Derivatization of a bioorthogonal protected trisaccharide linker - Toward multimodal tools for chemical biology

Article abstract of DOI:10.1021/bc300160a

When cross-linking biomolecules to surfaces or to other biomolecules, the use of appropriate spacer molecules is of great importance. Mimicking the naturally occurring spacer molecules will give further insight into their role and function, possibly unveil important issues regarding the importance of the specificity of carbohydrate-based anchor moieties, in e.g., glycoproteins and glycosylphosphatidylinositols. Herein, we present the synthesis of a lactoside-based trisaccharide, potentially suitable as a heterobifunctional bioorthogonal linker molecule whereon valuable chemical handles have been conjugated. An amino-derivative having thiol functionality shows promise as novel SPR-surfaces. Furthermore, the trisaccharide has been conjugated to a cholesterol moiety in combination with a fluorophore which successfully assemble on the cell surface in lipid microdomains, possibly lipid-rafts. Finally, a Cu^I-catalyzed azide-alkyne cycloaddition reaction (CuAAC) confirms the potential use of oligosaccharides as bioorthogonal linkers in chemical biology. (Chemical Equation Presented)

Full text of DOI:10.1021/bc300160a

Article
pubs.acs.org/bc
Derivatization of a Bioorthogonal Protected Trisaccharide
LinkerToward Multimodal Tools for Chemical Biology
Timmy Fyrner,† Karin Magnusson,† K. Peter R. Nilsson,† Per Hammarstrom,† Daniel Aili,‡
̈
and Peter Konradsson*,†
‡
†Division of Chemistry and Division of Molecular Physics, IFM, Linkoping University, SE-581 83 Linkoping, Sweden
̈
̈
S
* Supporting Information
ABSTRACT: When cross-linking biomolecules to surfaces or
to other biomolecules, the use of appropriate spacer molecules
is of great importance. Mimicking the naturally occurring
spacer molecules will give further insight into their role and
function, possibly unveil important issues regarding the
importance of the specificity of carbohydrate-based anchor
moieties, in e.g., glycoproteins and glycosylphosphatidylinosi-
tols. Herein, we present the synthesis of a lactoside-based
trisaccharide, potentially suitable as a heterobifunctional
bioorthogonal linker molecule whereon valuable chemical
handles have been conjugated. An amino-derivative having
thiol functionality shows promise as novel SPR-surfaces.
Furthermore, the trisaccharide has been conjugated to a
cholesterol moiety in combination with a fluorophore which
successfully assemble on the cell surface in lipid microdomains, possibly lipid-rafts. Finally, a CuI-catalyzed azide−alkyne
cycloaddition reaction (CuAAC) confirms the potential use of oligosaccharides as bioorthogonal linkers in chemical biology.
orthogonally protected molecule enables conjugation and
elongation in a great variety of sequences, independent of
each other, thereby serving as an ideal framework for
heterobifunctional cross-linking. In this work, we report such
a heterobifunctional rod-like trisaccharide cross-linking mole-
cule for applications in chemical biology. We foresee that future
applications of these derivatives will entail, e.g., glyco-lipid−
protein or glyco-lipid−fluorophore conjugates with variable
linker lengths (tri-, penta-, and heptasaccharide moieties) to
probe, e.g., for protein stability as a function of spacer length in
relation to the cell membrane, biomembrane organization, and/
or glyco-lipid distribution dependence.
INTRODUCTION
■
To investigate biomolecules in situ, their functionality and
possible applications, the choice of linker molecule is often
focused on availability,1,2 (i.e., commercially available or easily
accessible via chemical synthesis) and bioorthogonality.3 The
understanding of the specific role of spacers, as well as the
excavation of suitable bioavailable spacers have been an
intensive area of research.4−9 The use of oligo(ethylene)
glycols (OEGs) as linker molecules have been shown to be
fruitful in many fields.10−13 However, OEGs have a rather high
degree of variability in comparison to the naturally occurring
oligosaccharides as glycosylphosphatidylinositols or lipopoly-
saccharides. Our interest is to investigate how a more rigid, rod-
like14 carbohydrate-based linker can be adequate as a cross-
linking derivative. In spite of carbohydrates' abundance and
biological significance, their use as biofunctional molecules in
nanotechnology lags behind those of protein and nucleic
acids,15 mainly due to their complexity and challenging
availability.16 In surface plasmon resonace (SPR), however, a
caboxymethylated polysaccharide (dextran) is often used to
form a three-dimensional matrix on the sensor surface, enabled
for conjugation.17 The synthesized trisaccharide 1 (Figure 1)18
has been utilized to generate a variety of cross-linking
derivatives presenting reactive groups, e.g., (i) amino group
for biotinylation,19 (ii) thiol group for assembly upon gold
surfaces,20−22 (iii) alkyne residue for “click-chemistry” con-
jugation,23−25 (iv) Dansyl for fluorescence microscopy, and (iv)
N-cholesterol moiety as a lipid anchor.26 Employing a
RESULTS AND DISCUSSION
■
Synthesis. Starting from the known 3-β-Azido-5-cholestene
2,27 (Scheme 1) lipid-anchor derivative 5 was synthesized by
reducing the azido-group to the corresponding amine using
standard NaBH4/NiCl2 condition. The crude aminosteroid was
further coupled to the mono-tert-butyl-succinate 328 to give
compound 4. The tert-butyl was deprotected by refluxing with
formic acid in diethyl ether giving the lipid anchor 5.
The synthesis of the different conjugates commences from
trisaccharide 1, previously synthesized.18 The thioacetylated
derivative 7 (Scheme 2) was synthesized from 1 using catalytic
hydrogenolysis followed by coupling with 3-acetylthio-
Received: April 5, 2012
© XXXX American Chemical Society
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Figure 1. Target cross-linking trisaccharide, having chemically tailored properties to address a diversity of biophysical applications or biological
systems.
a
Scheme 1
a
Reagents and conditions: (i) NaBH4, NiCl2·6H2O (cat.), EtOH/CH2Cl2; (ii) 3,28 DIPEA TBTU, DMF; (iii) HCOOH, Et2O, reflux.
a
Scheme 2
a
Reagents and conditions: (i) Pd/C (10%), H2 (g), MeOH; (ii) 6,29 NaHCO3 (s), MeOH/H2O (1:1); (iii) NaOMe, MeOH; (iv) HCl 1 M (aq.);
(v) 5, 0.5 M HOAt (DMF), N-methylmorpholine, EDC·HCl, MeOH; (vi) 10,30 NaHCO3 (s), MeOH/H2O (1:1); (vii) 12, CuSO4·5H2O, sodium
ascorbate.
propionic acid N-hydroxysuccinimide ester 6.29 A two-step
deprotection procedure comprising a Zemplen deacetylation
The alkyne derivative 11 was synthesized using protocols in
accordance as for the earlier mentioned compounds by
hydrogenolysis followed by coupling with 4-pentynoic acid N-
hydroxysuccinimide ester 10.30 In comparison, yields for
compound 7 and 11 were improved using the preactivated
N-hydroxysuccinimide ester rather than the corresponding
carboxylic acids.
́
followed by an acid deprotection of the N-Boc using 1 M HCl
(aq.) yielded the target compound 8. The lipid-anchoring
derivative 9 was synthesized by hydrogenolysis and subsequent
coupling with steroid derivative 5 using the same method as
optimized earlier.18 It should be noted that the amphiphilic
character of compound 9 is reflected by its low solubility in
solvents for standard purifications. In our experience, a highly
concentrated solution can only be acquired using either MeOH
or a combination of solvent, i.e., chloroform/MeOH/H2O
(7:4:1).
A CuI-catalyzed azide−alkyne cycloaddition (CuAAC) with
azidohexa(ethylene) glycol 12,32 was performed on a 2 mg
scale; hence, characterization data are incomplete. The
following compounds 14 and 16 have been synthesized on a
1−2 mg scale (Scheme 3), i.e., full characterization data not
available, as a possible lipid raft dye or for protein conjugation
B
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a
Scheme 3
a
Reagents and conditions: (i) 1 M HCl (aq.), MeOH; (ii) Dansyl chloride, NaHCO3 (s), MeOH/H2O (1:1); (iii) 15,31 NaHCO3 (s), MeOH/H2O
(1:1).
studies, respectively. From intermediate 9, the dansyl structure
14, as well as the 2-iodoacetamido derivative 16, was
synthesized. The two-step conversion was commenced by a
deprotection of 9 followed by subsequent coupling with dansyl
chloride (→14) or iodoacetic acid N-hydroxysuccinimide ester
1531 to give target compound 16.
Application of 8 for Surface Plasmon Resonance. The
intention to improve and refine biosensors is of great interests.
We set out to synthesize a bifunctional trisaccharide having
both amine as well as thiol functionality 8 (Scheme 2). This
derivative was successfully assembled onto gold surfaces and
able to perform as a novel biosensor surface. The thiol moiety
Figure 3. Sensorgram showing the binding of avidin to the
biotinylated SAM.
in compound 8 enabled formation of self-assembled mono-
layers (SAMs) on gold surfaces. Biotin-NHS was coupled to the
terminal amine group in order to render a flexible biosensor
surface. Infrared reflection absorption spectroscopy (IRAS) was
employed to confirm the derivatization of the SAMs with
avidin, resulting in an additional increase of 500 RU in the
biotinylated channel (not shown).
Application of 14 for Selective Binding to Lipid
Microdomains in Living Cells. To generate a molecular tool
for studies of glyco-conjugated lipids and for understanding
their molecular distributions, we have synthesized a cholesterol
biotin. No significant changes were observed in the fingerprint
region after biotinylation, but a slight increase in the amide I
band (1657 cm−1) and the appearance of a new vibrational
mode at 1711 cm−1 originating from the biotin CO stretch
indicate successful coupling (Figure 2). The ellipsometric
containing glycoside 9 (Scheme 2).34 It was further derivatized
with either a fluorophore (Dansyl) containing derivative 14, for
the monitoring of lipid-microdomains (lipid rafts) or an iodo-
acetamide derivative 16 (Scheme 3), for site-specific con-
jugation to cysteine residues in proteins.35,36 It was shown by
Peterson and co-workers that having an amide bond compared
to a secondary amine on the N-cholesterol residue essentially
abolished the internalization.26 Hence, derivatives 14 and 16
should be predicted to accumulate on the cell surface.
thickness of SAMs of compound 8 was 15.6
0.5 Å. After
biotinylation, the thickness increased to 16.7 0.2 Å.
To investigate the binding and distribution of 14 in live cells,
we stained human fibroblasts with the probe. When adding 14
to living cells for 18 h of incubation, the probe clearly binds to a
compartment of the cell (Figure 4a). Comparing with different
compartments of the cell, such as the nucleus, mitochondria,
and α-tubulin, the probe seems to attach exclusively to the cell
membrane. The staining was uneven and punctuated, likely
reflecting binding to lipid-microdomains (lipid-rafts). The
staining pattern was very similar to that reported previously
for lipid-rafts on fibroblasts.37 An analogous staining procedure
was also performed using free Dansyl (5-dimethylamino)-1-
naphthalenesulfonamide (DNSA) instead of 14. No cellular
staining was observed from DNSA even though a 15-fold
higher staining concentration than that used for 14 was
employed (not presented herein). It is most plausible that the
cholesterol tag of 14 interacts through hydrophobic partitioning
with the target and hence shows specificity toward the
membrane lipid bilayer. Lipid rafts are compartments on the
cell surface enriched in monosialotetrahexosylganglioside
Figure 2. (a) IRAS spectra of the fingerprint and amide regions before
(bottom) and after (top) biotinylation. (b) Magnified view of the
amide I region.
Compound 8 was subsequently immobilized on SPR-gold
substrates. After biotinylation, a 5 min injection of 1 nM avidin
at a flow rate of 20 μL/min resulted in the binding of 1500 RU
(Figure 3) corresponding to 1.5 ng/mm2 (+biotin). No binding
was observed in the reference channel pretreated with acetic
acid N-hydroxysuccinimide33 (−biotin). The surface was
saturated with avidin after a second 5 min injection of 10 nM
C
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Figure 4. Compound 14 stains lipid-microdomains in living cells. Cultured lung fibroblasts were incubated with 0.3 μM of 14 for 18 h. (a) Single
fibroblast with small punctuate microdomains visible at the top of the cell (blue), counterstaining of cells were performed using Mitotracker (green)
and ToPro3 (red). (b) Fluorescence emission spectra of punctuate microdomains from three regions of interest showing emission peaks at 521.6−
524.8 nm (blue). Background emission was very low (red). (c) Confocal microscope image showing clustered fibroblasts stained as in (a). (d)
Computer-generated surface enhanced (3D) image of (c).
(GM1), cholesterol, and GPI-anchored proteins.38 Hence,
specificity of 14 toward lipid-rafts can be assumed and is
possibly also reinforced through glycolic interactions with
GM1. The cell staining of 14 was weaker in the antitubulin
antibody staining protocol (not presented herein), which could
be a result of the probe being washed away during several
incubation steps which were performed in the presence of the
detergent Triton-X.
With fluorescence microspectroscopy, the emission spectrum
of the punctuate microdomains where 14 was accumulated
showed typical Dansyl emission spectra with emission peaks at
523.7 1.5 nm (Figure 4b). This emission peak distribution of
compound 14 reflects a Stokes shift of the probe to reflect a
slightly hydrophobic environment.39 This shows that the probe,
when enriched within lipid microdomains on the cell surface, is
partially shielded from water. Using confocal scanning
fluorescence microscopy, we found that the fluorescently
tagged compound 14 showed prevalent labeling of micro-
domains in clustered fibroblasts (Figure 4c,d), which was more
pronounced than for solitary cells (c.f., Figure 4a and Figure
4c,d). This could indicate that lipid rafts are up-regulated
during cell−cell interactions.
to MALDI-TOF) which establishes the potential use of
oligosaccharides as spacer molecules as an alternative to the
commonly used oligo(ethylene) glycols.
CONCLUSION
■
In conclusion, an orthogonally protected trisaccharide has been
demonstrated as a general spacer/linker molecule by
conjugation with different chemical functionalities. It has
been illustrated that defined carbohydrate-based compounds
have potential use in biosensing applications, i.e., novel SPR
surfaces. The result from live cell incubation experiments
indicates that the trisaccharide-based linker incorporates into
cell surfaces, and was specifically enriched in microdomains.
These findings also show promise for conjugating biomolecules
onto cell surfaces, thereby mimicking glycoproteins. Using
compound 14 as an example of lipid-raft specificity, this opens
up the possibility of using compound 16 as either a “fishing
hook” for protein conjugation in living cells or a tool for
directed interaction of a recombinant protein toward these
cellular structures. Furthermore, a successful “click chemistry”
cycloaddition confirms the potential of using oligosaccharides
as unique bioorthogonal cross-linking derivatives.
Application of 11 for Click Chemistry. Finally, to ensure
the possible usefulness of derivative 11 (Scheme 2), bearing an
alkyne functionality for further “click chemistry” conjugations, a
test reaction (2 mg) was performed with hexa(ethylene) glycol
12 having an azido-functionality.32 The result displayed
complete consumption of the alkyne derivative 11 (according
EXPERIMENTAL SECTION
■
Synthesis. General. CH2Cl2 and toluene was distilled over
calcium hydride and collected onto predried 4 Å MS. Thin
layer chromatography (TLC) was carried out on Merck 60 F254
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plates and visualized by UV light and/or developed with PAA
[EtOH (95%, 744 mL), H2SO4 (conc., 27.6 mL), AcOH
(100%, 8.4 mL), p-anisaldehyde (20.4 mL)]. Flash column
chromatography (FC) was carried out on silica gel Merck 60
(40−63 μm). Reverse-phase chromatography (RP) was carried
out on Merck LiChroprep (RP-18). Gel permeation was
performed using Sephadex LH-20. Dialysis was performed
using Spectra/Por MWCO = 500 or MWCO = 1000. Proton
nuclear magnetic resonance (1H) and carbon nuclear magnetic
resonance (13C) was recorded on a Varian 300 MHz
spectrometer; multiplicities are quoted as singlet (s), doublet
(d), doublet of doublets (dd), triplet (t), apparent doublet (ad),
apparent triplet (at). ESI-MS (recorded at Medivir AB,
Huddinge, Sweden) was performed on a Water Synapt
HDMS instrument equipped with electrospray interface.
Matrix-assisted laser desorption ionization − time of flight
(MALDI-TOF) mass spectroscopy was recorded on a Voyager-
DE STR Biochemistry Workstation in a positive mode using
using α-cyano-4-hydroxycinnamic acid (CHCA) as matrix.
Optical measurements were recorded at 20 °C with a Perkin-
Elmer 141 polarimeter. FT-IR was recorded on a Perkin-Elmer
Spectrum 1000 using KBr pellets; appearances are quoted as
strong (s), medium (m), and weak (w). Melting points were
recorded on a Stuart melting point apparatus.
tert-Butyl-N-succinyl-3β-amino-5-cholestene (4). The
3-β-Azido-5-cholestene27 2 (1.00 g, 2.43 mmol) was dissolved
in EtOH/CH2Cl2 (20 mL, 3:1) whereupon NaBH4 (180 mg,
4.86 mmol) and NiCl2·6H2O (cat.) were added. After 1 h, the
solution was diluted with CH2Cl2 and washed with NaCl (sat.
aq.), dried, and concentrated. Without further purification, the
amine was dissolved in DMF (10 mL) whereupon mono-tert-
butyl-succinate 328 (850 mg, 4.86 mmol), DIPEA (1.70 mL,
9.72 mmol), and TBTU (1.56 g, 4.86 mmol) were added. After
1 h, the solution was diluted with toluene and washed
sequentially with NaCl (sat. aq.), NaHCO3 (sat. aq.), dried, and
concentrated. FC (toluene/EtOAc 4:1) followed by crystal-
lization from MeOH afforded 4 (1.11 g, 2.04 mmol, 84%) as
white needles. Rf = 0.57 (toluene/EtOAc 2:1); mp = 146−148
°C (MeOH); [α]D − 35 (c 1, CHCl3); IR (KBr) νmaxcm−1:
2934 (s), 1732 (s), 1644 (s), 1547 (s), 1366 (m), 1153 (s);
NMR: 13C (75.4 MHz, CDCl3) δ 11.9, 18.8, 19.4, 22.6, 22.9,
28.1, 28.2, 28.3, 29.2, 31.1, 31.6, 31.9, 31.9, 35.9, 39.6, 42.4,
49.7, 50.2, 56.2, 56.8, 80.7, 121.9, 140.4, 170.8, 172.4 (overlaps
occur in spectra); 1H (300 MHz, CDCl3): δ 0.67 (s, 3H), 0.85
(d, 3H, J = 0.6 Hz), 0.87 (d, 3H, J = 0.6 Hz), 0.91 (d, 3H, J =
6.3 Hz), 0.94−1.58 (m, 33H), 1.76−1.86 (m, 3H), 1.90−2.11
(m, 3H), 2.25−2.31 (m, 1H), 2.38 (t, 2H, J = 6.7 Hz), 2.56 (t,
2H, J = 6.7 Hz), 3.60−3.74 (m, 1H), 5.33−5.35 (m, 1H), 5.54
(d, 1H, J = 8.1 Hz); ESI-MS: [M+H]+ calcd for C35H60NO3,
542.4495; found 542.4505.
0.68 (s, 3H), 0.86 (d, 3H, J = 1.3 Hz), 0.88 (d, 3H, J = 1.3 Hz),
0.92 (d, 3H, J = 6.4 Hz), 0.95−1.63 (m, 24H), 1.78−1.88 (m,
3H), 1.92−2.06 (m, 2H), 2.10−2.15 (m, 1H), 2.23−2.30 (m,
1H), 2.44 (d, 2H, J = 6.8 Hz), 2.63 (d, 2H, J = 6.8 Hz), 3.58−
3.68 (m, 1H), 5.34−5.37 (m, 1H); ESI-MS: [M+H]+ calcd for
C31H52NO3, 486.3869; found 486.3844.
N-(3-Acetylthio-propanoyl)-2-aminoethyl (4-Deoxy-4-
N-tert-butyloxycarbonyl-glycyl-glucopyranosyl)-(1→3)-
(β-D-galactopyranosyl)-(1→4)-β-D-glucopyranoside (7).
To a solution of 1 (0.15 g, 0.18 mmol) in MeOH (5 mL),
10% Pd/C was added and stirred for 4 h under H2(g)
atomsphere (1 atm) followed by filtration though Celite and
evaporation. The residue was dissolved in MeOH/H2O (10
mL) whereupon NaHCO3 (s) (0.15 g, 0.18 mmol) and 3-
acetylthio-propionic acid N-hydroxysuccinimide ester 629 (46
mg, 0.19 mmol) were added and stirred overnight. The solution
was neutralized with Dowex-H+, filtered, and evaporated. RP
(H2O → H2O/MeOH 1:1) gave 7 (0.10 g, 0.12 mmol, 67%) as
a white solid. Rf = 0.69 (chloroform/MeOH/H2O 7:4:1); [α]D
- 10 (c 0.1, H2O); IR (KBr) νmaxcm−1: 2933 (w), 1693 (s),
1546 (m), 1368 (w), 1160 (w), 1072 (s); NMR: 13C (75.4
MHz, CD3OD): δ 25.9, 28.7, 30.5, 36.6, 40.6, 44.8, 53.2, 62.0,
62.5, 62.6, 69.6, 69.6, 71.6, 74.6, 74.8, 75.9, 76.3, 76.4, 76.7,
80.8, 84.5, 104.3, 104.7, 105.6, 158.5, 173.5, 173.7, 197.2,
(several overlaps occur in spectra); 1H (300 MHz, CD3OD): δ
1.46 (s, 9H), 2.32 (s, 3H), 2.50 (t, 2H, J = 7.0 Hz), 3.11 (t, 2H,
J = 7.0 Hz), 3.25−3.90 (m, 23H), 4.10 (ad, 1H, J = 3.0 Hz),
4.33 (d, 1H, J = 8.0 Hz), 4.43 (d, 1H, J = 7.6 Hz), 4.56 (d, 1H, J
= 7.8 Hz); ESI-MS: [M+H]+ calcd for C32H56N3O20S,
834.3100; found 834.3096.
N-(3-Thio-propanoyl)-2-aminoethyl (4-deoxy-4-N-
glycyl-glucopyranosyl)-(1→3)-(β-D-galactopyranosyl)-
(1→4)-β-D-glucopyranosyl-ammonium hydrochloride
(8). To a solution of 7 (50 mg, 60 μmol) in MeOH (5 mL),
NaOMe (7 mg, 0.12 mmol) was added. After 1.5 h, the mixture
was neutralized with Dowex-H+, filtered, and evaporated. RP
(H2O → H2O/MeOH (1:1)) gave the thiol which was used
subsequently in the following step. The deacetylated compound
was dissolved in H2O (5 mL) whereupon 1 M HCl (0.1 mL)
was added. After 1 h, the solution was evaporated and
coconcentrated repeatedly with MeOH giving the title
compound 8 (40 mg, 55 mmol, 92%) as an off-white solid.
Rf = not available; [α]D + 6 (c 0.1, H2O); IR (KBr) νmaxcm−1:
2882 (w), 1692 (s), 1643 (s), 1648 (s), 1566 (m), 1434 (m),
1158 (w), 1071 (s), 1021 (s), 950 (s); NMR: 13C (75.4 MHz,
CD3OD): δ 19.8, 39.2, 39.6, 40.4, 51.8, 60.3, 60.9, 61.0, 68.3,
68.4, 70.2, 73.2, 73.4, 74.2, 74.7, 75.0, 75.2, 79.0, 82.7, 102.7,
1
103.0, 103.9, 167.0, 173.2 (overlap occur in spectra); H (300
MHz, CD3OD): δ 2.54 (t, 2H, J = 6.7 Hz), 2.76 (t, 2H, J = 6.7
Hz), 3.33−3.98 (m, 23H), 4.12−4.13 (m, 1H), 4.39 (d, 1H, J =
7.8 Hz), 4.46 (d, 1H, J = 7.6 Hz), 4.63 (d, 1H, J = 7.7 Hz); ESI-
MS: [M+H]+ calcd for C25H46N3O17S, 692.2470; found
692.2491.
3β-Amino-5-cholestene-N,N-succinyl-2-N-aminoethyl
(4-Deoxy-4-N-tert-butyloxycarbonyl-glycyl-glucopyra-
nosyl)-(1→3)-(β-D-galactopyranosyl)-(1→4)-(β-D-gluco-
pyranoside (9). Compound 1 (42 mg, 50 μmol) was dissolved
in MeOH (10 mL) followed by the addition of Pd/C (10%,
cat.). The mixture was stirred under H2 pressure (1 atm) for 2
h, then filtered through Celite and evaporated. The residue was
dissolved in MeOH (10 mL), followed by the addition of 5 (26
mg, 53 μmol), 0.5 M (DMF) HOAt (10 μL, 5 μmol), N-
methylmorpholine (6 μL, 53 μmol), and stirred for 10 min
N-Succinyl-3β-amino-5-cholestene (5). The tert-butyl-
succinyl-3β-amino-5-cholestene 4 (0.50 g, 0.92 mmol) was
added to a solution of formic acid/Et2O (1:1, 20 mL) and
heated to reflux. After 1 h, the reaction mixture was evaporated
and coconcentrated subsequently with toluene affording 5
(0.44 g, 0.92 mmol, quant.) as a white solid. Rf = 0.66 (EtOAc/
MeOH 4:1); [α]D − 20 (c 0.1, chloroform/MeOH/H2O
7:4:1); IR (KBr) νmaxcm−1: 2963 (m), 2936 (s), 1731 (s), 1625
(s), 1555 (s), 1187 (m), 1158 (s), 822 (w); NMR: 13C (75.4
MHz, CDCl3/CD3OD 9:1) δ 11.9, 18.8, 19.4, 21.0, 22.6, 22.8,
23.9, 24.3, 28.1, 28.3, 28.9, 29.9, 31.1, 31.9, 31.9, 35.9, 36.3,
36.6, 37.9, 39.0, 39.6, 39.8, 42.4, 49.9, 50.2, 56.2, 56.8, 122.0,
1
140.3, 171.7, 175.3; H (300 MHz, CDCl3/CD3OD 9:1): δ
E
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whereupon EDC·HCl (10 mg, 53 μmol) were added and
stirred overnight. The mixture was evaporated and the obtained
residue was purified on a sephadex LH-20 column using MeOH
to give 9 (32 mg, 27 μmol, 55%) as a white solid. Rf = 0.14
(EtOAc/MeOH/H2O 7:1:0.5); [α]D − 31 (c 0.1, chloroform/
MeOH/H2O 7:4:1); IR (KBr) νmaxcm−1: 2933 (s), 1643 (s),
1547 (s), 1367 (m), 1160 (m), 1074 (s); NMR: 13C (75.4
MHz, CDCl3/CD3OD/D2O 7:4:1): δ 11.4, 18.2, 18.7, 20.6,
22.0, 22.2, 23.4, 23.9, 27.6, 27.8, 28.1, 29.3, 31.1, 31.2, 31.4,
31.5, 35.4, 35.8, 36.2, 37.6, 38.3, 39.0, 39.1, 39.4, 42.0, 43.3,
49.5, 49.9, 51.3, 55.8, 56.4, 60.2, 60.7, 60.8, 68.0, 68.2, 69.8,
72.7, 72.8, 73.8, 74.3, 74.6, 74.8, 74.9, 77.4, 78.8, 80.0, 82.6,
102.4, 102.7, 103.7, 121.3, 140.2, 156.8, 172.1, 173.4 (overlaps
occur in spectra); 1H (300 MHz, CDCl3/CD3OD/D2O 7:4:1):
δ 0.41 (m, 3H), 0.57−1.35 (m, 40H), 1.46−1.76 (m, 6H),
1.82−1.97 (m, 3H), 2.12−2.22 (m, 4H), 2.34−2.49 (m, 1H),
2.96−3.72 (m, 23H), 3.81 (ad, 1H, J = 2.9 Hz), 4.05−4.1 (m,
2H), 4.29 (d, 1H, J = 7.8 Hz), 5.06 (m, 1H); ESI-MS: [M+H]+
calcd for C58H99N4O20, 1171.6775; found 1171.6777.
overnight. The mixture was diluted in H2O (15 mL) and
purified with dialysis (MWCO = 1000) over 3 days in the dark
(4 × 2 L, H2O) followed by evaporation. Rf = 0.80
(chloroform/MeOH/H2O 7:4:1); ESI-MS: [M+H]+ calcd for
C65H102N5O20S, 1304.6761; found 1304.6731.
3β-Amino-5-cholestene-N,N-succinyl-2-N-aminoethyl
[4-deoxy-4-N-(2-iodoacetyl)-glycyl-glucopyranosyl]-(1→
3)-(β-D-galactopyranosyl)-(1→4)-(β-D-glucopyranoside
(16). To a solution of 9 (1 mg, 1 μmol) in MeOH (2 mL), 1 M
HCl (aq.) (10 μL) was added. After 15 min the solution was
evaporated and co-concentrated repeatedly with MeOH.
Without further purification the amine was dissolved in H2O/
MeOH (2 mL, 1:1) whereupon NaHCO3 (s) (2 mg, 0.02
mmol) and iodoacetic acid N-hydroxysuccinimide ester 1531 (3
mg, 0.01 mmol) were added and stirred overnight. The mixture
was diluted in H2O (4 mL) and purified with dialysis (MWCO
= 500) over 3 days in the dark (4 × 2 L, H2O) followed by
evaporation. Rf = 0.74 (chloroform/MeOH/H2O 7:4:1); ESI-
MS: [M+H]+ calcd for C55H92IN4O19, 1239.532; found
1239.5408.
Gold Surface Preparation and Characterization. SAMs
for IRAS and ellipsometry were prepared on gold-coated pieces
of silicon wafers. A wafer was cut into 40 × 20 mm2 pieces,
cleaned in a mixture of H2O, 30% H2O2, and 25% NH3 in a
5:1:1 volume ratio, at 80 C for 10 min (TL1-cleaning), rinsed
with Milli-Q (MQ) water, and then blown dry with nitrogen
gas. The pieces were mounted in a custom-built electron-beam
evaporation system with a base pressure of about 10−9 Torr,
where a 25 Å Ti layer was deposited immediately before a 2000
Å gold layer. Gold-coated glass slides for SPR were obtained
from GE Healthcare. Gold surfaces were TL1-cleaned before 24
h incubation in 100 μM aqueous solutions of the
oligosaccharide. After incubation, the SAMs were rinsed with
water, sonicated for 2 min, rinsed again, and finally dried before
characterization.
IRAS spectra were recorded using a Bruker IFS 66 system,
equipped with a liquid nitrogen cooled mercury cadmium
telluride (MCT) detector unit and a grazing angle (85°)
infrared reflection accessory. The measurement chamber was
continuously purged with nitrogen gas during the measure-
ments to reduce the amount of interfering water. 3000 scans
were collected at a resolution of 2 cm−1. Before Fourier
transformation a three-term Blackmann-Harris apodization
function was applied to the interferrograms. Deuterated
hexadecanthiol (HS(CD2)15CD3) immobilized on gold was
used as a reference to record the background spectrum.
Null-ellipsometric measurements were conducted on an
automatic Rudolph Research AutoEL III ellipsometer with a
He−Ne laser light source operating at 632.8 nm at an angle of
incidence of 70°. An optical model based on isotropic optical
constants for the peptide layer Npep = n + ik = 1.50, with n =
refractive index and k = extinction coefficient, was used for the
evaluation of the film thickness. Results from at least five
measurement spots for each sample were averaged.
Sensorgrams were recorded by a Biacore 3000 instrument
(GE Healthcare, Sweden) operating at a wavelength of 760 nm
and equipped with four flow channels. The temperature was 25
°C and HBS-EP (10 mM hepes, 0.15 M NaCl, 3 mM EDTA,
0.005% surfactant P20, pH 7.4, from GE Healthcare) was used
as a running buffer. Avidin from hen egg white (Thermo
Scientific) and (+)-Biotin N-hydroxysuccinimide ester (Sigma
Aldrich) were used.
N-(4-Pentynoyl)-2-aminoethyl (4-Deoxy-4-N-tert-bu-
tyloxycarbonyl-glycyl-glucopyranosyl)-(1→3)-(β-D-gal-
actopyranosyl)-(1→4)-β-D-glucopyranoside (11). Com-
pound 1 (38 mg, 45 μmol) was dissolved in MeOH (2 mL)
whereupon 10% Pd/C (cat.) was added. The solution was
stirred for 3 h under H2 (g) pressure (1 atm) followed by
filtration though Celite and evaporation. The residue was
dissolved in MeOH/H2O (2 mL, 1:1) whereupon NaHCO3(s)
(10 mg, 0.12 mmol) and 4-pentynoic acid N-hydroxysuccini-
mide ester 1030 (10 mg, 47 μmol) were added and stirred
overnight followed by concentration. RP (H2O → H2O/
MeOH 1:1) gave 11 (21 mg, 26 μmol, 60%) as a white solid. Rf
= 0.73 (Chloroform/MeOH/H2O 7:4:1); [α]D + 3 (c 0.1,
H2O); IR (KBr) νmaxcm−1: 2933 (w), 1702 (s), 1692 (s), 1660
(s), 1552 (s), 1369 (m), 1159 (m), 1073 (s); NMR: 13C (75.4
MHz, CD3OD): δ 15.7, 28.7, 36.0, 40.6, 44.8, 53.2, 61.9, 62.5,
62.6, 69.6, 69.7, 71.6, 74.6, 74.7, 75.8, 76.2, 76.4, 76.7, 76.8,
80.7, 81.0 84.4, 104.3, 104.6, 105.5, 158.6, 173.6, 174.2
1
(overlaps occur in spectra); H (300 MHz, CD3OD): δ 1.45
(s, 9H), 2.37−2.49 (m, 4H), 3.24−3.94 (m, 24H), 4.09 (ad,
1H, J = 2.9 Hz), 4.32 (d, 1H, J = 7.7 Hz), 4.42 (d, 1H, J = 7.6
Hz), 4.55 (d, 1H, J = 7.8 Hz); ESI-MS: [M+H]+ calcd for
C32H54N3O19, 784.3274; found 784.3236.
N-[(4-Propanoyl)-2-aminoethyl [(4-deoxy-4-N-tert-bu-
tyloxycarbonyl-glycyl-glucopyranosyl)-(1→3)-(β-D-gal-
actopyranosyl)-(1→4)-β-D-glucopyranosyl]-1H-1,2,3-tria-
zol-1-yl-hexa(ethylene) glycol (13). The alkyne-derivative
11 (2 mg, 3 μmol) was dissolved in H2O/MeOH (1.5 mL, 2:1)
whereupon azidohexa(ethylene) glycol 1232 (1 mg, 3 μmol),
CuSO4·5H2O (6 mg, 0.02 mmol), and sodium ascorbate (2 mg,
0.01 mmol) were added and stirred overnight. According to
MALDI-TOF, complete consumption of alkyne 11 was
detected to give compound 13. MALDI-TOF (CHCA): [M
+ Na]+ calcd for C44H78NaO25, 1113.50; found 1113.32.
3β-Amino-5-cholestene-N,N-succinyl-2-N-aminoethyl
[4-Deoxy-4-N-(5-(dimethylamino)naphthalene-1-sulfon-
yl)-glycyl-glucopyranosyl]-(1→3)-(β-D-galactopyrano-
syl)-(1→4)-(β-D-glucopyranoside (14). To a solution of 9 (1
mg, 0.9 μmol) in MeOH (2 mL), 1 M HCl (aq.) (10 μL) was
added. After 15 min, the solution was evaporated and co-
concentrated repeatedly with MeOH. Without further
purification the amine was dissolved in H2O/MeOH (2 mL,
1:1) whereupon NaHCO3 (s) (2 mg, 0.02 mmol) and
Dansylchloride (3 mg, 0.01 mmol) were added and stirred
F
dx.doi.org/10.1021/bc300160a | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
Notes
Cell Biology. Cell Culture. MRC-5 cells (human lung
fibroblast, ATCC CCL-171) were maintained in BIOAMF 2,
complete medium, (Biological Industries; Beit Haemek, Israel)
at 37 °C in humidified air with 5% CO2 (g). These cells were
seeded in a 12 well plate on round glass coverslips with a
diameter of 18 mm.
Reagents. MitoTracker Orange CMTMRos, Alexa Fluor
488 antirabbit IgG (H+L) and ToPro3 were purchased from
Invitrogen. Rabbit polyclonal antibody for alpha Tubulin
(ab18251) was purchased from Abcam and Fluorescence
Mounting Medium was from DAKO.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from the Swedish Research Council (VR),
the Swedish Foundation for Strategic Research (SSF) and Knut
and Alice Wallenbergs Stiftelse (KAW) is gratefully acknowl-
edged.
REFERENCES
■
(1) Derda, R., Wherritt, D. J., and Kiessling, L. L. (2007) Solid-phase
synthesis of alkanethiols for the preparation of self-assembled
monolayers. Langmuir 23, 11164−11167.
Staining of Cells with 14 and Mitotracker. Derivative 14
(96 μM, 50% DMSO in deionized water) was diluted with cell
culture medium to 0.3 μM and added to the cells for 18 h.
MitoTracker Orange CMTMRos (1 mM in DMSO) was
diluted with cell culture medium to 150 nM. The diluted
solution with compound 14 was discarded and the mitochon-
dria were stained with the mitotracker solution for 30 min.
After three washings with PBS (pH 7.3, Dubecco without Ca/
Mg), the cells were fixed in 4% formaldehyde in 37 °C. The
fixed cells were washed three times in PBS. ToPro3 (1 mM in
DMSO) was diluted with PBS to a final concentration of 15
μM. The nucleus was stained with this solution for 15 min.
After washing two times with deionized water, the coverslips
were mounted on glass slides with Fluorescence Mounting
Medium and dried overnight. The coverslips were sealed with
nail polish before further analysis. An analogous staining
procedure was also performed using 5 μM free Dansyl (5-
Dimethylamino)-1-naphthalenesulfonamide (SigmaAldrich
CAS: 1431−39−6) instead of 14.
Staining of Cells with 14 and Antialpha-Tubulin. The
staining with derivative 14 was performed equivalent to the
procedure with the mitochondria staining, except that the
staining concentration of 14 was 0.6 μM. The cells were
washed three times in PBS and fixed in 4% formaldehyde at 37
°C. After washing two times in PBS, the cells were
permeabilized for 30 min in 0.2% Triton X-100 in PBS.
Three washings in PBS for five minutes were performed
followed by incubation in blocking solution containing 10%
normal goat serum in PBS. The diluted primary antibody, alpha
Tubulin, (1:5000 in blocking solution) was added and
incubated for one hour. After three washings in PBS for five
minutes, the secondary antibody, Alexa Fluor 488 antirabbit
IgG (H+L) (1:1000 in blocking buffer), was added and
incubated for one hour. The cells were washed three times in
PBS for five minutes. The nucleus was stained with ToPro3 and
the cells were mounted as previously described in the
mitotracker staining procedure.
(2) Houseman, B. T., and Mrksich, M. (1998) Efficient solid-phase
synthesis of peptide-substituted alkanethiols for the preparation of
substrates that support the adhesion of cells. J. Org. Chem. 63, 7552−
7555.
(3) Sletten, E. M., and Bertozzi, C. R. (2011) From Mechanism to
Mouse: A Tale of Two Bioorthogonal Reactions. Acc. Chem. Res. 44,
666−676.
(4) Degennes, P. G. (1992) Soft Matter. Science 256, 495−497.
(5) Polyak, S. W., Forsberg, G., Forbes, B. E., McNeil, K. A., Aplin, S.
E., and Wallace, J. C. (1997) Introduction of spacer peptides N-
terminal to a cleavage recognition motif in recombinant fusion
proteins can improve site-specific cleavage. Protein Eng. 10, 615−619.
(6) Matsuyama, A., and Kato, T. (1998) Weakly nematic-highly
nematic phase transitions in main-chain liquid-crystalline polymers.
Phys. Rev. E 58, 585−594.
(7) Wong, J. Y., Kuhl, T. L., Israelachvili, J. N., Mullah, N., and
Zalipsky, S. (1997) Direct measurement of a tethered ligand-receptor
interaction potential. Science 275, 820−822.
(8) Moreira, A. G., and Marques, C. M. (2004) The role of polymer
spacers in specific adhesion. J. Chem. Phys. 120, 6229−6237.
(9) Zalipsky, S. (1995) Chemistry of polyethylene-glycol conjugates
with biologically-active molecules. Adv. Drug Delivery Rev. 16, 157−
182.
(10) Harris, J. M. (1992) Poly(ethylene glycol) chemistry: biotechnical
and biomedical applications, Plenum, New York.
(11) Andersson, M., Oscarson, S., and Oberg, L. (1993) Synthesis of
oligosaccharides with oligoethylene glycol spacers and their conversion
into glycoconjugates using N,N,N′,N″-tetramethyl(succinimido)-
uronium tetrafluoroborate as coupling reagent. Glycoconjugate Journal
10, 197−201.
(12) Dziadek, S., Jacques, S., and Bundle, D. R. (2008) A novel linker
methodology for the synthesis of tailored conjugate vaccines
composed of complex carbohydrate antigens and specific T(H)-cell
peptide epitopes. Chem.Eur. J. 14, 5908−5917.
(13) Moreau, J., and Marchand-Brynaert, J. (2011) Modular synthesis
of bifunctional linkers for materials science. Eur. J. Org. Chem., 1641−
1644.
(14) Schneider, M. F., Mathe, G., Tanaka, M., Gege, C., and Schmidt,
R. R. (2001) Thermodynamic properties and swelling behavior of
glycolipid monolayers at interfaces. J. Phys. Chem. B 105, 5178−5185.
(15) Narain, R. (2011) Engineered carbohydrate-based materials for
biomedical applications: polymers, surfaces, dendrimers, nanoparticles, and
hydrogels, Wiley, Hoboken, NJ.
(16) Hederos, M., and Konradsson, P. (2006) Synthesis of the
Trypanosoma cruzi LPPG heptasaccharyl myo-inositol. J. Am. Chem.
Soc. 128, 3414−3419.
(17) Lofas, S. (1995) Dextran modified self-assembled monolayer
surfaces for use in biointeraction analysis with surface-plasmon
resonance. Pure Appl. Chem. 67, 829−834.
(18) Fyrner, T., Svensson, S. C. T., Konradsson, P. Synthesis of tri-,
penta-, and hepta-saccharides, functionalized with orthogonally N-
protected amino residues at the reducing and non-reducing ends.
Tetrahedron, DOI: 10.1016/j.tet.2012.05.118.
Cellular Imaging. The fluorescence from the cell prepara-
tions was recorded with a confocal laser scanning microscope,
LSM 780. The lasers used were at 405 nm (14), 550 nm
(mitotracker), and 633 nm (ToPro3). Filters were set up at
375−539 nm, 543−630 nm, and 638−735 nm.
ASSOCIATED CONTENT
* Supporting Information
■
̈
̊
S
Figure 4 and NMR spectra of synthesized compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: petko@ifm.liu.se.
■
(19) Huang, N. P., Voros, J., De Paul, S. M., Textor, M., and Spencer,
N. D. (2002) Biotin-derivatized poly(L-lysine)-g-poly(ethylene
G
dx.doi.org/10.1021/bc300160a | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
glycol): A novel polymeric interface for bioaffinity sensing. Langmuir
18, 220−230.
(39) Ren, B. Y., Gao, F., Tong, Z., and Yan, Y. (1999) Solvent
polarity scale on the fluorescence spectra of a dansyl monomer
copolymerizable in aqueous media. Chem. Phys. Lett. 307, 55−61.
(20) Fyrner, T., Lee, H. H., Mangone, A., Ekblad, T., Pettitt, M. E.,
Callow, M. E., Callow, J. A., Conlan, S. L., Mutton, R., Clare, A. S.,
Konradsson, P., Liedberg, B., and Ederth, T. (2011) Saccharide-
functionalized alkanethiols for fouling-resistant self-assembled mono-
layers: synthesis, monolayer properties, and antifouling behavior.
Langmuir 27, 15034−15047.
(21) Hederos, M., Konradsson, P., and Liedberg, B. (2005) Synthesis
and self-assembly of galactose-terminated alkanethiols and their ability
to resist proteins. Langmuir 21, 2971−2980.
(22) Ekeroth, J., Borgh, A., Konradsson, P., and Liedberg, B. (2002)
Synthesis and monolayer characterization of phosphorylated amino
acid analogs. J. Colloid Interface Sci. 254, 322−330.
(23) Lallana, E., Riguera, R., and Fernandez-Megia, E. (2011)
Reliable and efficient procedures for the conjugation of biomolecules
through Huisgen azide-alkyne cycloadditions. Angew. Chem., Int. Ed.
50, 8794−8804.
(24) Grabosch, C., Kleinert, M., and Lindhorst, T. K. (2010) Glyco-
SAMs by ’dual click’: thiourea-bridged Glyco-OEG azides for
cycloaddition on surfaces. Synthesis-Stuttgart, 828−836.
(25) Bryan, M. C., Fazio, F., Lee, H. K., Huang, C. Y., Chang, A.,
Best, M. D., Calarese, D. A., Blixt, C., Paulson, J. C., Burton, D.,
Wilson, I. A., and Wong, C. H. (2004) Covalent display of
oligosaccharide arrays in microtiter plates. J. Am. Chem. Soc. 126,
8640−8641.
(26) Boonyarattanakalin, S., Martin, S. E., Dykstra, S. A., and
Peterson, B. R. (2004) Synthetic mimics of small mammalian cell
surface receptors. J. Am. Chem. Soc. 126, 16379−16386.
(27) Sun, Q., Cai, S., and Peterson, B. R. (2009) Practical synthesis of
3 beta-amino-5-cholestene and related 3 beta-halides involving i-
steroid and retro-i-steroid rearrangements. Org. Lett. 11, 567−570.
(28) Guzzo, P. R., and Miller, M. J. (1994) Catalytic, asymmetric-
synthesis of the carbacephem framework. J. Org. Chem. 59, 4862−
4867.
(29) Liu, L., Rozenman, M., and Breslow, R. (2002) Hydrophobic
effects on rates and substrate selectivities in polymeric transaminase
mimics. J. Am. Chem. Soc. 124, 12660−12661.
(30) Galibert, M., Dumy, P., and Boturyn, D. (2009) One-pot
approach to well-defined biomolecular assemblies by orthogonal
chemoselective ligations. Angew. Chem., Int. Ed. 48, 2576−2579.
(31) Schechter, I., Clerici, E., and Zakepitzki, E. (1971) Distinct
antigenic specificities of alanine peptide determinants attached to
protein carriers via terminal amino or carboxyl groups. Eur. J.
Biochem./FEBS 18, 561−72.
(32) Svedhem, S., Hollander, C. A., Shi, J., Konradsson, P., Liedberg,
B., and Svensson, S. C. T. (2001) Synthesis of a series of
oligo(ethylene glycol)-terminated alkanethiol amides designed to
address structure and stability of biosensing interfaces. J. Org. Chem.
66, 4494−4503.
(33) Jacobson, K. A., Kirk, K. L., Padgett, W. L., and Daly, J. W.
(1985) Functionalized congeners of adenosine - preparation of analogs
with high-affinity for A1-adenosine receptors. J. Med. Chem. 28, 1341−
1346.
(34) Seeberger, P. H., and Werz, D. B. (2007) Synthesis and medical
applications of oligosaccharides. Nature 446, 1046−1051.
(35) Davis, N. J., and Flitsch, S. L. (1991) A novel method for the
specific glycosylation of proteins. Tetrahedron Lett. 32, 6793−6796.
(36) Hammarstrom, P., Owenius, R., Martensson, L. G., Carlsson, U.,
̈
̊
and Lindgren, M. (2001) High-resolution probing of local conforma-
tional changes in proteins by the use of multiple labeling: Unfolding
and self-assembly of human carbonic anhydrase II monitored by spin,
fluorescent, and chemical reactivity probes. Biophys. J. 80, 2867−2885.
(37) Ishitsuka, R., Sato, S. B., and Kobayashi, T. (2005) Imaging lipid
rafts. J. Biochem. 137, 249−254.
(38) Simons, K., and Gerl, M. J. (2010) Revitalizing membrane rafts:
new tools and insights. Nat. Rev. Mol. Cell Biol. 11, 688−699.
H
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