Stereoselective Synthesis of Fluorinated Galactopyranosides as Potential Molecular Probes for Galactophilic Proteins: Assessment of Monofluorogalactoside–LecA Interactions

Article abstract of DOI:10.1002/chem.201806197

The replacement of hydroxyl groups by fluorine atoms on hexopyranoside scaffolds may allow access to invaluable tools for studying various biochemical processes. As part of ongoing activities toward the preparation of fluorinated carbohydrates, a systematic investigation involving the synthesis and biological evaluation of a series of mono- and polyfluorinated galactopyranosides is described. Various monofluorogalactopyranosides, a trifluorinated, and a tetrafluorinated galactopyranoside have been prepared using a Chiron approach. Given the scarcity of these compounds in the literature, in addition to their synthesis, their biological profiles were evaluated. Firstly, the fluorinated compounds were investigated as antiproliferative agents using normal human and mouse cells in comparison with cancerous cells. Most of the fluorinated compounds showed no antiproliferative activity. Secondly, these carbohydrate probes were used as potential inhibitors of galactophilic lectins. The first transverse relaxation-optimized spectroscopy (TROSY) NMR experiments were performed on these interactions, examining chemical shift perturbations of the backbone resonances of LecA, a virulence factor from Pseudomonas aeruginosa. Moreover, taking advantage of the fluorine atom, the ¹⁹F NMR resonances of the monofluorogalactopyranosides were directly monitored in the presence and absence of LecA to assess ligand binding. Lastly, these results were corroborated with the binding potencies of the monofluorinated galactopyranoside derivatives by isothermal titration calorimetry experiments. Analogues with fluorine atoms at C-3 and C-4 showed weaker affinities with LecA as compared to those with the fluorine atom at C-2 or C-6. This research has focused on the chemical synthesis of “drug-like” low-molecular-weight inhibitors that circumvent drawbacks typically associated with natural oligosaccharides.

Full text of DOI:10.1002/chem.201806197

A Journal of
Accepted Article
Title: Stereoselective synthesis of fluorinated galactopyranosides as
potential molecular probes on galactophilic proteins: assessment
of monofluorogalactosides-LecA interactions
Authors: Vincent Denavit, Danny Lainé, Chahrazed Bouzriba,
Elena Shanina, Émilie Gillon, Sébastien Fortin, Christoph
Rademacher, Anne Imberty, and Denis Giguére
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201806197
Link to VoR: http://dx.doi.org/10.1002/chem.201806197
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Chemistry - A European Journal
10.1002/chem.201806197
FULL PAPER
Stereoselective synthesis of fluorinated galactopyranosides as
potential molecular probes on galactophilic proteins: assessment
of monofluorogalactosides-LecA interactions
Vincent Denavit,[a] Danny Lainé,[a] Chahrazed Bouzriba,[b, c] Elena Shanina,[d] Émilie Gillon,[e] Sébastien
Fortin,[b, c] Christoph Rademacher,[d] Anne Imberty,[e] and Denis Giguère*[a]
Abstract: The replacement of hydroxyl groups by fluorine atoms on
hexopyranoside scaffolds may allow access to invaluable tools to
study various biochemical processes. As part of ongoing activities
toward the preparation of fluorinated carbohydrates, a systematic
investigation involving the synthesis and biological evaluations of a
series of mono- and polyfluorinated galactopyranosides is described.
The preparation of all the monofluorogalactopyranosides, one
molecular weight inhibitors that circumvent drawbacks typically
associated with natural oligosaccharides.
Introduction
The most widespread application of fluorinated
carbohydrates is as radiopharmaceuticals for cancer imaging
trifluorinated
galactopyranoside,
and
the
tetrafluorinated
galactopyranoside was achieved using a Chiron approach. The
synthetic challenge they present combined with the scarcity of some
of these compounds prompted us to evaluate their biological profile.
Firstly, our fluorinated compounds were investigated as
antiproliferative agents using normal human and mouse cells and
compared with cancerous cells. Most of the fluorinated compounds
showed no antiproliferative activity. Secondly, we used these
carbohydrate probes as potential inhibitors for galactophilic lectins.
We performed the first TROSY NMR, chemical shift perturbations of
[
1]
technique. Fluorinated carbohydrates are also invaluable tools
as mechanistic probes to study lectin-carbohydrate interactions
[
2]
and to decipher the mechanisms of glycosidases. To that end,
deoxyfluoro sugars provide useful insights into the role of
hydrogen bonding interactions in order to identify the key active
site of enzyme-substrate interactions.
Lectins, carbohydrate binding proteins, are found in all
[
3]
organisms from animals and plants, to bacteria and viruses.
Lectins are involved in various biological processes, and their
properties are in relation with their carbohydrate binding
the backbone resonances of LecA,
a virulence factor from
Pseudomonas aeruginosa. Moreover, taking advantage of the fluorine
atom, we achieved the direct detection of the 19F NMR resonance of
[4]
domain. Hence, it is crucial to elucidate and understand the
[5]
sugar binding properties of lectins. Among them, galactose-
specific lectins are of high interest as they bind for example to β-
galactose present on branches of N-linked glycans, or to α-
galactose epitopes of some human and non-human blood
the monofluorogalactopyranosides in presence and absence of LecA
to monitor ligand binding. Lastly, these results were corroborated with
the binding potency of the monofluorinated galactopyranoside
derivatives by isothermal titration calorimetry experiments. Analogs
with fluorine atoms at C-3 and C-4 have weaker affinities with LecA
as compared to compounds with fluorine atom at C-2 and C-6. This
research focused on the chemical synthesis of “drug-like” low-
[
6, 7]
groups.
include for example LecA (PA-IL lectin from the pathogenic
Pseudomonas aeruginosa), galectins, asialoglycoprotein
Galactose-specific lectins of therapeutical interest
receptor, and macrophage galactose-binding lectin. Intense
investigations have been devoted to the understanding of these
lectins ability to regulate numerous biological processes. Since
thorough understanding of biochemical pathways is hampered by
the natural complexity of carbohydrates, chemical or chemo-
enzymatic synthesis of glycomimetics ligands is likely to remain a
valuable option in glycoscience. Therefore, there is a major need
to develop new tools that target galactoside-specific lectins in
order to decipher their binding characteristics.
[
[
a]
b]
V. Denavit, D. Lainé, Prof. D. Giguère
Département de Chimie, 1045 Avenue de la Médecine,
Université Laval, Quebec City, Qc, Canada G1V 0A6,
PROTEO, RQRM
E-mail: denis.giguere@chm.ulaval.ca
C. Bouzriba, Prof. S. Fortin
CHU de Québec-Université Laval Research Center, Oncology
Division, Hôpital Saint-François d’Assise, 10 rue de l’Espinay,
Quebec City, Qc, G1L 3L5, Canada
C. Bouzriba, Prof. S. Fortin
Faculté de pharmacie, Université Laval, Quebec City, QC, Canada
G1V 0A6
The carbohydrate binding capability of numerous lectins has
been demonstrated by determining their ability to bind glycan
arrays and by inhibiting their interaction with glycoconjugates or
[
[
c]
d]
[
8]
E. Shanina, Prof. C. Rademacher
glycomimetics. This allows insight into the nature of the
hydrogen bonding involved between the hydroxyl groups of
carbohydrates and the binding sites of the proteins. The
monosaccharides used in these studies generally include fluoro-,
Max Planck Institute of Colloids and Interfaces, Department of
Biomolecular Systems, Am Mühlenberg 1, 14424 Potsdam,
Germany
E. Gillon, Prof. A. Imberty
Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France
[
e]
[
9]
deoxy-, and thio-hexoses, along with uronic acids and alditols.
The major drawbacks to use such diverse glycomimetic libraries
are their poor synthetic availability Consequently, the synthesis of
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Chemistry - A European Journal
10.1002/chem.201806197
FULL PAPER
fluoro-, deoxy-, and thio-glycosides is of high interest. In this
context, we wish to replace the carbohydrate hydroxyl groups with
fluorine atoms to generate new fluorine-substituted glycoside
analogs.[ The rationale to synthesize such compounds emerges
from the likenesses between hydroxyl group and fluorine atom in
regard to polarity and isosteric relationships.[ Also, the loss of
hydrogen donating capacity for the fluorine atom and the high C−F
HaCat primary epidermal keratinocyte, human HDFn neonatal
dermal fibroblast and mouse 3T3 embryonic fibroblast normal
cells. These results were compared with human HT-29 colon
adenocarcinoma and human M21 skin melanoma cancer cells.
Also, our synthetic library could be useful as stable molecular
probes with galactophilic lectins. This is exemplified by the first
TROSY NMR monitoring chemical shift perturbation of LecA from
the virulence factor Pseudomonas aeruginosa. Moreover, taking
advantage of the properties of fluorine atom, the direct detection
of the 19F NMR resonance of monofluorogalactopyranosides were
performed on LecA to identify chemical shift changes of the
ligands as well. Finally, these results were corroborated by the
binding potency of the monofluorinated galactopyranoside
derivatives by isothermal titration calorimetry.
10]
11]
[
12]
bond energy render them resistant to rapid in vivo degradation.
Finally, addition of a fluorine group on a hydroxypyranose core
can modulate the lipophilicity, which in turn can increase cell
permeability.[
13]
Definitely, fluorinated carbohydrates could
represent more “drug-like” tools that circumvent drawbacks
typically associated with natural oligosaccharides, such as low
affinity, limited metabolic stability, and high polarity leading to low
bioavailability.
As part of our ongoing program related to the synthesis of
fluorinated carbohydrates,[14] our attention was turned toward the
preparation of monofluorogalactopyranosides 1−4, along with the
tetrafluorinated galactopyranoside congener 6 (Figure 1). On our
way to prepare the polyfluoro derivative, we were also able to
access trifluorinated galactopyranoside 5. Heavily fluorinated
hexopyranosides (replacement of hydroxyl groups by fluorine
atoms) are particularly interesting because of the synthetic
challenge they present. We developed convenient synthetic
methodologies for further biological investigations. In the
expectation of increasing the molecular diversity of our library, two
aglycones were installed at the anomeric position, a β-O-benzoic
acid and a β-S-(2-naphthyl). This choice is no coincidence since
some galactoside-specific lectins are known to bind such
structural motifs.[
Results and Discussion
Our synthetic endeavours started with the preparation of 2-
fluorogalactopyranosides. Compound such as 2F-glycosides are
important tools used as specific mechanism-based glycosidase
[16a,
22]
inhibitors.
The
synthesis
of
2-deoxy-2-fluoro-
galactopyranoside derivatives is summarized in Scheme 1. Thus,
acetylated galactoside 7 was transformed in two steps into known
2,3,6-tri-O-D-galactal 8 in 91% yield. Treatment of compound 8
with Selectfluor® on large scale exclusively led to 2-deoxy-2-
fluoro-
D
-galactopynoside
9
after O-acetyl protection of the
[23]
anomeric position (α/β = 1.5:1). Stereoselective installation of
the anomeric aglycone proceeded through the α-galactosyl
bromide 10. The crude bromide product underwent a phase
transfer catalyzed nucleophilic displacement with methyl 4-
hydroxybenzoate or 2-naphthalenethiol, leading to derivative 11
15]
monofluoro
HO OH
O
HO OH
F
OH
O
HO
F
O
O
(63% yield over 2 steps) and 12 (55% yield over 2 steps)
HO
R
F
R
HO
R
HO
R
F
HO
HO
HO
respectively. The β configurations were determined by direct NMR
1
: 2F-Gal
2: 3F-Gal
3: 4F-Gal
4: 6F-Gal
coupling of the anomeric proton with the H-2 proton, and the
1
3
O
fluorine at C-2 ( H NMR (500 MHz) δ 5.23 (dd, J
H1-H2
= 7.4 Hz,
F
OH
O
R =
R =
F
F
F
O
3
H1-F2 = 3.8 Hz) for 11 and 4.82 (dd, JH1-H2 = 9.6 Hz, 3JH1-F2 = 2.8
3
J
F
R
2
CO H
R
Hz, 1H, H1) for 12). This methodology will be applied for the
determination of the anomeric configuration of all the
galactopyranoside derivatives prepared in this study.
F
S
F
5
: 2,3,4F-Gal
6: 2,3,4,6F-Gal
[
24]
trifluoro
tetrafluoro
Our next challenge involved the preparation of 3-deoxy-3-
fluorogalactopyranoside derivatives, which was based on our
Figure 1. Mono- and polyfluorinated galactopyranosides prepared in this work.
[
14b]
previously described method and summarized in Scheme 2.
In the context of this study, it is important to point out that
heavily fluorinated carbohydrates have been only used in kinetic
[
25]
Briefly, Cerny’s epoxide 13
was treated with potassium
studies,[ to improve protein-carbohydrate interactions, in the
16]
[17]
hydrogen fluoride in ethylene glycol at 200 °C for 5 hours and
yielded the desired 3-deoxy-3-fluoroglucopyranose 14 in 65%
yield as the sole isomer. With the required 3-deoxy-3-fluoro
derivative in hand, the next task involves inversion of
configuration at C-4. Thus, benzoylation of the free hydroxyl group
development of chemically modified analogs with improved
antigenicity,[
18]
and have been evaluated for their ability to cross
erythrocyte membranes.[
19]
The interesting properties of
polyfluorinated carbohydrates can partly be explained by
desolvation effect, together with attractive dipolar interactions
was followed by deprotection of the 4-O-benzyl group using TiCl
4
.
mediated by polar C−F bonds.[
20]
Then, compound 15 was subjected to a Lattrell-Dax epimerization
Herein, we present the first
[
26]
on gram scale through formation of triflate 16.
mixture was treated with KNO in DMF and generated the desired
1,6-anhydro-β- -galactopyranose derivative 17 in 72% yield over
steps. Acetolysis using a mixture of H SO and Ac O allowed
The crude
library and biological investigation of synthetic monofluorinated
galactopyranosides. To the best of our knowledge, only one report
2
[
21]
D
described the hydrolysis of a series of fluorogalactopyranoside.
2
2
4
2
However, the synthetic preparation of these compounds was not
described. Here, we disclosed the first antiproliferative screening
of mono- and polyfluorinated galactopyranosides on human
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Chemistry - A European Journal
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FULL PAPER
AcO OAc
AcO OAc c) Selectfluor®
isolated in 36% yield over 4 steps (78% per step) and product 34
was isolated in 49% yield over 4 steps (83% per step).
O
a) HBr/AcOH
O
d) Ac O
2
AcO
AcO
AcO OAc
b) Zn, NH4Cl
91% (2 steps)
64% (2 steps)
α/β = 1.5:1
8
7
O
O
b) BzCl, pyridine
81%
O
[
gram scale]
[gram scale]
a) KHF , 200
o
C
F
F
2
O
O
O
65%
c) TiCl
4
, CH
2%
gram scale]
2 2
Cl
AcO OAc
AcO OAc
O
BnO
13
[gram scale]
BnO 14 OH
8
HO
OBz
e) HBr/AcOH
O
O
15
[
AcO
AcO
FBr
F
9
OAc
2
d) Tf O, pyridine
10
f) TBAHS, Na2CO3
g) TBAHS, Na2CO3
AcO OAc
f) H
then NaOAc
2
SO
4
, Ac
2
O
O
O
F
F
e) KNO
2% (2 steps)
[gram scale]
2
, DMF
O
O
O
F
HO
HO
CO2Me
3% (2 steps)
58%
7
BzO
18
HS
OAc
OBz
TfO
OBz
α/β = 3:1
17
16
6
67% (2 steps)
AcO OAc
g) HBr/AcOH
h) TBAHS, Na
HO CO Me
63% (2 steps)
AcO OAc
2
CO
3
O
O
AcO OAc
AcO
O
AcO
S
AcO OAc
O
2
F
2
CO Me
F
O
F
O
F
1
2
BzO
1
1
BzO
2
CO Me
Br
Scheme 1. Synthesis of 2-deoxy-2-fluoro-galactopyranosides 11 and 12. a)
HBr/AcOH, CH Cl , rt, 2 h; b) Zinc (7.5 equiv), NH Cl (7.5 equiv), CH CN, 60 °C,
5 min, 91% over 2 steps; c) Selectfluor® (1.2 equiv), CH NO /H O (5:1), rt, 18
O/pyridine (1:5), rt, 20 h, 64% over 2 steps; e) HBr/AcOH, CH Cl , rt,
20
19
2
2
4
3
2 3
i) TBAHS, Na CO
4
3
2
2
AcO OAc
h; d) Ac
2
2
2
HS
7% (2 steps)
O
4
2 h; f) methyl 4-hydroxybenzoate (3.0 equiv), TBAHS (1.0 equiv), AcOEt/1M
Na CO (1:1), rt, 18 h, 63% over 2 steps; g) 2-thionaphthyl (3.0 equiv), TBAHS
1.0 equiv.), AcOEt/1M Na CO (1:1), rt, 18 h, 55% over 2 steps. AcOH = acetic
acid, Ac O = acetic anhydride, Selectfluor® = 1-chloromethyl-4-fluoro-1,4-
diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate); TBAHS
F
S
2
3
6
BzO
(
2
3
2
1
2
Scheme 2. Synthesis of 3-deoxy-3-fluoro-galactopyranosides 20 and 21. a)
KHF (6.1 equiv), ethylene glycol, 200 °C, 5 h, 65%; b) BzCl (3.0 equiv), pyridine,
CH Cl , rt, 1 h, 81%; c) TiCl (1.1 equiv), CH Cl , 0 °C, 1 h, 82%; d) Tf O (2.3
equiv), pyridine, CH Cl , 0 °C to rt, 0.5 h; e) KNO (3.0 equiv), DMF, rt, 24 h,
2% over 2 steps; f) H SO (10.0 equiv), Ac O (30.0 equiv), rt, 18 h, then NaOAc
(20.0 equiv), rt, 0.3 h, 58% (α/β = 3:1); g) HBr/AcOH, CH Cl , 0 °C to rt, 2 h; h)
methyl 4-hydroxybenzoate (3.0 equiv), TBAHS (1.0 equiv), AcOEt/1M Na CO
1:1), rt, 18 h, 63% over 2 steps; i) 2-thionaphthyl (3.0 equiv), TBAHS (1.0 equiv),
AcOEt/1M Na CO (1:1), rt, 18 h, 67% over 2 steps. Ac O = acetic anhydride,
BzCl = benzoyl chloride, DMF = N,N-dimethylformamide, NaOAc = sodium
acetate, TBAHS tetrabutylammonium hydrogen sulfate, Tf
=
tetrabutylammonium hydrogen sulfate.
2
2
2
4
2
2
2
2
2
2
the generation of protected 3-deoxy-3-fluoro-D-galactopyranose
8 in 58% yield (α/β = 3:1). The stereoselective functionalization
7
2
4
2
1
2
2
2
3
of the anomeric position was easily achieved as described above.
Thus, the mixture HBr/AcOH generated bromide 19, which was
subsequently subjected to the phase transfer catalyzed reactions.
Methyl 4-hydroxybenzoate 20 and 2-naphthalenethiol 21 were
isolated in 63% and 67% yields, respectively over 2 steps.
The syntheses of 4-deoxy-4-fluoro-galactopyranoside
(
2
3
2
=
2
O
=
trifluoromethanesulfonic anhydride.
O
O
OR
O
OR
c) H
then NaOAc
2% (2 steps)
2 4 2
SO , Ac O
derivatives were initiated by the use of known 4-O-p-
toluenesulfonyl 22 (Scheme 3).[
b) TBAF, THF
31%
O
F
27]
Protection of the residual
OR
4: R = MOM
5
TsO
OR
22: R = H
hydroxyl group allowed the preparation of compound 23 in high
yield and the latter was subjected to nucleophilic fluorination using
TBAF in boiling THF. Compound 24 was isolated together with an
unidentifiable impurity, consequently it was subjected to the next
reaction. The mixture was directly subjected to acetolysis under
acidic condition and the concomitant removal of the MOM
protecting group proceeded as expected, generating intermediate
α/β = 3:1
2
a) MOMCl
97%
23: R = MOM
F
OAc
O
F
OAc
O
d) HBr/AcOH
AcO
AcO
AcO OAc
25
AcO
Br
2
6
e) TBAHS, Na
HO CO
40% (2 steps)
2
CO
3
f) TBAHS, Na
2
CO
3
[
16c]
2
5 in 52% yield over 2 steps (α/β = 3:1).
Finally, the anomeric
2
Me
groups were installed using the strategy mentioned above,
allowing the isolation of products 27 and 28 via bromide
intermediate 26.
The easiest fluorinated analog to prepare was undoubtedly
the 6-deoxy-6-fluoro-galactopyranose. The synthetic route was
HS
69% (2 steps)
F
OAc
O
F
OAc
O
AcO
O
AcO
S
AcO
27
2
CO Me
AcO
straightforward and initiated with 1,2:3,4-di-O-isopropylidene-α-
D
-
28
galactopyranose 29 as inexpensive starting material (Scheme 4).
Fluoro derivative 30 was isolated in 87% yield using Me-DAST
with microwave irradiation (80 °C) for 1 hour. This method
Scheme 3. Synthesis of 4-deoxy-4-fluoro-galactopyranoside 27 and 28. a)
MOMCl (10.0 equiv), DIPEA (11.0 equiv), CH Cl , 40 °C, 18 h, 97%; b) TBAF
10.0 equiv), THF, 66 °C, 72 h, 31%, based on 76% purity; c) H SO (10.0 equiv),
O (30.0 equiv), rt, 18 h; then NaOAc (20.0 equiv), rt, 0.3 h, 52 % over 2 steps,
Cl , rt, 1 h; e) methyl 4-hydroxybenzoate (3.0 equiv),
TBAHS (1.0 equiv), AcOEt/1M Na CO (1:1), rt, 18 h, 40% over 2 steps; f) 2-
thionaphthyl (3.0 equiv), TBAHS (1.0 equiv), AcOEt/1M Na CO (1:1), rt, 18 h,
69% over 2 steps. Ac O = acetic anhydride, DIPEA = N,N-diisopropylethylamine,
2
2
(
Ac
2
4
2
[
14b]
provided higher yield as compared to conventional heating.
α/β = 3:1; d) HBr/AcOH, CH
2
2
2
3
Next, isopropylidene hydrolysis was followed by acetyl protection
allowing the preparation of anomeric mixture of 6-deoxy-6-fluoro-
galactopyranose 31. The latter was subjected to acidic conditions
followed by phase transfer catalyzed reactions. Product 33 was
2
3
2
MOMCl = chloromethyl methyl ether, NaOAc = sodium acetate, TBAF =
tetrabutylammonium fluoride, TBAHS = tetrabutylammonium hydrogen sulfate.
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Chemistry - A European Journal
10.1002/chem.201806197
FULL PAPER
O
OH
O
O
F
O
AcO
AcO
F
O
explained by a trans-diaxially positioned benzyloxy group at C-4
a) DAST
b) AcOH/H
2
O
O
O
capable of participation through an oxiranium intermediate
O
87%
gram scale]
O
c) Ac O, pyridine
2
AcO OAc
31
[29]
species.
4
A TiCl -mediated benzyl deprotection allowed the
O
O
[
generation of compound 38 in 66% yield, which was the ideal
precursor to generate the 1,6-anhydro-2,3,4-trideoxy-trifluoro-β-
29
30
d) HBr/AcOH
e) TBAHS, Na CO3
D
-galactopyranose 39. Thus, the free O-4 hydroxyl group on
2
AcO
AcO
F
AcO
F
O
compound 38 was activated as a triflate and subjected to a
nucleophilic fluorination using TBAF allowing the generation of
compound 39 with complete inversion of configuration. The
isolation and purification of trifluoro 39 proved to be difficult due
to its high volatility. Consequently, the crude mixture was treated
O
HO
CO Me
2
O
AcO
AcO
CO Me
AcO
Br
2
36% (4 steps)
33
32
f) TBAHS, Na
2 3
CO
AcO
AcO
F
O
under acidic conditions (H
product 40 in 63% yield over 3 steps. The configurations of the
2 4 2
SO , Ac O) generating the acetolysis
HS
S
[
30]
AcO
4
9% (4 steps)
1
9
34
fluorine atoms were ascertained based on F NMR spectroscopy
1
9
3
3
3
Scheme 4. Synthesis of 6-deoxy-6-fluoro-galactopyranoside 33 and 34. a) Me-
DAST (1.2 equiv), 2,4,6-collidine (2.4 equiv), CH Cl 80 °C, microwave
irradiation, 1 h, 87 %; b) AcOH/H O (4:1), reflux, 18 h; c) Ac O, pyridine, rt, 72
h; d) HBr/AcOH, CH Cl , rt, 2 h; e) methyl 4-hydroxybenzoate (3.0 equiv),
TBAHS (1.0 equiv), AcOEt/1M Na CO (1:1), rt, 18 h, 36% over 4 steps; f) 2-
thionaphthyl (3.0 equiv), TBAHS (1.0 equiv), AcOEt/1M Na CO (1:1), rt, 18 h,
9% over 4 steps. AcOH = acetic acid, Ac O = acetic anhydride, Me-DAST =
( F NMR (470MHz): J
= 12.8 Hz, J
= 13.4 Hz, J
F3-H4
=
F2-H3
F3-F4
2
2
,
3
3
6.4 Hz, JF4-H3 = JF4-H5 = 27.0 Hz). The aglycones were installed
2
2
using the same strategy as before, allowing the preparation of
compounds 42 and 45 via bromide 41. In both reactions, side
2
2
2
3
2
3
product
A was isolated from the mixture originating from
4
2
dimethylaminosulfur trifluoride, TBAHS = tetrabutylammonium hydrogen sulfate.
elimination of the anomeric bromine (Scheme 5). The last task
was the deoxofluorination at C-6 and represented a more
[
14a]
challenging task
experimentation,
than expected.
Upon
extensive
In order to increase the molecular diversity of our library, our
a
DAST-mediated deoxofluorination on 43
next task focused on the preparation of tetrafluorinated
Along the way, it became obvious that we
could also access one trifluorinated galactopyranoside. Hence,
allowed the generation of polyfluorohexopyranose 44 in 57% yield
[together with 32% of the L-arabino-hex-5-enopyranoside
derivative B]. For compound 45, due to the instability of the
galactopyranosides.[
14a]
[
31]
Scheme 5 described our synthetic endeavours toward this end
thionaphthyl moiety under the deoxofluorination conditions,
a
and starts with easily accessible Cerny’s epoxide 35 obtained in
4
anhydro derivative 35 was achieved upon exposure to potassium
hydrogen fluoride in 73% yield. Treatment of 36 with Deoxo-
Fluor® furnished 2,3-dideoxy-difluoro-glucose 37 in high yield
with complete retention of configuration. This result can be
different approach was followed. After de-O-acetylation, the free
hydroxyl group of 46 was activated as a triflate and a nucleophilic
fluorination using TBAF gave a disappointing 9% yield. The major
side product of this transformation was the elimination of the C-6
steps from levoglucosan.[ Nucleophilic fluorination of the 2,3-
28]
O
O
O
O
O
O
, 200 o
OH
F
F
F
a) KHF
2
C
b) Deoxo-Fluor®
87%
c) TiCl
4
, CH
2
Cl
2
d) Tf
2
O, pyridine
O
O
O
O
O
F
.
7
3%
[gram scale]
66%
e) TBAF 3H
2
O
BnO 3
BnO
F
BnO
F
HO
F
F
[
gram scale]
5
36
37
39
38
f) H
then NaOAc
2
SO
4
, Ac
2
O
63%
(3 steps)
h) TBAHS, Na
2
CO
3
F
OR
O
O
F
F
F
O
F
F
OAc
O
F
OAc
O
HO CO
2
Me
j) DAST
57%
F
g) HBr/AcOH
F
O
F
CO
2
Me
60% (2 steps)
F
F
F
OAc
CO
2
Me
Br
4
2: R = Ac
i) NaOMe
9%
41
40
44
9
4
3: R= H
k) TBAHS, Na CO3
2
F
OAc
O
F
B
O
F
F
F
OR
O
F
F
O
2
d) Tf O, pyridine
O
HS
F
2
CO Me
F
S
A
F
S
l) TBAF, THF
% (2 steps)
F
F
94% (2 steps)
9
F
C
47
45: R = Ac
6: R = H
Scheme 5. Synthesis of trifluorinated 43 and 46 and tetrafluorinated 44 and 47 galactopyranoside derivatives. a) KHF
3%; b) Deoxo-Fluor® (2.0 equiv), THF, microwave irradiation, 100 °C, 1.5 h, 87%; c) TiCl (1.1 equiv), CH Cl , 0 °C, 0.5 h, 66%; d) Tf
equiv), 0 °C, 0.2 h; e) TBAF⋅3H O (1.5 equiv), CH Cl , rt, 15 h; f) Ac O (30.0 equiv), H SO (10.0 equiv), 0 °C to rt, 18 h, then NaOAc (20.0 equiv), rt, 0.3 h, 63%
over 3 steps, α/β = 5:1; g) HBr/AcOH, CH Cl , rt, 66 h; h) methyl -hydroxybenzoate (3.0 equiv), TBAHS (1.0 equiv.), EtOAc/ 1M Na CO (1:1), rt, 18 h; 60% over
steps; i) 1M NaOMe, MeOH, rt, 1 h, 99% for 43, 99% for 46; j) DAST (3.0 equiv), CH Cl , microwave irradiation, 100 °C, 1 h, 57%; k) 2-thionaphthyl (3.0 equiv),
TBAHS (1.0 equiv), EtOAc/1M Na CO (1:1), rt, 18 h; 94% over 2 steps; k) Tf O (2.0 equiv), pyridine (10.0 equiv), 0 °C, 0.5 h; l) 1M TBAF in THF (15 equiv), −78 °C
to rt, 15 h, 9% over 2 steps. Ac O = acetic anhydride, DAST = diethylaminosulfurtrifluoride, Deoxo-fluor® = bis(2-methoxyethyl)aminosulfurtrifluoride, TBAF =
tetrabutylammonium fluoride, TBAHS = tetrabutylammonium hydrogen sulfate, Tf O = trifluoromethanesulfonic anhydride.
O
i) NaOMe
9%
F
S
9
4
F
2
(7.0 equiv), ethylene glycol, 200 °C, 2.5 h,
O (2.0 equiv), pyridine (3.0
7
4
2
2
2
2
2
2
2
2
4
2
2
p
2
3
2
2
2
2
3
2
2
2
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Chemistry - A European Journal
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leaving group leading to derivative C in 83% yield. This example
clearly shows the limitation of deoxyfluorination with aryl
thiohexopyranoside analogs.
Table 1. Deprotection of acetylated fluorogalactopyranosides generating
analogs 48−57.
The complete deprotection of our target products was
achieved according to two distinct protocols, depending on the
substrates (Table 1). In the case of the methyl p-(O-
galactosyl)benzoate analogs, lithium hydroxide was used for
concomitant de-O-acetylation and generation of the acid moiety,
allowing preparation of compounds 48, 50, 52, 54, 56, and 57. For
thiogalactoside analogs, a classical Zemplén de-O-acetylation
Method[a]
Entry
Starting
material
Product
Yield
(%)[
b]
(NaOMe, MeOH) provided the desired analogs 49, 51, 53, and 55.
Both methods furnished clean products in high yields.
1
11
12
20
21
27
28
33
34
43
44
A
89
97
92
98
86
94
93
98
94
97
In order to complement this study, we investigated key
physical properties of some of our fluorinated carbohydrates.
Firstly, in order to establish unambiguously the configuration of
the fluorine atoms of the polyfluorinated galactopyranoside
2
3
4
5
6
7
8
9
B
A
B
A
B
A
B
A
B
derivatives, an X-ray crystallographic analysis of 57 was achieved
32]
(
Figure 2).[ We obtained a crystalline polymorph with different
[14a, 33]
space group than what was previously reported.
In the solid
state, compound 57 is a dimer and the hexopyranose ring adopts
4
19
1
a C conformation. Secondly, we compared the F NMR spectra
of compound 48, 50, 52, 54, 56, and 57 (Figure 3). The
assessment of coupling constants in 19F and H NMR represents
a reliable tool for determination of the absolute configuration and
conformation of fluorinated carbohydrates. Figure 3 shows that
the multiplicities are very consistent, and depend on the relative
spatial relationships between neighbouring atoms. All fluorinated
1
carbohydrates adopts the 4
1
C conformation. For polyfluorinated
analogs 56 and 57, a comparison of fluorine signals with their
monofluorinated counterparts reveals that the F-H coupling
constants are similar with a slight change in chemical shift. Finally,
the C-6 fluorine atom in compound 54 adopt either a TG or GT
1
conformation. This was confirmed after analysis of the H NMR
spectrum (500 MHz). The proton at C-5 has a chemical shift of
3
4
.02 ppm with, amongst others, a coupling constant JH5-F6 of 15.2
Hz, corresponding to a gauche conformation with F-6. A similar
result is obtained for compound 57 (proton at H-5 = 4.52 ppm with
3
J
H5-F6 = 12.9 Hz). This result is in opposition with the conformation
of the fluorine atom at C-6 of compound 57 in the solid state
Figure 2). The GG conformer, corresponding to the one from the
(
crystal structure, adopts one of the highest energetic
conformation.[
14a,34]
This demonstrates that fluorine NMR are an
invaluable tool to analyse structural conformation of
organofluorine compounds.
1
0
[a] Method A: 1M LiOH (10.0 equiv), H
2
O/MeOH/THF (2:3:5); Method B: NaOMe
in MeOH.
[b] Yields refer to isolated pure products.
Figure 2. X-ray crystallographic analysis derived ORTEP of 57 showing 50%
thermal ellipsoid probability, carbon (gray), oxygen (red), fluorine (light green),
hydrogen (white).
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Chemistry - A European Journal
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Figure 3. Direct comparison of 19F resonances of compounds 48, 50, 52, 54, 56, and 57 (19F NMR, 470 MHz). Expansions from the spectrum display coupling
constant of each signals.
epidermal keratinocyte, human HDFn neonatal dermal fibroblast
Antiproliferative activity
and mouse 3T3 embryonic fibroblast normal cells as compared
with human HT-29 colon adenocarcinoma and human M21 skin
melanoma cancer cells (Table 2). Most of the fluorinated
compounds (43 and 48−56) showed no antiproliferative activity
against normal or cancer cell lines and could therefore be used in
various cells assays. In contrast, trifluorinated galactose
We wished to use our library in various biological systems.
Consequently, we evaluated the antiproliferative profile of
fluorinated galactopyranosides. Compounds 43, 46, 48−56 were
tested for their antiproliferative activity on human HaCaT primary
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Chemistry - A European Journal
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derivative 46 with a thionaphthyl moiety presented some activity
with no selectivity towards normal cell lines (IC50 = 45−69 µM) and
cancer cell lines (IC50 = 34−38 µM). This is a weak antiproliferative
agent when compared to Topotecan, a known chimiotherapeutic
active compound.
protein. This is exemplified in Figure 4a for the spectrum of 15N-
labelled LecA in presence of 2-deoxy-2-fluoro-galactopyranoside
48 (orange) and absence (blue). Multiple perturbations of
backbone chemical shifts and line broadening of these
resonances were visible particularly related to amino acid
constituting the carbohydrate recognition domain as inferred from
experiments with unsubstituted galactose. In contrast, 4-deoxy-4-
Table 2. Antiproliferative activity (IC50
) of molecular probe (48−56)
derivatives on human HaCaT primary epidermal keratinocyte, human HDFn
neonatal dermal fibroblast and mouse 3T3 embryonic fibroblast normal cells
as compared with human HT-29 colon adenocarcinoma and human M21
skin melanoma cancer cells.
fluoro-galactopyranoside
52
induces
significantly
less
perturbations in the protein backbone indicative for a reduced
interaction (Figure 4b). Clearly, the fluorine atom at C-4
abrogates binding to LecA.
IC50 (µM)[a]
A)
Normal cell lines
Cancer cell lines
Compound
HaCaT
HDFn
> 100
45
3T3
> 100
56
HT-29
> 100
38
M21
4
4
3
6
> 100
69
> 100
34
4
8−56
> 100
0.18
> 100
0.24
> 100
0.48
> 100
0.34
> 100
2.0
Topotecan
[a] IC50 is expressed as the concentration of drug inhibiting cell proliferation
by 50% after 48 h of treatment.
Biophysical measurements
Pseudomonas aeruginosa is a Gram-negative bacterium
and a leading pathogen for infections of immune-compromised
patients and patients suffering from cystic fibrosis.[ LecA is a
virulence factor crucial for biofilm formation by P. aeruginosa and
35]
B)
has been demonstrated to be involved in adhesion to lung
tissues.[
36]
This lectin has
a
strong specificity for α-
galactopyranose terminating oligosaccharides, however β-
galactopyranoside possessing an aromatic aglycon were reported
to be efficient binders.[
15b,37]
Moreover, 2-naphthyl-1-thio-β-D-
galactopyranoside were reported to have high affinity with LecA
of 6.3 µM), an affinity increase nicely supported by the
(K
D
available X-ray crystal structure with the lectin (PDB code
ZYF).[ In the context of this study, we proposed that our library
15b]
3
of fluorinated galactosides could represent efficient synthetic
glycomimetics of natural α-linked oligosaccharides with improved
properties. To unveil such interactions, we first looked at the
1
15
chemical shift perturbations in H, N-TROSY (transverse
relaxation-optimized spectroscopy) spectra of the backbone
15
amide resonances of
N-labelled LecA introduced by the
interaction with our fluorinated probes. It is important to note, that
we performed the first TROSY NMR experiments of LecA and this
milestone could be useful for future related experiments.
Complementary to these studies, we followed the perturbation of
the isolated and sensitive 19F resonances of the compounds.
Finally, these NMR experiments were corroborated using
isothermal titration calorimetry experiments.
The 1H, N-TROSY- NMR spectrum of 15N-labelled LecA
clearly demonstrated that the position of the fluorine atom on the
pyranose ring strongly influence the binding of galactose to the
Figure 4. Chemical shift perturbations of backbone resonances of LecA upon
1
15
binding to galactose derivatives 48 and 52 using H, N-TROSY NMR
15
experiments. Spectra of 350 µM N-labelled LecA in the absence (blue) and
presence (orange) of monofluorinated galactoside are shown for A) 0.2 mM 48
and B) 1.0 mM 52. Fluorination in C2 (48) induces multiple chemical shift
perturbation and line broadening while fluorine atom at C-4 (52) abrogates
binding to LecA.
15
Following-up on these results, we thus performed the
H, N-TROSY experiments over all the monofluorinated
1
15
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Chemistry - A European Journal
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galactosides. The results are shown in Figure 5 and summarize
there is a broadening of the peak associated with a reduction of
the peak intensity by about 40% indicating binding to the lectin.
All monofluorinated galactopyranosides 48−55 were analysed
analogously (Figure 6b). Taken together, derivatives with fluorine
atoms at C-2 (48 and 49) and C-6 (54 and 55) showed large
changes in peak intensity (35−80%). Also, for these derivatives,
compounds with the β-S-(2-naphthyl) aglycone (49 and 55)
experienced largest changes in peak intensity of up to 80%.
Finally, while no changes in peak intensity could be observed for
compound 50, significant changes in chemical shift were recorded,
indicative of a faster exchange rate on the NMR chemical shift
time scale.
To evaluate the affinity of the best compounds, we
performed isothermal titration calorimetry experiments on our
monofluorinated galactopyranoside analogues (48−49). A typical
thermogram of LecA interacting with compound 49 is given in
Figure 7a. First of all, no binding was observed for compounds
with fluorine atom at position 3 and 4, which are involved in
coordination of calcium ion. Fluorination at position 2 resulted in
a slight decrease in affinity. Fluorination at position 6 has a
stronger effect with decrease of one or two order of magnitude.
This is in agreement with the role of each hydroxyl group in the
complex between LecA and galactose (1OKO) (Figure 7b).
Oxygens at position 3 and 4 are crucial for the interaction, being
involved in direct coordination of the bridging calcium ion and in
hydrogen bonds with amino acid side chains. Oxygen O-6 is
involved in two direct hydrogen bonds with the protein, and
oxygen O-2 in only one.
the perturbed backbone resonances upon addition of
monofluorinated galactopyranosides 48−55.[
38]
Since no
assignment backbone resonance is available, labeling of the
peaks is arbitrary and we utilized these perturbations for
fingerprinting the interaction patterns. Similar perturbation
patterns would indicate similar binding patterns. First of all,
analogs with fluorine atoms at C-3 (50 and 51) and C-4 (52 and
53) showed limited chemical shift perturbations or changes in
peak intensities, thus suggesting no or weak affinities with LecA.
Hydroxyl group at C-3 and C-4 of galactose are involved in a
coordination to the calcium ion bound to LecA.[ Consequently,
fluorine atoms at these positions abrogate efficient binding to
LecA. Furthermore, as for compounds with fluorine atom at C-2
39]
(48 and 49) and C-6 (54 and 55), strong change in peak intensity
or chemical shift perturbation were noticed. In fact, the largest
changes were observed for residues involved in the carbohydrate
recognition domain as inferred from galactose binding (Figure 5,
top).[ Overall, a consensus of perturbed resonances can be
deduced from all compounds suggesting that a common binding
site is shared (Figure 5, bottom).[
39]
40]
Thus far, perturbations of the protein resonances were used
to monitor the interaction. Since, the incorporation of a fluorine
offers exquisite possibilities in NMR we chose to complement our
studies and to take advantage of this and perform 19F NMR of
monofluorinated galactoside derivatives in presence and absence
of LecA (Figure 6). Changes in peak intensity in the 19F NMR
spectra were followed as exemplified with 2-deoxy-2-fluoro-
galactopyranoside 48 (Figure 6a). Upon addition of LecA to 48,
Figure 5. Summary of backbone resonances of LecA perturbed by the presence of monofluorinated galactosides (48-55) and galactose using 1H,15N-TROSY NMR.
Resonance IDs are arbitrary and cannot be associated with amino acid residues in absence of an assignment, but serve as fingerprints to visualize interaction
patterns. If the change in peak intensity is more than 20% or if there is a chemical shift perturbation exceeding 0.025 ppm, the residue was attributed a blue color.
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A)
B)
Table 3.
K
d
values and thermodynamics for the binding LecA to selected
fluorinated galactopyranoside derivatives and reference compounds in ITC
assays. Experiments were performed twice and standard deviations was
lower than 10%.
−
∆
H
°
−
T
∆
S
°
−∆
G
°
K
d
Ligand
[
KJ mol−1]
[KJ mol−1
]
[KJ mol−1
]
[µM]
α-Gal-
β-Gal-
β-Gal-
β-Gal-
O
-Me
-Me
40.9
19.0
38.9
45.1
31.9
35.3
-
16.3
5.3
11.1
14.8
6.9
8.1
-
24.6
24.3
27.8
30.3
25.0
27.2
20.1
22.3
50.0
55.7
13.0
4.8
O
O
-p
-benzoic acid
S
-2-thionaphthyl
4
4
8
9
41
17
5 [
5 [
4
a]
303
124.2
a]
5
-
-
[a] Due to low affinity, a sigmoid curve could not be obtained for compounds
5
4 and 55, which precluded the determination of reliable value for ∆H.
Figure 6. Direct observation of 19F resonances of monofluorinated galactosides
upon binding to LecA. A) 19F NMR spectrum of compound 48 (blue) and in
presence of LecA (orange): Reduction of the signal intensity of the galactoside
indicates binding to LecA. Both spectra were normalized to reference
provided analogues with higher affinity as compared to their β-O-
benzoic acid counterpart (48 and 54). Futhermore, it is important
to point out that compound 49 constitute a privilege class of LecA
1
9
trifluoroacetic acid (TFA: −75.6 ppm). B) Percent peak intensity changes of
F
monofluorinated galactosides 48−55 in presence of LecA (blue) compared to
spectra recorded in absence of LecA. Compound 50 was arbitrarily assigned
with 100% to indicate binding inferred from chemical shift perturbation (orange)
in absence of intensity reduction.
inhibitors (3 times more potent than methyl β-D-galactopyranose).
For one thing, the aromatic thioglycoside represent a stable
functional group in biological medium by the glycosidically stable
thio linkage. Also, not only the high C−F bond energy renders
them resistant to in vivo degradation, fluorine group can increase
lipophilicity, which in turn can increase cell permeability. Finally,
in the context of this study, the limitation of our library are the low
water solubility of polyfluorinated analogues (especially for
compounds with the β-S-(2-naphthyl aglycone) and the low affinity
between fluorine atoms with the calcium cation. One can assume
that our library of fluorinated analogues would be more suitable
as ligand for other galactophilic proteins that does not bear a
cationic metal in the carbohydrate recognition domain.
It is also of interest to evaluate the effect of fluorination on
the thermodynamics, by analysing the enthalpy and entropy
contribution of the 2F-derivative compared to native compounds.
[41]
Fluorination at position
2 decreases strongly the enthalpy
contribution (loss of 20%) but the entropy barrier is also
significantly decreased (Table 3). As a result, the decrease in
affinity is limited, corresponding to a loss of about 10% in free
energy. The decrease in binding enthalpy can be correlated to the
loss of the hydrogen bond to Asn107, and the gain in entropy
contribution to a modification of the water network. Moreover, as
previously observed, the β-S-(2-naphthyl) aglycone (49 and 55)
A)
B)
Figure 7. A) Thermogram of LecA interacting with compound 49. The ITC plot (measure by VP-ITC Microcal) in the lower panel shows the total heat released as a
function of total ligand concentration for the titration shown in the upper panel. The solid line denotes the best least square fit to experimental data using a one site
model. B) D
-Galactose in the carbohydrate recognition domain of LecA (1OKO).[
39]
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Chemistry - A European Journal
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on a JASCO DIP-360 digital polarimeter at 589 nm, and are reported in
−1
2
−1
units of 10 (deg cm g ).
Conclusion
The preparation and characterization of the first library of
fluorinated galactopyranosides was achieved with two aglycones
at the anomeric position: β-O-benzoic acid and β-S-(2-naphthyl).
Most of the fluorinated compounds showed no antiproliferative
activity against normal or cancer cell lines. Also, our library of
stable glycomimetics can be used as molecular probes with
galactophilic lectins. The first TROSY NMR following chemical
shift perturbation of LecA and 19F NMR in presence and absence
of LecA suggested that analogs with fluorine atoms at C-3 and C-
Cell lines culture
HaCaT primary epidermal keratinocyte and human HDFn neonatal
dermal fibroblast human cells were purchased from Thermo Fisher
Scientific, while mouse 3T3 embryonic fibroblast and human HT-29 colon
adenocarcinoma cells were purchased from the American Type Culture
Collection (Manassas, VA). M21 human skin melanoma cells were kindly
provided by Dr. David Cheresh (University of California, San Diego School
of Medicine). HaCaT and 3T3 cells were cultured in high-glucose
Dulbecco’s minimal essential medium (DMEM, Gibco, Thermo Fisher
Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco,
Thermo Fisher Scientific) and 1% antibiotic penicillin-streptomycin (5,000
U/mL). HDFn cells were cultured in DMEM supplemented with 2% (v/v)
FBS, 1% penicillin-streptomycin, fibroblast growth factor (3 ng/mL),
epidermal growth factor (10 ng/mL), hydrocortisone (1 ng/mL) and heparin
4
seems to have weak affinities with LecA. Furthermore,
compounds with fluorine atom at C-2 and C-6 showed strong
changes in peak intensity or chemical shift perturbations. This
results was corroborated with isothermal titration calorimetry
experiments. Compound 49 was a high affinity ligand for LecA
with dissociation constants of 17 µM. The present investigation
clearly shows the importance of systematic investigations in the
search of stable and potent glycomimetics as lectin ligand. By
blending organic synthesis and biological studies endeavors, we
strongly believe that the resulting molecules could serve as useful
tools to deepen investigations on the use of stable glycomimetics
and to underscore their relevance and their yet underestimated
potential to medicine.
(
10 ng/mL). HT-29 and M21 cells were cultured in DMEM supplemented
with 5% of FBS. Cells were maintained at 37 °C in a moisture-saturated
atmosphere containing 5% CO
2
.
Antiproliferative activity assay
The growth inhibition potency of all compounds was assessed using
the procedure recommended by the National Cancer Institute (NCI)
Developmental Therapeutics Program for its drug screening program with
[42]
slight modifications. Briefly, 96-well Costar microtiter clear plates were
3
seeded with 75 µL of a suspension of either HaCaT (4.5 × 10 ), 3T3 (3 ×
3
3
3
3
1
0 ), HDFn (3 × 10 ), HT-29 (5 × 10 ), or M21 (3 × 10 ) cells per well in
the appropriate medium. Plates were incubated for 24 h. Freshly
solubilised drugs in DMSO (40 mM) were diluted in fresh medium and 75
µL aliquots containing serially diluted concentrations of the drug were
added. Final drug concentrations ranged from 100 µM to 78 nM. DMSO
concentration was kept constant at < 0.5% (v/v) to prevent any related
toxicity. Plates were incubated for 48 h, after which growth was stopped
by the addition of cold trichloroacetic acid to the wells (10% w/v, final
concentration). Afterward, plates were incubated at 4 °C for 1 h. Then,
plates were washed 5-times with distilled water and a sulforhodamine B
solution (0.1% w/v) in 1% acetic acid was added to each well. After 15 min
at room temperature, the exceeding dye was removed and was washed 5-
times with a solution of 1% acetic acid. Bound dye was solubilized in 20
mM Tris base and the absorbance was read using an optimal wavelength
Experimental Section
Chemical synthesis
All reactions were carried out under an argon atmosphere with dry
solvents under anhydrous conditions, unless otherwise noted. Dry
tetrahydrofuran (THF), toluene, benzene, diethyl ether (Et
dimethylformamide (DMF), and methylene chloride (CH
obtained by passing commercially available pre-dried, oxygen-free
formulations through activated alumina columns. Yields refer to
chromatographically and spectroscopically (NMR) homogeneous
materials, unless otherwise stated. Reagents were purchased at the
highest commercial quality and used without further purification, unless
otherwise stated. Reactions were monitored by thin-layer chromatography
2
O), N,N′-
Cl were
2
2
)
(530-580 nm) with a SpectraMax® i3x (Molecular Devices). Data obtained
from treated cells were compared to the control cell plates fixed on the
treatment day and the percentage of cell growth was thus calculated for
each drug. The experiments were done at least twice in triplicate. The
assays were considered valid when the coefficient of variation was < 10%
for a given set of conditions within the same experiment.
(
TLC) carried out on 200 µm SiliaPlate™ Aluminium backed TLC (indicator
F-254) using UV light as visualizing agent and an ethanolic solution of
phenol and sulphuric acid, and heat as developing agents. SiliaFlash®
P60 (particle size 40 – 63 µm, 230 – 400 mesh) was used for flash column
chromatography. NMR spectra were recorded on Agilent DD2 (at 500 MHz
1
19
13
for H, 470 MHz for F and 126 MHz for C) instruments and calibrated
1
15
H, N TROSY NMR
Fluorinated galactopyranosides binding to LecA were validated
using residual undeuterated solvent (Chloroform-d: δH = 7.26 ppm, δC =
7
2
7.16 ppm; DMSO-d
.05 ppm, δC = 29.84 ppm; Methanol-d
6
: δH = 2.50 ppm, δC = 39.52 ppm; Acetone-d
6
: δH =
1
15
1
15
using protein-observed H,
N TROSY NMR. All H, N TROSY
4
: δH = 3.31 ppm, δC = 49.0 ppm)
measurements were performed at 310 K in Norell S-3-800-7 3mm tubes
19
as an internal reference. Calibration of F NMR were done using
hexafluorobenzene, which have been measured at –162.29 ppm
compared to the chemical shift of the reference compound CFCl
(
Norell) on a Bruker Ascend 700 MHz spectrometer (Bruker, Billerica, MA,
TM
USA) equipped with a 5mm TCI700 CryoProbe
.
3
. The
Briefly, H, N TROSY spectra of 350 µM (U: 15N) LecA were
1
15
following abbreviations were used to designate multiplicities: s = singlet, d
recorded in 20 mM HEPES, 150 mM NaCl, pH 7.4 buffer containing 10 mM
=
=
doublet, t = triplet, q = quartet, p = quinted, h = sextet, m = multiplet, br
broad. Infrared (IR) spectra were recorded on a Thermo Nicolet 380 FT-
CaCl
fluorinated
the respective spectrum was compared to a spectrum with the same
amount (v/v) of DMSO-d to factor out changes caused by the solvent. A
H, N TROSY pulse sequence with WATERGATE solvent suppression,
28 increments, and 16 scans per increment was applied. Data were
2
, 100 µM DSS as internal reference and 10% D
2
O. 0.2-1.5 mM of
D
-galactose compound dissolved in DMSO-d
6
was added and
IR spectrometer, with ZnSe crystal plate. High-resolution mass spectra
HRMS) were recorded on an Agilent serial 1200 TOF (time of flight) 6210
(
6
mass spectrometer using ESI (electrospray ionization). Melting points are
uncorrected and were recorded on a Stanford Research Systems Optimelt
MPA100 automated melting point system. Optical rotations were recorded
1
15
1
[43]
[44]
processed in NMRpipe and further analysed in CCPN.
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Chemistry - A European Journal
10.1002/chem.201806197
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1H,15N TROSY resonances were indexed with IDs from 1 to 118 due
0048) and CBH-EUR-GS (ANR-17-EURE-0003). Finally, the
authors would like to thanks Dr. Yoann M. Chabre for useful
discussions.
to lack of protein backbone resonance assignment. Based on this
reference spectrum, resonance IDs were transferred to the spectrum with
compound in order to compare changes in presence of a compound. In
case of ambiguities caused by strongly overlapping or disappearing peaks
among reference spectra, resonance IDs were not transferred. Chemical
shift perturbation (CSP) were calculated according to:
Keywords: organic synthesis • fluorinated glycoside • isothermal
titration calorimetry • LecA • TROSY NMR
ͥ
ͦ
ͦ
∆
ꢀ Ɣ ǯ ʞ∆ꢀ ƍ ʚꢂ∆ꢀ
ꢃ
ʛ ʟ
ꢁ
ͦ
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weighing factor of 0.14 for all amino acid backbone resonances. The
threshold value was set to 0.015 ppm based on four independent
measurements of reference spectra. In addition to CSPs, peaks that
reduced at least 20% in normalized signal intensity compared to reference
spectrum were used as indicators for carbohydrate binding.
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F NMR measurements
The measurement using ligand-observed 19F NMR was performed
to validate binding of fluorinated galactopyranosides to LecA. Briefly, a
spectrum of 50 µM compound alone and with 100 µM LecA in TBS buffer
(
2 2
25 mM Tris, 150 mM NaCl, pH 7.8, 10 mM CaCl , 100 µM TFA, 10% D O)
was recorded at 310 K in Norell S-3-800-7 3mm tubes (Norell) on a Bruker
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a 5mm TCI700 CryoProbe . All spectra were normalized to internal
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Engineering Research Council of Canada (NSERC), the Fonds
de Recherche du Québec – Nature et Technologies, CHU de
Québec-Université Laval Research Center and the Université
Laval. D. L. thanks the Fonds de Recherche du Québec – Nature
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FULL PAPER
Vincent Denavit, Danny Lainé,
Chahrazed Bouzriba, Elena Shanina,
Émilie Gillon, Sébastien Fortin,
Christoph Rademacher, Anne Imberty
and Denis Giguère*
Page No. – Page No.
Stereoselective synthesis of
fluorinated galactopyranosides as
potential molecular probes on
galactophilic proteins: assessment of
monofluorogalactosides-LecA
interactions
Sweet Teflon! The synthesis and biological evaluation of mono- and polyfluorinated
galactopyranosides are described. These stable probes are useful lectin ligand as
demonstrated with the first TROSY NMR following chemical shift perturbation of
LecA and isothermal titration calorimetry experiments.
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