Regulating competing supramolecular interactions using ligand concentration

Article abstract of DOI:10.1021/jacs.6b03421

The complexity of biomolecular systems inevitably leads to a degree of competition between the noncovalent interactions involved. However, the outcome of biological processes is generally very well-defined often due to the competition of these interaction

Full text of DOI:10.1021/jacs.6b03421

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Article
Regulating Competing Supramolecular
Interactions Using Ligand Concentration
Abraham J.P. Teunissen, Tim F.E. Paffen, Gianfranco Ercolani, Tom F. A. de Greef, and E.W. Meijer
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b03421 • Publication Date (Web): 10 May 2016
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Regulating Competing Supramolecular Interactions Using Ligand Concentration
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Abraham J. P. Teunissen,†,‡ Tim F.E. Paffen,†,‡ Gianfranco Ercolani,∥ Tom F.A. de Greef,*,†,§ and
E.W. Meijer*,†,‡
†Institute for Complex Molecular Systems, ‡Laboratory of Macromolecular and Organic Chemistry, and
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§Computational Biology, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands.
∥Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata, Via della Ricerca Scientifica,
00133 Roma, Italy.
ABSTRACT: The complexity of biomolecular systems inevitably leads to a degree of competition
between the non-covalent interactions involved. However, the outcome of biological processes
is generally very well-defined often due to the competition of these interactions. In contrast,
specificity in synthetic supramolecular systems is usually based on the presence of a minimum
set of alternative assembly pathways. While the latter might simplify the system, it prevents the
selection of specific structures and thereby limits the adaptivity of the system. Therefore,
artificial systems containing competing interactions are vital to stimulate the development of
more adaptive and life-like synthetic systems. Here, we present a detailed study on the self-
assembly behavior of a C2v-symmetrical tritopic molecule, functionalized with three self-
complementary ureidopyrimidinone (UPy) motifs. Due to a shorter linker connecting one of
these UPys, two type of cycles with different stabilities can be formed, which subsequently
dimerize intermolecularly via the third UPy. The UPy complementary 2,7-diamido-1,8-
naphthyridine (NaPy) motif was gradually added to this mixture in order to examine its effect
on the cycle distribution. As a result of the C2v-symmetry of the tritopic UPy, together with
small differences in binding strength, the cycle ratio can be regulated by altering the
concentration of NaPy. We show that this ratio can be increased to an extent where one type of
cycle is formed almost exclusively.
KEYWORDS. Multivalency, Supramolecular, Non-covalent, Competition, Computational model,
Ureidopyrimidinone.
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■ INTRODUCTION
Competition plays a dominant role in all biological processes; it controls the evolution of
species,1 the growth of organs,2 and even memory formation.3 Especially on the molecular scale
the presence of competing pathways allows a delicate regulation of various processes, e.g., the
repair of double-strand DNA in eukaryotic cells depends on an interplay between two repair
mechanisms4 and RNA transcription in E. Coli is regulated by the competition of seven different
RNA polymerase σ-subunits.5 While the presence of competition allows more flexibility in
controlling the pathway desired, it also allows the possibility of undesired processes. Nature
typically uses additives such as chaperones in protein folding6 or competitive binders to
regulate gene expression.7,8 Multivalent scaffolds are another important way through which
competing processes are controlled. Coupling multiple receptors together enhances their
combined binding strength as a result of chelate cooperativity9 and this can have profound
effects on the kinetics.10 By regulating the number of mono- and multivalent receptors,
equilibria can be influenced without the need of molecular changes in the binding motifs
themselves, which is for example used by cells to probe ligand density.11 Competing assembly
pathways play an important role in non-linear processes such as feedback-loops and regulatory
networks.12 As a result of their complex and often counterintuitive behavior a combined
experimental and computational approach is often vital to gain an in-depth understanding, as
shown by recent advances in systems biology.13,14
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In recent years multiple synthetic systems displaying semi-biological behavior have been
reported, such as logic gates,15,16 self-sorting systems17,18 and the activation of a reaction by
outcompeting a supramolecular protecting group.19 While competing species are present in
such systems, the large differences in binding strengths make them function in an on/off
manner, without possessing the delicate regulation observed in biological systems. Other
chemical systems have mimicked the multivalency found in biomolecules. In such systems, the
binding motifs are generally not self-complementary20 and positioned in a symmetrical manner,
e.g., C3-symmetrical in trivalent pseudorotaxanes,21,22 C4-symmetrical on the 4 sides of a
porphyrin23,24 or C6-symmetrical in a hexavalent pyridine construct.25 While these systems can
form receptor-ligand contacts at different positions on the molecule, their symmetry makes the
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resulting species identical, preventing any competition. Additionally, the close proximity of the
binding motifs often introduces a chelate cooperative effect,9,26,27 limiting the amount of
populated species.
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By delicately regulating competing processes via external stimuli, identical building
blocks can be used for the synthesis of a variety of complexes,28,29 in contrast to the more
common approach of modifying binding constants by changing the molecular structure.30 An
elegant example is given by Otto et. al., who has demonstrated that regulating the template
concentration in a dynamic combinatory library can lead to the amplification of specific
receptors, and the strongest receptor is not necessary amplified the most.31 This shows that,
similar as in natural systems, the introduction of competition can lead to complex behavior, but
it does not necessary lead to a lack of selectivity. For competition to be present, it is vital that
different structures are formed as a result of a certain group that binds at different positions.
Secondly, to act as a regulatory mechanism, the different structures formed in such manner
must be of comparable stability, potentially influenced by external stimuli.32
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Our group has recently reported on a system where competing intra- and
intermolecular interactions between supramolecular building blocks result in the buffering of
catalytic activity over a broad concentration range.33 This system is based on the self-
complementary ureidopyrimidinone (UPy) motif and its binding to 2,7-diamido-1,8-
naphthyridine (NaPy)34, of which the latter can act as a phase-transfer catalyst in its unbound
state.35 It has been shown that the competition between linear and cyclic contacts formed by a
ditopic UPy lead to a buffered concentration of NaPy chain-stopper, i.e., the active catalyst. This
work demonstrates that a combined experimental and computation approach is also vital in
synthetic systems to thoroughly understand competing processes.36 To better mimic the way
nature uses competition as a regulatory mechanism, we have now designed a system where the
competition between structures of comparable stability is regulated via the binding of a small
additional ligand.
We report the study of a C2v-symmetrical tritopic UPy (Figure 1). As a result of its
symmetry this compound can form two mutually exclusive types of cycles, which subsequently
dimerize via the pendant third UPy, resulting in three different structures. Then the NaPy ligand
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is titrated to the mixture and its effect on the cycle distribution is examined. We show that by
altering the concentration or selectivity of NaPy, the ratio between the two types of cycles can
be regulated. Using this approach it is possible to exclusively form one type of cycle only,
without requiring changes to the molecular structure of the tritopic UPy.
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Figure 1. Molecular structure of the UPy-UPy and UPy-NaPy dimers (Top). Schematic representation of
the cyclization and subsequent dimerization of a C2v-symmetrical tritopic supramolecular building block,
as well as its binding to a ligand (Bottom). The tritopic UPy consists of one short linker connecting the
UPy on the “A” position and two longer linkers connecting the UPys on position “B”. Due to this
symmetry, two mutually exclusive type of cycles can be formed, which dimerize via the remaining 3rd
UPy. By varying the concentration of NaPy (N) the cycle distribution can be regulated, leading to a
structure with all UPys bound to NaPy at higher ligand concentrations.
■
RESULTS AND DISCUSSION
Design and synthesis of tritopic UPy 1 and its reference compounds. We have designed
a C2v-symmetrical tritopic UPy molecule that is functionalized with one short linker connecting
the UPy at position “A” (light blue Figure 1) and two longer linkers of equal length connecting
the UPy at the “B-positions” (dark blue Figure 1). As a result of this architecture, it is possible to
form two mutually exclusive type of cycles, i.e., between an A and B-type UPys, or between
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both B-type UPys. The abundance of either cycle will depend on its relative stability, while the
symmetric nature of the compound provides two ways of forming the A-B cycle, making its
formation entropically more favourable.37 Additionally, a more indirect factor influencing the
cycle distribution is predicted. Due to the high binding constant of the UPy motif (Kdim = 6*107
M-1 in chloroform at 25 ˚C),38 unbound UPys are generally unfavorable, therefore the cyclic
structures will dimerize via the pendent binding position. Depending on the type of dimerized
cycle that is formed, three different types of linear dimers are created (i.e., A-A, A-B, and B-B).
The different molecular connectivity of the A and B-type UPys results in small differences in the
stability of these dimers (vide infra), which influences the cycle distribution as well, e.g. a
relatively high stability of the A-A dimers favors the formation of B-B cycles. Similarly,
association of a NaPy (N) that can bind to the UPy motif will be governed by the stabilities of
the A-N and B-N dimers. Additionally, ligand binding is governed by statistical factors i.e., there
are twice as many B as A groups, making it more likely to form B-N contacts. Such ligand binding
competes with intramolecular cycle formation, giving rise to a complex interplay of
interactions.
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As mentioned, to alter the stability of both type of cycles, the linker connecting the A-
type UPy is slightly shorter than that connecting the B-type UPys. In addition to this, we
synthesized the A-type UPy in close proximity to an ester moiety, with the goal to change its
1
chemical shift compared to the B-type UPys and thereby simplify characterization by H-NMR
(Figure 2). To further aid characterization of the different configurations adopted by 1,
reference compounds SCref 2, LCref 3, Aref 4, and Bref 5 are synthesized as well. While SCref 2 acts
as a model for the smaller A-B cycle, LCref 3 has a UPy group on the A position covalently
protected by an UV-labile o-nitrobenzyl group and therefore acts a reference compound for the
larger B-B cycle. The linear, intermolecular, interactions between the A and B-type UPys
potentially formed by 1 are represented by Aref 4 and Bref 5, respectively. In addition to these
reference compounds, UPy complementary NaPy 6 is used in this study. Finally, NaPy is unable
to form stable homodimers in CDCl3.
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Figure 2. Molecular structures and schematic depiction of tritopic UPy 1 and reference compounds SCref
2, LCref 3, Aref 4, and Bref 5, as well as UPy complementary NaPy 6. Roman numerals are used to denote
the specific protons referred to in the text.
Tritopic UPy 1 was synthesized via identical route as LCref 3, starting from the formation
of UPy alcohol 8 (Scheme 1). UPy 8 was then coupled to Boc-protected amino acid 9 via EDC
mediated esterification, resulting in UPy 10. Subsequent deprotection using HCl resulted in UPy
amine 11. The alcohol moiety of commercially available dimethyl 5-hydroxyisophthalate was
protected using 1-(chloromethyl)-2-nitrobenzene and the methylester was hydrolyzed using
aqueous KOH, resulting in 12. Compound 12 was subsequently converted in the di-acylchloride
using SOCl2 and coupled to 11, resulting in ditopic UPy 13 which was deprotected using UV-
light, resulting in phenol 14 containing both B-type UPys. To introduce the UPy on the A
position, CDI activated isocytosine 7 was coupled to commercially available 4-(tert-butoxy)-4-
oxobutan-1-aminium chloride resulting in UPy 15. This UPy 15 was then protected using 1-
(chloromethyl)-2-nitrobenzene yielding 16, which was deprotected by TFA to give protected
UPy 17. Ester formation between 14 and 17 resulted in LCref 3, which upon UV-irradiation gave
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tritopic UPy 1. Further details, as well as the synthesis and characterization of SCref 2, Aref 4, Bref
5, and NaPy 6 can be found in the ESI.
7
8
9
N
O
NH
O
a) DMF
93 %
H2N
OH
R
4
O
N
H
N
N
4
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H
O
N
N
N
OH
4
7
H
H
N
8
d) HCl
Dioxane
O
c) EDC
DMAP
O
O
HN
O
O
HN
Cl
H
b) Boc2O
82 %
OH
OH
H
3N
O
N
H2N
O
N
O
N
H
N
N
O
O
N
N
N
O
9
9
95 %
H
84 %
4
9
H
9
9
H
H
O
O
11
10
O
e)
1) K2CO3
Cl
O
O
O
O
NO2
O
O
NO2
f)
O
O
NH
R
OH
OH
NH
R
1) SOCl2
2) TEA
NO2
2) KOH
g) UV
HO
O
O
HO
14
84 %
77 %
40 %
13
12
NH
R
NH
R
O
O
O
N
h) CDI
N
O
j)
91 %
Cl
N
O
N
N
O
N
O
NH2
H
H
NO2
N
HN
NH
R
H
N
l) EDC
DMAP
7
O
NO2
O
N
N
O
H
LCref
3
K2CO3
NH
O
71 %
i) TEA
O
O
NH
Cl
H3N
O
92 %
R
90 %
O
N
N
N
O
H
H
15
O
29 %
m) UV
O
OH
HN
NO2
N
k) TFA
94 %
NO2
N
HN
R
O
NH
HN
O
O
O
O
O
N
N
O
N
N
O
O
H
H
O
N
N
N
16
17
H
H
O
Tritopic UPy 1
O
NH
R
Scheme 1. Synthesis of LCref 3 and tritopic UPy 1. Reagents and conditions: a) DMF, room temperature,
16 h, 93 %; b) TEA, Boc2O, 60 ˚C, 16 h, 82 %; c) EDC, DMAP, room temperature, 60 ˚C, 16 h, 84 %; d) 3 M
HCl in dioxane, room temperature, 16 h, 95 %; e) K2CO3, 1-(chloromethyl)-2-nitrobenzene, 85 ˚C, 2 h,
KOH, H2O/THF, room temperature, 16 h, 77 %; f) SOCl2, DMF, room temperature, 4 h, TEA, room
temperature, 16 h, 40 %; g) DCM, UV (λ = 315-400 nm), room temperature, 10 h, 84 %; h) CDI, 80 ˚C, 16
h, 91 %; i) TEA, room temperature, 16 h, 92 %; j) K2CO3, 1-(chloromethyl)-2-nitrobenzene, 80 ˚C, 16 h, 90
%; k) TFA, room temperature, 16 h, 94 %; l) EDC, DMAP, room temperature, 16 h, 71 %; m) DCM, UV (λ =
315-400 nm), room temperature, 5 h, 29 %.
1H-NMR analysis of tritopic UPy 1 and its reference compounds. A solution of tritopic
1
UPy 1 was analyzed by H-NMR (c = 2 mM, CDCl3), the sample was cooled to -15 ˚C to improve
signal analysis. Multiple signals were observed for all UPy N-H protons, as well as the protons of
the aromatic core, suggesting that 1 forms a complex mixture of species in solution (Figure 3).
No changes were observed over the course of three hours, suggesting thermodynamic
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1
Figure 3. Partial H-NMR spectrum of tritopic UPy 1 in CDCl3 at -15 ˚C and 2 mM, showing multiple
signals for protons I-IV due to the different conformations adopted by 1. The UPy N-H signals originate
from the combined A and B-type UPys in tritopic UPy 1.
equilibrium was reached fast, which could be expected considering the short life-time of the
UPy-UPy interaction in CDCl3 (0.12 sec at 25 ˚C).38 Small changes in the concentration (c = 1-7
mM) did not lead to any significant changes in the 1H-NMR spectrum (See Figure S7 for 1H-NMR
spectra).
1
1
In order to interpret the H-NMR of 1 correctly, we chose to compare its H-NMR
spectrum to that of the reference compounds taken under identical conditions, i.e., c = 2 mM at
1
-15 ˚C in CDCl3 (Figure 4). Study of the H-NMR spectra of the reference compounds revealed
that all UPy-UPy and UPy-NaPy interactions can be identified using a unique chemical shift
(Figure 4B and Figure S1). Interestingly, using the respective protons I and II in Aref 4 and Bref 5,
it even proved possible to discriminate between linear A-A, A-B, and B-B UPy contacts (Figure
4C). Deconvolution of these signals showed that in an equimolar mixture, Aref-Aref and Bref-Bref
dimers are more abundant than Aref-Bref dimers (See Figure S3 for more details). This suggests
that the different connectivity of the A and B-type UPys has led to different stabilities of the
various types of contacts. Likely, the close proximity of the A-type UPys to an ester moiety and
aromatic core has led to a reduced binding strength, as has been shown for UPys with other
types of polar side-chains.39,40
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Figure 4. A Schematic overview of all UPy reference compounds and their interactions, as well as the
NaPy ligand and its interactions with the A and B-type UPys. B Partial 1H-NMR spectra of proton I-III of all
UPy reference compounds as well as compound 1 in CDCl3, T = -15 ˚C, c = 2 mM each. C Partial 1H-NMR
spectra of proton I and II of Aref 4 and Bref 5 in CDCl3, showing that it is possible to distinguish between
Aref-Aref, Aref-Bref and Bref-Bref UPy dimers, T = -15 ˚C, c = 2 mM each.
Since tritopic UPy 1 has a similar molecular architecture as its reference compounds, it is
expected that identical interactions will lead to similar chemical shifts, thereby providing means
to determine the speciation of tritopic UPy 1. In addition to this, quantitative knowledge about
the stability of the cycles formed by 1 is vital for obtaining a complete understanding of its
behavior.
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Figure 5. A and B Partial 1H-NMR spectra of proton III of the B-type UPy in SCref 2 and proton IV in LCref 3
at various concentrations in CDCl3 at -15 ˚C. Signals associated with monomeric cycles are annotated
with MC, signals associated with higher order cycles and polymers with Pol. This signal assignment was
based on the expected concentration dependent behavior of ditopic molecules.41 C and D Concentration
of monomeric cycles versus the total concentration. The plateau concentration that is reached is equal
to the effective molarity (EM = Kintra/Kinter) of the monomeric cycles (8.2 mM for SCref 2 and 5.8 mM for
LCref 3). The fraction of monomeric cycles was calculated by dividing the area of the peak assigned to MC
by the total peak area corresponding to the specific proton. Considering the errors on weighing (1 %),42
pipetting (1 %)43 and NMR integration/deconvolution (5 %),44 we used standard error propagation
techniques to determine the standard deviation to be ≈ 5 %. The errors bars depicted two times the
standard deviation i.e., 10 %. The solid line is used to guide the eye. E Expected speciation of tritopic UPy
1 at 2 mM in CDCl3 based on the EMs of SCref 2 and LCref 3. Assuming equal binding constants of UPy
dimerization, the expected fraction of A-B cycles was calculated as 2*EMAB/(2*EMAB+EMBB) and the
fraction of B-B cycles as EMBB/(2*EMAB+EMBB). The distribution of dimeric cycles was calculated as
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follows: fraction of dimeric A-B cycles = (fraction A-B cycles)^2, fraction of dimer consisting of an A-B
cycle connected to an B-B cycle = 2*(fraction A-B cycles)*(fraction B-B cycles), fraction of dimeric B-B
cycles = (fraction B-B cycles)^2.
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Using SCref 2 and LCref 3, the effective molarities (denoting the stability of the cycles and
defined as EM = Kintra/Kinter) of the monomeric A-B and B-B cycle were determined to be 8.3 mM
and 5.8 mM, respectively. This was indicated by the maximal attainable concentration of these
species in CDCl3 (Figure 5).41 These effective molarities, together with the symmetry factor of
two for the formation of the A-B cycle, results in an expected 74 % of A-B cycles and 26 % B-B
cycles for compound 1 at 2 mM. Assuming that the linear contacts connecting these cycles are
of equal stability, dimerization of the cycles will result in the statistical distribution depicted in
Figure 5E. In conclusion, by studying the reference compounds we obtained an estimate of the
1
speciation of 1, as well as the means of interpreting its H-NMR spectrum. This allows us to
study the titration of tritopic UPy 1 with NaPy 6 and to record the changes in composition of
the afforded species of 1 when 6 is added.
Determining the speciation of tritopic UPy 1 in the presence of different amounts of
NaPy 6. The UPy-complementary supramolecular unit 2,7-diamido-1,8-naphthyridine (NaPy) 6
was titrated to tritopic UPy 1 in order to study its influence on the cycle distribution of 1. Since
UPy proton III gave the best signal separation it was used to determine the exact speciation of
tritopic UPy 1. During this titration 6 different signals could be distinguished (Figure 6A). By
comparison with the reference compounds, signals a and c could be assigned to the A and B-
type UPys in the monomeric A-B cycle. Signal c also contained the linear and/or cyclic B-B
interactions, while these could be distinguished in LCref 3, signal overlap prevented this for
tritopic UPy 1. In a similar fashion, signals d and e were assigned to A-N heterodimers.
As shown in Figure 6B, the addition of NaPy first leads to the appearance of signal d,
which is converted into signal e upon further addition of NaPy. This behavior strongly suggests
that signal d originates from an A-N contact connected to a B-B cycle, while signal e originates
from an A-N contact in a tritopic UPy with all UPys bound to NaPy. Signal f most likely originates
from a B-N contact. As a result, the five most abundant signals that are observed during the
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NaPy titration can be identified using the reference compounds, with only signal b lacking. After
considering several explanations for signal b, we have concluded that it most likely originates
from linear A-B and small amounts of linear A-A interactions (See ESI for further details). By
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comparing the H-NMR spectra of the reference compounds with that of tritopic UPy 1, we
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have obtained a likely assignment of the signals observed during the titration of NaPy to 1
(Figure 6C). Since some of these signals are attributed to multiple types of contacts, it is difficult
to infer the species distribution directly from the data. To overcome this difficulty and validate
our assignment, we have constructed a thermodynamic binding model and subsequently fitted
it to the titration data.
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Figure 6. A Partial 1H-NMR spectrum of UPy proton III in 1 with various amounts of NaPy 6 in CDCl3 at -
15 ˚C and c = 2 mM. B Zoom-in of Figure 6A, depicting the shape of signal d and e as a function of the
equivalents of NaPy 6 added. C Schematic depiction of the species present in solution showing the
specific interactions assigned to the various 1H-NMR signals.
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Modelling the speciation of Tritopic UPy 1 as a function of NaPy concentration. The
thermodynamic binding model used to fit the NaPy titration data includes all possible
monomeric and dimeric species of tritopic UPy 1, cyclized and NaPy bound species (for details
see ESI). The model input parameters are binding constants for A-A, A-B, and B-B type UPy
contacts (KAA, KAB, and KBB, respectively), binding constants for both types of UPy-NaPy contacts
(KAN and KBN), and the effective molarities of both small A-B and large B-B monomeric cycles
(EMSC and EMLC, respectively). Parameter bounds were applied to avoid the outcome of
unrealistic parameter values. Given the simple aliphatic chain connecting the B-type UPys, we
assumed the binding constant of linear B-B and B-N contacts to be similar to literature values
(6*107 M-1 and 5*106 M-1, respectively)38,34 and allowed a relatively small deviation of 5 %. Since
it is unlikely that the other type of UPy and NaPy contacts are significantly stronger, an upper
limit of 108 M-1 was introduced for these contacts. Furthermore, KAA and KAB were given lower
limits of 105 M-1 and KAN a lower limit of 103 M-1 (in line with reference experiments, see ESI for
further details). Lastly, the effective molarities were constrained to the values that were
obtained from the ditopic reference compounds SCref 2 and LCref 3, allowing for a 10 % deviation
due to potential experimental error.
A large number of non-linear least square optimizations with different initial values were
performed to ensure that the global minimum was obtained (Figure 7A). Since the model
parameters are highly correlated this results in a collection of parameter values that give
equally good fits (Figure 7B). Nonetheless, using this collection of optimized parameter values
to calculate the molecular speciation plots resulted in almost identical species distributions
(Figure 7C and D). Thus, while the parameter values cannot be determined exactly, the species
distribution can. The measured speciation of tritopic UPy 1 in the absence of NaPy 6 is quite
similar to the one predicted, assuming equal binding strengths (Figure 5E and 7C). However, as
a result of the differences in UPy-UPy binding strengths, i.e., B-B > A-B > A-A, the fraction of
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dimerized A-B cycles is slightly higher than expected, at the expense of the B-B cycles. Upon the
addition of NaPy 6 the relatively weak linear UPy-UPy contacts connecting the dimerized cycles
are disrupted first, resulting in monomeric cycles with the 3rd UPy bound to NaPy. At higher
equivalents of NaPy the cycles open up, resulting in a tritopic UPy with NaPy bound to all UPys.
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Figure 7. A Normalized peak intensities of H-NMR spectra of signals observed for proton III during
titration of NaPy 6 to tritopic UPy 1 (symbols) and the best fit based on the thermodynamic model
(lines). B Fit parameter values of all non-linear least square optimizations that have a squared 2-norm
residual within 5% of the optimal parameter set. C Calculated distribution of cyclized tritopic UPy dimers
based on the parameter values of the best fits. D Average of the calculated speciation during NaPy
titration, based on the parameter values of the best fits. Note that the 95% confidence interval is smaller
than the linewidth of the plot, thus it was omitted.
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Interestingly, signal “a” first increases and subsequently decreases during the NaPy
titration, implying that the fraction of small A-B cycles is increased upon addition of small
amounts of NaPy. Using the fitted species distribution, the fractions of small and large cycles
during the titration were calculated, confirming this observation (Figure 8A). The fraction of
small cycles increases from 0.80 to 0.86, which is attributed to KBN being approximately one
order of magnitude higher than KAN, along with the fact that there are two B-type UPys to every
A-type UPy. Both of these effects increase the likelihood of NaPy binding to a B-type UPy, which
subsequently stabilizes the small A-B cycle with respect to the larger B-B cycle. Thus, at low
equivalents, NaPy 6 acts as a promoter for the formation of the small A-B cycle in tritopic UPy 1.
Having demonstrated the amplifying effect of NaPy 6 on the cycle ratio, we investigated
the extent to which NaPy can influence the cycle distribution. Interestingly, decreasing the
value of KAN in the simulation even further allows the exclusive formation of small A-B cycles,
while increasing KAN allows the exclusive formation of large B-B cycles (Figure 8B). In this
manner, the concentration and selectivity of NaPy can be used to regulate the fraction of each
type of cycle, without altering the molecular structure of tritopic UPy 1.
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Figure 8. A Calculated fractions of small and large cycles during NaPy titration, based on the model
parameters of the best fits. The 95% confidence interval is smaller than the linewidth of the plot, thus it
was omitted. B Average simulated fractions of small and large cycles during NaPy titration using various
values of KAN and the model parameters of the best fits. The exact values of KAN are shown next to the
color bar. The error bars denote the 95% confidence interval, calculated as two times the standard
deviation.
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CONCLUSIONS
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We report on the self-assembly behavior of a C2v-symmetrical tritopic UPy building block, with
special emphasis on the competition between two modes of intramolecular cycle formation
and how this equilibrium is influenced by ligand binding. We have shown that with the correct
reference compounds and molecular design it is possible to quantify the complex intra- and
intermolecular interactions formed by this molecule. The resulting speciation stems from
differences in cycle stability, symmetry factors and small differences in binding strength, as
corroborated by detailed computational modeling. By varying the concentration and selectivity
of the ligand, the fraction of each type of intramolecular cycle can be precisely controlled. We
show that using this approach it is possible to exclusively form either type of cycle, without
changing the molecular structure of the tritopic UPy. Our study also highlights the difficulties
associated with characterizing multitopic systems at the molecular level and shows that even a
relatively simple multitopic building block requires a combined experimental and
computational approach. We foresee that the work presented in this paper is an important
contribution to the further understanding and development of multivalent systems with
unexpected emerging properties.
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ASSOCIATED CONTENT
Supporting Information
Detailed experimental procedures, complete characterization of compounds and
supramolecular interactions, as well as further information on the computational model, is
available free of charge via the internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Authors
* e.w.meijer@tue.nl
* a.palmans@tue.nl
Notes
The authors declare no competing financial interest
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ACKNOWLEDGEMENTS
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We like to thank Dr. Anja Palmans for useful discussions about synthesis. This work is financed
by the Dutch Organization for Scientific Research (NWOTOP and ECHO-STIP) grant: No.
10007851 and No. 717.013.001), the Dutch Ministry of Education, Culture and Science (Gravity
program 024.001.035), and the European Research Council (FP7/2007-2013 and H2020/2014-
2020, ERC Advanced Grant No. 246829 and ERC Starting Grant No. 677313).
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