Templating a polymer-scaffolded dynamic combinatorial library

Article abstract of DOI:10.1039/c1cc11998b

A water soluble polymer-scaffolded dynamic combinatorial library whose members can interconvert through acylhydrazone exchange was prepared and shown to re-equilibrate in the presence of macromolecular templates.

Full text of DOI:10.1039/c1cc11998b

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COMMUNICATION
Templating a polymer-scaffolded dynamic combinatorial libraryw
Clare S. Mahon, Alexander W. Jackson, Benjamin S. Murray and David A. Fulton*
Received 8th April 2011, Accepted 27th April 2011
DOI: 10.1039/c1cc11998b
A water soluble polymer-scaffolded dynamic combinatorial
library whose members can interconvert through acylhydrazone
exchange was prepared and shown to re-equilibrate in the
presence of macromolecular templates.
addition of a template in the same manner as that demon-
strated by other DCLs,5,6 inducing the system to re-equilibrate
and amplifying the concentration of library members which
best bind the template. This wholly thermodynamically
controlled templation process provides a mechanism to correct
and refine binding sites, a virtue absent in molecularly
imprinted polymers8,9 (MIPs) where the molecular imprinting
process is kinetically controlled and there is no scope for error-
correction during the templating procedure. This deficiency
leads to significant heterogeneity and binding pockets which
are not optimised, with detrimental effects on molecular
recognition.10–12 The PS-DCL concept can be contrasted to
the conventional combinatorial approach utilised by Schrader
and co-workers13 for the discovery of polymers for use in
protein recognition. In this work, a small set of functionalized
monomers was used to prepare libraries of copolymers which
were screened successfully to find selective binders for arginine-
rich proteins in aqueous solution, a process requiring extensive
synthesis and screening. As with MIPs, the recognition units
are incorporated irreversibly into the polymer scaffold and
there is no available mechanism for the optimisation of
binding sites. Recently Ghadiri and co-workers14 have
arguably demonstrated the validity of aspects of the PS-DCL
concept within their impressive work on sequence-adaptive
oligonucleotides. We report here the generation of an aqueous
PS-DCL based on acylhydrazone exchange upon a simple
synthetic polyacrylamide scaffold, and demonstrate that this
library can re-equilibrate in the presence of macromolecular
templates, work which we believe represents important first
steps towards the realisation of the PS-DCL concept as a
general method for the discovery of truly macromolecular
receptors.
Dynamic combinatorial chemistry1 (DCC) has emerged in
recent years as a powerful tool for the discovery of molecular
receptors. DCC uses reversible covalent reactions to link
together building blocks, forming libraries of compounds
whose product distributions are under thermodynamic
control. The reversible nature of the linkages enables the
library members to reconfigure their structures by exchanging
and reshuffling their building blocks. Equilibrium perturbations,
such as the addition of a template, can result in the structural
adaptation of the library, amplifying the concentration of
those library members which are stabilized by the template.
The library can be kinetically ‘fixed’—often by simply
quenching the catalyst involved in catalysing the dynamic
exchange—allowing library members to be isolated and
characterized. The appeal of the dynamic combinatorial
approach lies in its inherent ability to combine library
synthesis and affinity screening into a single step, making it
a potentially rapid, simple and cost-effective method for the
discovery of receptors. DCC has demonstrated great promise
for the discovery of small molecule receptors,2–6 but its
potential for the discovery of macromolecular counterparts
has yet to be investigated.
Several years ago, one of us described7 a new class of
DCL—the so-called ‘‘polymer-scaffolded dynamic combinatorial
library’’ (PS-DCL)—which was designed to apply concepts
from the field of DCC towards the discovery of macromolecular
receptors. PS-DCLs are based upon a synthetic polymer
scaffold where functionalized residues are grafted onto the
scaffold through dynamic covalent linkages. The reversibility
of these linkages allows residues to exchange with one another
and reshuffle their positions upon the polymer scaffold,
presenting a mechanism for the members of the library
to adapt their primary structures. We hypothesize that a
polymer-scaffolded DCL should be able to respond to the
Acylhydrazone exchange was chosen as the dynamic reaction
to append organic residues to suitably functionalized polymer
scaffolds as it is a well-studied and successful reaction in
dynamic covalent chemistry.1 Acylhydrazone linkages are
formed from the acid-catalysed condensation of acylhydrazides
with aldehydes, and the resulting acylhydrazone bonds can
readily undergo component exchange, with optimum rates of
formation and exchange observed at pH 4.5.15 Many DCLs
have been reported based around acylhydrazone exchange
in organic media,2–6 however, there have been relatively
few reports of dynamic systems based upon acylhydrazone
exchange in aqueous solution.16,17
Chemical Nanoscience Laboratory, School of Chemistry,
Newcastle University, Newcastle upon Tyne, NE1 7RU,
United Kingdom. E-mail: d.a.fulton@ncl.ac.uk;
Fax: +44 (0) 191 222 6929; Tel: +44 (0) 191 222 7065
w Electronic supplementary information (ESI) available: Experimental
procedures, spectroscopic data, GPC traces. See DOI: 10.1039/
c1cc11998b
Our water-soluble PS-DCL was constructed upon the polymer
scaffold P1, which is a polyacrylamide derivative featuring
c
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indirectly by using 1H NMR spectroscopy to measure the
relative concentrations of unconjugated residues 1 and 2 in
solution, thus allowing the residual composition upon the
polymer scaffolds to be ascertained. Equilibrium was reached
after 16 h, with 1H NMR spectroscopy revealing both
unconjugated acylhydrazides were present in solution in a
1.0 : 1.0 ratio, implying the residual composition upon the
polymer scaffolds is also 1.0 : 1.0. No aldehyde signal was
observed, indicating that the polymer is fully functionalized
with acylhydrazone residues. The PS-DCL composition was
monitored over a period of 48 h, with no further deviation
from this composition observed. This observation suggests
that in the absence of any template, the polymer scaffold
displays no particular preference for the incorporation of
either residue 1 or 2.
We hypothesized that a macromolecular template with the
capacity to participate in multivalent interactions with a
polymeric receptor would be most suited to our system and
so a 70 kDa poly(sodium-4-styrene sulphonate) and the
protein Bovine Serum Albumin (BSA) were chosen as macro-
molecular templates. Upon addition of poly(sodium-4-styrene
sulphonate),z changes in the residual composition of the
1
PS-DCL as a function of time were monitored by H NMR
spectroscopy, which revealed (Fig. 1) an increase in the ratio of
free acylhydrazide 2 relative to 1 of 1.2 : 1.0 from an initial
ratio of 1.0 : 1.0. This observation suggests that the PS-DCL
has responded to the addition of the template, re-equilibrating
to incorporate a greater proportion of residue 1 onto the
polymer scaffold and rejecting residue 2 (Fig. 2). We postulate
that the observed templating effect is a consequence of
favourable multivalent ion-ion interactions between residues
of 1 conjugated to the polymer scaffold and the poly(sodium-
4-styrene sulphonate) template.
The templating effect of BSA upon the PS-DCL was also
investigated. BSA has an isoelectronic point (pI) of 5.518
indicating its surface is positively charged under the
experimental conditions. Upon addition of BSA, 1H NMR
Scheme 1 Preparation of a PS-DCL.
reactive aromatic aldehyde functions. Polymer P1 was prepared
(see ESIw) by the RAFT copolymerization of N,N0-dimethyl-
acrylamide and the monomer N-ethylacrylamide-2-(4-formyl-
benzamide), affording a polymer possessing a degree of
polymerization of approximately 85 and displaying approx-
1
imately 14 aldehyde functions (by H NMR spectroscopy).
Because of the limited water-solubility of this polymer, our
PS-DCL was prepared (Scheme 1) by a two stage process.
Conjugation of excess Girard’s reagent T (1) onto P1 through
hydrazone bonds drives the acylhydrazone formation equilibrium
to completion and produces a water-soluble polymer (P2).
Upon addition of a second acylhydrazide derivative (2), the
polymer undergoes component exchange to produce the
PS-DCL which is composed of an inter-converting mixture
of polymers adorned with varying amounts of the residues 1
and 2. All experiments were performed using 50 mM concen-
trations of 1 and 2 in buffered D2O (NH4OAc/AcOH pH 4.5).
The composition of the residues conjugated to the polymer
scaffolds cannot be monitored directly by 1H NMR spectroscopy
because the diagnostic signals corresponding to conjugated
residues overlap. Instead, we determined the residual composition
Fig. 1 1H NMR spectroscopic analysis (500 MHz, D2O, pH 4.5) of
PS-DCL before (t = 0 h) and after (t = 17 h) addition of poly(sodium-
4-styrene sulphonate) highlighting the changes in intensity of the
diagnostic signals within the residues 1 and 2 17 h after the addition
of poly(sodium-4-styrene sulphonate) template.
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has been observed indirectly using 1H NMR spectroscopy.
The PS-DCL has also been shown to respond to the addition
of two macromolecular template species in a manner that may
be rationalised. It could be argued that the actual effects
of templating on the polymer composition are small in
comparison to the large amplifications of binders associated
with macrocyclic DCLs.1 However, interactions between
polymer and macromolecular templates are likely to involve
localised regions, analogous to protein-protein binding
through ‘hot spots,’ where the substitution of a small number
of residues has been observed to drastically reduce binding
affinity.19 We are now attempting to establish if the
re-equilibration process upon templation leads to an enhancement
in binding affinity between the members of the PS-DCL
and the templates, and developing more complex PS-DCLs
and HPLC methods to study changes in the library during
templation. Our strategy could lead to the development
of wholly synthetic receptors for the selective recognition
of proteins, or to the generation of low-cost antibody mimics.
We wish to thank EPSRC and One North East for
considerable support, and acknowledge the assistance of the
EPSRC National Mass Spectrometry Service Centre,
Swansea. We also thank Dr C. Y. Wills for assistance with
1H NMR experiments.
Fig. 2 Effect of addition of poly(sodium-4-styrene sulphonate) to a
PS-DCL upon the concentrations of unconjugated 1 and 2 as a
function of time (diamonds). There is no observed in change in the
ratio of 1 and 2 in the absence of template (squares) or in the absence
of polymer (triangles).
spectroscopy revealed an increase in the concentration of
unconjugated acylhydrazide 1 relative to 2 of 1.0 : 0.8 from
an initial ratio of 1.0 : 1.0 (Fig. 3). This observation suggests
the PS-DCL has re-equilibrated to incorporate a greater propor-
tion of residue 2 onto the polymer scaffold and rejected residue 1.
We postulate that this templating effect may be a result of
favourable ion–dipole interactions between BSA and ethylene
glycol residues on the polymer scaffold, or in avoidance of
unfavourable cation–cation interactions between BSA and
residues of 1 present on the polymer scaffold. To verify that
the effects observed were indeed as a consequence of the
templating effects of poly(sodium-4-styrene sulphonate) or
BSA, control experiments were performed. For PS-DCLs to
which no template was added, the library maintained a 1.0 : 1.0
composition of 1 and 2 in solution over a period of 17 h, as
monitored by 1H NMR spectroscopy (Fig. 2 and 3). Templates
were also added to 50 mM solutions of 1 and 2 in the absence
of polymer scaffold and monitored for 17 h by 1H NMR
spectroscopy. We observed no differences in chemical shifts or
signal broadening, evidence which suggests there are no
significant interactions between either template and the acyl-
hydrazides 1 and 2. These observations indicate that the
re-equilibriation processes observed are indeed as a consequence
of the PS-DCL interacting with the template.
Notes and references
z Addition of a small aliquot of MeOH-d4 was necessary to prevnt
precipitation of the PS-DCL and poly(sodium-4-styrene sulphonate).
MeOH-d4 was also added to relevant control reactions.
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Fig. 3 Effect of addition of bovine serum albumin to a PS-DCL upon
the concentrations of unconjugated residues 1 and 2 as a function of
time (diamonds). There is no observed change in the ratio of 1 and 2 in
the absence of template (squares) or in the absence of polymer
(triangles).
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 7209–7211 7211