Teaming up synthetic chemistry and histochemistry for activity screening in galectin-directed inhibitor design

Article abstract of DOI:10.1007/s00418-016-1525-5

A hallmark of endogenous lectins is their ability to select a few distinct glycoconjugates as counterreceptors for functional pairing from the natural abundance of cellular glycoproteins and glycolipids. As a consequence, assays to assess inhibition of le

Full text of DOI:10.1007/s00418-016-1525-5

Histochem Cell Biol
DOI 10.1007/s00418-016-1525-5
ORIGINAL PAPER
Teaming up synthetic chemistry and histochemistry for activity
screening in galectin‑directed inhibitor design
1
1
2
1
1
René Roy · Yihong Cao · Herbert Kaltner · Naresh Kottari · Tze Chieh Shiao ·
1
2
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Karima Belkhadem · Sabine André · Joachim C. Manning · Paul V. Murphy ·
Hans‑Joachim Gabius
2
Accepted: 23 November 2016
©
Springer-Verlag Berlin Heidelberg 2016
Abstract A hallmark of endogenous lectins is their ability
to select a few distinct glycoconjugates as counterreceptors
for functional pairing from the natural abundance of cellular
glycoproteins and glycolipids. As a consequence, assays to
assess inhibition of lectin binding should necessarily come as
close as possible to the physiological situation, to character-
ize an impact of a synthetic compound on biorelevant bind-
ing with pharmaceutical perspective. We here introduce in
a proof-of-principle manner work with sections of paraffin-
embedded tissue (jejunum, epididymis) and labeled adhesion/
growth-regulatory galectins, harboring one (galectin-1 and
galectin-3) or two (galectin-8) types of lectin domain. Six
pairs of synthetic lactosides from tailoring of the headgroup
of reduction in staining intensity by synthetic compounds
relative to unsubstituted/free lactose proved the applicabil-
ity and sensitivity of the method. Flanking cytofluorimetric
assays on lectin binding to native cells gave similar grad-
ing, excluding a major impact of tissue fixation. The experi-
ments revealed cell/tissue binding of galectin-8 preferentially
via one domain, depending on the cell type so that the effect
of an inhibitor in a certain context cannot be extrapolated to
other cells/tissues. Moreover, the work with the other galec-
tins attests that this assay enables comprehensive analysis
of the galectin network in serial tissue sections to determine
overlaps and regional differences in inhibitory profiles.
(3′-O-sulfation) and the aglycone (β-methyl to aromatic S-
Keywords Agglutinin · Glycocluster · Glycoprotein ·
and O-linked extensions) as well as three bi- to tetravalent
Glycosylation · Lectin
glycoclusters were used as test compounds. Varying extents
Introduction
Yihong Cao and Herbert Kaltner have contributed equally to this
work.
The glycan part of cellular glycoconjugates is increasingly
considered to harbor physiologically relevant information
Electronic supplementary material The online version of this
article (doi:10.1007/s00418-016-1525-5) contains supplementary
material, which is available to authorized users.
(Gabius 2009, 2015; Gabius and Roth 2017). In this sense,
the molecular characterization of its complexity and of the
dynamics of regulation is a step toward elucidating the range
of implied functions (Kopitz 2009, 2017; Zuber and Roth
*
*
René Roy
roy.rene@uqam.ca
2
009; Corfield 2015, 2017; Corfield and Berry 2015; Hen-
net and Cabalzar 2015; Ledeen and Wu 2015; Schengrund
015; Bhide and Colley 2017; Roth and Zuber 2017).
Hans-Joachim Gabius
gabius@tiph.vetmed.uni-muenchen.de
2
1
2
3
Pharmaqam and Nanoqam, Department of Chemistry,
University du Québec à Montréal, P.O. Box 8888, Succ.
Centre-Ville, Montreal, QC H3C 3P8, Canada
Toward this end, the application of plant/fungal lectins for
glycan profiling has considerably advanced our status of
knowledge on glycan localization and sites of glycosyla-
tion (Roth 1978, 2011; Roth et al. 1983; Danguy et al.
1994; Manning et al. 2017). Although such work with a
heterologous combination (plant lectin/mammalian tissue)
is mostly of descriptive merit and cannot predict respective
Faculty of Veterinary Medicine, Institute of Physiological
Chemistry, Ludwig-Maximilians-University Munich,
Veterinärstr. 13, 80539 Munich, Germany
School of Chemistry, National University of Ireland Galway,
University Road, Galway, Ireland
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3

Histochem Cell Biol
reactivity for tissue lectins, as recently emphasized (Topin
et al. 2016), the broadly successful mapping with these tools
provided the incentive to proceed to introduce tissue lectins
to cyto- and histochemistry (for information on animal lec-
tin classes, structure and functions, please see Gabius 1987,
with respect to the ligand side, binding 3′-sulfated lactose
is also documented for galectin-1, galectin-3 and galectin-4
(Gal-1, Gal-3 and Gal-4) (Allen et al. 1998; Ideo et al. 2002;
for information on the galectin network, please see Kalt-
ner and Gabius 2012; García Caballero et al. 2016; Kaltner
et al. 2017). Shared reactivity, indeed, is of physiological
relevance. The synthetic preparation of sulfated lactosides
was conducive to delineate the cooperation of Gal-1 and
Gal-8 in differentiating B cells into plasma cells via block-
ing assays, illustrating functional overlap within the galec-
tin network (Tsai et al. 2011; Tu et al. 2013). Such a coop-
eration (or also antagonism) is possible for other processes
involving galectins such as pathogenesis of osteoarthritis
or tumor growth regulation (Kopitz et al. 2001; Sanchez-
Ruderisch et al. 2010; Toegel et al. 2014).
This collective evidence prompted us to give emphasis
in this pilot study to Gal-8 and also consider Gal-1 and Gal-
3. In detail, we explored comparatively the effect of agly-
conic extension of lactosides without/with 3′-O-sulfation
on their ligand properties to these human galectins, first by
screening in a solid-phase assay and then for selected cases
in cell assays. The question whether and how staining of
tissue sections by labeled Gal-8 is affected by such deriva-
tives was then addressed. Finally, three bi- to tetravalent
glycoclusters, known to reduce Gal-1 and Gal-3 binding in
solid-phase and cell assays (Wang et al. 2012), were tested
in the histochemical setting.
1997, 2002; Solís et al. 2015; Bhide and Colley 2017; Kalt-
ner et al. 2017; Manning et al. 2017; Mayer et al. 2017).
When it has been accomplished, this approach offers the
potential to examine characteristics of the functional pairing
in situ and strategies to block disease-associated binding.
A key finding from biochemical analysis of cellular bind-
ing partners of tissue lectins is that only a few glycoconju-
gates qualify as counterreceptors: when faced with the abun-
dance of cell glycans, endogenous lectins have a particular
selectivity among the diversity of glycoconjugates, engag-
ing few distinct counterreceptors to complete their mission
(Gabius et al. 2015, 2016; for examples and signaling routes
triggered by this interaction, please see Kaltner et al. 2017). In
addition to the structure of the target glycan(s), not yet fully
delineated topological parameters of their presentation on
the scaffold (glycoprotein, glycolipid or a complex thereof)
or in a microdomain appear to matter (Murphy et al. 2013;
Roy et al. 2016). As first critically discussed in the case of the
selectins, common assay systems “can give a positive result
that may not be relevant to the natural situation” (Varki 1994)
so that the question of identifying the ‘real’ ligands becomes
highly relevant (Varki 1997). This said, cultured cells with
their natural display of glycans are coming into focus as
platform to examine lectin binding and then the potency of
synthetic compounds to interfere with a functional pairing.
In the quest of taking this testing even closer to the level of
the in vivo situation, sections of tissues deserve attention and
efforts. After all, the actual microenvironment is known to
have a profound influence on cell properties. Having recently
documented the graded capacity of bi- to tetravalent glyco-
clusters to reduce the extent of binding of two plant lectins
to tissue sections (André et al. 2016), we here introduce this
assay concept to human galectins and three types of synthetic
compounds, i.e., free glycans with headgroup modification/
aglyconic extension and glycoclusters. The tandem-repeat-
type galectin-8 (Gal-8) is a challenging test case.
Materials and methods
General synthetic methods
All reactions in organic medium were performed in stand-
ard oven-dried glassware under an inert atmosphere of
nitrogen using freshly distilled solvents. Solvents and rea-
gents were deoxygenated, when necessary by purging with
nitrogen. All reagents were used as supplied without prior
purification unless otherwise stated, and obtained from
Sigma-Aldrich Chemical Co. Ltd. Reactions were moni-
tored by analytical thin-layer chromatography (TLC) using
silica gel 60 F254 precoated plates (E. Merck), and com-
pounds were visualized with a 254-nm UV lamp, a mix-
ture of iodine/silica gel and/or mixture of ceric ammonium
molybdate solution [100 mL H SO , 900 mL H O, 25 g
Structurally, a linker peptide connects the two different
carbohydrate recognition domains (CRDs) of Gal-8 (Hadari
et al. 1997; Zick et al. 2004). They have high affinity for
either 3′-sialylated/sulfated lactosides (Gal-8N) or histo-
blood group A/B epitopes (Gal-8C) and recognize N-acetyl-
lactosamine (LacNAc) in high-density presentation, both
CRDs being involved in letting the lectin associate with
cells (Hirabayashi et al. 2002; Ideo et al. 2003, 2011; Carls-
son et al. 2007; Stowell et al. 2008; Vokhmyanina et al.
2
4
2
(NH ) Mo O H O, 10 g Ce(SO ) ] and subsequent spots’
4
6
7
24
2
4 2
development by gentle warming with a heat gun. Purifi-
cations were performed by silica gel flash column chro-
matography using Silicycle (60 Å, 40–63 µm) with the
indicated eluent. NMR spectroscopy was used to record
1
13
2012; Yoshida et al. 2012; Ruiz et al. 2014, 2015). Thus, the
H NMR and C NMR spectra at 300 or 600 MHz and at
individual contributions of each CRD to a binding process
require to be elucidated in each case. Equally interesting
75 or 150 MHz, respectively, on Bruker (300 MHz) and
Bruker Avance III HD 600-MHz spectrometers. Proton
1
3

Histochem Cell Biol
O
O
OH
O
OH
O
S
Na O-
+
O
O
HO
X
R
HO
OH
HO
2
3
. NaOMe, MeOH
rt,1h
. i) Bu2SnO
DMF/Tol
OAc 1. HXR, TBAHS
OAc
OAc
O
AcO OAc
OAc
O
EtOAc, 1M Na2CO3
OH
O
AcO
O
O
OH
O
HO OH
or
AcO
O
X
90°C, 6h
O
AcO
AcO
R
YO
O
X
AcO
HO
R
AcO OAc
AcO
ii) SO NMe
3.
DMF
3
Br KOH 1M / DCM 1:1
TBAB, rt, 2h30
HO
1
rt, 17h
O
O
1
0 Y = H;
XR =
6
XR =
1
1 Y = SO3Na; XR =
OR
O
OR
S
S
YO
O
X
7 XR =
12 Y = H;
XR =
O
RO
13 Y = SO Na; XR =
3
RO
RO OR
O
O
O
2
3
4
4
5
X = OMe, Y = H
X = OMe, Y = SO3Na
a X = N3, Y = R = Ac
b X = N3, Y = H, R = H
X = N3, Y = SO3Na, R = H
8
9
XR =
XR =
14 Y = H;
XR =
1
5 Y = SO3Na; XR =
O
O
O
O
OMe
OMe
16 Y = H;
XR =
O
17 Y = SO3Na; XR =
CO2Me
CO2Me
Fig. 1 The two sites of lactose modifications highlighted in blue and red (top) and the synthetic steps leading to lactoside derivatives with six
types of aglyconic extension and their 3′-O-sulfated derivatives
and carbon chemical shifts (δ) are reported in ppm relative
the corresponding aromatic alcohol (1 equiv.), tetrabutyl-
ammonium bromide (TBAB, 0.5 equiv.) and KOH (1 M, 6
equiv.). The mixture was stirred at room temperature for 2 h
30 min and then washed successively with water and brine.
The organic layer was dried over Na SO and concentrated
under reduced pressure. Purification by silica gel column
chromatography (Hex/AcOEt: 7/3) afforded the correspond-
ing compounds 6–9 as yellow oil (yield 70–81%).
to the chemical shift of residual CHCl , which was set at
3
1
13
7
(
.26 ppm ( H) and 77.16 ppm ( C). Coupling constants
J) are reported in Hertz (Hz), and the following abbrevia-
tions are used for peak multiplicities: singlet (s), doublet
d), doublet of doublets (dd), doublet of doublet with equal
2
4
(
coupling constants (t ), triplet (t), multiplet (m). Analysis
ap
and assignments were carried out using correlated spectros-
copy (COSY) and heteronuclear single quantum coherence
(
(
HSQC) experiments. High-resolution mass spectrometry
HRMS) data were measured with a liquid chromatogra-
General synthetic procedure B: Zemplén transesterification
reaction
phy–mass spectrometry–time of flight, Agilent Technolo-
gies (LC–MS–TOF), in positive and/or negative electro-
spray mode(s) at the analytical platform of UQAM.
To a solution of lactosides 6–9 in dry methanol was added
a solution of sodium methoxide (1 M in MeOH, 0.1 equiv.).
After stirring at room temperature for 1 h, the reaction
was completed and then neutralized by addition of ion-
Synthesis of the ligand panel
+
exchange resin (Amberlite IR 120 H ). The solution was
General synthetic procedure A: phase‑transfer catalysis
filtered and evaporated in vacuo to afford the de-O-acety-
(
PTC) reaction
lated lactosides as white powders (yield 95% quant).
PTC reactions were performed following the previously
established protocols (Roy 1997; Roy et al. 1997; Tropper
et al. 1991, 1992a, b; Cao et al. 1994; Carrière et al. 2000) or
under the slightly modified procedure as follows: to a solu-
tion of peracetylated lactosyl bromide 1 (Fig. 1; Shiao et al.
General synthetic procedure C: preparation
of 3′‑O‑sulfated lactosides
A mixture of deacetylated lactosides (1 equiv.) and dibutyl-
tin oxide (1.15 equiv.) in DMF/toluene (6 mL/3 mL) was
stirred at 90 °C for 6 h. The solution was then concentrated,
2014) (1 equiv.) in dichloromethane (6 mL) were added
1
3

Histochem Cell Biol
and sulfur trioxide–trimethylamine complex (Me N·SO )
2,3,4,6‑Tetra‑O‑acetyl‑β‑d‑galactopyranosyl‑
3
3
(
1.3 equiv.) and dry DMF (6 mL) were added. After stirring
(1 → 4)‑2,3,6‑tri‑O‑acetyl‑β‑d‑glucopyranosyl azide
at room temperature for 17 h, the reaction was quenched
with water and evaporated under vacuum. The residue was
(compound 4a)
+
purified through a column of DOWEX Marathon C (Na )
Toasolutionof2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl-
(1 → 4)-2,3,6-tri-O-acetyl-α-d-glucopyranosyl bromide 1
(515 mg, 0.736 mmol, 1.0 equiv.) in ethyl acetate
and eluted with H O to obtain the pure 3′-O-sulfated lacto-
2
sides as white powder after lyophilization (yields 80–84%).
(1.0 mL/100 mg of sugar, 5.2 mL) were added sodium
2
,3,4,6‑Tetra‑O‑acetyl‑β‑d‑galactopyranosyl‑
azide (NaN , 239 mg, 3.68 mmol, 5.0 equiv.), tetrabutylam-
3
(
(
1 → 4)‑2,3,6‑tri‑O‑acetyl‑α‑d‑glucopyranosyl bromide
monium hydrogen sulfate (TBAHS, 255 mg, 0.736 mmol,
1.0 equiv.) and a saturated aqueous solution of Na CO
acetobromolactose) (compound 1)
2
3
(1.0 mL/100 mg of sugar, 5.2 mL). The mixture was vig-
To a solution of per-O-acetylated lactose (Shiao et al. 2014)
14.2 g, 21 mmol) in anhydrous CH Cl (63 mL) was added
hydrobromic acid (33% in AcOH, 47.9 mL). The reaction
mixture was stirred at room temperature for 1 h, then neu-
orously stirred for 2 h until disappearance of the starting
material, as judged by TLC (hexane–EtOAc 2:3). The
organic layer was separated, washed with a saturated aque-
ous NaHCO solution (3 × 15 mL), H O (15 mL), brine
(
2
2
3
2
tralized with saturated aqueous NaHCO and washed with
(15 mL), then dried over Na SO , filtered and concentrated
3
2 4
brine. The organic layer was dried over Na SO and con-
under reduced pressure. The white solid crude product was
then purified by crystallization in a mixture of EtOH and
petroleum ether at −20 °C for 16 h. The white solid was
filtered and dried under vacuum to give the title compound
2
4
centrated under reduced pressure to give lactosyl bromide
(13.6 g, 93%) as a white solid. Its spectroscopic data
1
agreed well with those of the literature (Tropper et al. 1991;
Shiao et al. 2014).
4a (OAc) (453 mg, 93%). R = 0.34 hexanes/EtOAc (1:1);
f
mp 73.5–75.0 °C (EtOH–petroleum ether); [α]D −19.4 (c
1
Methyl β‑d‑lactopyranoside (methyl
1, CHCl ); H NMR (CDCl ) δ 5.33 (dd, 1H, J = J
3
3
3,4
4,5
(
(
β‑d‑galactopyranosyl)‑(1 → 4)‑β‑d‑glucopyranoside)
3.4 Hz, H-4′), 5.19 (dd, 1H, J2,3 = J 9.3 Hz, H-3), 5.09
3,4
compound 2)
(dd, 1H, J1,2 7.9 Hz, J2,3 10.4 Hz, H-2′), 4.94 (dd, 1H, H-3′),
4
.84 (dd, 1H, J1,2 = 8.9 Hz, J 9.4 Hz, H-2), 4.62 (d, 1H,
2,3
To a solution of lactosyl bromide 1 (350 mg, 0.5 mmol,
equiv.) in dry methanol (10 mL) was added dropwise a
solution of sodium methoxide (1 M in MeOH, 50 μL, 0.1
equiv.) over a period of 10 min. After stirring at room tem-
perature for 1 h, the reaction was completed and then neu-
H-1), 4.49 (dd, 1H, J6 12.1 Hz, J 2.1 Hz, H-6a), 4.47
a,6b 5,6a
1
(d, 1H, H-1′), 4.13–4.047 (m, 3H, H-6ª’, H-6b and H-6b’),
3.86 (m, 1H, H-5′), 3.77 (dd, 1H, J4,5 9.2 Hz, H-4), 3.69
(ddd, 1H, J5,6a 2.1 Hz, J5,6b 5.1 Hz, H-5), 2.13, 2.12, 2.06,
1
3
2.05, 2.03, 2.03 and 1.95 ppm (7s, 21H, COCH ); C NMR
3
tralized by addition of ion-exchange resin (Amberlite IR
(CDCl ) δ 170.3, 170.2, 170.1, 170.0, 169.6, 169.4, 169.0
3
+
1
20 H ). The solution was filtered and evaporated in vacuo
(CO), 101.0 (C-1′), 87.6 (C-1), 75.7 (C-4), 74.7 (C-5), 72.4
(C-3), 70.9 (C-2), 70.9 (C-3′), 70.7 (C-5′), 69.0 (C-2′),
66.5 (C-4′), 61.7 (C-6), 60.7 (C-6′), 20.7, 20.7, 20.6, 20.6,
to give methyl lactoside 2 (154 mg, 87%) as a white pow-
der R 0.33 (CH Cl /MeOH: 7/3). Spectroscopic data agree
f
2
2
−
20.5, 20.5 and 20.4 ppm (COCH ). IR ν (CHCl ) cm :
3 max 3
1
with those described previously (Koto et al. 2004).
+
2
120s (N ). ESI–MS: [M + NH ] calcd for C H N O ,
3
4
26 39
4
17
Methyl 3′‑O‑sulfo‑β‑d‑lactopyranoside, sodium salt
679.2305; found, 679.2316. The spectroscopic data agreed
with those published (Tropper et al. 1992a).
(
compound 3)
β-d-Lactopyranosyl azide (compound 4b) was synthe-
sized according to a literature procedure followed by the
Zemplén transesterification procedure described above
(procedure B) (Shiao et al. 2014; Pintal et al. 2015).
Following the general procedure C, compound 3 was
obtained as a white powder; yield: 108 mg (80%), R 0.25
f
1
(
CH Cl /MeOH: 7/3). H NMR (300 MHz, MeOD): δ 4.53
2
2
(
d, 1H, J = 7.77 Hz, H-1), 4.27 (m, 1H, H-3′), 4.26 (d, 1H,
J = 7.83 Hz, H-1′), 4.24 (m, 1H, H-4′), 3.93 (m, 2H, H-6),
3′‑O‑Sulfo‑β‑d‑lactopyranosyl azide, sodium salt
(compound 5)
3
3
1
.82–3.69 (m, 5H, H-6′,2,2′,5′), 3.64–3.61 (m, 1H, H-4),
.59–3.56 (m, 1H, H-3), 3.55 (s, 3H, OMe), 3.47–3.44 (m,
1
3
H, H-5). C NMR (75 MHz, D O): δ 103.8 (C-1′), 103.6
Following the general procedure C, compound 5 was
2
1
(
(
(
4
C-1), 80.2 (C-3′), 79.6 (C-4), 75.3 (C-5′), 75.0 (C-5), 74.9
C-3), 73.3 and 69.4 (C-2,2′), 67.2 (C-4′), 61.1 (C-6′), 60.5
C-6), 56.0 (OMe). ESI-HRMS: m/z calcd for C H O S,
36.0887; found 435.0811 [M − H] .
obtained as a white powder, H NMR (600 MHz, D O): δ
2
4.77 (d, 1H, J = 8.8 Hz), 4.56 (d, 1H J = 7.8 Hz), 4.33
(ddd, 1H, J = 9.9, 3.3, 0.5 Hz), 4.28 (d, 1H, J = 3.3 Hz),
3.98 (dd, 1H, J = 12.3, 1.9 Hz), 3.84 (dd, 1H, J = 12.4,
1
3
24 14
−
1
3

Histochem Cell Biol
4
.6 Hz), 3.82–3.74 (m, 3H), 3.74–3.65 (m, 4H), 3.31 (t, 1H,
1H, H-5), 2.18 (s, 3H), 2.18–1.99 (21H, 7OAc). 13C NMR
1
3
J = 9.1 Hz). C NMR (150 MHz, D O): δ 103.1, 90.6,
(75 MHz, CDCl ): δ 170.3, 170.2, 170.1, 170.0, 169.7,
2
3
8
0.6, 8.3, 77.3, 75.6, 75.0, 73.2, 69.7, 67.5, 61.6, 60.5.
169.5, 169.1, 166.5, 150.0, 149.8, 126.0, 122.9, 117.9,
113.4, 101.1 (C-1′), 99.6 (C-1), 76.1 (C-4), 72.9 (C-5), 72.4
(C-3), 71.3 (C-2), 70.9 (C-3′), 70.7 (C-5′), 69.0 (C-2′), 66.6
ESI-HRMS: m/z calcd for C H N O S, 447.0722; found
4
1
2
21
3
13
−
46.0718 [M − H] .
(
C-4′), 61.9 (C-6), 60.8 (C-6′), 56.1 (OMe), 52.2 (CO Me),
2
2
‑Naphthyl 2,3,6,2′,3′,4′,6′‑hepta‑O‑acetyl‑β‑d‑lacto‑
20.8, 20.6, 20.5. ESI-HRMS: m/z calcd for C H O ,
3
5
44 21
+
pyranoside (compound 6)
800.2375; found 818.2675 [M + NH ] .
4
Following the general procedure A, compound 6 was
2‑Naphthyl β‑d‑lactopyranoside (compound 10)
obtained as a yellow oil; yield: 556 mg (73%), R 0.5 (Hex/
f
1
EtOAc: 1/1). H NMR (300 MHz, CDCl ): δ 7.74–7.64 (m,
Following the general procedure B, compound 10 was
obtained as a white powder; yield: 209 mg (95%), Rf
3
3
(
H, Ar), 7.40–7.32 (m, 2H, Ar), 7.26 (d, 1H, Ar), 7.12–7.08
dd, 1H, Ar), 5.37–5.36 (m, 1H, H-4′), 5.33–5.27 (m, 1H,
H-3), 5.27–5.18 (m, 2H), 5.0–5.96 (dd, 1H), 4.55–4.52
1
0.55 (CH Cl /MeOH: 7/3). H NMR (300 MHz, MeOD):
2
2
δ 7.82–7.78 (m, 3H, Ar), 7.50–7.29 (m, 4H, Ar), 5.14 (d,
1H, J = 7.62 Hz, H-1), 4.45 (d, 1H, J = 7.5 Hz, H-1′),
4.02–3.90 (m, 2H, J = 10.86 Hz, H-6′), 3.86 (m, 1H, H-4′),
3.84–3.75 (m, 2H, J = 11.4 Hz, H-6), 3.73–3.71 (m, 3H,
H-3,4,5′), 3.66 (m, 1H, J = 7.62 Hz, H-2), 3.64 (m, 1H,
H-5), 3.60 (m, 1H, J = 7.5 Hz, H-2′), 3.55–3.50 (m, 1H,
(
(
m, 2H), 4.20–4.07 (m, 3H), 3.96–3.85 (m, 3H), 2.17–1.98
21H, 7OAc). Spectroscopic data agree with those of the
literature (Johnsson et al. 2005).
2‑Naphthyl 2,3,6,2′,3′,4′,6′‑hepta‑O‑acetyl‑1‑thio‑β‑d‑lacto‑
1
3
pyranoside (compound 7)
H-3′). C NMR (75 MHz, MeOD): δ 155.3 (Cq Ar), 134.5
(Cq Ar), 129.9 (Cq Ar), 128.9, 127.2, 126.7, 125.9, 123.8,
Following the general procedure A, compound 7 was
118.5, 110.6, 103.6 (C-1′), 100.7 (C-1), 78.8 (C-5′), 75.7
(C-5), 75.3 and 74.9 (C-3,4), 73.4 (C-3′), 73.2 (C-2), 71.1
(C-2′), 68.9 (C-4′), 61.1 (C-6), 60.3 (C-6′). ESI-HRMS:
m/z calcd for C H O , 468.1632; found 468.1608.
obtained as a yellow oil; yield: 650 mg (83%), R 0.52
f
1
(
Hex/EtOAc: 1/1). H NMR (300 MHz, CDCl ): δ 7.95 (d,
3
1H, J = 1.2 Hz, Ar), 7.81–7.73 (m, 3H, Ar), 7.53–7.45 (m,
3H, Ar), 5.31–5.28 (m, 1H, H-4′), 5.21 (t, 1H, H-3), 5.10–
5.04 (dd, 1H, H-2′), 4.95–4.93 (m, 1H, H-3′), 4.90 (t, 1H,
2
2
28 11
2‑Naphthyl 3′‑O‑sulfo‑β‑d‑lactopyranoside, sodium salt
H-2), 4.75 (d, 1H, J = 10.0 Hz, H-1), 4.54–4.49 (m, 1H,
H-6a), 4.47 (d, 1H, J = 7.8 Hz, H-1′), 4.12–4.10 (m, 1H,
H-6b), 4.08–4.04 (m, 2H, H-6′ab), 3.85 (t, 1H, J = 6.7 Hz,
H-5′), 3.72 (t, 1H, H-4), 3.69–3.65 (m, 1H, H-5), 2.13–1.93
(compound 11)
Following the general procedure C, compound 11 was
obtained as a white powder; yield: 136 mg (80%), Rf
1
(
21H, 7OAc). Spectroscopic data agree with those previ-
0.48 (CH Cl /MeOH: 7/3). H NMR (300 MHz, D O): δ
2
2
2
ously reported (Rodrigue et al. 2013).
7.85–7.77 (m, 3H, Ar), 7.5–7.42 (m, 3H, Ar), 7.27 (d, 1H,
J = 9 Hz, Ar), 5.15 (d, 1H, J = 7.41 Hz, H-1), 4.51 (d, 1H,
J = 7.7 Hz, H-1′), 4.28 (dd, 1H, J = 3.3, 9.7 Hz, H-3′),
4.22 (m, 1H, J = 3.3 Hz, H-4′), 3.95 (d, 1H, J = 11.58 Hz,
H-6a), 3.75 (d, 1H, J = 11.58 Hz, H-6b), 3.71–3.66 (m, 6H,
H-3,4,5,5′,6′ab), 3.64 (dd, 1H, J = 7.7, 9.7 Hz, H-2′), 3.59
4
‑Methylumbelliferyl 2,3,6,2′,3′,4′,6′‑hepta‑O‑acetyl‑β‑d‑
lactopyranoside (compound 8)
This compound was prepared as already described in the
literature (Wang et al. 1998).
1
3
(m, 1H, H-2). C NMR (75 MHz, D O): δ 154.3 (Cq Ar),
2
1
33.8 (Cq Ar), 129.9, 129.7 (Cq Ar), 127.7, 127.1, 126.9,
Methyl 3‑methoxy‑4‑(2,3,6,2′,3′,4′,6′‑hepta‑O‑acetyl‑β‑d‑
lactopyranosyloxy)benzoate (compound 9)
124.9, 118.5, 110.7, 102.5 (C-1′), 99.9 (C-1), 80.0 (C-3′),
78.0 (C-5), 74.9 (C-3,5′), 74.1 (C-4), 72.6 (C-2), 69.1
(C-2′), 66.8 (C-4′), 60.8 (C-6′), 60.0 (C-6). ESI-HRMS:
Following the general procedure A, compound 9 was
m/z calcd for C H O S, 548.1216; found 547.1144
[M − H] .
2
2
28 14
−
obtained as a yellow oil; yield: 573 mg (71%), R 0.24
f
1
(
Hex/EtOAc: 45/55). H NMR (300 MHz, CDCl ): δ 7.76–
3
7
.50 (m, 2H, Ar), 7.10 (d, 1H, J = 8.3 Hz, Ar), 5.38–5.37
2‑Naphthyl 1‑thio‑β‑d‑lactopyranoside (compound 12)
Following the general procedure B, compound 12 was
(
m, 1H, H-4′), 5.37–5.28 (m, 1H, H-3), 5.25–5.20 (m, 1H,
H-2), 5.17–5.12 (m, 1H, H-2′), 5.05 (d, 1H, J = 7.4 Hz,
H-1), 4.99–4.96 (m, 1H, H-3′), 4.52–4.51 (m, 2H, H-1′,6a),
obtained as a white powder; yield: 401 mg (quant.), R 0.57
f
1
4
3
.20–4.08 (m, 3H, H-6b, 6′ab), 3.94–3.88 (m, 2H, H-4, 5′),
(CH Cl /MeOH: 7/3). H NMR (300 MHz, MeOD): δ 8.10
2
2
.92 (s, 3H, CO Me), 3.88 (s, 3H, OMe), 3.80–3.75 (m,
(s, 1H, Ar), 7.85–7.80 (m, 3H, Ar), 7.68–7.65 (m, 1H, Ar),
2
1
3

Histochem Cell Biol
7
.53–7.48 (m, 2H, Ar), 4.76 (d, 1H, J = 9.0 Hz, H-1), 4.39
1H), 5.21 (d, 1H, J = 7.7 Hz), 4.61 (d, 1H, J = 7.8 Hz),
4.35 (dd, 1H, J = 9.9, 3.2 Hz), 4.30 (d, 1H, J = 3.3 Hz),
4.08–3.98 (m, 1H), 3.94–3.74 (m, 7H), 3.70 (m, 2H), 2.35
(
d, 1H, J = 7.38 Hz, H-1′), 3.99–3.85 (m, 2H, H-6′), 3.82
(
m, 1H, H-4′), 3.79–3.67 (m, 2H, H-6), 3.61–3.59 (m, 3H,
1
3
H-3,4,5), 3.57 (m, 1H, H-2′), 3.53 (m, 1H, H-5′), 3.51–3.47
(s, 3H). C NMR (150 MHz, D O + MeOD): δ 165.4,
2
1
3
(
m, 1H, H-3′), 3.37–3.36 (m, 1H, H-2). C NMR (75 MHz,
160.3, 157.1, 154.8, 127.6, 116.2, 114.8, 112.2, 104.5,
103.5, 100.5, 81.0, 78.8, 76.0, 75.9, 75.1, 73.4, 70.1, 67.8,
61.9, 60.7, 18.9. ESI-HRMS: m/z calcd for C H O S
MeOD): δ 132.6 (Cq Ar), 130.9 (Cq Ar), 130.3 129.1 (Cq
Ar), 129.0, 127.8, 127.2, 127.1, 126.1, 125.8, 103.5 (C-1′),
2
2
28 16
+
8
7
7.6 (C-1), 79.2 (C-5′), 78.7 (C-5), 76.6 and 75.7 (C-3,4),
3.4 (C-3′), 72.0 (C-2), 71.1 (C-2′), 68.9 (C-4′), 61.1
580.1171; found 581.1166 [M + H] . Spectroscopic data
agreed with those described in the literature (Wang et al.
1998).
(
C-6), 60.6 (C-6′). ESI-HRMS: m/z calcd for C H O S,
22 28 10
4
84.1403; found 484.1446.
Methyl 3‑methoxy‑4‑(β‑d‑lactopyranosyloxy)benzoate
2
‑Naphthyl 3′‑O‑sulfo‑1‑thio‑β‑d‑lactopyranoside, sodium
(compound 16)
salt (compound 13)
Following the general procedure B, compound 16 was
Following the general procedure C, compound 13 was
obtained as a white powder; yield: 283 mg (quant.),
1
obtained as a white powder; yield: 119 mg (84%), R 0.5
R 0.26 (CH Cl /MeOH: 75/25). H NMR (300 MHz,
f
f
2
2
1
(
(
CH Cl /MeOH: 7/3). H NMR (300 MHz, D O): δ 7.92
D2O + DMSO-d ): δ 7.76–7.74 (m, 2H, Ar), 7.31 (d, 1H,
2
2
2
6
s, 1H, Ar), 7.79–7.72 (m, 3H, Ar), 7.5–7.4 (m, 3H, Ar),
J = 9.2 Hz, Ar), 5.32 (d, 1H, J = 7.7 Hz, H-1), 4.53 (d,
1H, J = 7.4 Hz, H-1′), 4.07–3.90 (m, 9H, H-4′,6′,OMe
4
.74 (d, 1H, J = 8.82 Hz, H-1), 4.42 (d, 1H, J = 7.83 Hz,
H-1′), 4.23 (dd, 1H, J = 3.2, 9.66 Hz, H-3′), 4.19 (t, 1H,
and CO Me), 3.90–3.69 (m, 6H, H-3,4,5,5′,6), 3.66 (m,
2
1
3
J = 3.2 Hz, H-4′), 3.85 (d, 1H, J = 11.88 Hz, H-6a), 3.74
2H, H-2,3′), 3.55 (m, 1H, H-2′). C NMR (75 MHz,
(
(
(
d, 1H, J = 11.88 Hz, H-6b), 3.66 (s, 2H, H-6′ab), 3.63
D2O + DMSO-d ): δ 157.3 (Cq Ar), 150.2 (Cq Ar), 148.5
6
m, 1H, H-5′), 3.61 (d, 1H, J = 7.83 Hz, H-2′), 3.57–3.54
(Cq Ar), 123.7, 114.8, 112.8, 103.4 (C-1′), 99.3 (C-1),
78.8; 75.6; 75.1 and 74.5 (C-3,4,5,5′), 72.9 (C-3′), 72.7
(C-2), 70.9 (C-2′), 68.5 (C-4′), 61.0 (C-6′), 60.0 (C-6),
1
3
m, 2H, H-3,4), 3.47 (s, 1H, H-5), 3.35 (m, 1H, H-2).
C
NMR (75 MHz, D O): δ 133.2, 132.1, 130.2, 129.5, 128.7,
2
1
27.6, 127.4, 126.9, 126.7, 102.46 (C-1′), 87.0 (C-1), 80.0
56.1 (OMe), 52.7 (CO Me). ESI-HRMS: m/z calcd for
2
+
(
(
C-3′), 78.6 (C-5), 77.9 (C-3), 75.7 (C-4), 74.8 (C-5′), 71.5
C-2), 69.0 (C-2′), 66.8 (C-4′), 60.8 (C-6′), 60.0 (C-6).
C H O , 506.1635; found 529.1521 [M + Na] .
2
1
30 14
ESI-HRMS: m/z calcd for C H O S , 564.0982; found
Methyl 3‑methoxy‑4‑(3′‑O‑sulfo‑β‑d‑lactopyranosyloxy)
benzoate, sodium salt (compound 17)
2
2
28 13 2
−
5
63.0909 [M − H] .
4
‑Methylumbelliferyl β‑d‑lactopyranoside (compound 14)
Following the general procedure C, compound 17 was
obtained as a white powder; yield: 45 mg (81%), R 0.12
f
1
Following the general procedure B, compound 14 was
(CH Cl /MeOH: 75/25). H NMR (300 MHz, MeOD): δ
2
2
1
obtained as a white powder; H NMR (600 MHz, DMSO-
7.65 (m, 2H, Ar), 7.26 (d, 1H, Ar), 5.13 (d, 1H, J = 7.7 Hz,
d + D O): δ 7.83–7.52 (m, 1H), 7.17–6.89 (m, 2H), 6.23
H-1), 4.56 (d, 1H, J = 7.8 Hz, H-1′), 4.37–4.18 (m, 2H,
6
2
(
d, 1H, J = 5.8 Hz), 5.12 (d, 1H, J = 7.8 Hz), 4.23 (d, 1H,
H-3′,4′), 3.98–3.87 (m, 8H, H-6,OMe and CO Me), 3.85–
2
J = 7.2 Hz), 3.82–3.44 (m, 8H), 3.41 (t, 1H, J = 9.1 Hz),
.36–3.29 (m, 3H), 2.39 (s, 3H). C NMR (150 MHz,
DMSO-d ): δ 160.7, 160.3, 154.7, 154.0, 126.9, 114.6,
3.77 (m, 2H, H-6′), 3.75 (m, 1H, H-2′), 3.73–3.71 (m,
1H, H-4), 3.70–3.61 (m, 3H, H-3,5,5′), 3.61–3.57 (m, 1H,
1
3
3
1
3
H-2). C NMR (75 MHz, MeOD): δ 156.8 (Cq Ar), 150.6
(Cq Ar), 149.1 (Cq Ar), 123.1, 115.0, 112.7, 103.5 (C-1′),
100.2 (C-1), 80.3 (C-3′), 79.0 (C-4), 75.4 and 75.3 and 74.7
(C-3,5,5′), 73.0 (C-2), 69.5 (C-2′), 67.1 (C-4′), 61.0 (C-6′),
6
1
7
13.8, 112.0, 104.0, 103.5, 99.8, 80.1, 75.8, 75.3, 74.8,
3.3, 73.0, 70.8, 68.4, 60.8, 60.2, 18.5. ESI-HRMS: m/z
+
calcd for C H O 500.1530; found 523.1413 [M + Na] .
2
2
28 13
Spectroscopic data agreed with those described in the lit-
erature (Wang et al. 1996).
60.1 (C-6), 55.3 (OMe), 51.2 (CO Me). ESI-HRMS:
2
m/z calcd for C H O S, 586.1204; found 585.1130
2
1
30 17
−
[
M − H] .
4
‑Methylumbelliferyl 3′‑O‑sulfo‑β‑d‑lactopyranoside,
sodium salt (compound 15)
Glycoclusters
Following the general procedure C, compound 15 was
The bi-, tri- and tetravalent glycoclusters with lactose
(compounds 18, 19) or 2-fucosyllactose (compound 20) as
headgroup (Fig. 2) were prepared using copper-catalyzed
1
obtained as a white powder; H NMR (600 MHz, D O):
2
δ 7.64 (d, 1H, J = 8.8 Hz), 7.16–6.88 (m, 2H), 6.17 (s,
1
3

Histochem Cell Biol
Fig. 2 Bi-, tri- and tetravalent glycoclusters with lactose (18, 19) and 2′-fucosyllactose (20) as headgroups (Wang et al. 2012)
azide alkyne cycloaddition as described (Wang et al.
012).
same cell suspension in parallel. Controls included moni-
2
toring of viability and assessment of lectin-independent
staining. The quantitative data are expressed in the terms
of percentage of positive cells/mean fluorescence intensity.
Galectins
Human galectins and their separate CRDs were purified
after recombinant production by affinity chromatography
as a key step and rigorously checked for purity as described
Galectin histochemistry
Tissue specimen of jejunum and epididymis of four six-
week-old C57BL/6 mice was processed to obtain sections
(about 5 µm) as described (André et al. 2016). Following
saturation of sites for non-specific binding of proteins and
systematic titrations to identify a concentration of bioti-
nylated galectin yielding a strong signal with minimal
background to be able to discern quantitative differences
in the inhibition studies, incubation, visualization of bound
probe, semiquantitative grading of staining and recording
of microphotographs were carried out as described (Kaltner
et al. 2015, 2016; André et al. 2016).
(
André et al. 2008, 2014; Giguère et al. 2011). Labe-
ling under activity-preserving conditions was carried out
with the N-hydroxysuccinimide ester derivative of biotin
(
Sigma) or the fluorescent dyes fluorescein isothiocyanate
®
or Alexa Fluor 488 (Invitrogen, Darmstadt, Germany).
Solid‑phase and cell assays
Binding of labeled galectins in the absence or presence of
test compounds to surface-presented asialofetuin was mon-
itored spectrophotometrically as described (André et al.
2
008, 2014). Cytofluorimetric analysis of galectin bind-
−5
ing to Chinese hamster ovary (CHO) wild-type (Pro ) as
well as pancreatic (Capan-1) and colon (SW480) adeno-
carcinoma cells was performed as described (André et al.
Results and discussion
Design of the test compounds
2
003, 2008; Amano et al. 2012). Experimental series with
the synthetic test compounds and lactose as reference were
routinely performed for each cell line with aliquots of the
Carbohydrate chemistry offers three possibilities to tune
affinity and selectivity of lectin ligands, i.e., the structure
1
3

Histochem Cell Biol
of the headgroup including physiological and bio-inspired
substitutions, the characteristics of the aglycone (spacer)
and the properties of glycoclusters including valency and
topology of ligand presentation (Roy et al. 2016). Our
panel of test compounds covers examples for each case,
starting from lactose as reference. The monovalent lacto-
sides are arranged as pairs of unsubstituted and 3′-O-sul-
fated lactose (Fig. 1). Introduction of β-linked extensions
from methyl and azide to four types of O- and S-conjugated
aromatic substituents yielded a total of six pairs of lacto-
sides (Fig. 1). Lactose as part of bi- and tetravalent glyco-
clusters (compounds 18, 19) and 2′-fucosyllactose as part
of a trivalent glycocluster 20 (Fig. 2) have already proved
potent as inhibitor for Gal-3 in solid-phase and cell assays
Inhibition of galectin binding to glycoprotein/cells
Asialofetuin was selected as matrix in solid-phase assays,
because the LacNAc termini of its three complex-type
N-glycans are potent ligands for human galectins (Dam
et al. 2005). As indicated previously for lactosides with
aglyconic extensions (Giguère et al. 2011), the extent of
carbohydrate-dependent binding of labeled human Gal-8
to this surface-presented glycoprotein was responsive to
both structural modifications in the compound panel of
monovalent lactosides in solid-phase assays. The presence
of aromatic aglycones increased the inhibitory capacity of
the β-methyl derivative markedly in the enhancements, as
reported previously for Gal-8N and spacered conjugation
with fluorescein (Carlsson et al. 2007) and also for full-
length Gal-8 and an isoxazole derivative (Giguère et al.
2011). The sensitivity of the reactivity was independent
of the length of the linker peptide between the two CRDs
arising from alternative splicing and resides mostly in the
N-terminal CRD, when testing Gal-8N and Gal-8C in par-
allel. 3′-O-Sulfation further enhanced the extent of inhibi-
tion, as expected based on the respective literature listed in
the introduction. Comparatively reduced but still notable
extents of inhibition were observed for Gal-1 and Gal-3, as
expected. These results using an assay of a model glyco-
protein adsorbed to a plastic surface thus ascertained activ-
ity of the compounds for galectin binding, competing with
LacNAc, and confirmed the so far collected evidence on
Gal-8, Gal-1 and Gal-3. Because Gal-8 binding to cells can
involve both domains to varying extents by engaging differ-
ent types of glycans from surface glycomes, we proceeded
to assess the impact of the presence of test compounds on
galectin binding to cultured cells by cytofluorimetry.
The CHO wild-type line was tested as representative
of a cell surface presenting α2,3-sialylated N-glycans,
to which the N-terminal domain of Gal-8 binds well in a
carbohydrate-dependent manner (Kaltner et al. 2009).
Consequently, lactose (at 2 mM) gave a modest inhibition,
which was enhanced by aglycone presence (compound
10 tested at 0.5 mM) (Fig. 3a). The 3′-sulfated compound
11 (at 0.1 mM) of this pair surpassed that level of inhibi-
tion (Fig. 3a). In order to examine inhibitory capacity of
Gal-8 binding, when the degree of sialylation is reduced,
we turned to a respective cell model, i.e., a pancreatic
adenocarcinoma line with a tumor-suppressor-dependent
decrease in this property (André et al. 2007; Amano et al.
2012).
(
Wang et al. 2012), thus serving here as internal control for
the validity of the histochemical procedure.
The synthesis of the first part of the panel followed a
strategy to combine deprotection of peracetylated glycosyl
halide with inversion of the anomeric configuration of the
halide under classical Zemplén transesterification conditions
(
NaOMe, MeOH) (Wang and Lee 1995). In detail, treatment
of the known peracetylated lactosyl bromide (compound 1)
Shiao et al. 2014) under the conditions given above pro-
(
vided, directly and in one step, methyl lactoside 2 in 87%
yield (Fig. 1). The 3′-O-sulfation was based on the regiose-
lective enhancement of hydroxyl group nucleophilicity using
well-established tin chemistry (Wang et al. 1996). Treatment
of compound 2 with dibutyltin oxide followed by quenching
of the labile tin acetal with sulfur trioxide-trimethylamine
complex led to the desired 3′-O-sulfated methyl lactoside 3
in 80% yield. By stereoselective introduction of the agly-
cones under PTC conditions, the synthesis of the six lac-
toside pairs shown in Fig. 1 was completed. For instance,
peracetylated lactosyl azide 4a and aryl derivatives 6–9 were
obtained from peracetylated lactosyl bromide 1 by S 2-type
N
reaction conditions using our standard procedures. Com-
pounds 4a and 6–9 were obtained in yields ranging from 71
1
to 95%. All compounds were fully characterized by H-NMR
spectroscopy, mass spectrometry and the data sets agreed
with the literature data when known (see experimental sec-
tion). The peracetylated intermediates were de-O-acetylated
under the Zemplén conditions described above in essentially
quantitative yields in all cases to give free lactosides 10, 12,
14 and 16. Following sequential treatment of the polyols
with the dibutyltin oxide reagent and sulfation with the com-
mercially available sulfur trioxide–trimethylamine complex
(
Me N·SO ), the 3′-O-sulfated lactosides 11, 13, 15 and 17
3
3
were obtained in good to excellent yields (Fig. 1). Position-
ing of the sulfate group was readily detectable on the basis of
the C-NMR chemical shift displacement of the 3′-carbon,
which usually appeared 7 ppm downfield (~δ 80 ppm) from
the hydroxylated precursors at δ ~ 73 ppm.
In this case, surface staining of cells by Gal-8, too,
was strong. Its sensitivity to the presence of inhibitors
with aglyconic extension and to 3′-O-sulfation, however,
was reduced (Fig. 3b). This result indicates that a shift to
involvement of the C-terminal domain in this situation is
13
1
3

Histochem Cell Biol
type of CRD in each case so that a synthetic compound will
not face the problem to compete with the cellular glycan(s)
at two structurally different lectin sites. Corroborating the
literature and solid-phase assay data, stepwise increase in
inhibitory activity by aglyconic extension and subsequent
3
′-O-sulfation could be obtained, benzoate presence along
with 3′-O-sulfation proving less favorable than other aro-
matic extensions (Fig. 3c–f). These data reveal that the
presence of different CRDs in a galectin (here tandem-
repeat-type Gal-8) can make the response to an inhibitor in
the context of cell surface binding hardly predictable and
that a cross-reactivity of the synthetic lactosides with other
galectins is still possible. In order to be able to measure the
effect of an inhibitor on galectin binding in the context of a
tissue, we proceeded to analyzing galectin-dependent stain-
ing in sections.
Inhibition of galectin binding to tissue sections
Building on the previous experience with two plant lectins
and sections of murine jejunum/epididymis (André et al.
2
016), probe-independent staining was rigorously excluded
and a galectin concentration resulting in strong staining
at low background was defined by systematic titrations
in each case, i.e., Gal-8 at 0.5 µg/mL, Gal-1 and Gal-3 at
1
µg/mL. Because generation of the signals could then be at
least drastically impaired by high concentrations of lactose,
as in the other assay types, direct comparisons between
the reference with lactose and data obtained for each test
compound in titrations were possible. To document this
internal reference, the effect of the presence of lactose in
increasing concentrations on staining distribution and
intensity in sections of jejunum is presented in Fig. 4a–d.
The overall assessment by semiquantitative evaluation of
the processed specimen (i.e. by grading staining into the
categories from ++++ to −; for details, please see legend
of Fig. 4) is given in the bottom left part of each micropho-
tograph. Compound 11 (Fig. 4e–h) and also compound 13
(Fig. 4i–l) were more effective than lactose, as illustrated
for compound 11 in Fig. 3a in cytofluorimetric analysis
of cells with high-level α2,3-sialylation. Reinforcing the
importance of the type of cell shown in Fig. 3a, b, process-
ing of sections of epididymis yielded a different picture:
lactose proved to be more active than compound 11 (Online
Resource 1). As with cells in culture (Fig. 3a, b), the activ-
ity profile of an inhibitor, mostly acting on the N-terminal
domain, in this assay is governed by the nature of the con-
tact sites in the tissue for tandem-repeat-type Gal-8.
Fig. 3 Semilogarithmic representation of fluorescent surface stain-
ing of wild-type CHO (a) as well as human pancreatic adenocarci-
noma (Capan-1; b, c) and colon adenocarcinoma (SW480; d–f) cells
by human galectins. Percentage of positive cells/mean fluorescence
intensity is given for each scan, along with the control value (back‑
ground) obtained by cell processing without the incubation step
with lectin-containing solution (gray‑shaded area). The 100% value
(
in the absence of test compound) is given as solid black line. Their
numbers (for background/100%-control) are given at the top/bottom
of the respective listing. Biotinylated Gal-8 (5 µg/mL) was incubated
with 2 mM lactose, 0.5 mM compound 10 and 0.1 mM compound
1
1 (a). Fluorescent Gal-8 (2 µg/mL) was incubated with 2 mM lac-
tose and 2.5 mM of compounds 10 or 11, respectively (b). Fluores-
cent Gal-1 (20 µg/mL) was incubated with 0.5 mM Lac, compound
1
4 or compound 15 (c). Biotinylated Gal-3 (10 µg/mL) was incubated
with solutions containing 0.1, 0.2 or 10 mM lactose (d), 0.1 mM
compound 14 or 0.1 mM compound 15 (e) and 0.1 mM compound 17
or 0.1 mM compound 16 (f). Numbers of staining parameters (from
bottom to top) are given with the 100% value (bottom) and negative
control (top) as references in the given order
likely. Evidently, the susceptibility of inhibition of Gal-8
binding to cells can depend on the actual glycome profile
of the target cells, which determines preferences for con-
tact via the N- or the C-terminal domain. In contrast to the
heterobivalent Gal-8, Gal-1 and Gal-3 have only a single
When tested for Gal-3, the effect of lactose was evi-
dent (Fig. 5a, b). Compounds 16 and 17 were active in a
markedly (16) or slightly better manner (17) (Fig. 5c, d).
As in these two cases and Gal-3 (Fig. 3d, f), correspond-
ence between cytofluorimetry and histochemistry data
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Histochem Cell Biol
Fig. 4 Histochemical staining profiles of labeled Gal-8 in sections
of murine jejunum (a–l) in the absence or presence of the cognate
sugar (lactose or derivatives, as shown in Fig. 1). Strong reactiv-
ity for Gal-8 (0.5 µg/mL) was found in the deep parts of the glan-
dulae intestinales and, to a weaker extent, in the epithelial lining of
villi intestinales. The extent of staining remained at the 100% level
even in the presence of 0.5 mM (free) lactose (a). Stepwise reduc-
tion in signal intensity occurred in the presence of 1 mM (b), 2 mM
of 0.5 mM (e), 1 mM (f), 2 mM (g) and 5 mM (h) compound 11. This
inhibitory effect was slightly enhanced in the presence of 0.5 mM (i),
1 mM (j), 2 mM (k) and 5 mM (l) compound 13 relative for the series
with compound 11. Category of semiquantitative grading of the stain-
ing intensity is given in the bottom left part of each microphotograph.
The intensity of staining in sections is grouped into the following cat-
egories: −, no staining; (+), weak but significant staining; +, weak
staining; ++, medium staining; +++, strong staining; ++++, very
strong staining. Scale bars 20 µm
(
c) and 5 mM (d) lactose. By comparison, the extent of positivity for
labeled Gal-8 was seen to be reduced more strongly in the presence
was revealed for Gal-1, too, when tested at identical con-
centration with Gal-1, compounds 14 and 15 proved to be
more potent than lactose (Figs. 3c, 5e–h). For these two
galectins, i.e., Gal-1 and Gal-3, the presence of a single
type of CRD makes inhibitory capacity less dependent on
cell type than for the tandem-repeat-type protein. Having
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Histochem Cell Biol
Fig. 5 Histochemical staining profiles of labeled Gal-3 (a–d) and
Gal-1 (e–h) in sections of murine jejunum in the absence or pres-
ence of the cognate sugar (lactose or derivatives, as shown in Fig. 1).
Strong reactivity for labeled Gal-3 (1 µg/mL) was found in glandu-
lae intestinales and, comparatively less, in the epithelial lining of villi
respectively, most efficiently with compound 16 (c). Intense staining
by labeled Gal-1 (0.5 µg/mL) was observed in the epithelial cells of
the villi intestinales (e). When comparing the effect of the presence
of 5 mM lactose (f) with those compounds 14/15 (g, h), a more effec-
tive reduction in staining intensity by compounds 14/15 was obtained.
Scale bars 20 µm
(
a). The extent of binding of biotinylated Gal-3 was reduced in the
presence of 5 mM lactose (b), compound 16 (c) or compound 17 (d),
tested monovalent lactosides, we next explored the inhibi-
tory activity of the glycoclusters 18–20, in one case with
structural extension from lactose (compounds 18, 19) to its
systematic titrations were examined comparatively and pre-
sented here for the concentrations at 0.25 mM (Fig. 6a–d)
and 0.5 mM (Fig. 6e–h) of lactose (free or conjugated).
Compared to the effect of free sugar (Fig. 6a, e), the pres-
ence of the bivalent (compound 18 with lactose; Fig. 6b, f),
trivalent (compound 20 with 2′-fucosyllactose; Fig. 6c, g)
and tetravalent (compound 19 with lactose; Fig. 6d, h) gly-
coclusters reduced staining. Increase in valency and struc-
tural complexity led to increased inhibitory capacity, in full
agreement with the solid-phase and cell assay data reported
previously (Wang et al. 2012). Interestingly, staining by
Gal-1 in the same setting was also very susceptible to
inhibition in sections of jejunum (Fig. 6i–p). Although the
homologues of Gal-1 tested previously were less respon-
sive than Gal-3 in solid-phase assays (Wang et al. 2012),
these results (please see Fig. 6m–p) advise caution and the
avoidance of simple extrapolations when moving from an
assay with a glycoprotein to that with tissue sections.
2
-fucosyl derivative (20), the histoblood group H (type II)
trisaccharide.
Gal-3 is known to form aggregates upon complex for-
mation with multivalent ligands due to intermolecular con-
tacts via its N-terminal tail and CRD (Ahmad et al. 2004;
Kopitz et al. 2014; Ippel et al. 2016). The presentation of
ligands in spatial vicinity can thus convey strong inhibitory
potency to glycoclusters, as shown in solid-phase and cell
assay for compounds 18–20 (Wang et al. 2012). Because
the degree of N-glycan branching of the tested glycopro-
tein can affect glycocluster efficiency (André et al. 2009),
a monitoring in tissue sections can answer the question on
relative inhibitory capacity. These three glycoclusters were
thus tested in the histochemical assay. Staining profiles of
sections of murine jejunum by labeled Gal-3 obtained by
1
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Histochem Cell Biol
1
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Histochem Cell Biol
◂
Fig. 6 Histochemical staining profiles of labeled Gal-3 (a–h) and
Gal-1 (i–p) in sections of murine jejunum in the absence or presence
of the cognate sugar (free or conjugated to scaffolds, as shown in
Fig. 2). Reduction in staining intensity in the presence of glycoclus-
ters 18, 20 and 19 is compared to the effect of the same concentra-
tions of free lactose. Inhibition of binding of labeled Gal-3 (1 µg/mL)
to epithelial cells of glandulae intestinales is shown for free lactose at
the perspective of drug design, the resulting data can give
incentive to iterative refinements of the structure of the test
compound.
The presented evidence validates the hypothesis of the
assay’s applicability for human galectins. Especially the
test case of the tandem-repeat-type Gal-8 highlights its
practical value by revealing strong dependence of inhibitor
activity on the tissue type. Obviously, caution needs to be
exercised when considering extrapolations. The presence of
the two different CRDs of Gal-8 equips this protein with
capacity to let each CRD target certain counterreceptors,
e.g., promatrix metalloproteinase-9 (Gal-8N) and CD11b
0
.25 mM (a) and for compound 18 at this concentration (b), further
enhanced for the trivalent glycocluster 20 (c) and for tetravalent 19
d). Inhibition of lectin binding was similarly different when applying
.5 mM of the sugar, free or attached to the scaffold (e, lactose; f, 18;
(
0
g, 20; h, 19). Intense staining of epithelial cells of villi intestinales by
labeled Gal-1 (0.5 µg/mL) was only slightly reduced in the presence
of 1 mM (i) or 2 mM (m) (free) lactose. Compared to lactose, the
ligand-presenting glycoclusters 18, 20, 19 reduced the staining inten-
sity to a greater extent, with enhancements when increasing the con-
centration of sugar from 1 mM (j, 18; k, 20; l, 19) to 2 mM (n, 18; o,
(MAC-1, α -integrin) (Gal-8C) in the case of neutrophils
M
(Nishi et al. 2003). On the grounds of differences in gly-
2
0; p, 19). Scale bars 20 µm
can reactivity between the two CRDs (Ideo et al. 2003;
Carlsson et al. 2007; Stowell et al. 2008), our respective
data attest that the inhibitory capacity of a blocking rea-
gent against Gal-8 with preferential activity to one type of
CRD cannot be simply calculated for a certain cell/tissue
type, unless information on features of in situ contact part-
ners is available. Equally important, the aspect of require-
ment of a network analysis is emphasized by revealing
that inhibitors can interfere with binding of more than one
galectin, a source of undesired side effects when envision-
ing the development of a galectin-based therapy (Smetana
et al. 2013). Changing the backbone to type 1 LacNAc of
a sulfated disaccharide and altering the site of sulfation to
the 6-position of GlcNAc, for example, could reduce such
side reactivity to Gal-1, while still exerting strong effects
on Gal-8 (Carlsson et al. 2007; Tu et al. 2013). A switch
back to 3′-O-sulfation and substituting GlcNAc by Gal-
NAc to prepare sulfated CD176 (T(F) antigen) will direct
respective compounds to Gal-4 (Ideo et al. 2002), the trans-
lator of glycan-encoded signals in apical and axonal trans-
port of glycoproteins (Stechly et al. 2009; Velasco et al.
2013; Higuero et al. 2017). By the way, the members of
the siglec family require sialylation for contact building
so that 3′-O-sulfation is not sufficient for binding (Rapo-
port et al. 2006). Because galectins mediate cell adhesion
and migration, for Gal-8 with potential biomedical rel-
evance (Levy et al. 2001; Nagy et al. 2002; Friedel et al.
2016), the emerging possibility to program glycan presen-
tation on surfaces of biomimetic vesicles (glycodendrim-
ersomes) will make versatile test models for respective
assays with such glycotopes available (Percec et al. 2013;
Zhang et al. 2014, 2015a). Due to the feasibility of prepar-
ing even complex glycans (Oscarson 2009) and of design-
ing structural variants of galectins, e.g., by modular con-
jugations or transplantations (Vértesy et al. 2015; Zhang
et al. 2015b; Ludwig et al. 2016), the lectin histochemical
assay can complement structure–activity investigations, by
studying the staining profiles comparatively and by letting
Conclusions and perspectives
The emerging wealth of data on the broad range of func-
tions of endogenous lectins, as summarized in Table 5 in
Manning et al. (2017), is teeming with information that
could inspire novel ideas for a more thorough under-
standing of cell biological systems and routes for drug
design, as indicated in this issue’s introduction (Gabius
and Roth 2017). In order to fulfill their missions, these
lectins appear to select distinct glycoconjugates as their
physiological counterreceptors. When acting as effectors
on different cell types, the biochemical nature of these
target sites in terms of scaffold (protein or lipid) or gly-
can structure can differ between various cells. Also, due
to their cooperation in a network (Kaltner and Gabius
2
012; Kaltner et al. 2016, 2017), the response profile to a
synthetic reagent may in vivo be complex. This situation
calls for an assay that comes as close as possible to the
natural profiles of glycans to assess the capacity of such
a compound as physiologically relevant inhibitor in dif-
ferent cells/tissues and its range of side reactivities in the
lectin network.
Toward this end, we have tested the hypothesis that
lectin histochemistry with labeled human galectins and
synthetic sugar derivatives/glycoclusters will be helpful.
Compared to cells adapted to growth in culture, tissue sec-
tions present cells in their microenvironment, along with
the extracellular matrix and stroma, albeit after tissue fixa-
tion and processing. As illustrated previously (Plzák et al.
2
004; Smetana et al. 2006; Dawson et al. 2013; Kaltner
et al. 2015) and in this issue (Kaltner et al. 2017), extra-
and intracellular binding of the probes can then be mapped
in the same section (or cytological specimen). Hereby, sus-
ceptibility of galectin binding at different sites to the pres-
ence of synthetic compounds will be evaluated, and this in
serial sections for the entire lectin network. Viewed from
1
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Histochem Cell Biol
custom-made test compounds help characterize the molec-
ular nature of binding sites.
André S, Wang GN, Gabius H-J, Murphy PV (2014) Combining gly-
cocluster synthesis with protein engineering: an approach to
probe into the significance of linker length in a tandem-repeat-
type lectin (galectin-4). Carbohydr Res 389:25–38
André S, Kaltner H, Kayser K, Murphy PV, Gabius H-J (2016) Merg-
ing carbohydrate chemistry with lectin histochemistry to study
inhibition of lectin binding by glycoclusters in the natural tissue
context. Histochem Cell Biol 145:185–199
Bhide GP, Colley KJ (2017) Sialylation of N-glycans: mechanism,
cellular compartmentalization and function. Histochem Cell
Biol 147(2). doi:10.1007/s00418-016-1520-x
Cao S, Meunier SJ, Andersson FO, Letellier M, Roy R (1994) Mild
stereoselective syntheses of thioglycosides under PTC condi-
tions and their use as active and latent glycosyl donors. Tetrahe-
dron: Asymmetry 5:2303–2312
Carrière D, Meunier SJ, Tropper FD, Cao S, Roy R (2000) Phase
transfer catalysis toward the synthesis of O-, S-, Se-, and C-gly-
cosides. J Mol Catal A Chem 154:9–22
Carlsson S, Oberg CT, Carlsson MC, Sundin A, Nilsson UJ, Smith
D, Cummings RD, Almkvist J, Karlsson A, Leffler H (2007)
Affinity of galectin-8 and its carbohydrate recognition domains
for ligands in solution and at the cell surface. Glycobiology
In sum, using labeled tissue lectins as tools in histo-
chemistry enables the mapping of profiles of accessible
binding sites in sections, as reviewed in this issue by Kalt-
ner et al. (2017). By maintaining aspects of the natural
glycan complexity and mode of presentation in sections,
this experimental platform has advantages relative to arti-
ficial systems, tissue processing and the presence of lectins
in situ notwithstanding.
Acknowledgements R.R. thanks the Natural Sciences and Engineer-
ing Research Council of Canada (NSERC) for a Canadian Research
Chair for financial support. P.V.M. thanks Science Foundation Ire-
land for financial support (08/SRC/B1393 and 12/IA/1398), the lat-
ter being co-funded under the European Regional Development Fund
under Grant No. 14/SP/2710. H.-J.G. thanks the excellence pro-
gram of the Ludwig-Maximilians-University Munich, the Verein zur
Förderung des biologisch-technologischen Fortschritts in der Medizin
e.V. (Heidelberg, Germany) and the EC (for ITN network funding;
GLYCOPHARM) for generous support and also Drs. B. Friday, G.
Ippans and A. Leddoz for inspiring discussions.
1
7:663–676
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s00418-016-1526-4
Corfield AP, Berry M (2015) Glycan variation and evolution in the
eukaryotes. Trends Biochem Sci 40:351–359
Dam TK, Gabius H-J, André S, Kaltner H, Lensch M, Brewer CF
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