A new chemical probe for the detection of the cancer-linked galectin-3

Article abstract of DOI:10.1039/b611050a

A chemical probe was developed for the detection of the emerging cancer marker galectin-3. The probe contains a benzophenone moiety which covalently attaches itself to the protein upon binding and irradiation. Introduction of a fluorescent label via 'clic

Full text of DOI:10.1039/b611050a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A new chemical probe for the detection of the cancer-linked galectin-3†
Lluis Ballell, Monique van Scherpenzeel, Katerina Buchalova, Rob M. J. Liskamp and Roland J. Pieters*
Received 31st July 2006, Accepted 12th October 2006
First published as an Advance Article on the web 30th October 2006
DOI: 10.1039/b611050a
A chemical probe was developed for the detection of the emerging cancer marker galectin-3. The probe
contains a benzophenone moiety which covalently attaches itself to the protein upon binding and
irradiation. Introduction of a fluorescent label via ‘click’ chemistry allows the labelled proteins to be
visualized in a gel. With the probe, selective visualization of galectin-3 in protein mixtures was shown
and remarkably even in cell lysates.
binding and processing ones, with detection limits in the low
Introduction
nanogram range per gel lane. Furthermore detection was possible
The development of methods that allow the study of specific
in complex cell lysates. The method aims to provide an alternative
subsections of the proteome is an important research goal.1
Current methods typically reduce the sample complexity through
multidimensional separations, followed by identification and
quantification by mass spectrometry. Complementary to this,
synthetic molecular probes can tag a particular protein class,
typically based on catalytic activity. The advantage of this path
is that information about the quantities of functional proteins
is obtained. Probes designed for enzymes can covalently capture
their target proteins by taking advantage of their catalytic
mechanisms with the use of suicide inhibitors.2 The appeal of
these probes is that the captured set of proteins is a reflection of
the enzyme activity present in a certain sample, rather than the
protein abundance that most other methods report on. For this
reason the inhibitor-probe method is called activity-based protein
profiling (ABBP).3 However, this cannot be done for proteins that
merely bind their target molecules or for enzymes for which no
suicide inhibitors exist. For these cases photoreactive groups4 have
been linked to the ligands in order to attach to nearby protein
residues. Such probes3e can thus capture proteins as a function of
their binding activity within a complex sample as was shown for
kinases,5,6 metalloproteases,7,8 HMG-CoA reductase,9 and aspar-
tic proteases.10 An example of a non-catalytic protein group are the
carbohydrate binding proteins: the lectins. The galectins, a group
of 15 b-galactoside binding lectins, are a particularly interesting
subgroup thereof due to their involvement in many health related
biological processes.11 In particular, galectin-3 expression has been
correlated, with notable exceptions, with cancer aggressiveness,12
metastasis,13 and apoptosis.14 In this context galectin-3 can be
considered an emerging cancer marker.
to antibody based methods.11b In order to arrive at the improve-
ments, multivalency was introduced into the system in order
to increase galectin-3 affinity16 and lower the detection limit.17
Multivalency is known to be of great importance in the biological
mechanism of action of the galectins which often involves cross-
linking or aggregation. For galectin-3 this is particularly true as
this predominantly monomeric lectin was shown to aggregate
into a pentameric form of the lectin in the presence of divalent
ligands.18 A divalent lactoside, similar to the one presented here
was shown to increase binding 15-fold in a heamagglutination
inhibition study.16b The other factor favouring galectin-3 detection
is the presence of an aromatic group linked to the 3ꢀ position of
the used lactose ligand. Such substituents were shown to greatly
enhance the binding to galectin-3, i.e. over 200-fold, due to cation–
p interactions of the aryl group to a protein arginine residue.19
The detection principle and the used compounds are shown in
Scheme 1andChart1. The scheme shows the non-covalentbinding
of the probe (1) to a partial aggregate of galectin-3 followed by
photo-labelling. In the final step a fluorescent reporter molecule
(2) is introduced to allow visualization of the captured proteins
in a gel. Such a two step protocol has advantages such as fewer
artefacts due to non-specific effects of bulky labels in the crucial
We previously reported15 on a photoaffinity probe for the
profiling of galectins within samples of low protein complexity and
high galectin concentration. We here report on a significant leap
in development. Our focus has become the most cancer relevant
galectin-3. Our approach allowed the detection of galectin-3
in more complex mixtures of proteins, including carbohydrate
Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute
for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB,
Utrecht, The Netherlands
† Electronic supplementary information (ESI) available: Details of labeling
studies and MALDI MS and 1H and 13C NMR spectra. See DOI:
10.1039/b611050a
Scheme 1 Depiction of the capture strategy of galectin-3 in which
photocovalent attachment and chemoselective ligation are combined. The
incubation of a divalent ligand and galectin-3 leads to aggregate formation
and enhanced affinity.
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Chart 1 Structures of used probes 1 and 3 and reporter molecule 2.
photoreaction step. The alkyne moiety on the probe allows for
‘click chemistry’20 to introduce the fluorescent reporter molecule.
Subsequently an alkyne group was introduced into the molecule
by means of a BOP coupling of propargyl amine to the scaffold
carboxylic acid function. Deprotecting the lactose hydroxyls
yielded 1. In order to get an idea about the magnitude of the
operative multivalency effect for 1 a monovalent reference probe 3
was also prepared as shown in Scheme 3. This synthesis involved
the monofunctionalization of the divalent scaffold 7, followed by
Synthesis
The synthesis of probe 1 started by linking the previous probe
415 to the divalent scaffold 521 using ‘click’ chemistry (Scheme 2).
Scheme 2 Reagents and conditions: a) CuSO4, Na ascorbate, DMF–H2O, MW, 20 min, 84%; b) propargyl amine, BOP, DIPEA, DMF, 14 h, 94%; c)
MeONa–MeOH, quant.
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Scheme 3 Reagents and conditions: a) propargyl bromide (1 equiv.), K2CO3, DMF, 16 h, 33%; b) 4, CuSO4, Na ascorbate, DMF–H2O, MW, 20 min,
89%; c) propargyl bromide (1 equiv.), K2CO3, DMF, 14 h, 61%; d) MeONa–MeOH, quant.
linking of the photoligand 4. Finally incorporation of the alkyne
group and sugar deprotection yielded 3.
Probe evaluation
To evaluate the probe, protein mixtures were prepared. The first
mixture consisted of six common (marker) proteins and the
second mixture contained a series of seven carbohydrate binding
and processing proteins, including several galactose specific ones
(protein mix I and II22). To these mixtures various amounts of
galectin-3 were added and also various amounts of probe were
applied. Following the above protocol, the fluorescent gel images
obtained showed a strong and dominant band corresponding to
galectin-3, indicating good selectivity of the probe in both protein
mixtures (Fig. 1A). In the first mixture some minor labelling
of phosphorylase B (lane 5, 94 kDa) was observed. Strikingly,
in the second protein mixture the probe exhibited selectivity
for galectin-3 over galectin-1, as indicated by the absence of a
fluorescent band at 14 kDa in lane 8. A faint band at twice
the molecular weight of galectin-3 (≈60 kDa) was seen in a
sample only containing galectin-3 (lane 7), indicating the capture
of two copies of this protein. In order to confirm that binding
of the carbohydrate portion of the probe was a prerequisite for
detection, an experiment was performed with increasing amounts
of the competing lactose ligand (Fig. 1B). The experiment clearly
Fig. 1 (A) Evaluation of probe (1, 5 lM) in prepared protein mixtures.
showed that the signal of fluorescently labelled galectin-3 disap-
pears with the addition of larger quantities of the weak lactose
ligand.
Lane 1: protein mix I22 and lane 2: galectin-3, both silver stained. Lanes
3–5: Fluorescence images, galectin-3 (120 ng) in protein mix I (400 ng per
protein). Lane 3: no probe, lane 4: no light irradiation. Lanes 6 and 7:
galectin-3 (67 ng), silver stained and fluorescence image respectively; lane
8 fluorescence image of galectin-3 (40 ng) with protein mix II22 (40 ng
per protein). (B) Competition experiment between lactose and probe 1. A
sample of 50 ng galectin-3 was incubated with 10 lM divalent probe in
the presence of different concentrations of lactose. Top row: fluorescent
picture of labeled galectin-3. Bottom row: silver stain of the same gel, as a
loading control.
The most favourable probe (1) concentration was determined by
using a concentration range and was found to be around 5 lM (see
supporting information†) with a two-fold excess of fluorescein–N3
construct 2. Higher concentrations of 2 gave rise to saturated
fluorescence gels and thus poorly visible protein signals. With
these concentrations of 1 and 2 the minimal detection limit for
galectin-3 was found to be 1–5 ng which is clearly sufficient for
application.23 With the related monovalent probe 3 around 50 ng of
galectin-3 could be detected, thus confirming the benefits of multi-
valency.
Once the optimal probe concentration and the lower detection
limit were known, we proceeded to apply the methodology to an E.
coli and a human cell line lysate to explore the probe sensitivity in a
biologically complex environment (Fig. 2). The bacterial lysate was
spiked with various concentrations of galectin-3. Following the
protocol, fluorescent images of the gel clearly show that galectin-
3 can be detected under these conditions. In the corresponding
experiments with the CaCo2 lysate, which is known to express
galectin-3,24 the spiked sample showed a band corresponding to
the expected galectin-3 position (lane 9), but also in the non-
spiked sample a band is visible (lane 10). In order to confirm
the presence of endogenous galectin-3 in the CaCo2 cell line,
the presumed galectin-3 band was excised from the gel, digested
and analysed by MS. The analysis confirmed the presence of
galectin-3. Again experiments were run with increasing amounts of
competing lactose (lanes 5–8). At the higher lactose concentration
(1–10 mM) the galectin-3 lane fades while non-targeted ligands
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in biology and especially cancer and may ultimately aid in its
diagnosis and prognosis.
Experimental
General
All reagents were purchased from commercial sources and used
without further purification apart from solvents that were distilled
before use. Dowex 50 × 8 (H+ form, 20–50 mesh) from Fluka.
Analytical thin layer chromatography (TLC) was performed on
Merck pre-coated silica gel 60 F254 (0.25 mm) plates. Spots were
visualized with UV light, ninhydrin, or Cl2-TDM. Column chro-
matography was carried out using Merck Kieselgel 60 (40–63 mm).
1H NMR and 13C NMR were obtained on a Varian 300 MHz
spectrometer. Chemical shifts are given in ppm with respect to
internal TMS for 1H NMR. 13C NMR spectra were recorded using
the attached proton test (APT) pulse sequence. Two-dimensional
1H–1H correlation and total correlation spectroscopy (COSY and
TOCSY) and 1H–13C correlated heteronuclear single quantum
coherence (HSQC) NMR spectra (500 MHz) were recorded at
300 K with a Varian Unity INOVA 500 spectrometer.
Low resolution ES-MS experiments were performed on a Shi-
madzu LCMS QP8000 system. Exact masses were measured by na-
noelectrospray time-of-flight mass spectrometry on a Micromass
LCToF mass spectrometer at a resolution of 5000 fwhm. Gold-
coated capillaries were loaded with 1 lL of sample (concentration
20 lM) dissolved in a 1 : 1 (v/v) mixture of CH3CN–H2O with
0.1% formic acid. NaI or poly(ethylene glycol) (PEG) was added
as internal standard. The capillary voltage was set between 1100
and 1350 V, and the cone voltage was set at 30 V.
Fig. 2 Lane 1: labeling of 20 ng galectin-3. Lane 2–4: labeling of an E. coli
lysate with different amounts of galectin-3 and with no galectin-3 added.
The box indicates the galectin-3 position. Lane 9–10: fluorescent image
of human colon carcinoma lysate, with and without galectin-3 added.
The asterisk indicates labeling of galectin-3 present in this lysate, which
was confirmed by MALDI analysis. Lane 5–8: Competition experiment
between 10 lM divalent probe and increasing concentrations of lactose;
50 ng galectin-3 was added to 3 lg lysate. The box indicates the band of
galectin-3, which decreases in intensity with increasing concentrations of
lactose.
remained equally visible, clearly indicating that specific binding is
responsible for the galectin-3 detection.
Discussion/conclusions
We here present a probe-based system for the detection of the
cancer-linked galectin-3, which is considered an emerging cancer
marker. The chemical probe based method, as presented here,
may ultimately prove to be a valid alternative to the often used
antibody based methods as a detection method for active galectin-
3. However, the protein–carbohydrate interactions, governing the
probe–galectin-3 binding are notoriously weak, illustrating the
tremendous challenge to realize this goal. Two features of the
probe presented here have allowed a significant step forwards.
The first is the multivalency of the system and the second is the
use of a C3ꢀ aromatic substituent on the lactose ligand that is
known to greatly enhance binding to galectin-3. With probe 1
it proved possible to strongly and selectively identify galectin-3
from a mixture of carbohydrate binding and processing proteins,
including galactose binding ones and even the close cousin
galectin-1. Furthermore in this study it was possible to visualize
galectin-3 in spiked lysates of bacterial and human origin, despite
the fact that non-targeted, non-specifically bound and likely highly
abundant proteins were also labelled by the probe. Gratifyingly, in
an experiment with a non-spiked caco2 cell lysate, which should
contain significant amounts of endogenous galectin-3, it proved
possible to excise the corresponding fluorescent band and after
digestion and mass spectrometric analysis, it was unambiguously
identified as galectin-3. This experiment bodes well for further
development of the principle. In conclusion, we have shown the
utility of a galectin-3 specific divalent photoaffinity probe for the
visualization of galectin-3 in biological protein mixtures. This type
of probe in combination with various reporter molecules may
become a valuable tool in further deciphering the galectin-3 roles
3,5-Bis-{8-(1H-1,2,3-triazol-4-yl)-(3,6-dioxa)octyl-[2,4,6-tri-O-
acetyl-3-O-4-methylbenzophenon-b-D-galactopyranosyl-(1→4)-
2,3,6-tri-O-acetyl-b-D-glucopyranoside]methoxy}benzoic acid (6)
To a solution of 4 (172 mg, 0.182 mmol) and 7 (10.5 mg,
0.046 mmol) in DMF (2 mL), CuSO4·5H2O (3.4 mg, 0.014 mmol),
sodium ascorbate (5.4 mg, 0.028 mmol) and three drops of water
were added. The reaction mixture was exposed to microwave
irradiation for 20 min at 80 ◦C. TLC (DCM : MeOH, 9 : 1)
and showed at this point total consumption of 5. The mixture was
concentrated and purified on silica gel (DCM : MeOH, 20 : 1) to
afford 6 as a slightly yellow viscous oil (82 mg, 84%). Excess 4 was
recovered by chromatography. 1H NMR (500 MHz, CDCl3): 1.98,
2.01, 2.03, 2.06, 2.07, 2.13 (6 × s, 6 × 6H), 3.55–3.62 (m, 16H), 3.72
(t, J = 9.3, 2H), 3.80–3.89 (m, 8H), 4.06–4.11 (m, 6H), 4.40 (d, J =
8.3, 2H), 4.43–4.45 (m, 4H), 4.51 (d, J = 8.0, 2H), 4.54 (t, J = 4.9,
4H), 4.78 (d, J = 12.7, 2H), 4.85 (m, 2H), 5.06 (dd, J = 8.3, J =
9.8, 2H), 5.16 (t, J = 9.3, 2H), 5.20 (s, 4H), 5.49 (d, J = 2.9, 2H),
6.85 (s, 1H), 7.31–7.33 (m, 6H), 7.47 (m, 4H), 7.58 (t, 2H, J = 7.3),
7.74 (d, J = 7.8, 4H), 7.77 (d, J = 7.3, 4H), 7.85 (s, 2H); 13C NMR
(125 MHz, CDCl3): 20.9, 21.0, 21.1, 50.5, 61.6, 62.4, 65.6, 69.3,
69.6, 70.4, 70.7, 70.8, 70.9, 71.0, 71.2, 71.9, 72.9, 73.1, 76.4, 77.8,
100.8, 101.3, 107.3, 109.1, 124.6, 127.3, 128.5, 130.2, 130.4, 132.7,
137.2, 137.7, 142.3, 143.5, 159.6, 168.0, 169.5, 169.9, 170.1, 170.5,
170.7, 170.8; HR-ESI-MS calculated for C101H120N6O44 2121.742
[M + H]+ and 2143.724 [M + Na]+, found 2121.730 and 2143.702.
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slightly yellow viscous oil (71 mg; 89% yield). 1H NMR (500 MHz,
CDCl3): 1.98, 2.02, 2.03, 2.06, 2.07, 2.13 (6 × s, 6 × 3H), 3.53–3.55
(m, 8H), 3.64 (m, 1H), 3.70 (t, 1H, J = 9.3), 3.80–3.88 (m, 7H),
4.05–4.11 (m, 3H), 4.4–4.5 (m, 4H), 4.53 (t, 2H, J = 4.9), 4.77 (d,
1H, J = 12.2), 4.85 (t, 1H, J = 8.8), 5.04 (dd, 1H, J = 8.3, J = 9.3),
5.14 (t, 1H, J = 9.3), 5.2 (s, 2H), 5.49 (d, 1H, J = 2.5), 6.73 (s, 1H),
7.15 (s, 2H), 7.32 (d, 2H, J = 7.8), 7.47 (dd, 2H, J = 7.3, J = 7.8),
7.58 (t, 1H, J = 7.3), 7.72 (br s, 1OH) 7.75 (d, J = 7.8, 2H), 7.77 (d,
J = 7.8, 2H), 7.87 (s, 1H); 13C NMR (125 MHz, CDCl3): 20.9, 21.0,
21.1, 50.6, 52.5, 61.6, 62.2, 62.3, 65.6, 69.4, 69.5, 70.4, 70.7, 70.8,
70.9, 71.0, 71.2, 71.8, 72.9, 73.1, 76.4, 77.8, 100.8, 101.4, 107.5,
107.6, 110.4, 124.7, 127.3, 128.6, 130.3, 130.5, 132.4, 132.8, 137.3,
137.7, 142.3, 143.6, 158.0, 159.5, 167.0, 169.7, 170.1, 170.2, 170.6,
170.8, 170.9; HR-ESI-MS calculated for C55H65N3O24 1152.404
[M + H]+ and 1174.386 [M + Na]+, found 1152.410 and 1174.380.
3,5-Bis-{8-(1,2,3-triazol-4-yl)-(3,6-dioxa)octyl-[3-O-4-
methylbenzophenone)-b-D-galactopyranosyl-(1→4)-
b-D-glucopyranoside]methoxy}benzoic acid propargyl amide (1)
Carboxylic acid 6 (97 mg, 0.045 mmol) and propargylamine
hydrochloride (7 mg, 0.09 mmol) were dissolved in DMF (2 mL)
and the pH was adjusted to approximately 8 using DIPEA. BOP
(20 mg, 0.045 mmol) was added and the pH was checked and
adjusted again. The mixture was stirred overnight when TLC
(DCM : MeOH, 9 : 1) indicated total conversion of the starting
material. The mixture was concentrated and loaded onto a silica
column for chromatography (DCM : MeOH, 20 : 1). The amide
was obtained as a colorless viscous oil (91 mg; 94% yield). Part
of this material compound (49 mg, 0.023 mmol) was treated with
a 5 M NaOMe solution (0.2 mL) in MeOH (2 mL). The reaction
was neutralized with Dowex H+ after 3 h of stirring at room
temperature. The solution was filtered off and concentrated to
dryness to give to give 1 in quantitative yield. 1H NMR (500 MHz,
MeOD): 2.62 (s, 1H), 3.26 (m, 2H), 3.35 (m, 2H), 3.44 (dd, J =
2.9, J = 9.8, 2H), 3.50 (m, 4H), 3.57–3.64 (m, 16H), 3.67–3.84 (m,
10H), 3.87–3.92 (m, 6H), 4.09 (m, 2H), 4.13 (m, 2H), 4.28 (d, J =
7.8, 2H), 4.36 (d, J = 7.8, 2H), 4.6 (t, J = 4.9), 4.78 (d, J = 12.6,
2H), 4.86 (m, 12OH and 2H), 5.23 (s, 4H), 6.89 (s, 1H), 7.13 (d,
J = 1.9, 2H), 7.53 (t, J = 7.8, 4H), 7.62–7.66 (m, 6H), 7.74–7.76
(m, 8H), 8.19 (s, 2H); 13C NMR (125 MHz, CD3OD): 29.1, 50.4,
60.8, 61.3, 61.6, 65.9, 68.6, 69.1, 70.1, 70.2, 70.6, 70.7, 71.2, 73.5,
75.1, 75.2, 75.7, 79.7, 81.6, 103.1, 103.9, 105.6, 106.8, 125.4, 127.5,
128.4, 129.8, 130.0, 132.7, 136.2, 136.6, 137.7, 143.2, 144.2, 159.8,
167.8; HR-ESI-MS calculated for C80H99N7O31 1654.646 [M + H]+
and 1676.628 [M + Na]+, found 1654.641 and 1676.621.
Methyl 3-(prop-2-ynyl)-5-{8-(1H-1,2,3-triazol-4-yl)-(3,6-dioxa)-
octyl-[2,4,6-tri-O-acetyl-3-O-4-methylbenzophenon)-b-D-
galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-b-D-
glucopyranoside]methoxy}benzoate (9)
To a solution of compound 8 (72 mg, 0.063 mmol) and K2CO3
(17 mg, 0.12 mmol) in DMF (1 mL), propargyl bromide (80%
solution in toluene, 14 ll, 0.12 mmol) was added. The mixture
was stirred overnight. After TLC (DCM : MeOH, 9 : 1) showed
completion, the mixture was loaded onto silica gel for column
chromatography (DCM : MeOH, 50 : 1 to 20 : 1) to give 9 as a
1
colorless oil (46 mg, 61%). H NMR (500 MHz, CDCl3): 2.00,
2.03, 2.04, 2.08, 2.09, 2.10 (6 × s, 6 × 3H), 2.55 (s, 1H), 3.53–3.58
(m, 9H), 3.68 (m, 1H), 3.74 (dd, J = 8.8, J = 9.8, 1H), 3.80–3.82
(m, 1H), 3.87–3.91 (m, 6H), 4.08–4.12 (m, 3H), 4.40 (d, J = 8.3,
1H), 4.40–4.46 (m, 2H), 4.53 (d, J = 8.3, 1H), 4.56 (t, J = 4.9,
2H), 4.72 (d, J = 2.4, 2H), 4.79 (d, J = 12.7, 1H), 4.87 (dd, J =
8.3, J = 9.3, 1H), 5.07 (dd, J = 8.3, J = 9.8, 1H), 5.18 (t, J = 9.7,
1H), 5.23 (s, 2H), 5.50 (d, J = 2.5), 6.84 (t, J = 2.4, 1H), 7.27 (s,
1H), 7.33–7.35 (m, 3H), 7.49 (t, J = 7.3, 2H), 7.60 (t, J = 7.3, 1H),
7.76–7.80 (m, 4H), 7.83 (s, 1H); 13C NMR (125 MHz, CDCl3): 20.9,
21.0, 21.1, 50.6, 52.6, 56.3, 61.6, 62.3, 62.5, 65.5, 69.3, 69.7, 70.5,
70.5, 70.8, 71.0, 71.2, 71.9, 72.9, 73.1, 76.3, 76.5, 77.9, 78.2, 100.8,
101.4, 107.5, 108.8, 109.2, 124.4, 127.3, 128.6, 130.2, 130.5, 132.5,
132.7, 137.3, 137.8, 142.2, 143.5, 158.8, 159.6, 166.7, 169.3, 169.9,
170.0, 170.5, 170.6, 170.7; HR-ESI-MS calculated for C58H67N3O24
1190.419 [M + H]+ and 1212.401 [M + Na]+, found 1190.416 and
1212.399.
Methyl 3-hydroxy-5-{8-(1H-1,2,3-triazol-4-yl)-(3,6-dioxa)octyl-
[2,4,6-tri-O-acetyl-3-O-4-methylbenzophenone)-b-D-galacto-
pyranosyl-(1→4)-2,3,6-tri-O-acetyl-b-D-glucopyranoside]-
methoxy}benzoate (8)
To a solution of methyl 2,4-dihydroxybenzoate (1 g, 5.95 mmol) in
DMF (20 mL) K2CO3 (0.822 g, 5.9 mmol) and propargyl bromide
(80% solution in toluene) (0.66 mL, 5.9 mmol) were added and
the mixture was stirred for 16 h at rt. The crude product was
then filtered through Hyflo SuperCell, concentrated to dryness,
dissolved in EtOAc and washed 3 × with water. The organic
layer was dried (K2SO4) and concentrated again. TLC (hexane :
EtOAc, 1 : 1) showed a mixture of starting material and mono- and
dialkylated products. Silica gel column chromatography (hexane :
EtOAc, 3 : 1) gave monoalkylated product methyl 3-hydroxy-5-
(prop-2-ynyloxy)benzoate (0.40 g; 33% yield) as a white solid. 1H
NMR (500 MHz, CDCl3): 2.55 (s, 1H), 3.92 (s, 3H), 4.72 (d, 2H,
J = 2.4), 5.25 (s, 1H), 6.71 (d, 1H, J = 2.4), 7.2 (s, 1H), 7.24 (d, 1H,
J <1); 13C NMR (125 MHz, CDCl3): 52.6, 56.3, 76.2, 78.2, 107.8,
108.4, 110.3, 132.4, 156.9, 158.9, 166.9; HR-ESI-MS calculated
for C11H10O4 207.0657 [M + H]+ and 229.0477 [M + Na]+, found
207.0661 and 229.0482. This compound (15 mg, 0.07 mmol)
and 4 (82 mg, 0.09 mmol) were dissolved in DMF (2 mL) and
CuSO4·5H2O (2.7 mg, 0.01 mmol) and sodium ascorbate (4.3 mg,
0.022 mmol) were added together with 3 drops of water. This
mixture was exposed to microwave irradiation at 80 ◦C for 20 min.
The mixture was concentrated and purified by silica column
chromatography (DCM : MeOH, 20 : 1 to 10 : 1) to afford 8 as a
3-(Prop-2-ynyl)-5-bis-{8-(1,2,3-triazol-4-yl)-(3,6-dioxa)octyl-[3-
O-4-methylbenzophenon)-b-D-galactopyranosyl-(1→4)-b-D-
glucopyranoside]methoxy}benzoic acid (3)
Compound 9 (30 mg, 0.025 mmol) was dissolved in a mixture of
dioxane (1.4 mL) and MeOH (0.5 mL) and 4 N NaOH (0.1 mL,
Tesser’s base) and the reaction was stirred for 5 h. The resulting
mixture was neutralized with Dowex H+, filtered and concentrated
in vacuo to give 3 (21 mg, 90%). 1H NMR (500 MHz, MeOD): 2.98
(t, J = 2.4, 1H), 3.26 (m, 1H), 3.41–3.43 (m, 1H), 3.44 (dd, J =
2.9, J = 9.8, 1H), 3.52–3.54 (m, 2H), 3.57–3.64 (m, 8H), 3.67–3.84
(m, 5H), 3.87–3.92 (m, 3H), 3.95–3.98 (m, 1H), 4.09 (d, J = 2.9,
1H), 4.30 (d, J = 7.8, 1H), 4.38 (d, J = 7.8, 1H), 4.60 (t, J = 4.9,
2H), 4.77 (d, J = 2.44, 2H), 4.77–4.90 (m, 6OH and 2H), 5.23
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(s, 2H), 6.86 (s, 1H), 7.27 (s, 1H), 7.3 (s, 1H), 7.54 (t, J = 7.8,
2H), 7.63–7.67 (m, 3H), 7.76–7.78 (m, 4H), 8.16 (s, 1H). 13C NMR
(125 MHz, CD3OD): 50.4, 55.8, 60.8, 61.3, 61.6, 65.9, 68.6, 69.1,
70.1, 70.2, 70.6, 70.7, 73.5, 75.1, 75.3, 75.7, 76.0, 78.3, 79.6, 81.6,
103.1, 103.9, 106.5, 108.7, 108.9, 125.3, 127.4, 128.3, 129.8, 129.9,
132.6, 134.2, 136.7, 137.8, 143.3, 144.2, 159.0, 159.5; HR-ESI-MS
calculated for C45H53N3O18 924.340 [M + H]+ and 946.322 [M +
Na]+, found 924.341 and 946.323.
from yeast (Serva 22828); b-galactosidase from E. coli (Boehringer
1510220, 105031); b-glucosidase from almonds (Sigma G4511);
galectin-1 (R & D Systems 1152-GA-CF), lectin from Arachis
hypogaea (peanut), peanut agglutinin, PNA (Sigma L0881); lectin
from Helix aspersa (garden snail), helix aspersa agglutinin, HAA
(Sigma L6635); galactosyl transferase from bovine milk (Fluka
48279) was added to each one of these samples. The resulting
solutions were irradiated for 30 min under a 366 nm UV lamp
at 4–5 cm distance at 4 ◦C. After photoincubation, 5 lL of
ligand (100 lM in DMSO : tert-butyl alcohol, 1 : 4), 5 lL of
CuSO4 (50 mM in water) and a two fold excess with respect
to probe quantity of fluorescein–azide conjugate 2 (10 lL of a
50 lM solution in water) were added and the volume adjusted
to 100 lL. The samples were then gently shaken for 1 h at rt
and denatured (50 lL of a 10% SDS, 40% glycerol and 2% DTT
solution) boiling at 95 ◦C for 5 min. From the total volume
of 150 lL, 20 lL were loaded onto a 15% Tris-HCl gel for
SDS-PAGE. After extensive washing of the developed gel (1–
2 h in water) to eliminate excess dye reagent, fluorescence was
detected with a Typhoon fluorescence scanner and each gel was
subsequently silver stained (in Fig. 1, only lane 8 is shown with
40 ng of galectin-3 i.e. 3 lL galectin-3, see supporting information
for the rest†).
Synthesis of fluorescein–azide adduct (2)
6-[Fluorescein-5(6)-carboxamido]hexanoic acid N-hydroxysucci-
nimide ester (Sigma) (20 mg, 0.025 mmol, 75% purity)
was dissolved in MeOH (0.6 mL) and 1-amino-11-azido-
3,6,9-trioxaundecane (Toronto Research Chemicals) (16 mg,
0.075 mmol) was added to the stirring solution. DIPEA was then
added dropwise until the solution reached pH 8–9 and the mixture
was stirred overnight. The resulting solution was concentrated to
dryness and loaded onto a silica gel for column chromatography
(DCM : MeOH, 10 : 1) to give fluorescein-azide adduct (2) as a
yellow solid (14 mg, 81%). 1H NMR (300 MHz, CD3OD) 8.45 (d,
J = 2.0 Hz, 1H), 8.06 (m, 1H), 7.67 (d, J = 2.0 Hz, 1H), 7.33 (d,
J = 8.0 Hz, 1H), 7.04 (m, 2H), 6.57 (m, 2H), 3.08–3.66 (m, 26H).
HR-ESI-MS calculated for C35H39N5O10 690.278, [M + H]+, found
690.279.
Lactose competition experiments
Galectin-3 labeling studies in presence of protein mixture I
Stock solutions of lactose (1, 10, 100 mM) in 50 mM HEPES buffer
pH 7.4, 150 mM NaCl were prepared. For a concentration range
of 0.1, 1 and 10 mM, 2 ll from the lactose stock solutions were
added, together with 1.2 ll galectin-3 stock solution (125 lg ml−1
in HEPES buffer), 4 ll probe 1 stock solution (50 lM in water :
DMSO 20 : 1) and in the case of the lysate experiment 2.4 ll
human cell lysate (4.3 mg mL−1 stock as determined by BCA
protein concentration assay), to a final sample volume of 20 lL
(in HEPES buffer). The samples were irradiated as described
above. After photoincubation, 3 lL of fluorescein azide conjugate
2 (100 lM stock in water), 0.8 lL CuSO4 and 0.8 lL TCEP (both
50 mM stock in water) were added and the final sample volume
was adjusted to 40 lL. The samples were gently shaken for 1 h
at rt and denatured (20 lL of a 10% SDS, 40% glycerol and 2%
DTT solution) boiling at 95 ◦C for 5 min. From the total sample
volume of 60 lL, 20 lL was loaded onto a 15% Tris-HCl gel for
SDS-PAGE. After extensive washing of the developed gel (1–2 h
in water), fluorescence was detected with a Typhoon fluorescence
scanner and each gel was subsequently silver stained.
Solutions of probe 1 (10, 5, 2, 1, and 0.5 lL of a 50 lM stock
solution in water : DMSO 20 : 1) were diluted to 35 lL total
volume in HEPES buffer pH 7.4, 150 mM NaCl. A 5 lL aliquot
of galectin-3 stock solution (human, recombinant form, carrier
free, R & D Systems Europe, Abingdon, UK) (125 lg mL−1 in
HEPES buffer) together with 10 lL of protein mix I (200 lg
mL−1 of each protein: a-lactalbumin, trypsin inhibitor, carbonic
anhydrase, ovalbumin, albumin and phosphorylase B) was added
to each one of these samples and the resulting solutions were
irradiated for 30 min under a 366 nm UV lamp at 4–5 cm distance
at 4 ◦C. After photoincubation, 5 lL of ligand (100 lM in DMSO :
tert-butyl alcohol, 1 : 4),25 5 lL of CuSO4 (50 mM in water) and a
two fold excess with respect to probe quantity of fluorescein–azide
conjugate 2 (20, 10, 4, 2, and 1 lL respectively of a 50 lM solution
in water) were added and the volume adjusted to 80 lL. The
samples were then gently shaken for 1 h at rt and denatured (20 lL
of a 10% SDS, 40% glycerol and 2% DTT solution) boiling at 95 ◦C
for 5 min. From the total volume of 100 lL, 20 lL were loaded onto
a 15% Tris-HCl gel for SDS-PAGE. After extensive washing of
the developed gel (1–2 h in water) to eliminate excess dye reagent,
fluorescence was detected with a Typhoon fluorescence scanner
and each gel was subsequently silver stained (only the lane with
5 lL probe (5 lM) is shown in Fig. 1 (lane 5), see supporting
information for the rest†).
Labelling of galectin-3 spiked in bacterial and human cell extract
by probe 1
To 40 lL cell lysate (with a protein concentration of 3 lg mL−1)
(or, in lane 1, 40 lL 50 mM HEPES buffer pH 7.4) 0, 1.2 or
3 lL (respectively 0, 20 or 50 ng lane−1) of a 125 lg mL−1 stock
solution of galectin-3 (in HEPES buffer pH 7.4) was added. An
aliquot of 10 lL of probe 1 was added and the total volume was
adjusted to 55 lL. The resulting solutions were photoactivated
and submitted to conjugation to the fluorescein–azide dye 2 and
20 lL from a total sample volume of 150 lL was loaded onto a
gel and visualized as described above.
Galectin-3 labeling studies in presence of protein mixture II
Probe 1 (5 lL of a 50 lM stock solution in water : DMSO
20 : 1) was diluted to 35 lL total volume in HEPES buffer
pH 7.4, 150 mM NaCl. Decreasing amounts of galectin-3 were
added to each of the samples: 5, 3, 2, 1 and 0.5 lL of galectin-
3 stock solution (100 lg mL−1 in HEPES buffer) together with
3 lL of protein mixture II (100 lg mL−1 in each a-glucosidase
4392 | Org. Biomol. Chem., 2006, 4, 4387–4394
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Table 1 Characteristics of the identified galectin-3, notably the number of peptides used for identification of the protein, the Mascot score and the
percentage sequence coverage
Protein ID
Galectin-3
Accession NCBI
gi|48145911
MW (Da)
26057
pI
# peptides
9
Mascot score
346
SQ (%)
35.3
8.60
Bacterial protein extraction
Fellowship to L.B. under contract MCFI-2002-00249, and a Marie
Curie Training Site Fellowship to K.B. under contract QLK2-CT-
2000-60047. M.v.S. was supported by the Netherlands Proteomics
Centre (NPC).
E. coli cells (BL21(DE3) from Novagen, OD600 2.2, 1.5 ml) were
pelleted by centrifugation at 5000 rpm for 10 min. The cells were
resuspended in 300 ll of B-Perꢀ Reagent (Pierce, no. 78243) and
R
vortexed for 2 min. The homogeneous mixture was centrifuged at
13000 rpm for 5 min. The supernatant was taken for the labeling
studies. Protein concentrations of the solutions were determined
by BCA protein concentration assay, using bovine serum albumin
as a standard.
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©