Supramolecular autoregulation

Article abstract of DOI:10.1021/ja4006833

Enzyme activity in biological systems is often governed by control mechanisms in which the catalytic properties are made sensitive or insensitive to differences in enzyme or substrate concentration. Here, we report the first supramolecular system where th

Full text of DOI:10.1021/ja4006833

Article
pubs.acs.org/JACS
Supramolecular Autoregulation
Francisco Rodríguez-Llansola and E. W. Meijer*
Institute for Complex Molecular Systems and Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of
Technology, P.O. Box 513, 5600 MB, Eindhoven, The Netherlands
S
* Supporting Information
ABSTRACT: Enzyme activity in biological systems is often
governed by control mechanisms in which the catalytic
properties are made sensitive or insensitive to differences in
enzyme or substrate concentration. Here, we report the first
supramolecular system where the catalytic activity is made
concentration independent through the use of newly designed
inhibitor molecules. The precise concentration dependence of
coupled supramolecular equilibriums between free catalyst,
inhibited catalyst, active inhibitor, and inactive inhibitor allows
to keep the concentration of free catalyst at 1 mM in a broad
concentration range, yielding an autoregulated catalytic system.
supramolecular systems. We will use organocatalysis here to
illustrate the concept of supramolecular autoregulation, also
because this concept is often found in controlling enzymatic
conversions in nature.
1. INTRODUCTION
Autoregulation is a fascinating adaptive process found within
many different biological systems and is used to adjust or
mitigate the response a system has to a stimulus. A large variety
of sophisticated regulation mechanism is found in natural
systems that permits this internal control upon stimuli. For
example, catalytic activities in biochemical pathways are often
regulated through complex feedback loops, in which they are,
e.g., made insensitive to differences in the enzyme concen-
tration.1 The importance of understanding these phenomena in
biology is gaining full attention in the field of systems biology,
and recently some initial approaches have been undertaken to
mimic processes in artificial systems.2−4 One of the chemical
processes that potentially can benefit from an autoregulation
mechanism is found in the field of organocatalysis.5−8
Organocatalysis is an intriguing way to synthesize complex
molecules. Such reactions are catalyzed using specific
interactions between the catalysts and substrates without
using (transition) metals. This type of catalysis often leads to
excellent yields with high stereoselectivity and is mimicking in
many aspects its natural counterpart. The efficiency and the
selectivity of these organocatalysts are in general concentration-
dependent; i.e., the turnover numbers, chemical selectivity, and
stereoselectivity are dependent on the concentration of catalyst
used in the reaction.9−13 This phenomenon is mainly thought
to be related to (i) the catalytic mechanism that involves more
than one molecule of catalyst in the catalytic cycle (reaction
order with respect the catalyst is higher than 1) and (ii) catalyst
aggregation, which is obviously concentration-dependent. To
remove the concentration-dependent catalytic properties has
been described as a profound challenge for organocatalysis and
requires the autoregulation of the active catalyst concen-
tration.14 Therefore, the introduction of autoregulation in
organocatalysis is a challenging next step to provide robustness
in the process, and it involves mastering of the complexity in
In biological systems, supramolecular interactions are often
used to control the enzyme concentration and, thus, the
catalytic activity. A simple but highly effective biological
strategy for controlling catalytic activity is the use of
complexation, yielding supramolecular complexes that are
catalytically inactive or less active.15 These can be formed by
individual enzymes (oligomeric complexes) or by the
combination of enzymes and specific scaffold proteins
(heterogenic complexes). The final complex entity acts as a
reservoir for single enzymes and allows their release after
dilution, keeping the active enzyme concentration constant and
thereby having the catalytic activity autoregulated.
Research in our group over the past 15 years has focused on
the design and study of well-defined supramolecular systems
and their application in different fields such as materials
science,16 biomedicine,17 electronics,18 etc. A well-defined
multicomponent system developed and used by us and others
is the concentration-dependent dynamic equilibrium as shown
in Figure 1.19 The 2-ureido-4[1H]-pyrimidinone (UPy) unit
shows self-complementarity in forming highly stable dimers
(Kdim = 6 × 107 M−1 in CDCl3). After tautomerization to the 2-
ureido-6[1H]-pyrimidinone, it becomes complementary to the
2,7-diamido-1,8-naphthyridine (NaPy) unit, yielding a UPy-
NaPy complex (Ka = 5 × 106 M−1 in CDCl3). This equilibrium
is regulated by Le Chatelier’s principle, and as a consequence,
the distribution of species can be controlled as a function of the
total concentration. Selectivity toward the formation of UPy-
Received: January 21, 2013
Published: April 2, 2013
© 2013 American Chemical Society
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While the bifunctional UPy scaffolds with short spacers
(1a,b) were insoluble in chloroformprobably due to the
formation of supramolecular linear polymersscaffolds 1c−e
1
presented a remarkable solubility. Analysis of the H NMR
spectra confirmed that the tautomeric behavior of UPy units
present in the scaffold structures 1c−e is in agreement with
previously reported results, having 4[1H]-pyrimidinone as the
major tautomer present in CDCl3 and the 6[1H]-pyrimidinone
as the major tautomer in DMSO-d6.26
Initial evidence for the intramolecular dimerization came
from comparing the 1H NMR spectra (CDCl3, 298 K) of 1c−e
with the spectrum of the model compound 2. In the spectrum
of 2 the signal corresponding to the resonance from the
urethane NH proton (g in Figure 2) appeared at 4.87 ppm and
Figure 1. Top: Dynamic supramolecular equilibrium between UPy
dimers, NaPy, and UPy-NaPy complex. Bottom: Calculated fractions
of UPy in UPy dimers, UPy monomers, and UPy-NaPy complex.19
NaPy complexes has been applied, for example, in the selective
synthesis of well-defined supramolecular block copolymers.20
Here, we introduce such a supramolecular system based on
UPy-NaPy motifs capable of keeping the concentration of free
NaPy constant upon several dilutions, thereby engineering the
first supramolecularly autoregulated system. Remarkably, we
found in preliminary studies that the basic character of the 1,8-
naphthyridine unit in the NaPy motif makes it an efficient
catalyst for different mild base-catalyzed reactions. Thus, the
catalytic site of NaPy can be a priori supramolecularly
deactivated after UPy-NaPy complexation. In this Article, we
report the preliminary catalytic results of this autoregulated
system in a model Michael reaction, showing the clear benefits
of bringing together complex supramolecular systems and
organocatalysis.
Figure 2. Chemical structures for scaffolds 1a−e, model compound 2,
and organocatalyst 3.
was found to be quite insensitive to concentration (Figure S1,
Supporting Information). In dilute samples of the bifunctional
UPy scaffolds 1 (0.5 mM), the signals for the urethane NH
protons (labeled “g”) were found to be downfield shifted (1c,
5.04 ppm; 1d, 5.21 ppm; and 1e, 5.17 ppm) relative to that of
model compound 2 (Figure S3). These results indicated that
urethane protons g in the compounds 1c−e are most likely
involved in intramolecular H-bonding and the cyclic monomer
arrangement presents a different local chemical environment. In
addition, NOE studies were carried out in order to evaluate the
size of the species of 1c−e detected in CDCl3 under dilute
conditions. Small, positive NOEs were obtained in all three
cases, suggesting a low molecular weight for the folded species
2. RESULTS AND DISCUSSION
2.1. Design of the Cyclic Supramolecular Scaffolds.
Mathematical models suggest that the use of cyclic scaffolds,
formed upon UPy intramolecular dimerization of bifunctional
molecules, can increase the concentration dependence of the
equilibrium shown in Figure 1. Where normally these
bifunctional molecules form supramolecular polymers, some
design rules exists to make them preferentially exist in cyclic
forms.21−24 In order to achieve the synthesis of stable cyclic
monomers that only polymerize at very high concentrations, we
decided to prepare different scaffolds of the optimized length
functionalized with the UPy motif at both ends. Earlier
molecular studies showed that bifunctional UPy derivates with
short spacers (shorter than 14) are not able to dimerize in an
intramolecular way due to the antiparallel fashion of the UPy
association.24 One unexplored and easy synthetic route to
access scaffolds with long, flexible linkers between the UPy
units is by direct reaction of a mono-UPy isocyanate25 and the
corresponding aliphatic diol in the presence of dibutyltin
dilaurate. Scaffolds 1a−e were prepared using this strategy.
Compound 2 was prepared as a model system in which the
UPy dimerization takes place in an intermolecular way,
compared to the scaffolds 1 where dimerization takes place
intramolecularly. NaPy derivative 3 was made by a standard
route to act as the organocatalyst.
(Figure S4).21 At low concentrations (0.5−4 mM), the H
1
NMR signals of compounds 1c−e were concentration
independent, suggesting that mainly cyclic monomers are
present. At higher concentrations, new signals related to the
formation of larger supramolecular complexes were detected in
1
the H NMR spectra (Figure 3; also see Figures S5−S7). The
main differences between resonances of the monomeric species
and the larger aggregates are found in the signals corresponding
to the UPy motif (protons c and d in Figure 2), NH of the
urethane moiety (proton g), and the alkyl chain (mainly proton
e). The fact that nonviscous solutions are obtained in the
concentration range from 10−2 to 10−1 M suggests a low
concentration of linear polymers even at these high
concentrations.
1
Using these H NMR spectroscopy data (vide supra), we
were able to determine the maximum concentration of
monomeric cycles (MCMC) of the different scaffolds 1c−e.
Figure 3 shows plots of the high concentration of monomers
within monomeric cycles as a function of overall concentration
for scaffolds 1c−e. The higher value of MCMC obtained for
scaffold 1c (10 1 mM) with respect to those obtained with
scaffolds 1d (8 1 mM) and 1e (4 1 mM) suggests that the
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Figure 4. Top: Schematic representation of the equilibrium between
the dimeric form of compound 2, compound 3, and complex 2·3.
Bottom: (a) Measured fractions determined by 1H NMR spectroscopy
in CDCl3 (filled points) and calculated (lines) for free 3 and the
complex 2·3 as a function of concentration in an equimolar mixture of
2 and 3. (b) Measured concentration of free 3 in solution (filled
points) and calculated (red line) as a function of total concentration.
Kdim(UPy) = 6 × 107 M−1, and Ka(UPy-NaPy) = 3 × 106 M−1.
Figure 3. Top: 1H NMR spectra (400 MHz, CDCl3, 298 K) of scaffold
1c at different concentrations. Asterisks indicate the distinguishable
signals originating from the cyclic monomer. Bottom: MCMC values
as a function of total concentration in CDCl3 at 298 K for scaffolds
1c−e (panels a−c, respectively) and molecular models proposed in
solution. The models correspond to energy minima obtained with
molecular mechanics calculations using the AMBER* force field in
CHCl3 (MACROMODEL 9.9). Nonpolar hydrogen atoms have been
omitted for clarity.
M−1, Figure 4a and Figure S9) is in good agreement with
previously reported results in CDCl3. Simulation of the
equilibrium using different dimerization and association
constants predicts that a change in distribution of the species
at a given concentration (selectivity) is possible; however, no
relevant effect on the concentration dependence is observed
(Figures S10 and S11). In a second study, changing the molar
ratio between the UPy and NaPy units showed an effect on the
equilibrium upon dilution; however, total autoregulation of the
NaPy concentration was not achieved (Figure S12).
monomeric cyclic species becomes less stable as the length of
the linker increases. These results are in full agreement with
those previously reported in similar systems and can be directly
related to a decrease in the effective concentration.27 After the
synthesis and assembly studies of the scaffolds 1c−e and the
model compound 2, we studied how the concentration-
dependent equilibrium between the free NaPy and UPy-NaPy
complex is affected.
2.2. Concentration-Dependent Studies. The position of
the equilibrium between UPy dimers, NaPy, and the UPy-NaPy
complex and hence the selectivity of the heterocomplexation
are regulated by Le Chatelier’s principle. Therefore, at high
concentrations the formation of UPy-NaPy heterodimer is
favored, while upon dilution the formation of UPy dimers
becomes more important, with the consequent release of free
NaPy molecules.19 As an example, the observed concentration
dependence of equimolar mixtures of the model compound 2
with the NaPy-based molecule 3 in CDCl3 is shown in Figure
4a.
Upon dilution of a concentrated sample containing the 2·3
complex from 80 to 0.12 mM (Figure 4a), the selectivity for
formation of the heterodimer complex was found to be
decreased from 95% to 62%, respectively. By plotting the
concentration of free 3 as a function of the total concentration
of 3, it can be appreciated that dilution (from 80 to 10 mM)
results in a decrease in concentration of free 3 (from 4.2 to 1.1
mM).
Employing the cyclic monomeric scaffolds 1c−e, it is
expected that the competition between intra- and intermo-
lecular interactions will yield a higher concentration depend-
ence associated with Le Chatelier’s principle (Figure 5). Indeed,
1
when we performed a H NMR dilution experiment in CDCl3
employing scaffolds 1c−e, the concentration dependence was
remarkably increased in all cases (Figure 5a; also see Figure
S13). For example, it can be seen in Figure 5a that, in the case
of scaffold 1d, the system is composed almost exclusively of the
complex 1d·3 at high concentrations (C = 80 mM) and that
after dilution to 0.12 mM, all NaPy 3 is released in its free form.
Fine adjustment of the effective molarity in the scaffolds 1 by
changing the length of the spacer between the two UPy units
allows for complete autoregulation of free catalyst 3. Indeed, in
the presence of scaffold 1c or 1d, the concentration of free 3 is
buffered at 1.0 0.2 mM in a broad concentration range of 4−
80 mM of total 3 (Figure 5b). Interestingly, scaffold 1e is not
able to promote the total autoregulation of free 3. This different
behavior is most likely related to the lower stability of its
monomeric cycle observed in the conformational studies
(Figure 3). The high concentration dependence observed for
scaffolds 1c−e can be simulated in good agreement by
employing a simplified mathematic model (Figures S14 and
S15) and assuming that only the cyclic monomer of the
scaffolds is formed (Figure 5c). It is remarkable that the
autoregulation of free 3 found experimentally with 1c,d was
even better than predicted by the model (Figure 5d).
Therefore, the internal supramolecular mechanism mitigates
the effect that dilution has on the concentration of free 3. The
2.3. Organocatalysis. After the design of the scaffold and
the catalyst, we studied the effect that the presence of 1d has on
the catalytic properties of 3 in order to test the internal
mechanism of autoregulation. The basic character of the NaPy
unit in 3 makes it an efficient organocatalyst for the well-
1
results obtained from the concentration-dependent H NMR
experiment on 2 and 3 were fitted to a mathematical model
assuming the reported28 Kdim(UPy) = 6 × 107 M−1. The
calculated selectivity for the UPy-NaPy complex (Ka = 3 × 106
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Figure 5. Top: Schematic representation of the equilibrium between
cyclic monomers of 1, compound 3, and complex 1·3. Bottom: (a)
Figure 6. Top: Michael addition of 2,4-pentanedione to trans-β-
nitrostyrene. Bottom: (a) Conversion as a function of time employing
different concentrations of 3. (b) Catalytic activity of 3 in the Michael
addition at different concentration of catalyst. (c) Conversion as a
function of time in a catalytic system formed by compound 3 (20 mM)
with different equivalents of scaffold 1d. (d) Catalytic activity of a
system formed by 3 and 1d in a equimolar ratio of UPy:NaPy units at
different concentrations.
1
Measured fractions determined by H NMR in CDCl3 of free 3 and
the complex 1d·3 as a function of the total concentration. (b)
Measured concentrations of free 3 as a function of the total
concentration of 3 in a 1:2 mixture of either scaffold 1c (filled
circles), 1d (circles), or 1e (triangles) and 3, respectively. (c)
Calculated fractions of free 3 and the complex 1·3 as a function of total
concentration of 3. (d) Calculated concentration of free 3 as a function
of total concentration. The UPy:NaPy ratio employed in the
calculations was 1:1 (KI(UPy) = 1 × 107 and Ka(UPy-NaPy) = 3 ×
106 M−1).
regulates the final catalytic activity.30 However, in this system
the TOF has become concentration independent and strikingly
different from that of the non-autoregulated system (Figure
6d). Cyclic scaffold 1d is able to keep constant the
concentration of the free catalyst 3 and to mitigate the effect
that a dilution has on the catalytic properties, yielding the first
example of supramolecular autoregulation in organocatalysis.
studied Michael addition of 2,4-pentanedione to trans-β-
nitrostyrene. The catalytic activity of 3 is expressed in turnover
frequency (TOF = [Product]/[Catalyst]·h−1) and is strongly
related to the specific concentration of 3 (Figure 6a,b). Such
concentration dependence is proposed to be related to the
hydrogen-bonding donor and acceptor properties of 3, which
are also involved in the activation of both reagents via hydrogen
bonding.29 When the reaction was carried out in the presence
of 1 equiv of 1d, a clear decrease of reactivity was found (Figure
6c). After the addition of 1d, the starting reaction rate was
decreased by a factor of 12 (Figures S17 and S18). Remarkably,
the reaction can be stopped after addition of another equivalent
of 1d; showing the selectivity and efficiency of this scaffold for
the supramolecular inhibition of the catalytic properties of 3.
Under the catalytic conditions employed, the system
composed of an equimolar mixture of the UPy and NaPy
units presented 95% of compound 3 in the form of the complex
1d·3 and 5% of free 3. This selectivity is in agreement with the
data obtained in the absence of reagents, suggesting that the
equilibrium is not affected under the catalytic conditions. It is
worth mentioning that although the 1H NMR data under these
catalytic conditions suggest a concentration of free 3 of around
1 mM, the reactivity observed was similar to that of a system
with a 7 mM concentration of free 3. This discrepancy is
proposed to be related to the inherent complexity and dynamic
nature of this supramolecular system. As a consequence,
scaffold 1d nonlinearly deactivates 3 in a way that resembles the
results observed in recent studies carried out with enzymes,
where the lifetime of the active and inactive configuration
3. CONCLUSIONS
In summary, we report a unique supramolecular system that is
able to keep the concentration of an organic catalyst constant
over a broad overall concentration regime, due to the presence
of a scaffold. This autoregulation is based on two distinct
competitive molecular interactions in a multicomponent
system. These molecular interactions have different concen-
tration-dependent profiles, based on Le Chatelier’s principle.
This difference in concentration-dependent behavior is able to
compensate the decreased concentration of a given species after
dilution due its lower association to the scaffold. The high
concentration dependence achieved with the cyclic scaffolds
1c,d in the equilibrium of free and complexed 3 mitigates the
effect that dilution has on the final catalytic properties. The
combination of supramolecular chemistry with organocatalysis
has created a complex catalytic behavior that mimics one of the
enzymatic autoregulation mechanisms found in nature. This is a
starting point to develop new complex systems with positive
and negative feedback loops. It will open new ways to study
well-defined models or to mimic biological systems.
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(22) Li, S. L.; Xiao, T.; Xia, W.; Ding, X.; Yu, Y.; Jiang, J.; Wang, L.
Chem.Eur. J. 2011, 17, 10716−10723.
ASSOCIATED CONTENT
■
S
* Supporting Information
(23) Xiao, T.; Li, S. L.; Zhang, Y.; Lin, C.; Hu, B.; Guan, X.; Yu, Y.;
Jiang, J.; Wang, L. Chem. Sci. 2012, 5, 1417−1421.
Additional spectroscopic data, concentration-dependent stud-
ies, details on the equilibrium model and the curve-fitting
procedure, catalytic methods, and catalytic results. This material
is available free of charge via the Internet at http://pubs.acs.org.
(24) Sontjens, S. H. M.; Sijbesma, R. P.; van Genderen, M. H. P.;
̈
Meijer, E. W. Macromolecules 2001, 34, 3815−3818.
(25) Folmer, B. J. B.; Sijbesma, R. P.; Versteegen, R. M.; van der Rijt,
J. A. J.; Meijer, E. W. Adv. Mater. 2000, 12, 874−878.
(26) Beijer, F. H.; Sijbesma, R. P.; Kooijman, H.; Spek, A. L.; Meijer,
E. W. J. Am. Chem. Soc. 1998, 120, 6761−6769.
AUTHOR INFORMATION
■
Corresponding Author
e.w.meijer@tue.nl
(27) De Greef, T. F. A.; Smulders, M. M. J.; Wolffs, M.; Schenning,
A. P. H. J.; Sijbesma, R. P.; Meijer, E. W. Chem. Rev. 2009, 109, 5697−
5754.
Notes
(28) Sontjens, S. H. M.; Sijbesma, R. P.; van Genderen, M. H. P.;
̈
The authors declare no competing financial interest.
Meijer, E. W. J. Am. Chem. Soc. 2000, 122, 7487−7493.
(29) Okino, T.; Hoashi, Y.; Furukawa, T.; Xu, X.; Takemoto, Y. J.
Am. Chem. Soc. 2004, 127, 119−125.
ACKNOWLEDGMENTS
■
(30) Benkovic, S. J.; Hammes-Schiffer, S. A. Science 2003, 301, 1196−
1202.
We are supported by the National Research School of Catalysis
(NRSC-C), the Ministry of Education, Culture and Science
(Gravity program 024.001.035), the Netherlands Organization
for Scientific Research (NWO), and the European Research
Council (FP7/2007-2013) ERC Grant Agreement. We thank
Dr. Tom F. A. de Greef for stimulating discussions. Illustrations
are credited to ICMS Animation Studio, TU/e.
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