of surface
chemical structural variations on protein binding were evaluated.
The binding performances of O-linked glycoconjugates and its
S-linked analogs were compared for the first time by the SPRi
technique, indicating the effect of that element variation on
protein binding is negligible in the present case. The intermediate
linker length proved to be optimal, and the linker structure
difference with regard to the incorporated triazole-ring and the
linear feature showed little influence on lectin binding in the
surface system used. The PFPA-based photochemistry proved
versatile, and reliable for surface modifications and biosensor
production. During the synthesis of the glycoconjugates, several
different synthetic pathways were developed, resulting in good
yields in few steps. The method utilizing glycosyl thiols proved
especially efficient (up to 70% of total yield), highlighting its
potential in various glycobiological studies, also owing to the fact
that the products are as effective as their O-linked analogs while
more resistant towards hydrolytic enzymes. Anomerization in the
thioglycosylation process occurred, but excellent stereocontrol
could be accomplished.
Fig. 4 SPRi responses of Compounds 1–6 toward Con A interrogation.
Each data point ( SEM) represents the average response of six replicates
for each compound. D%R in the Y-axis means the percentage of change in
reflectivity.
The concentration of each compound was fixed at 10 mM, and
10 mM of interrogating Con A solution was then applied. First
of all, the data clearly shows that Con A selectively binds to its
cognate mannose ligands (compounds 1–5), but not to the control
galactose-derivative (compound 6) on the sensor. This matches the
established specificity of the lectin.58,59 Good lectin selectivity and
minimum nonspecific binding for the photogenerated biointer-
faces were thus demonstrated. Moreover, the effects of structural
variations among the mannose-derivatives (compounds 1–5) on
lectin binding performances were also shown.
The performances of the S-linked glycoconjugates and their O-
linked analogues were compared by Con A interrogation towards
glycoconjugates 1 and 2. The results in Fig. 4 indicate that
glycoconjugates 1 and 2 show relatively similar binding capacities
toward Con A, with that of 1 being slightly higher. To the best of
our knowledge, this is the first report on the biological evaluation
of S-linked glycoconjugates in comparison with their O-linked
analogs using the SPRi technique.
The effects of different linker lengths and linker structures on
protein binding were further addressed. Three different linker
lengths (3, 6 and 9 EG units) and two different linker structures
(linear and triazole-containing) were evaluated for protein binding
on the same sensor with the conditions developed. Previous
studies showed that a reduced distance between the PFPA moiety
and the carbohydrate group resulted in less efficient interactions,
and the TEG-spacer linked PFPA-carbohydrates yielded the best
binding results.16 Thus, only linkers longer than TEG moiety
were targeted in the present study. Glycoconjugate 2, 3 and 4
contained spacers of three, six and nine ethylene glycol units,
respectively. Glycoconjugate 5 had an equivalent chain length
to 3 however possessing a triazole-ring within the molecule. As
shown in Fig. 4, the lectin binding capacity decreases as the
spacer length increases over the tested range under the system
used. This observation is to some extent expected, likely due to
a combination of entropic effects, potential entanglement, and
increased intramolecular photoinsertion during UV irradiation.
On the other hand, compared to the linear-linker glycoconjugate
Experimental
General
Water used for analysis was obtained from a Millipore Milli-
Q system with at least 18.2 MX resistivity. Concanavalin A
(Con A) from jack bean Canavalia ensiformis, and poly(2-ethyl-
2-oxazoline) (PEOX) (Mw ca. 500,000) were from Sigma–Aldrich
(St Louis, MO). All other solvents and commercially available
compounds were of HPLC, certified ACS, or reagent grade and
used as received. PBS (pH 7.4) buffer was prepared with KH2PO4
(1.69 mM), Na2HPO4 (8.00 mM), and NaCl (154 mM) in Milli-
Q water (1.00 L). H, 13C and 19F NMR data were recorded on
1
a Bruker Avance 400 instrument at 400 MHz (1H) or a Bruker
DMX 500 instrument at 500 MHz (1H), 125 MHz (13C) or 470
MHz (19F). Chemical shifts are reported as d values (ppm) with
either CDCl3 (1H: d = 7.26, 13C = 77.16), DMSO-d6 (1H: d =
2.50, 13C = 39.52) or D2O (1H: d = 4.79) as internal standard.
1
J values are given in Hz. H peak assignments were made by
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first order analysis of the spectra supported by standard 1H–
1H correlation spectroscopy (COSY). Thin layer chromatography
(TLC) was performed on precoated Cromatofolios AL Silica
gel 60 F254 plates (Merck, Darmstadt, Germany). Flash column
chromatography was performed on silica gel 60, 0.040–0.063 mm
(SDS, Val de Reuil, France). All experiments containing penta-
/tetrafluorophenylazide groups were conducted in the absence of
light with all reaction flasks covered with aluminum foil to prevent
decomposition.
H, OCH2), 3.82 (dd, 1 H, J = 3.5 and 9.4 Hz, H-3), 3.78–3.71
(m, 9 H, 4 ¥ OCH2, H-5), 3.71–3.61 (m, 3 H, H-4, H-6a, H-6b),
3.50 (t, 2 H, J = 5.1 Hz, CH2N3). 13C NMR (125 MHz, D2O): d
99.92, 72.71, 70.46, 69.92, 69.60, 69.52, 69.47, 69.23, 66.70, 66.37,
60.88, 50.12. The spectroscopic data are in agreement with the
literature.63
Compound 1
Compound 10 (135 mg, 0.4 mmol) was dissolved in dry MeOH
(4 mL), and Pd/C (40 mg) was added. Then the flask was purged
with N2 and filled with H2. The solution was stirred vigorously at
RT for 1 h. And then the reaction mixture was filtered through
celite and the solvent evaporated to give the amino product. The
crude amino product and compound 11 (133 mg, 0.4 mmol) were
dissolved in dry DMF (4 mL), and the reaction mixture was stirred
in dark at RT for 2.5 h under nitrogen atmosphere. The solvent
was evaporated under reduced pressure. Then the crude product
was purified by flash column chromatography using solvent system
DCM/MeOH (11 : 1 v/v), giving compound 1 as a colorless oil
(62%). 1H NMR (500 MHz, D2O): d 4.87 (d, 1 H, J = 1.5 Hz, H-1),
3.95–3.93 (m, 1 H, H-2), 3.89–3.59 (m, 17 H, 6 ¥ OCH2, H-3, H-4,
H-5, H-6a, H-6b). 13C NMR (125 MHz, D2O): d 160.73, 144.49,
142.56, 141.32, 139.39, 122.43, 110.11, 99.91, 72.71, 70.48, 69.94,
69.68, 69.49, 68.52, 66.69, 66.33, 60.88, 39.69. The spectroscopic
data are in agreement with the literature.17
2-(2-(2-Azidoethoxy)ethoxy)ethanol (8)
2-(2-(2-Chloroethoxy)ethoxy)ethanol (5.0 g, 30 mmol) and
sodium azide (3.9 g, 60 mmol) were dissolved in DMF (40 mL).
The mixture was stirred at 90 ◦C overnight under nitrogen
atmosphere. The solvent was evaporated under reduced pressure.
Then the residue was washed with water and extracted with
ethyl acetate (EtOAc) for three times. The organic phase was
combined, washed with brine, dried over Na2SO4 and evaporated.
The crude product was purified by flash column chromatography
using solvent system Hexanes (Hex)/EtOAc (1 : 1 v/v), giving
1
compound 8 as a colorless oil (quant.). H NMR (500 MHz,
CDCl3): d 3.74 (t, 2 H, J = 4.4 Hz, CH2CH2OH), 3.70–3.66 (m, 6 H,
2 ¥ OCH2 and CH2CH2N3), 3.62 (t, 2 H, J = 4.4 Hz, CH2CH2OH),
3.40 (t, 2 H, J = 5.1 Hz, CH2N3). 13C NMR (125 MHz, CDCl3):
d 72.61, 70.81, 70.55, 70.22, 61.93, 50.80. The spectroscopic data
are in agreement with the literature.14
2-(2-(2-Azidoethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (13)
Compound 9
Compound 8 (1.0 g, 5.7 mmol) was dissolved in THF (30 mL),
triethylamine (4.6 g, 45.6 mmol) and DMAP (77 mg, 0.6 mmol)
were added. The mixture was stirred under N2 and cooled down to
0 ◦C. Then a solution of tosyl chloride (4.4 g, 22.8 mmol) in THF
(20 mL) was added portion by portion to the mixture. The reaction
was allowed to warm up to room temperature. The mixture was
washed by water and extracted by DCM three times. The organic
phase was combined, washed with 1 M HCl, brine, dried by
Na2SO4 and evaporated. The crude product was purified by flash
column chromatography using solvent system Hex/EtOAc (2 : 1
v/v), giving compound 13 as a colorless oil (78%). 1H NMR (500
MHz, CDCl3): d 7.80 (d, 2 H, J = 8.3 Hz, OC6H4CH3), 7.34 (d,
2 H, J = 8.3 Hz, OC6H4CH3), 4.16 (t, 2 H, J = 4.8 Hz, CH2OTs),
3.70 (t, 2 H, J = 4.8 Hz, CH2CH2OTs), 3.64 (t, 2H, J = 5.1 Hz,
CH2CH2N3), 3.60 (s, 4 H, 2 ¥ OCH2), 3.36 (t, 2 H, J = 5.1 Hz,
CH2N3), 2.45 (s, 3 H, OC6H4CH3). 13C NMR (125 MHz, CDCl3):
d 144.97, 133.15, 129.97, 128.12, 70.95, 70.77, 70.24, 69.38, 68.94,
50.81, 21.79. The spectroscopic data are in agreement with the
literature.64
Penta-O-acetate-a-D-mannose 7 (3.0 g, 7.7 mmol) and compound
8 (2.0 g, 11 mmol) in dry dichloromethane (DCM, 30 mL) was
cooled to 0 ◦C. BF3·Et2O, (5.5 g, 38 mmol) was added dropwise,
and the solution was stirred overnight at RT. After completion
of the reaction, the product solution was poured into ice water
(30 mL), extracted with DCM, and the organic phase washed
with saturated NaHCO3, brine, dried over Na2SO4 and the solvent
evaporated. Then the crude product was purified by flash column
chromatography using solvent system Hex/EtOAc (2 : 1 v/v),
giving compound 9 as a colorless oil (60%). 1H NMR (500 MHz,
CDCl3): d 5.35 (dd, 1 H, J = 3.5 and 9.9 Hz, H-3), 5.28 (t, 1 H,
J = 9.9 Hz, H-4), 5.26 (dd, 1 H, J = 1.6 and 3.5 Hz, H-2), 4.86 (d,
1 H, J = 1.6 Hz, H-1), 4.28 (dd, 1 H, J = 5.0 and 12.3 Hz, H-6a),
4.09 (dd, 1 H, J = 2.5 and 12.3 Hz, H-6b), 4.05 (m, 1 H, H-5),
3.81 (m, 1 H, OCH2), 3.71–3.64 (m, 9 H, OCH2), 3.39 (t, 2 H, J =
5.4 Hz, CH2N3), 2.14 (s, 3 H, OAc), 2.09 (s, 3 H, OAc), 2.03 (s,
3 H, OAc), 1.98 (s, 3 H, OAc). 13C NMR (125 MHz, CDCl3): d
170.85, 170.21, 170.06, 169.88, 97.89, 70.96, 70.86, 70.26, 70.23,
69.74, 69.23, 68.56, 67.57, 66.33, 62.58, 50.84, 21.06, 20.92, 20.86.
The spectroscopic data are in agreement with the literature.63
1-Azido-2-(2-(2-iodoethoxy)ethoxy)ethane (14)
Compound 13 (329 mg, 1 mmol) and sodium iodide (450 mg,
3 mmol) were dissolved in DMF (6 mL). The mixture was
stirred at 50 ◦C for five hours under nitrogen atmosphere. The
solvent was evaporated under reduced pressure. Then the residue
was washed with water and extracted with EtOAc three times.
The organic phase was combined, washed with brine, dried by
Na2SO4 and evaporated. The crude product was purified by
flash column chromatography using solvent system Hex/EtOAc
(7 : 1 v/v), giving compound 14 as a colorless oil (82%). Rf 0.15
Compound 10
Compound 9 (202 mg, 0.4 mmol) was dissolved in dry MeOH
(3 mL), and NaOMe (11 mg, 0.20 mmol) was added. The reaction
mixture was stirred at RT for 1 h. Amberlite IR-120 H+ resin
was added to adjust the pH to 7, then the solution was filtered
and the solvent evaporated, giving compound 10 as a colorless oil
(quant.). 1H NMR (500 MHz, D2O): d 4.88 (d, 1 H, J = 1.6 Hz,
H-1), 3.96 (dd, 1 H, J = 1.6 and 3.5 Hz, H-2), 3.91–3.85 (m, 2
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(6 : 1 hexane/EtOAc). IR (ReactIR iC10, MeOH): vmax/cm-1 2924,
2867, 2104, 1444, 1350, 1284, 1125, 1034, 993, 931, 853. 1H NMR
(500 MHz, CDCl3): d 3.77 (t, 2 H, J = 6.6 Hz, CH2CH2I), 3.70 (t,
2H, J = 5.1 Hz, CH2CH2N3), 3.68 (s, 4 H, 2 ¥ OCH2), 3.40 (t, 2 H,
J = 5.1 Hz, CH2N3), 3.27 (t, 2 H, J = 6.6 Hz, CH2I). 13C NMR (125
MHz, CDCl3): d 72.17, 70.84, 70.43, 70.30, 50.87, 3.01. HRMS:
calcd. for C6H12IN3O2 [M + Na]+ 307.9866; observed 307.9868.
(1 : 1 v/v), giving compound 18 as a colorless oil (81%). Rf 0.18
(1 : 1 hexane/EtOAc). IR (ReactIR iC10, MeOH): vmax/cm-1 2924,
2867, 2104, 1756, 1612, 1458, 1351, 1288, 1105, 1036, 998, 946, 854.
1H NMR (500 MHz, CDCl3): d 3.76 (t, 2 H, J = 6.6 Hz, CH2CH2I),
3.71–3.63 (m, 18H, 9 ¥ OCH2), 3.39 (t, 2 H, J = 5.1 Hz, CH2N3),
3.26 (t, 2 H, J = 6.6 Hz, CH2I). 13C NMR (125 MHz, CDCl3): d
72.11, 70.84, 70.81, 70.76, 70.72, 70.36, 70.16, 50.82, 3.04. HRMS:
calcd. for C12H24IN3O5 [M + Na]+ 440.0653; observed 440.0645.
1,8-Ditosyl-3,6-dioxaoctane (16)
1,17-Ditosyl-3,6,9,12,15-pentaoxaheptadecane (20)
Triethylene glycol (15) (1.5 g, 10 mmol) was dissolved in THF
(30 mL), triethylamine (4.0 g, 40 mmol) and DMAP (13 mg,
1.1 mmol) were added. The mixture was stirred under N2 and
cooled down to 0 ◦C. Then a solution of tosyl chloride (4.0
g, 21 mmol) in THF (20 mL) was added portion by portion
to the mixture. The reaction was allowed to warm up to room
temperature. The mixture was washed with water and extracted
with DCM three times. The organic phase was combined, washed
with 1 M HCl, brine, dried over Na2SO4 and evaporated. The
crude product was purified by flash column chromatography using
solvent system Hex/EtOAc (3 : 1 v/v), giving compound 16 as a
white powder (81%). 1H NMR (500 MHz, CDCl3): d 7.79 (d, 4 H,
J = 8.3 Hz, OC6H4CH3), 7.34 (d, 4 H, J = 8.3 Hz, OC6H4CH3),
4.14 (t, 4 H, J = 4.8 Hz, 2 ¥ CH2OTs), 3.65 (t, 4 H, J = 4.8 Hz,
2 ¥ CH2CH2OTs), 3.53 (s, 4 H, 2 ¥ OCH2), 2.45 (s, 6 H, 2 ¥
OC6H4CH3). 13C NMR (125 MHz, CDCl3): d 144.99, 133.16,
120.99, 128.12, 70.86, 69.34, 68.91, 21.79. The spectroscopic data
are in agreement with the literature.65
To a solution of hexaethyleneglycol (19) (2.82 g, 10 mmol) in DCM
(35 mL) at 0 ◦C were added triethylamine (3.03 g, 30 mmol), tosyl
chloride (4.78 g, 25 mmol) and DMAP (610 mg, 5 mmol). After
being stirred under N2 at RT for 2 h, the mixture was neutralized by
saturated NH4Cl solution and extracted with DCM three times.
The organic phase was combined, washed with brine, dried by
Na2SO4 and evaporated. The crude product was purified by flash
column chromatography using solvent system Hex/EtOAc (1 : 4
v/v), giving compound 20 as a colorless oil (84%). 1H NMR (500
MHz, CDCl3): d 7.79 (d, 4 H, J = 8.3 Hz, OC6H4CH3), 7.34
(d, 4 H, J = 8.3 Hz, OC6H4CH3), 4.15 (t, 4 H, J = 4.8 Hz,
2 ¥ CH2OTs), 3.68 (t, 4 H, J = 4.8 Hz, 2 ¥ CH2CH2OTs),
3.64–3.59 (m, 8 H, 4 ¥ OCH2), 3.58–3.56 (m, 8 H, 4 ¥ OCH2),
2.44 (s, 6 H, 2 ¥ OC6H4CH3). 13C NMR (125 MHz, CDCl3): d
144.93, 133.13, 129.96, 128.11, 70.88, 70.74, 70.68, 70.64, 69.39,
68.81, 21.78. The spectroscopic data are in agreement with the
literature.65
26-Azido-3,6,9,12,15,18,21,24-octaoxahexacosyl
4-methylbenzenesulfonate (21)
17-Azido-3,6,9,12,15-pentaoxaheptadecyl
4-methylbenzenesulfonate (17)
To a solution of compound 20 (1.89 g, 3.4 mmol) and compound 8
(368 mg, 2.1 mmol) was added 60% NaH in oil (337 mg, 8.4 mmol)
under nitrogen portion by portion. The mixture was stirred at RT
for 3 h. The solvent was evaporated under reduced pressure. Then
the residue was washed with water and extracted with DCM three
times. The organic phase was combined, washed with 1 M HCl,
brine, dried over Na2SO4 and evaporated. The crude product was
purified by flash column chromatography using solvent system
EtOAc/EtOH (10 : 1 v/v), giving compound 21 as a colorless oil
(63%). Rf 0.38 (8 : 1 EtOAc/EtOH). IR (ReactIR iC10, MeOH):
To a solution of compound 16 (3.7 g, 8 mmol) and compound 8
(700 mg, 4 mmol) was added 60% NaH in oil (640 mg, 16 mmol)
under nitrogen portion by portion. The mixture was stirred at RT
for 1.5 h. The solvent was evaporated under reduced pressure. Then
the residue was washed with water and extracted with DCM three
times. The organic phase was combined, washed with 1 M HCl,
brine, dried over Na2SO4 and evaporated. The crude product was
purified by flash column chromatography using solvent system
Hex/EtOAc (1 : 5 v/v), giving compound 17 as a colorless oil
(59%). 1H NMR (500 MHz, CDCl3): d 7.79 (d, 2 H, J = 8.3 Hz,
OC6H4CH3), 7.34 (d, 2 H, J = 8.3 Hz, OC6H4CH3), 4.15 (t, 2 H,
J = 4.8 Hz, CH2OTs), 3.72–3.55 (m, 20 H, 10 ¥ OCH2), 3.38 (t,
2 H, J = 5.1 Hz, CH2N3), 2.45 (s, 3 H, OC6H4CH3). 13C NMR
(125 MHz, CDCl3): d 144.80, 132.98, 129.83, 127.95, 70.71, 70.64,
70.59, 70.55, 70.54, 70.49, 70.00, 69.28, 68.64, 50.66, 21.62. The
spectroscopic data are in agreement with the literature.66
v
max/cm-1 2874, 2108, 1603, 1457, 1356, 1292, 1252, 1176, 1101,
1019, 925, 819, 781. 1H NMR (500 MHz, CDCl3): d 7.79 (d, 2 H,
J = 8.3 Hz, OC6H4CH3), 7.34 (d, 2 H, J = 8.3 Hz, OC6H4CH3), 4.16
(t, 2 H, J = 4.8 Hz, CH2OTs), 3.72–3.55 (m, 32 H, 16 ¥ OCH2), 3.39
(t, 2 H, J = 5.1 Hz, CH2N3), 2.45 (s, 3 H, OC6H4CH3). 13C NMR
(125 MHz, CDCl3): d 144.91, 133.14, 129.94, 128.11, 70.87, 70.82,
70.79, 70.75, 70.73, 70.69, 70.64, 70.15, 69.36, 68.80, 50.81, 21.76.
HRMS: calcd. for C25H43N3O11S [M + Na]+ 616.2511; observed
616.2494.
1-Azido-17-iodo-3,6,9,12,15-pentaoxaheptadecane (18)
Compound 17 (461 mg, 1 mmol) and sodium iodide (750 mg,
5 mmol) were dissolved in DMF (10 mL). The mixture was
stirred at 50 ◦C for five hours under nitrogen atmosphere. The
solvent was evaporated under reduced pressure. Then the residue
was washed with water and extracted with EtOAc three times.
The organic phase was combined, washed with brine, dried over
Na2SO4 and evaporated. The crude product was purified by
flash column chromatography using solvent system Hex/EtOAc
1-Azido-26-iodo-3,6,9,12,15,18,21,24-octaoxahexacosane (22)
Compound 21 (593 mg, 1 mmol) and sodium iodide (750 mg,
5 mmol) were dissolved in acetone (5 mL). The mixture was
stirred and refluxed for one hour under nitrogen atmosphere.
The solvent was evaporated under reduced pressure. Then the
residue was washed with water and extracted with EtOAc three
times. The organic phase was combined, washed with brine, dried
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over Na2SO4 and evaporated. The crude product was purified
by flash column chromatography using solvent system 100% of
EtOAc, giving compound 22 as a colorless oil (86%). Rf 0.36 (8 : 1
EtOAc/EtOH). IR (ReactIR iC10, MeOH): vmax/cm-1 2867, 2106,
Compound 2
Compound 24 (69 mg, 0.19 mmol) was dissolved in dry MeOH
(3 mL), and Pd/C (20 mg) was added. The flask was purged
with N2 and filled with H2 after which the solution was stirred
vigorously at RT for 1 h. The reaction mixture was subsequently
filtered through celite and the solvent evaporated to give the
amino product. To a solution of the crude amino product in
dry DMF (3 mL) was added compound 11 (63 mg, 0.19 mmol).
The reaction mixture was stirred in the dark at RT for 2.5 h
under nitrogen atmosphere. The solvent was evaporated under
reduced pressure, and the crude product was purified by flash
column chromatography using solvent system DCM/EtOH (12 : 1
v/v), yielding compound 2 as a colorless oil (78%). Rf 0.55 (6 : 1
DCM/MeOH). IR (ReactIR iC10, MeOH): vmax/cm-1 3300, 3099,
2933, 2884, 2129, 1657, 1562, 1489, 1423, 1354, 1334, 1272, 1231,
1
1458, 1351, 1298, 1256, 1108, 1043, 1000, 950, 854. H NMR
(500 MHz, CDCl3): d 3.76 (t, 2 H, J = 6.6 Hz, CH2CH2I), 3.71–
3.62 (m, 30 H, 15 ¥ OCH2), 3.39 (t, 2 H, J = 5.1 Hz, CH2N3),
3.26 (t, 2 H, J = 6.6 Hz, CH2I). 13C NMR (125 MHz, CDCl3):
d 72.11, 70.83, 70.80, 70.76, 70.72, 70.36, 70.16, 50.82, 3.05.
HRMS: calcd. for C18H36IN3O8 [M + Na]+ 572.1439; observed
572.1425.
Compound 24
Compound 14 (86 mg, 0.3 mmol) and compound 23 (78 mg,
0.36 mmol) were dissolved in dry DMF (2 mL). The mixture was
◦
1
1078, 996, 953, 920, 882, 853, 802. H NMR (500 MHz, D2O):
stirred at 50 C for 1 h under nitrogen atmosphere. The solvent
d 5.32 (d, 1 H, J = 1.6 Hz, H-1), 4.03 (dd, 1 H, J = 1.6 and
3.5 Hz, H-2), 3.96 (m, 1 H, H-5), 3.87 (dd, 1 H, J = 2.2 and
12.3 Hz, H-6a), 3.82–3.68 (m, 10 H, 4 ¥ OCH2, H-3, H-6b), 3.65
(t, 1 H, J = 9.8 Hz, H-4), 3.62 (t, 2 H, J = 5.4 Hz, CH2NH),
2.93–2.78 (m, 2 H, SCH2). 13C NMR (125 MHz, D2O): d 160.65,
144.52, 142.56, 141.40, 139.42, 122.44, 110.17, 84.90, 73.16, 71.66,
71.03, 69.58, 69.49, 69.47, 68.52, 67.07, 60.85, 39.74, 29.98.
HRMS: calcd. for C19H24F4N4O8S [M + Na]+ 567.1149; observed
567.1135.
was evaporated under reduced pressure. Then the crude product
was purified by flash column chromatography using solvent system
DCM/MeOH (10 : 1 v/v), giving compound 24 as a colorless oil
(87%). Rf 0.50 (6 : 1 DCM/MeOH). IR (ReactIR iC10, MeOH):
v
max/cm-1 3383, 2931, 2879, 2114, 1657, 1604, 1448, 1422, 1351,
1304, 1076, 972, 920, 882, 853, 804. 1H NMR (500 MHz, D2O): d
5.35 (d, 1 H, J = 1.6 Hz, H-1), 4.06 (dd, 1 H, J = 1.6 and 3.5 Hz,
H-2), 4.00 (m, 1 H, H-5), 3.89 (dd, 1 H, J = 2.2 and 12.3 Hz, H-6a),
3.84–3.69 (m, 10 H, 4 ¥ OCH2, H-3, H-6b), 3.67 (t, 1 H, J = 9.8 Hz,
H-4), 3.50 (t, 2 H, J = 5.1 Hz, CH2N3), 2.96–2.81 (m, 2 H, SCH2).
13C NMR (125 MHz, D2O): d 84.90, 73.15, 71.66, 71.00, 69.57,
69.50, 69.37, 69.23, 67.09, 60.86, 50.17, 29.98. HRMS: calcd. for
C12H23N3O7S [M + Na]+ 376.1149; observed 376.1143.
Compound 3
Compound 3 was prepared following the same procedure as for
compound 2. Colorless oil (81%). Rf 0.52 (6 : 1 DCM/MeOH). IR
(ReactIR iC10, MeOH): vmax/cm-13328, 3095, 2929, 2882, 2129,
1657, 1562, 1491, 1423, 1330, 1272, 1231, 1101, 1099, 1082, 994,
Compound 25
Compound 25 was prepared following the same procedure as for
compound 24. Colorless oil (84%). Rf 0.46 (6 : 1 DCM/MeOH).
IR (ReactIR iC10, MeOH): vmax/cm-1 3396, 2927, 2874, 2109,
1657, 1604, 1455, 1423, 1351, 1304, 1257, 1104, 1080, 953, 854,
1
953, 922, 882, 847, 802. H NMR (500 MHz, D2O): d 5.34 (d,
1 H, J = 1.6 Hz, H-1), 4.05 (dd, 1 H, J = 1.6 and 3.5 Hz, H-2),
3.98 (m, 1 H, H-5), 3.88 (dd, 1 H, J = 2.2 and 12.3 Hz, H-6a),
3.82–3.67 (m, 22 H, 10 ¥ OCH2, H-3, H-6b), 3.66 (t, 1 H, J =
9.8 Hz, H-4), 3.62 (t, 2 H, J = 5.4 Hz, CH2NH), 2.95–2.80 (m, 2
H, SCH2). 13C NMR (125 MHz, D2O): d 160.67, 144.50, 142.53,
141.39, 139.40, 122.47, 110.18, 84.92, 73.17, 71.67, 71.02, 69.68,
69.61, 69.59, 69.54, 69.50, 69.36, 68.51, 67.08, 60.86, 39.74, 29.96.
HRMS: calcd. for C25H36F4N4O11S [M + Na]+ 699.1930; observed
699.1918.
1
802. H NMR (500 MHz, D2O): d 5.35 (d, 1 H, J = 1.6 Hz, H-
1), 4.06 (dd, 1 H, J = 1.6 and 3.5 Hz, H-2), 4.00 (m, 1 H, H-5),
3.89 (dd, 1 H, J = 2.2 and 12.3 Hz, H-6a), 3.84–3.69 (m, 22 H,
10 ¥ OCH2, H-3, H-6b), 3.67 (t, 1 H, J = 9.8 Hz, H-4), 3.51 (t,
2 H, J = 5.1 Hz, CH2N3), 2.96–2.81 (m, 2 H, SCH2). 13C NMR
(125 MHz, D2O): d 84.91, 73.17, 71.67, 71.01, 69.60, 69.55, 69.38,
69.23, 67.10, 60.87, 50.16, 29.97. HRMS: calcd. for C18H35N3O10S
[M + Na]+ 508.1935; observed 508.1924.
Compound 4
Compound 26
Compound 4 was prepared following the same procedure as for
compound 2. Colorless oil (82%). Rf 0.25 (10 : 1 DCM/MeOH).
IR (ReactIR iC10, MeOH): vmax/cm-1 3352, 3093, 2929, 2879,
2129, 1657, 1558, 1492, 1423, 1353, 1330, 1272, 1231, 1103, 994,
953, 925, 884, 849, 802. 1H NMR (500 MHz, D2O): d 5.35 (d, 1 H,
J = 1.6 Hz, H-1), 4.05 (dd, 1 H, J = 1.6 and 3.5 Hz, H-2), 3.99 (m,
1 H, H-5), 3.88 (dd, 1 H, J = 2.2 and 12.3 Hz, H-6a), 3.83–3.67 (m,
34 H, 16 ¥ OCH2, H-3, H-6b), 3.66 (t, 1 H, J = 9.8 Hz, H-4), 3.62
(t, 2 H, J = 5.4 Hz, CH2NH), 2.96–2.81 (m, 2 H, SCH2). 13C NMR
(125 MHz, D2O): d 160.61, 144.53, 142.54, 141.35, 139.37, 122.42,
110.22, 84.92, 73.19, 71.68, 71.04, 69.69, 69.59, 69.51, 69.39, 68.52,
67.09, 60.87, 39.74, 29.97. HRMS: calcd. for C31H48F4N4O14S [M
+ 2Na]2 + 427.1304; observed 427.1297.
Compound 26 was prepared following the same procedure as for
compound 24. Colorless oil (84%). Rf 0.45 (6 : 1 DCM/MeOH).
IR (ReactIR iC10, MeOH): vmax/cm-1 3402, 2879, 2114, 1657,
1604, 1457, 1351, 1306, 1257, 1106, 946, 853, 802. 1H NMR (500
MHz, D2O): d 5.35 (d, 1 H, J = 1.6 Hz, H-1), 4.06 (dd, 1 H, J = 1.6
and 3.5 Hz, H-2), 4.00 (m, 1 H, H-5), 3.89 (dd, 1 H, J = 2.2 and
12.3 Hz, H-6a), 3.84–3.69 (m, 34 H, 16 ¥ OCH2, H-3, H-6b), 3.67
(t, 1 H, J = 9.8 Hz, H-4), 3.51 (t, 2 H, J = 5.1 Hz, CH2N3), 2.96–
2.81 (m, 2 H, SCH2). 13C NMR (125 MHz, D2O): d 84.91, 73.18,
71.67, 71.02, 69.59, 69.55, 69.38, 69.23, 67.10, 60.87, 50.15, 29.97.
HRMS: calcd. for C24H47N3O13S [M + Na]+ 640.2722; observed
640.2709.
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Glycoarray fabrication
Acknowledgements
SF 10 glass substrates (18 ¥ 18 mm, Schott Glass Technology,
Inc, Elmsford, NY) were cleaned with piranha solution (7 : 3 v/v
98% sulfuric acid/35% hydrogen peroxide; Caution: the piranha
solution reacts violently with organic solvents—take extreme care
when handling this solution), and titanium (2 nm) and gold films
(45 nm) were subsequently deposited on the surfaces using electron
beam (EB) evaporation (performed at Washington Technology
Center (WTC), Seattle). The gold substrates were cleaned using
piranha solution for 30 s, thoroughly washed using boiling water
for 20 min (3 times) and dried under a stream of nitrogen.
The cleaned substrates were immersed in a solution of PFPA-
disulfide16 (1.7 mM in CH2Cl2) at RT in the dark overnight. Then
the substrates were rinsed several times with CH2Cl2 to remove
excess PFPA-disulfide and dried under a stream of nitrogen. A
solution of PEOX in CHCl3 (10 mg mL-1) was spin-coated onto
the PFPA-activated substrates at 2000 rpm for 60 s using a model
P6204 spin-coater (Specialty Coating Systems, Indianapolis, IN).
The spin-coated substrates were then UV-irradiated for 5 min
through a 280 nm optical filter using a 450 W medium pressure
Hg lamp (Hanovia, ACE Glass Inc., Vineland, NJ). The intensity
at the sample location was measured to be 4.5 mW cm-2 using a
254-nm sensor. The irradiation time includes a 2 min warm-up
period of the lamp to reach its full intensity. The substrates were
subsequently soaked in ethanol for 3 h to remove unbound PEOX
and then dried under a stream of nitrogen.
Solutions of PFPA-derivatized carbohydrates (10 mM in wa-
ter) were printed on the PEOX coated gold substrates using
a BioOdyssey Calligrapher Miniarrayer (Bio-Rad Laboratories,
Hercules, CA). An MCP 360 capillary pin was used to dispense
the carbohydrate solutions of varying concentrations from a 96-
well plate onto the gold substrate using a robotic array spotter.
A 6 ¥ 6 array pattern of the PFPA-derivatized carbohydrates was
created either with 6 different concentrations of each compound
or 6 spots of the same concentration of each compound. The
spot size was 400 mm in diameter and the distance between the
spots 800 mm. The humidity level in the arraying chamber was
maintained at 60% and the temperature at 20 ◦C. The substrates
were then UV-irradiated for 5 min through a 280 nm optical filter.
The substrates were rinsed with water to remove any excess PFPA-
derivatized carbohydrates and washed with PBS containing 0.1%
Tween 20 for 30 min. The substrates were finally dried under a
stream of nitrogen.
This study was supported in part by the Swedish Research
Council (contract no. 622-2003-2343), the Royal Institute of
Technology, the National Institutes of General Medical Science
(NIGMS) under NIH Award Numbers R01GM080295 and
2R15GM066279, ARL-ONAMI Center for Nanoarchitectures
for Enhanced Performance. LD thanks the China Scholarship
Council for a special scholarship award.
Notes and references
1 A. Varki, Glycobiology, 1993, 3, 97.
2 R. A. Dwek, Chem. Rev., 1996, 96, 683.
3 A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C.
R. Bertozzi, G. W. Hart and M. E. Etzler, Essentials of glycobiology,
Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press,
2009.
4 D. G. Castner and B. D. Ratner, Surf. Sci., 2002, 500, 28.
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SPR imaging
SPR imaging was performed using an SPR imagerII (GWC
technologies, Madison, WI), operating at ambient temperature.
The flow rate of the solutions throughout the experiments was
maintained at 100 mL/min. The glycoarrays were first equilibrated
with PBS containing 0.1% (w/v) Tween 20 (running buffer),
then treated with 0.2% (w/v) BSA in PBS containing 0.1%
(w/v) Tween 20 in order to prevent nonspecific binding, and
subsequently re-equilibrated with running buffer. All protein
binding cycles were initiated by equilibrating the glycoarrays with
running buffer, followed by the specific protein solution, and
finally re-equilibration with running buffer. After protein binding,
the glycoarrays were regenerated with urea (8 M), and then re-
equilibrated with running buffer.
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