Potent divalent inhibitors with rigid glucose click spacers for Pseudomonas aeruginosa lectin LecA

Article abstract of DOI:10.1039/c2cc30234a

The synthesis of a new rigid spacer based on carbohydrate-triazole repeating units and their incorporation into divalent systems is described. Inhibition studies showed that a well-matched system with a rigid spacer with flexible ends leads to the most potent inhibition of Pseudomonas aeruginosa lectin LecA.

Full text of DOI:10.1039/c2cc30234a

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COMMUNICATION
Potent divalent inhibitors with rigid glucose click spacers for
Pseudomonas aeruginosa lectin LecAw
Francesca Pertici and Roland J. Pieters*
Received 11th January 2012, Accepted 1st March 2012
DOI: 10.1039/c2cc30234a
The synthesis of a new rigid spacer based on carbohydrate-triazole
repeating units and their incorporation into divalent systems is
described. Inhibition studies showed that a well-matched system
with a rigid spacer with flexible ends leads to the most potent
inhibition of Pseudomonas aeruginosa lectin LecA.
Fig. 1 A divalent ligand with a rigid spacer containing flexible ends
Carbohydrate–protein interactions play a key role in many
important biological processes and often involve multivalency
to increase binding to biologically relevant levels.1 Selective
interfering with these interactions may enable the development
of therapeutics for inflammation,2 bacterial toxins,3 bacterial
infections,4 cancer,5 flu,6 and AIDS.7 A logical approach
towards inhibitors of protein–carbohydrate interactions is to
make them multivalent. This approach has seen numerous
successful examples with multivalency effects of several orders
of magnitude e.g. with bacterial toxins and multisite lectins.8
For potent inhibition the length of the spacer connecting the
ligands is a known factor of importance.9 The calculation of
the ‘effective length’ is a useful optimization guide for the
commonly used flexible spacers that tend to fold.8a Typically
flexible PEG-based spacers have to be made three times longer
than the distance spanned by an extended conformation for an
optimal match.8a Besides the length, spacer rigidity is another
important factor, which has received far less attention in the
design of multivalent ligands.10 This is due to challenges in
creating a spacer that is of optimal length, maintains its
distance and is sufficiently soluble. We here explored these
issues in the design and synthesis of rigid spacers that contain
flexible ends to allow some adjustment of the attached ligands
(Fig. 1). The spacers were outfitted with galactose units and
their inhibition of the Pseudomonas aeruginosa adhesion lectin
and virulence factor LecA was evaluated.11
binding to a protein with two binding sites.
Fig. 2 Synthesis and elongation strategy of the spacer.
Rotation of bonds within the spacer is possible but the overall
shape remains close to linear according to modelling.12a Its
synthesis strategy involved a copper catalyzed azide–alkyne
cycloaddition (CuAAC) between a suitable azide B and a
building block A, followed by the conversion of the free axial
hydroxyl group present in product C to an equatorial azide in
D. Repeating this process allowed chain elongation.
The actual synthesis of building block 4 is shown in
Scheme 1 and started with 1.13 Its benzyl and trimethylsilyl
groups were removed using BCl3ÁSMe2 which was followed by
acetylation to give 2 in good yield. After removal of the acetyl
groups, 3 was selectively benzoylated at low temperature to
give 4 along with other isomers that were recycled.
In order to build the spacer, a starting point B (Fig. 2) was
needed. Compound 5 (Scheme 2) was used for this purpose
and it was linked to 4 to give the ‘click’ product in high yield.
In the design of the spacer, hydrophilic carbohydrates were
incorporated to enhance the solubility. In order to prevent
folded conformations, glucose moieties were connected by
1,4-triazole units using direct equatorial linkages at positions
1 and 4 of the sugar, as can be seen in structure E (Fig. 2).
Department of Medicinal Chemistry and Chemical Biology,
Utrecht Institute for Pharmaceutical Sciences, Utrecht University,
P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
E-mail: R.J.Pieters@uu.nl; Fax: +31-30-2536655
w Electronic supplementary information (ESI) available: Synthetic
procedures, spectral data and inhibition data. See DOI: 10.1039/
c2cc30234a
Scheme 1 Preparation of the building block 4. Reagent and conditions:
(a) (i) BCl3ÁSMe2, CH2Cl2, microwave, 80 1C, 20 min; (ii) Ac2O, Py, rt,
14 h, 70% over 2 steps; (b) MeONa, MeOH, quant.; (c) BzCl, Py,
À40 1C, 67%.
c
4008 Chem. Commun., 2012, 48, 4008–4010
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Table 1 Inhibitory potency of mono and divalent galactosides on
LecA bindinga
Relative potency
(per sugar)
Compound
Valency
IC50/M
20
14
18
19
1
2
2
2
12 (Æ6.0) Â 10À5
22 (Æ2.3) Â 10À8
38 (Æ6.1) Â 10À8
20 (Æ5.9) Â 10À7
1(1)
545(272)
315(158)
60(30)
22
12
16
1
2
2
92 (Æ37) Â 10À6
31 (Æ6.2) Â 10À5
17 (Æ3.4) Â 10À7
1(1)
0.30(0.15)
54(27)
FITC-labeled LecA, 20 mg mLÀ1 binding to a galactoside functio-
a
nalized surface.16
(Scheme 3). Since the target was LecA, a galactose binding
lectin, the two galactosides 2 and 21, differing in their aglycon,
were chosen for attachment. In 21 the alkyne moiety is
connected to the sugar via a flexible four atom chain, while
in 2 the alkyne moiety is directly connected. Respective ‘click’
reactions yielded the divalent ligands 12, 14, 16 and 18 in good
yield after purification by preparative HPLC.
Scheme 2 Synthesis of the spacers. Reagent and conditions: (a)
CuSO4Á5H2O, Na-ascorbate, DMF, 10% H2O, microwave, 80 1C,
30 min, 85–91%; (b) (i) Tf2O, Py, CH2Cl2, 0 1C, 1 h; (ii) NaN3, DMF,
rt, 4 h, 80–85% over 2 steps; (c) (i) TFA, CH2Cl2; (ii) imidazole-1-
sulfonyl azide, K2CO3, CuSO4Á5H2O, MeOH, 82–85% over 2 steps.
Its axial 4-hydroxyl was converted, via the triflate, to an
equatorial azido group, by sodium azide, thus yielding 6. By
reiteration of this procedure compounds 7 and 9 were synthesized
with two and three carbohydrate units, respectively. Finally,
the Boc-protected amino groups were deprotected and converted
to azido groups to give 8 and 10.14
These compounds were employed in LecA inhibition studies
and their inhibitory potencies were compared to that of the
monovalent reference compounds and the divalent 19. The
latter contains the long flexible PEG-based spacer, which is
expected to span the 26 A between neighbouring binding sites
of LecA (see ESIw).15 Inhibitory potencies were determined by
observing the binding of fluorescently labelled LecA to a
galactose-displaying chip surface16 (Table 1).
Both 8 and 10 contain two azido groups, allowing a double
‘‘click’’ reaction to introduce the carbohydrate ligands
Scheme 3 Reagent and conditions: (a) CuSO4Á5H2O, Na-ascorbate, DMF, 10% H2O, microwave, 80 1C, 30 min, 83–85%; (b) MeONa, MeOH,
70–87%.
c
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In this assay two monovalent galactosides were used as
reference compounds to account for the different aglycon part
of the divalent ligands. Compound 20, with a propylene
aglycon, and 22, with a triazole aglycon, showed IC50’s of
120 and 93 mM, respectively. With the exception of compound
12, all divalent ligands were more potent. Compound 14 was
the most potent with an IC50 of 220 nM, a 545-fold improve-
ment over the monovalent ligand 20. The related compound
18, containing an extra spacer unit, was almost as potent with
an IC50 of 380 nM. The flexible divalent PEG-based ligand 19
showed an IC50 of 2 mM which is 60 times better than the
monovalent ligand 20.
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A dramatic decrease in potency was observed with 12, a
relative of 14 just lacking 2 propyloxy units, which showed
an IC50 of 310 mM, clearly not showing divalent binding.
Compound 16, a relative of 12 just containing an extra spacer
unit, showed major improvement with an IC50 of 1.7 mM,
which is 182 times better than 12.
To conclude, a series of divalent inhibitors, based on
carbohydrate-triazole spacers, were synthesized in a multiple
step synthesis, using azide–alkyne ‘‘click’’ chemistry in high
yield. These compounds were evaluated as LecA inhibitors.
Considering that a triazole-glucose unit contributes ca. 7 A to
the spacer length, the prepared compounds were expected to
show potency variation. Multivalency effects were observed as
almost all divalent compounds showed improved potency over
the monovalent. It was shown that 14 and 18 both containing
rigid moieties were considerably more potent than the PEG
based 19. Compounds 14 and 18 contained more flexibility in
its spacer than 12 and 16, which feature a direct attachment to
the sugar. For 12 and 16 the potency is critically dependent on
the spacer length, where one spacer unit too few, as in 12, leads
to the abolishment of divalent binding.12b For such rigid
compounds a perfect design could lead to high potency and
more importantly to very high specificity. Naturally we can
only include whole building blocks and no partial ones. There-
fore not all distances can be prepared with a rigid design, so
flexible end groups are needed. In the compounds containing
the most flexible spacer ends, i.e. 14 and 18, more forgiving
inhibition behaviour was observed with respect to design
imperfections. Besides the enhanced potency of the compounds
containing rigid spacer units, effective non-folding spacers can
also be considerably shorter. There were 61 atoms present
between the sugar anomeric carbons of 19, and between 22
and 37 atoms for 12, 14, 16 and 18. Additionally, the use of
sugars in the spacer is likely to enhance the biocompatibility17
and their rigid nature should enhance their selectivity. Overall
the design strategy has led to some of the most potent LecA
inhibitors that rival those of greater valency and size.18
This research is supported by the Dutch Technology
Foundation STW, applied science division of NWO and the
Technology Program of the Ministry of Economic Affairs. We
thank Dr Johan Kemmink for the molecular modelling.
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15 Measured between the anomeric oxygens of bound galactosides.
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c
4010 Chem. Commun., 2012, 48, 4008–4010
This journal is The Royal Society of Chemistry 2012