Efficient synthesis of phenylene-ethynylene rods and their use as rigid spacers in divalent inhibitors

Article abstract of DOI:10.3762/bjoc.9.25

The synthesis of phenylene-ethynylene rods and their use as rigid spacers is described. Alternation of a Sonogashira reaction and silyl group cleavage was used to obtain rigid spacers with even and odd numbers of phenylene units. Preliminary applications of these rods in divalent systems are shown. Inhibition studies with Pseudomonas Aeruginosa lectin LecA showed that the rigid spacer proved greatly beneficial for the inhibitory potency.

Full text of DOI:10.3762/bjoc.9.25

Efficient synthesis of phenylene-ethynylene rods and
their use as rigid spacers in divalent inhibitors
Francesca Pertici1, Norbert Varga2, Arnoud van Duijn1, Matias Rey-Carrizo1,
Anna Bernardi2 and Roland J. Pieters*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 215–222.
1Department of Medicinal Chemistry and Chemical Biology, Utrecht
Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box
80082, 3508 TB Utrecht, The Netherlands and 2Dipartimento di
Chimica, Universita’ degli Studi di Milano, via Golgi 19, 20133 Milano,
Italy
doi:10.3762/bjoc.9.25
Received: 06 December 2012
Accepted: 09 January 2013
Published: 31 January 2013
Email:
Associate Editor: S. Flitsch
Roland J. Pieters* - R.J.Pieters@uu.nl.
© 2013 Pertici et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
multivalent carbohydrates; LecA inhibition; phenylene ethynylene;
rigid spacers; Sonogashira reaction
Abstract
The synthesis of phenylene-ethynylene rods and their use as rigid spacers is described. Alternation of a Sonogashira reaction and
silyl group cleavage was used to obtain rigid spacers with even and odd numbers of phenylene units. Preliminary applications of
these rods in divalent systems are shown. Inhibition studies with Pseudomonas Aeruginosa lectin LecA showed that the rigid spacer
proved greatly beneficial for the inhibitory potency.
Introduction
Linker or spacer molecules have a wide range of applications in formation [9], carbohydrate–triazole conjugation [10],
many areas of chemistry as bridging molecules between sepa- aryl–alkyne linked structures [11-15] and the use of DNA as a
rate functional units. Spacers are often flexible but depending rigid bridge between silver nanoparticles and quantum dots [5].
on the nature of the application, efforts have been made to make Among the rigid linking units the phenylene-ethynylene unit
rigid linkages between functional units. Examples of these have has seen considerable interest in sensor [16] and molecular-
been reported in areas such as nanoelectronics and nanooptics electronics applications [17]. This is due to the specific fluores-
[1], surfactants [2-4], photoelectrochemical detection [5], catal- cent, conducting and electrochemical properties that the conju-
ysis [6], glycosylation reactions [7], and carbohydrate–protein gated system confers to the molecule [18]. In the study of
interactions [8]. Many different strategies and molecule types carbohydrate–protein interactions, it is now well known that
have been used depending on the desired geometry, such as ring making a system multivalent increases the binding or inhibitory
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Beilstein J. Org. Chem. 2013, 9, 215–222.
potency of ligands, whose monovalent counterparts would
otherwise be too weak to have biological relevance [19-22]. The
spacer is an important factor in the design of an effective multi-
valent ligand [23]. While most systems reported thus far contain
flexible spacers, there is a major untapped potential for systems
based on rigid spacers, even though some flexibility may be
needed to overcome design imperfections [10].
Figure 2: Generic structure of spacers containing an even (n = 1, 3)
and an odd (n = 2, 4) number of units.
We herein describe the synthesis of rigid spacers of various
lengths based on phenylene-ethynylene units and their incorpo-
ration into divalent ligands. For design purposes this system has
the advantage that due to the high rigidity and linearity it is easy
Results and Discussion
to predict their length, as was recently shown by EPR measure- Synthetic strategies
ments [24]. The solubility of rigid hydrophobic spacers in an Depending on the number of units in the spacer, two different
aqueous environment is notoriously poor, and therefore PEG routes can be applied. The pathway followed to obtain rods
attachments have been employed. Such PEG units were also containing an even number of units is shown in Figure 3. The R
incorporated in glycopolymers based on the phenylene-ethynyl- group on the ring is used to increase the solubility of the
ene repeating units by Seeberger and co-workers (see schemati- system. The strategy relies on orthogonal protecting groups R1
cally in Figure 1a), who used them for the detection of bacteria and R2 of structure A, to enable the selective deprotection
[25]. The fluorescent properties of the polymer allowed a fast needed to make B. Its free alkyne moiety can undergo a
detection of the E. coli bacteria. Brewer et al. reported a diva- Sonogashira reaction with C to give D. At this point the
lent inhibitor with a short rigid spacer containing just a single removal of the protecting group R1 and R2 can be performed, to
phenylene-ethynylene [26]. The inhibition shown by this com- either couple the ligands or elongate the system by a double
pound was disappointing since it was less potent than its flex- Sonogashira reaction.
ible divalent counterpart. There are no other examples in the
literature that show inhibition studies of lectins using divalent The strategy to prepare spacers containing an odd number of
ligands directly connected by a rigid spacer based on phenyl- units is shown in Figure 4. The strategy is more straightforward
ene-ethynylene units (Figure 1b).
since it does not require any orthogonal deprotection step.
Starting with F, a double Sonogashira reaction with C should
yield the three-unit system G. Removal of the protecting groups
R1 can be performed, to either couple the ligands or elongate
the system by a double Sonogashira reaction.
Synthesis of the building blocks
The building blocks were prepared as shown in Scheme 1. In
the general structures shown in Figure 3 and Figure 4 the R
group is used to increase the solubility. For this purpose dieth-
ylene glycol was used as a side chain, which terminated as a
free hydroxy group for 1 and a methoxy group for 2. Silyl
groups were used as selective protective groups for the alkyne
moiety. Monoalkyne 3 and bisalkyne 4 were made from 1 [29]
Figure 1: Schematic depiction of (a) a rigid phenylene-ethynylene
polymer core with ligands attached via flexible chains, and (b) a diva-
lent ligand using phenylene-ethynylene as a rigid spacer connecting
the ligands.
In order to systematically study the effects of spacer lengths on by a Sonogashira reaction with TIPS-acetylene in 31% and
the binding potencies of divalent ligands, having access to a 50%, respectively. Similarly, 5 and 6 were obtained from 2, in
series of spacers of well-defined lengths was imperative. We agreement with a recent literature report [16]. The use of the
here report the synthesis of a series of spacers based on phenyl- microwave reactor allowed a shorter reaction time (20 min at
ene-ethynylene building blocks (Figure 2), with distinct 60 °C) than the one reported in the literature (24 h at 40 °C for 6
syntheses for the compounds containing an even and an odd and 24 h at 10 °C for 5).
number of aromatic rings. One of the spacers was incorporated
into the structure of a divalent galactoside ligand and was used Synthesis of a two-unit spacer
to inhibit the virulence-linked lectin LecA of Pseudomonas The strategy of Figure 3 was applied to the synthesis of the two-
aeruginosa [27,28].
unit spacer. In order to obtain our orthogonally protected inter-
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Beilstein J. Org. Chem. 2013, 9, 215–222.
Figure 3: Synthetic strategy for rigid spacers with an even number of units.
Figure 4: Synthetic strategy for rigid spacers with an odd number of units.
Scheme 1: Synthesis of building blocks; (a) from 1, Pd(PPh3)4 , CuI, PPh3, TEA, toluene, 50 °C, 5 h, 31% for 3 and 50% for 4; from 2, PdCl2(PPh3)2 ,
CuI, TEA, THF, microwave, 60 °C, 20 min, 35% for 5 and 53% for 6.
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Beilstein J. Org. Chem. 2013, 9, 215–222.
mediate 7, mono-iodo compound 5 was coupled with tert- mono-iodo derivative 3, because partial silyl migration to the
butyl(ethynyl)dimethylsilane (TBDMS-acetylene, Scheme 2). free hydroxy groups was observed while attempting mono-desi-
From 7 the more labile TMS group was removed by using lylation.
K2CO3 to give 8. This compound was coupled to 5 by a
Sonogashira reaction to give the protected two-unit spacer 9. Synthesis of three-unit spacers
Removal of its silyl protecting groups with TBAF yielded the To obtain the three-unit spacer, the odd strategy (Figure 4) was
desired two-unit spacer 10. A slightly different strategy (see applied starting with compound F. In order to make F, the silyl
Supporting Information, Scheme S1) was used starting from the protecting groups of both 4 and 6 were removed (Scheme 3).
Scheme 2: Synthesis of a two-unit spacer. (a) PdCl2(PPh3)2, CuI, TEA, THF, microwave, 60 °C, 20 min, 81% for 7, 79% for 9; (b) K2CO3, MeOH/
CH2Cl2, 45 min, 78%; (c) TBAF, THF, 84%.
Scheme 3: Synthesis of three-unit spacers. (a) from 4: TBAF, THF, 62%; from 6 and 14: K2CO3, MeOH/CH2Cl2, 45 min, 85–88%; (b) from 11:
Pd(PPh3)4, CuI, PPh3, TEA, toluene, 50 °C, 14 h, 50%; from 12: PdCl2(PPh3)2, CuI, TEA, THF, microwave, 60 °C, 20 min, 75%.
218

Beilstein J. Org. Chem. 2013, 9, 215–222.
For 4 the TIPS groups were removed with an excess of TBAF Preliminary application
to give 11. Compound 6, which bears the TMS group, was As part of our program on bacterial adhesion inhibition by
treated with K2CO3 to afford 12. Both 11 and 12 were elong- multivalent carbohydrates, the bacterial lectin LecA, a viru-
ated in a double Sonogashira reaction with 3 and 5, respective- lence factor of the problematic pathogen Pseudomonas aerugi-
ly. Of the products, 14 was deprotected by using K2CO3 to nosa is a target of interest [30,31]. This tetrameric lectin binds
yield the three-unit spacer 15, according to a recent literature galactosides and the shortest distance between two binding sites
report [16].
is around 26 Å [20,28]. We previously noted that the use of a
rigid spacer with some flexibility in the aglycon chain
connecting the galactose ligands, led to more potent inhibition
Synthesis of a four-unit spacer
The four-unit spacer was synthesized starting from the two- [10]. Since the distance to cover is ca. 26 Å, the best match in
unit spacer 10 and the iodo compound 5 through a double the phenylene-ethynylene series is the three-unit spacer 15. Just
Sonogashira reaction to give 16 (Scheme 4). Deprotection of the the spacer, without the aglycon linking moiety measures around
two alkyne moieties with K2CO3 afforded the four-unit spacer 22 Å. Coupling of the galactose ligand and the flexible aglycon
17.
part should result in a promising compound. CuAAC of 15 with
the ligand 20 was performed as shown in Scheme 6 to give 21.
The acetyl protecting groups were removed by using NaOMe in
Synthesis of a five-unit spacer
The synthesis of the five-unit spacer started with the elongation MeOH, yielding final product 22.
of the three-unit spacer 15 through a double Sonogashira reac-
tion with iodo compound 5 to give 18 (Scheme 5). The five-unit Compound 13, bearing hydroxy-terminated diethylene glycol
spacer 19 was obtained after deprotection of the alkyne moieties chains, has the potential of solubilizing in water constructs
with K2CO3.
bearing active spearheads less hydrophilic than simple sugars.
Scheme 4: Synthesis of a four-unit spacer. (a) PdCl2(PPh3)2, CuI, TEA, THF, microwave, 60 °C, 20 min, 73%; (b) K2CO3, MeOH/CH2Cl2, 45 min,
76%.
Scheme 5: Synthesis of a five-unit spacer. (a) PdCl2(PPh3)2, CuI, TEA, THF, microwave, 60 °C, 20 min, 70%; (b) K2CO3, MeOH/CH2Cl2, 45 min,
75%.
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Beilstein J. Org. Chem. 2013, 9, 215–222.
Scheme 6: Synthesis of divalent ligand 22. (a) CuSO4·5H2O, Na-ascorbate, DMF/H2O, microwave, 80 °C, 40 min, 85%; (b) NaOMe, MeOH, 41%.
We have been developing pseudo-disaccharide molecules such
as 23 [32] (Scheme 7) as mimics of mannose disaccharides for
the interaction with DC-SIGN and other C-lectins [33-35]. This
molecule and its derivatives [36] contain lipophilic moieties that
generally increase their affinity for the target proteins, but can
create solubility problems. Desilylation of 13 (TBAF, THF,
Scheme 7) followed by in situ CuAAC with the pseudo-disac-
charide 23 led to the divalent ligand 24, which was found to be
fully soluble in water, at least up to millimolar concentrations.
Compound 24 is a mimic for DC-SIGN inhibition and its bioac-
tivity will be tested elsewhere.
Inhibition studies
The inhibitory potency of 22 for LecA was studied in an ELISA
type assay by using a glycochip as the solid phase [10]. In this
assay an IC50 value of 0.9 μM was determined (Table 1). This
compared favorably with the monovalent reference compound
25 (Figure 5), which exhibited an IC50 of 120 μM [10]. Simi-
larly, the divalent compound 22 was a more potent inhibitor
than the higher valency compound 26, especially when
expressed as the relative potency per sugar, which is 11 for the
tetravalent 26 [37,38] and 67 for 22.
Conclusion
In this work a strategy for the synthesis of rigid spacers or rods
Scheme 7: Synthesis of divalent ligand 24. (a) 13, TBAF in THF, rt,
1 h, then H2O, TBTA, CuSO4·5H2O, Na-ascorbate, 23, rt, 18 h, 76%.
of different length based on phenylene-ethynylene units was
developed. On the phenyl ring, two versions of a solubilizing
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Beilstein J. Org. Chem. 2013, 9, 215–222.
with the divalent structure based on the phenylene-ethynylene
spacer, with an IC50 of 0.9 μM, i.e., a 133-fold potency increase
over a monovalent reference compound, clearly showing the
potential for spacers of this nature.
Table 1: Inhibitory potency of mono, di- and tetravalent galactosides
on LecA bindinga.
Relative
Compound
Valency
IC50/μM
Potency
(per sugar)
It was also shown that the three-unit spacer 13 could be used in
a one-pot desilylation and CuAAC reaction to give 24, which
was found to be fully soluble in water, despite the more
lipophilic nature of the active ligand. This finding paves the
way for the synthesis and evaluation of polyvalent glyco-
mimetics with regularly spaced cores to be used for Man-
specific C-lectin inhibition assays. With access to structural
data of target lectins, the design of multivalent inhibitors can be
performed on a customized level, where the selection of the
proper spacer is based on the information about the binding site,
such as its density, orientation and position. Furthermore, the
rigidity of the rods described above can contribute to over-
coming the entropic penalty of flexible multivalent scaffolds,
thus improving the overall activity of the ligands.
25
26
22
1
4
2
120
2.7
1
44 (11)
133 (67)
0.90
aFITC-labeled LecA, 20 μg mL−1 binding to a galactoside functional-
ized surface.
Supporting Information
Supporting Information File 1
Synthetic procedures and spectral data.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-9-25-S1.pdf]
Acknowledgements
This research is supported by the Dutch Technology Founda-
tion STW, applied science division of NWO and the Tech-
nology Program of the Ministry of Economic Affairs. N.V. was
supported by a European fellowship under the ITN project
Carmusys (FP7-MC-ITN-213592). We thank the COST Action
CM1102 Multiglyconano for support and Stefania Ordanini for
optimization of the synthesis of 30 (see Supporting Information
File 1, Scheme S1). M.R.C. acknowledges Fundació Crèdit
Andorrà for a masters grant.
Figure 5: Previously tested compounds.
diethylene glycol moiety were used, one terminating in a
hydroxy and one terminating in a methoxy group. The hydroxy
version led to some migration of the silyl protecting group upon
attempted monodeprotection, and alternative strategies had to
be devised for the synthesis of the two-unit spacer. Compounds
17 and 19, containing four and five phenylene-ethynylene units,
respectively, can still be further elongated, depending on the
need of the project, thus enabling the preparation of long
spacers with a well-defined number of monomeric units. The
CuAAC of the three unit spacer 15 with a galactose ligand gave
the divalent ligand 21 in good yield. After deprotection
this compound was used to inhibit the lectin LecA from
Pseudomonas aeruginosa. A major potency increase was seen
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