Combining Click Reactions for the One-Pot Synthesis of Modular Biomolecule Mimetics

Article abstract of DOI:10.1021/acs.orglett.9b02811

Here, we report on the first combined one-pot use of the two so-called "click reactions": The thiol-ene coupling and the copper-catalyzed alkyne-azide cycloaddition. These reactions were employed in an alternating and one-pot fashion to combine appropriately functionalized monomeric carbohydrate building blocks to create mimics of trisaccharides and tetrasaccharides as single anomers, with only minimal purification necessary. The deprotected oligosaccharide mimics were found to bind both plant lectins and human galectin-3.

Full text of DOI:10.1021/acs.orglett.9b02811

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Combining Click Reactions for the One-Pot Synthesis of Modular
Biomolecule Mimetics
Anne Brinkø,† Christian Risinger,‡ Annie Lambert,§ Ola Blixt,‡ Cyrille Grandjean,§
and Henrik H. Jensen*,†
†Department of Chemistry, Aarhus University, Langelandsgade 140, 8000 Aarhus C, Denmark
‡
Department of Chemistry, Chemical Biology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
§
́
́
́
́
́
́
Faculte des Sciences et des Techniques, Unite Fonctionnalite et Ingenierie des Proteines (UFIP), Universite de Nantes, UMR
̀
CNRS 6286, 2, rue de la Houssiniere, BP92208, 44322 Nantes Cedex 3, France
S
* Supporting Information
ABSTRACT: Here, we report on the first combined one-pot use of the two so-called
“click reactions”: the thiol−ene coupling and the copper-catalyzed alkyne−azide
cycloaddition. These reactions were employed in an alternating and one-pot fashion to
combine appropriately functionalized monomeric carbohydrate building blocks to create
mimics of trisaccharides and tetrasaccharides as single anomers, with only minimal
purification necessary. The deprotected oligosaccharide mimics were found to bind both
plant lectins and human galectin-3.
Scheme 1. (A) Thiol−Ene Click Reaction (TEC); (B) Cu-
he three biopolymers DNA/RNA, proteins, and saccha-
Trides are modular in nature and fundamentally consist of
monomeric units linked through phosphate diester, peptide,
and glycosidic bonds, respectively. Although the complete
synthesis of large representatives from each class has been
conducted,1−8 pitfalls and challenges in the preparation of
biopolymers are still present, making the synthesis of simplified
structures, or mimics, of the parent compounds desirable.
Often, molecules mimicking naturally occurring structures are
more easily accessible and have the potential to possess
improved characteristics, such as solubility, stability, and
affinity.9−13 To bypass the often cumbersome synthesis of
natural oligosaccharides, effort has been directed toward the
synthesis of oligosaccharide mimics, achieved by attaching the
monosaccharides in a way different from the naturally found
O-glycosidic linkage,14−18 which is a subject that has been well
reviewed by Werz et al.19
Catalyzed Azide−Alkyne Cycloaddition Click Reaction
a
(CuAAC)
a
DPAP = 2,2-dimethoxy-2-phenylacetophenone (photoinitiator).
It has been observed that often it is only a small part of the
oligosaccharide that interacts with receptor proteins, while the
rest of the molecule acts as a scaffold to orient the binding
determinants in the desired spatial arrangement.11,12 Con-
sequently, it should be possible to substitute the natural
glycosidic bond with noncarbohydrate motifs while conserving
the biological activity of the parent molecule.34 Our aim was to
prepare a series of oligosaccharides possessing non-natural
linkages in an iterative fashion, using bespoke building blocks
of commonly found monosaccharides. Both the TEC and the
CuAAC reactions have a large functional group tolerance, and
we hoped it would be possible to omit the use of protecting
groups, or at least keep their use to a minimum for easy
handling and convenient global deprotection in the final step.
Perhaps two of the most prominent click reactions20
namely, the thiol−ene coupling (TEC)21,22 and the copper-
catalyzed azide−alkyne cycloaddition (CuAAC)23,24 (Scheme
1)have become valuable tools in chemistry and chemical
biology for the conjugation of molecular fragments under mild
reaction conditions. While the two reactions have been
sporadically employed in combination to expand the space of
achievable structures, it has so far been necessary to perform
purifications between reactions.25−32
Therefore, we were keen to investigate whether the TEC33
and CuAAC34 reactions could be used to assemble larger
biomolecules mimicking naturally occurring motifs in a one-
pot sequential reaction to make only minimal purification
necessary.
Received: August 8, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b02811
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Letter
In addition, we aimed at developing the chemistry to work in
an aqueous medium to be later applicable on biomolecules
different from saccharides. As a starting point for the desired
oligosaccharide mimics it was decided to employ D-N-acetyl
glucosamine (GlcNAc), D-galactose (Gal), and D-mannose
(Man), since, together, these constitute more than 75% of all
monosaccharides found in mammals.35 Furthermore, as
established by Werz et al., these monosaccharides are most
frequently connected by linkages that are specific in terms of
both regiochemistry and anomeric stereochemistry. Accord-
ingly, in the present project, we chose to preserve the naturally
preferred linkage pattern found in mammalian glycans,35 which
is β-D-Gal (capping); (3 → 1)-β-D-Gal; α-D-Man (capping);
(2 → 1)-α-D-Man; β-D-GlcNAc (capping) and (4 → 1)-β-D-
GlcNAc. A series of building blocks possessing the desired
functionalities were then synthesized in their O-acetyl
protected form in order to ease chromatographic purification
(Figure 1).
Scheme 2. Linear and Block Synthesis Approach the
Synthesis of Tetrasaccharide Mimics
were synthesized specifically for this project. A detailed
description of their preparation can be found in the Supporting
Information.
During test experiments, it was observed that the
unprotected version of the Group C alkyne thioacetate
building blocks, containing the free thiol, decomposed during
the CuAAC reaction. However, this could be circumvented by
employing a thiol protecting group, and, for this purpose,
acetyl protection of sulfur was found to be optimal, since it was
sufficiently stable while also being easy to remove selectively
using dimethylamino propylamine (DMAPA).36
With all the desired building blocks in hand, synthesis of the
oligosaccharide mimics was undertaken using anaerobic
conditions with freshly degassed solvents, to prevent the
oxidation of Cu(I) to Cu(II) during the CuAAC reaction and
thiol to disulfide under the TEC reaction. Furthermore, a
standard UV lamp (365 nm) for TLC visualization was
employed for the TEC radical chemistry. Otherwise, the
reactions were performed on the bench.
The successful preparation of nine trisaccharide mimics
listed in Figure 2, using a linear approach, demonstrates the
compatibility of the TEC and CuAAC reactions in a one-pot
setup. The reported outcomes are after silica gel column
chromatography of the final product, since no purification of
the intermediate products was performed. An overall yield of
22%−65% was achieved using a ratio of 1:1:1 among the
reacting partners. The reactions were optimized utilizing this
1:1:1 stoichiometry as the building blocks were considered to
be equally precious. Customarily, for conjugation of smaller
decorations to larger scaffolds such as proteins37,38 and
polymers,25,26 a vast excess of the smaller fragment is used.
A representative synthesis of a trisaccharide mimic is shown
in Scheme 3. For the three tetrasaccharide mimics, both a
linear approach and a convergent 2 + 2 block synthesis were
employed (Scheme 2). Specifically, the linear approach was
employed when a Group A thiol was chosen as the capping
building block, whereas the convergent approach was utilized
when Group A azides were chosen as the capping unit. The
convergent approach was not employed with capping thiols
from Group A, because this would result in it being necessary
Figure 1. Building blocks for oligosaccharide mimic synthesis.
The building blocks were divided into four groups: A, B, C,
and D. Group A compounds only possess one functional
handle for extension (thiol or azide) to be used as a capping
unit at the nonreducing end. Group B compounds have both
an alkene and an azide functionality to enable attachment of a
Group A thiol and allow for further attachment to Group C or
D alkyne-containing building blocks. Group C compounds
possess an alkyne and a thioacetate to be able to undergo
reactions with either an azide from Group A, or a larger
structure with a Group B azide terminating group. Finally,
Group D monomers are reducing end terminating building
blocks possessing either an alkyne or alkene with a Boc-
protected amine to enable immobilization. These units are
suited for reactions with a glycosyl azide or thiol from Group B
or with a de-S-acetylated unit from Group C. The overall
strategy is outlined in Scheme 2.
Of the listed building blocks (Figure 1), A1−A6 were
known compounds, while B1−B3, C1−C3, and D1 and D2
B
DOI: 10.1021/acs.orglett.9b02811
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For the next synthetic step, it was found to be important to
increase the pH via the addition of NaOH to pH 8 for the acid-
labile Boc-protecting group to remain stable. After adjusting
the pH of the reaction, the addition of dD1 (deprotected D1),
together with CuSO4·5H2O and sodium ascorbate, converted
the intermediate to the desired trisaccharide mimic. Since it
proved problematic to purify the unprotected trisaccharide
mimic,41 it was acetylated to ease purification and structural
verification (see Scheme 4).
Scheme 4. One-Pot Synthesis of Trisaccharide Mimic dA2-
dB1-dD1 without Protecting Groups
Figure 2. Synthesized protected trisaccharide and tetrasaccharide
mimics. [Yields are given as a percentage of the isolated purified
targets molecules.]
Scheme 3. Representative Linear Synthesis of the Gal-
GlcNAc-Gal Trisaccharide Mimic
The ability of the prepared oligosaccharide mimics was next
to be investigated as lectin binding ligands. First, a series of
plant lectins were explored in a microarray setting. These were
Concanavalin A (ConA), wheat germ agglutinin (WGA),
Lycopersicon esculentum lectin (LEL), and Vicia villosa lectin
(VVL).
No binding was found to occur to the latter lectin (VVL),
which was included as a negative control that typically
recognized GalNAc that was not present among the
oligosaccharide mimics.
As shown in Figure 3. Con A exhibited binding to the three
mimics dA4-dB1-dD1, dA4-dB2-dD1, and dA4-dB3-dD1, all
possessing a terminal mannose unit as a thioglycoside. The
to perform a TEC reaction with a deprotected thiol from
Group C and an alkene from Group D. Since the Group C
building blocks also contain an alkyne functionality, it would
be able to undergo a thiol−yne coupling with a building block
from the same group, destroying the desired selectivity of the
reaction.
All the mimics could be globally deprotected in excellent
yields, using NaOMe in MeOH39 to obtain the deprotected
oligosaccharides.
To explore whether the reactions reported above also were
compatible with carbohydrate building blocks in their
deprotected form, dA2 (deprotected A2) was reacted with
alkene dB1 (deprotected B1) in acetic acid buffer (pH 4.0)
containing no organic cosolvent. The previously used radical
initiator DPAP was found to be insoluble, while the radical
initiator Vazo4440 developed for radical reactions in an
aqueous medium cleanly provided the desired intermediate,
as judged by TLC analysis.
Figure 3. Glycan mimic microarray showing binding to Concanavalin
A.
C
DOI: 10.1021/acs.orglett.9b02811
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Letter
compound showing the highest binding was dA4-dB3-dD1,
which had two neighboring mannose residues. GlcNAc has
previously been found to have substantial Con A inter-
actions,42 which could explain the observed cross-reactivity by
mannose lacking but GlcNAc-displaying trisaccharides dA6-
dB1-dD1 and dA6-dB2-dD1.
WGA was found to bind strongly and exclusively to the
oligosaccharide mimics possessing a terminal N-acetylglucos-
amine (GlcNAc) (dA6-dB1-dD1 and dA6-dB2-dD1), as well
as the positive control (GlcNAc itself).
In addition, weak LEL binding was observed to the same
two compounds with a slightly higher specificity for dA6-dB2-
dD1 that contains two GlcNAc building blocks. Contrary to
WGA, LEL did not bind significantly to the GlcNAc positive
control. For figures showing binding to WGA and LEL, see the
Supporting Information.
Given the positive results from the plant lectin studies, we
next turned our attention to a human lectin (galectin) involved
in health and disease. Galectins are a structurally related family
of proteins that binds to β-galactoside motifs through a highly
conserved carbohydrate recognition domain (CRD). They are
involved in a pleiotropic number of fundamental biological
processes, and they are key contributors to the homeostasis but
also take part in many pathologies such as cancer progression,
diabetes, and inflammatory and immune diseases.43 They are
classified in three groups: (i) dimeric galectins, which contain
two CRDs; (ii) monomeric galectins, which contain a single
CRD but form homodimers in vivo; and, finally, (iii) the so-
called chimeric galectin, galectin 3, which is the only one that
also contains a non-CRD region. This new region mediates the
polymerization of galectin-3. Thus, in summary, all galectins
are functionally multivalent in vivo. Since human galectin-3,
which is known to be sensitive to a multivalent ligand
presentation, is one of the prominent members of this lectin
family and has been identified as a potential therapeutic target,
it was evaluated as a possible target of the herein-synthesized
(oligo)-galactosides as lactosamine mimics. Evaluation of their
binding affinities toward the galectin-3 was performed by
competitive fluorescence polarization assay, using known 2-
[(fluoresceinyl)thioureido]ethyl 4-O-[3-O-(3-methoxybenzyl)-
β-D-galactopyranosyl]-β-D-glucopyranoside probe44 and 1,2-
diacetamido-4-O-(β-galactopyranosyl)-1,2-dideoxy-β-D-gluco-
pyranose45 as a positive control.
ides to the disaccharides, up to the tetrasaccharides, and were,
at least, higher by 1 order of magnitude. Disaccharide dA1-
dD1 was the least potent inhibitor, compared to other
oligomers. The two galactosides are connected to a triazole
scaffold which might confer too much rigidity or less than
optimal orientation to accommodate the CRD binding site.
Otherwise, characteristics of the spacers (nature or position)
do not seem to significantly influence the recognition for
higher oligomers. Noticeably, these linear oligo-galactosides
proved more potent than classical cluster galactosides that
were linked by their reducing end to a scaffold whose affinity
for the galectin-3 was found in the upper mM range.47,48
In summary we have reported the first combined one-pot
application of the CuAAC and TEC reactions and demon-
strated their use in the preparation of mimics of natural
oligosaccharides incorporating the most common oligosac-
charide building blocks and conserving the naturally preferred
linkage pattern. A collection of nine trisaccharide mimics and
three tetrasaccharide mimics possessing acetyl groups were
prepared in a mixture of tBuOH/H2O, before being
deacetylayed to provide the deprotected oligosaccharide
mimics. The synthesis of a trisaccharide mimic from the
deprotected building blocks was also demonstrated in aqueous
buffer under anaerobic conditions.
It was possible to show that the prepared compounds were
both recognized by plant lectin and human galectin-3.
Regarding the latter, a cluster effect was observed, suggesting
that these linear oligomers could be an alternative to the
dendronized or branched multivalent structures usually
proposed. The stable intermonomeric linkages should confer
higher survival to these oligomers in biological media,
designing them as valuable surrogates to natural oligosacchar-
ides.
The developed strategy combines the convenience of the
CuAAC and TEC reactions with a one-pot strategy and a
desirable stoichiometry of reactants and can be employed to
prepare an array of biomolecule mimics or for bioconjugation.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b02811.
In our assay, K of monogalactoside β-GalS(CH2)3NHBoc
d
Experimental synthetic procedure and characterization
with detailed description of lectin binding evaluation and
data (PDF)
was found to be above 5 mM, which is a value in accordance
with that usually reported for methyl β-D-galactopyranoside
(4.4 mM)46 (see Table 1). On the other hand, affinities
consistently increased when switching from the monosacchar-
AUTHOR INFORMATION
■
Table 1. Dissociation Constants for Ligands of Galactin-3 at
Room Temperature, Performed as Duplicates
Corresponding Author
*E-mail: hhj@chem.au.dk.
ORCID
a
b
ligand
Kd (mM)
valency
β-GalS(CH2)3NHBoc
dA1-dD1
dA2-dD2
dA1-dC1-dD2
dA2-dB1-dD1
dA1-dC1-dB1-dD1
dA2-dB1-dC1-dD2
>5
1
2
2
3
3
4
4
Ola Blixt: 0000-0003-4143-6276
Henrik H. Jensen: 0000-0003-2738-4502
Author Contributions
1.32
0.62
0.34
0.36
0.20
0.18
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
a
Ligands are ranked according to their number of galactose moieties.
Valency is determined based on the number of galactose moieties.
b
The authors declare no competing financial interest.
D
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Letter
(32) Yang, B.; Zhang, H.; Peng, H.; Xu, Y.; Wu, B.; Weng, W.; Li, L.
Polym. Chem. 2014, 5, 1945−1953.
ACKNOWLEDGMENTS
■
A.B. and H. H. J. are grateful for financial support from The
Villum Foundation (No. VKR023110).
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