Relationship between conformational flexibility and chelate cooperativity

Article abstract of DOI:10.1021/jo2000397

A family of four biscarbamates (AA) and four bisphenols (DD) were synthesized, and H-bonding interactions between all AA?DD combinations were characterized using ¹H NMR titrations in carbon tetrachloride. A chemical double mutant cycle analysis

Full text of DOI:10.1021/jo2000397

ARTICLE
pubs.acs.org/joc
Relationship Between Conformational Flexibility and Chelate
Cooperativity
M. Cristina Misuraca,† Tudor Grecu,† Zoraida Freixa,‡ Valentina Garavini,† Christopher A. Hunter,*,†
Piet W.N.M. van Leeuwen,‡ M. Dolores Segarra-Maset,‡ and Simon M. Turega†
†Department of Chemistry, University of Sheffield, Sheffield S3 7HF, United Kingdom
‡Institute of Chemical Research of Catalonia (ICIQ), Av. Països Catalans 16, 43007 Tarragona, Spain
S Supporting Information
b
ABSTRACT: A family of four biscarbamates (AA) and four
bisphenols (DD) were synthesized, and H-bonding interactions
between all AA•DD combinations were characterized using 1H
NMR titrations in carbon tetrachloride. A chemical double
mutant cycle analysis shows that there are no secondary
electrostatic interactions or allosteric cooperativity in these
systems, and the system therefore provides an ideal platform
for investigating the relationship between chemical structure
and chelate cooperativity. Effective molarities (EMs) were
measured for 12 different systems, where the number of rotors in the chains connecting the two H-bond sites was varied from 5
to 20. The association constants vary by less than an order of magnitude for all 12 complexes, and the variation in EM is remarkably
small (0.1ꢀ0.9 M). The results provide a relationship between EM and the number of rotors in the connecting chains (r): EM ≈
10rꢀ3/2. The value of 10 M is the upper limit for the value of EM for a noncovalent intramolecular interaction. Introduction of rotors
reduces the value of EM from this maximum in accord with a random walk analysis of the encounter probability of the chain ends
(rꢀ3/2). Noncovalent EMs never reach the very high values observed for covalent processes, which places limitations on the
magnitudes of the effects that one is likely to achieve through the use of chelate cooperativity in supramolecular assembly and
catalysis. On the other hand, the decrease in EM due to the introduction of conformational flexibility is less dramatic than one might
expect based on the behavior of covalent systems, which limits the losses in binding affinity caused by poor preorganization of the
interaction sites.
’ INTRODUCTION
There are good methods available for estimating the proper-
ties of a single point H-bond interaction,23,24 but when there are
multiple intermolecular contacts, communication between the
coupled binding sites makes prediction problematic.25ꢀ31 The
parameter which is used to quantify the chelate cooperativity
associated with the formation of an intramolecular interaction is
the product KEM, where K is the association constant for the
corresponding intermolecular binding interaction under the
same conditions, and EM is the effective molarity.8 When
KEM . 1, the intramolecular process is strongly favored, and
efficient assembly of the complex will take place. For relatively
rigid complexes with good geometric complementarity, where
there are no complications due to changes in conformational
flexibility or conformational strain, the values of EM are on the
order 10 M.24,32 For synthetic supramolecular systems that have
been designed using high affinity binding sites and highly
preorganized scaffolds, KEM is usually much greater than 1,
and intramolecular interactions are therefore formed quantita-
tively. Under these conditions, the free energy of binding is an
Cooperativity is a central concept in supramolecular science.
Systems that feature multiple noncovalent interactions show sig-
nificant increases in thermodynamic and kinetic stability compared
with the corresponding single point interactions.1ꢀ6 Although two
different forms of cooperativity have been identified, allosteric and
chelate,7,8 it is chelate cooperativity (or multivalency) that is usually
responsible for the all-or-nothing behavior that characterizes
biological processes like folding and self-assembly.9ꢀ12 Chelate
cooperativity is straightforward to implement in synthetic supra-
molecular design, and the multivalent approach to noncovalent
chemistry is therefore beginning to find applications in
nanotechnology.13ꢀ16 However, the quantitative relationship be-
tween chemical structure, supramolecular architecture, and non-
covalent cooperativity remains largely unexplored.17ꢀ21 Although
cooperative phenomena are widespread in biology, the complexity
ofthese systemsmakesitdifficult to dissect individual contributions
to the overall behavior of the system. Synthetic supramolecular
complexes, where structure and complexity can be controlled
through synthesis, provide the ideal model systems for establishing
quantitative structureꢀactivity relationships,22 and here we use this
approach to investigate chelate cooperativity.8
Received: January 13, 2011
Published: March 18, 2011
r
2011 American Chemical Society
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ARTICLE
additive function of the individual intermolecular contacts.32ꢀ34
When KEM ≈ 1, there are mixtures of partially bound states, as
well as the fully assembled complex, and the behavior of the
system may be strongly dependent on small changes in condi-
tions. This regime is common in biology but has been less widely
investigated in synthetic model systems. When KEM , 1, there
are no intramolecular interactions and no assembly of the desired
complex.
The relationship between chemical structure and EM has been
thoroughly explored for both the kinetics and the thermody-
namics of covalent cyclization reactions: EM decreases with an
increase in the conformational flexibility of the chain linking the
reactive end groups and the degree of conformational strain
introduced on cyclization.35,36 The thermodynamic conse-
quences of freezing a single rotor at room temperature have
been estimated at 5.6 kJ molꢀ1 for formation of a covalent bond,
and this is borne out by the experimental data for cyclization of
small rings.37 However, as the ring size increases, the incremental
effect of introducing additional rotors on EM decreases, due to
the increased conformational mobility of the cyclic product.35 In
the limit of very large rings and very flexible molecules
(polymers), the probability that an intramolecular reaction will
Figure 1. AA•DD complexes used to quantify the relationship between
the chemical structures of the linkers, L1 and L2, and chelate coopera-
tivity in formation of the intramolecular H-bond. The proton labeling
scheme is shown (R = 2-ethylhexyl).
take place between the two chain ends is found to vary as Nꢀ3/2
,
where N is the number of bonds in the chain.38 For noncovalent
interactions, there is less experimental data available. Estimates of
the thermodynamic cost of freezing a rotor by formation of a
noncovalent interaction range from 0.5 to 5 kJ molꢀ1 39ꢀ44
.
Whitesides studied the intramolecular binding of a protein to a
ligand, where the two binding sites were connected via a flexible
linker, and found a rather weak dependence of EM on linker
length.21 Reinhoudt obtained a similar result using theoretical
methods to estimate EM.17 Thus, it appears that noncovalent
EMs are insensitive to conformational flexibility. In this paper,
we quantify the effect of conformational flexibility on chelate
cooperativity by measuring noncovalent EMs using a synthetic
supramolecular model system.
Figure 2. Chemical double mutant cycle used to assess the chelate
cooperativity associated with the intramolecular H-bond in the doubly
H-bonded AA•DD complex, state A.
electrostatic interactions and allosteric cooperativity, which
usually complicates the analysis of multiply H-bonded
complexes.46 By synthesizing families of AA and DD com-
pounds, it is possible to measure the stabilities of all pairwise
AA•DD combinations, and hence quantify the relationship
between chelate cooperativity and the nature of the linker. In
this paper, we vary both the geometric complementarity and
conformational flexibility of the linkers L1 and L2 in Figure 1
and quantify the effects on the effective molarity (EM) for the
intramolecular H-bond interaction.
The free energy contribution of the intramolecular H-bond to
the stability of the AA•DD complex can be evaluated using a
chemical double mutant cycle (Figure 2).22,31 This requires the
measurement of four different association constants, using the
monofunctional reference compounds (A and D) and the
bifunctional compounds (AA and DD). The double mutant
cycle allows the quantification of any substituent effect, due to the
presence of the linkers in the bifunctional compounds, as well as
any allosteric cooperativity, due to complexation-induced
changes in the intrinsic properties of the binding sites. Thus,
the magnitude of the chelate cooperativity can be dissected from
the other factors that contribute to the overall stability of the
AA•DD complex. Equation 1 shows how the EM associated with
the intramolecularity of the second H-bond is determined from
the double mutant cycle experiment (the statistical factor of 2
accounts for the degeneracy of the symmetric AA•DD complex).
’ APPROACH
We have shown previously that the phenolꢀcarbamate
interaction is ideally suited to construction of simple model
systems for the quantification of cooperativity in H-bonded
complexes.31 Phenol is a poor H-bond acceptor (β = 2.7) and
one of the strongest H-bond donor functional groups (R = 3.8).
Carbamate is a poor H-bond donor (R = 2.4) and a good
H-bond acceptor (β = 7.3).24 Thus, the phenolꢀphenol and the
carbamateꢀcarbamate interaction are both too weak to occur
to any significant extent even in very nonpolar solvents, and the
single point phenolꢀcarbamate H-bonding interaction is suffi-
ciently strong to allow accurate measurement of the 1:1
association constant in nonpolar solvents like carbon tetra-
chloride (Kref). This provides a measurable noncooperative
reference point that can be compared with cooperative inter-
actions in systems that feature more than one H-bond. Here, we
will examine the properties of complexes that make two
phenolꢀcarbamate H-bonds (Figure 1). By choosing the
AA•DD motif, i.e., the 1:1 complex formed between a bi-
sphenol and a biscarbamate, it is possible to avoid the potential
problems of intramolecular folding and aggregation that are
common in multiply H-bonded motifs.19,45 The use of relatively
long linkers will ensure that the H-bonding sites in these
systems are sufficiently far apart to minimize secondary
KAKD
KBKC
KðAA•DDÞK2ðA•DÞ
2EM ¼
¼
ð1Þ
KðAA•D2ÞKðA2•DDÞ
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1H NMR Titration Experiments. Complexation between the
phenols and carbamates was investigated by H NMR titration
’ RESULTS
1
Synthesis. We prepared a set of four bisphenols and four
biscarbamates connected by linkers of varying length and flexibility
(Scheme 1). Bisphenol 3a is commercially available. Bisphenols
3bꢀd were prepared from dihaloalkanes and the monoprotected
bisphenol 1b, followed by removal of the benzyl group by hydro-
genation. The bisphenols were converted to the corresponding
biscarbamates 4aꢀd in one step. The monofunctional phenols 1a
and 1b are commercially available. These were converted to the
corresponding carbamates 2a and 2b to provide four reference
compounds for studying singly H-bonded complexes (Scheme 1).
1H NMR Dilution Experiments. Self-association of the com-
pounds was investigated using 1H NMR dilution experiments in
carbon tetrachloride. The phenols are significantly less soluble
than the carbamates, which are equipped with solubilizing
groups. The solubility of phenol 3d was too low even for use
as a host in titration experiments, and will not be discussed
further. No changes in chemical shift were observed for the
phenols (0.1ꢀ0.6 mM) or for the carbamates (40ꢀ400 mM) in
dilution experiments. Thus, self-association does not complicate
experiments in carbon tetrachloride. The titration data for the
reference A•D complexes all fit well to a 1:1 binding isotherm.
The association constants and limiting complexation-induced
changes in chemical shift (Δδ) are summarized in Table 2. The
titration data for the reference complexes A2•DD and AA•D2 fit
well to 2:1 and 1:2 binding isotherms with identical stepwise
microscopic association constants (Table 2). The phenol Δδ
1
Table 1. H NMR Chemical Shifts (ppm) Measured in CCl4
at 298 Ka
H1
H2
H3
H4
H5
1a
1b
3a
3b
3c
2a
2b
4a
4b
4c
4d
7.0
6.8
7.0
6.9
6.8
6.7
6.7
6.7
6.8
6.8
4.4
4.1
4.3
4.2
4.1
1
the titration experiments. Table 1 lists the H NMR chemical
7.1
7.0
7.2
7.1
7.0
6.9
7.0
6.9
7.0
7.0
6.9
6.8
shifts of the phenols (1 and 3) and the carbamates (2 and 4) in
carbon tetrachloride solution. The bifunctional compounds (3
and 4) have similar chemical shifts to the corresponding mono-
functional reference compounds (1 and 2, respectively), which
implies that there are no intramolecular H-bonds or aromatic
interactions in the free state; i.e., the molecules adopt extended
rather than folded conformations.
a See Figure 1 for the proton labeling scheme.
Scheme 1
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ARTICLE
Table 2. Association Constants and Limiting Complexation-
Induced Changes in Chemical Shift (ppm) for Formation of
1:1 and 2:1 Reference Complexes Determined from 1H NMR
a
Titrations at 298 K in CCl4
microscopic
Δδ (ppm)
macroscopic
complex K2 (Mꢀ1) K1 (Mꢀ1
)
Kobs
H1 H2 H3
D•A
D•A
D•A
D•A
1a•2a
1a•2b
1b•2a
1b•2b
17
17
19
21
20
17 Mꢀ1
17 Mꢀ1
19 Mꢀ1
21 Mꢀ1
39 Mꢀ1
ꢀ0.2 ꢀ0.3 2.6
ꢀ0.2 ꢀ0.3 2.6
ꢀ0.2 ꢀ0.3 2.7
ꢀ0.2 ꢀ0.3 2.6
ꢀ0.2 ꢀ0.3 2.7
D•AA 1b•4b
D2•AA (1b)2•4b
DD•A 3b•2b
DD•A2 3b•(2b)2
20
20
380 Mꢀ2 ꢀ0.2 ꢀ0.3 2.7
20
41 Mꢀ1
ꢀ0.1 ꢀ0.2 1.0b
420 Mꢀ2 ꢀ0.2 ꢀ0.4 2.1
a See Figure 1 for the proton labeling scheme. b Only one of the two OH
groups is H-bonded in this 1:1 complex, and so this value is the average
of a free and a bound OH chemical shift, i.e., 1/2(0.0 þ 2.0) ppm. The
same is true for the aromatic protons, H1 and H2.
Figure 3. Various complexes are possible for the interaction of a
bifunctional host (DD) with a bifunctional guest (AA). Under the
conditions used, [DD] , [AA] and [DD] , 1/Kref, only the species in
the box are significantly populated. Kref is the 1:1 association constant for
a complex featuring a single point phenolꢀcarbamate H-bond. EM is the
effective molarity for the intramolecular interaction. The statistical
factors of 4 and 1/2 account for the degeneracies of the complexes.
values are similar in all cases: small negative changes are observed
for the CH protons, and large positive changes (þ2ꢀ3 ppm) are
observed for the OH protons. These changes in chemical shift are
classic signatures of H-bonding interactions with the OH groups,
and the fact that the values are similar for the A2•DD and AA•D2
complexes indicates that both H-bonds are made in these com-
plexes. The microscopic association constants for all of the
complexes involving the monofunctional reference compounds
are very similar (Kref = 17ꢀ21 Mꢀ1). This result shows that the
linkers have no significant effect on the intrinsic binding properties
of the recognition units; i.e., there are no substituent effects on the
H-bond properties of the phenol or carbamate. In addition, the
observation of identical microscopic stepwise association con-
stants for the A2•DD and AA•D2 complexes implies that allosteric
cooperativity is negligible. Thus, the data in Table 2 imply that Kref
is constant for these systems, i.e. KB = KC = KD = Kref2, which
greatly simplifies the double mutant cycle analysis in Figure 2.
Under these conditions, K(AA•DD) = 2Kref2EM.
The AA•DD titrations are potentially more complicated,
because a number of different complexes are possible
(Figure 3).8 The titrations were carried out under conditions
where the guest (AA) was always present in a large excess, and
the concentration of host (DD) was low ([DD] , 1/Kref). This
ensures that open oligomeric complexes, o-(AA)N•(DD)N, are
not significantly populated, and higher order cyclic oligomers,
c-(AA)N•(DD)N, are unlikely to be populated. Thus, three
different complexes should be considered in the analysis of the
titration data: the singly H-bonded 1:1 complex o-AA•DD, the
doubly H-bonded 1:1 complex c-AA•DD, and the 2:1 complex
(AA)2•DD. The key parameter that determines speciation in this
system is 1/2KrefEM, the dimensionless equilibrium constant
between the open and closed 1:1 complexes. If 1/2KrefEM is
sufficiently large, then closed complex c-AA•DD will predomi-
nate, and a simple 1:1 binding isotherm will result.
Table 3. Association Constants and Limiting Complexation-
Induced Changes in Chemical Shifts (ppm) Obtained by
Fitting the Data from 1H NMR Titrations in CCl4 at 298 K to a
1:1 Binding Isotherma
Δδ (ppm)
complex
K1 (Mꢀ1
)
H1
H2
H3
3a•4a
3a•4b
3a•4c
3a•4d
3b•4a
3b•4b
3b•4c
3b•4d
3c•4a
3c•4b
3c•4c
3c•4d
200 ( 60
260 ( 130
290 ( 70
140 ( 30
320 ( 30
890 ( 260
630 ( 350
210 ( 10
280 ( 50
560 ( 80
280 ( 110
170 ( 10
ꢀ0.2
ꢀ0.2
ꢀ0.2
ꢀ0.2
ꢀ0.2
ꢀ0.2
ꢀ0.2
ꢀ0.2
ꢀ0.2
ꢀ0.3
ꢀ0.2
ꢀ0.2
ꢀ0.3
ꢀ0.4
ꢀ0.4
ꢀ0.3
ꢀ0.4
ꢀ0.5
ꢀ0.4
ꢀ0.3
ꢀ0.4
ꢀ0.5
ꢀ0.4
ꢀ0.4
2.2
2.2
2.3
2.3
2.3
2.4
2.3
2.3
3.1
2.3
2.3
2.3
a See Figure 1 for proton labeling scheme. All experiments were repeated
at least twice, and the average values are reported with errors at the 95%
confidence limit.
of the doubly H-bonded 1:1 complex c-AA•DD in all cases. The
limiting complexation-induced changes in chemical shifts (Δδ)
provide further evidence for the structures of the complexes
(Table 3). The signals due to the bisphenol OH protons (H3)
show large positive changes in chemical shift (þ2ꢀ3 ppm) that
are characteristic of H-bonding interactions, and comparison
with the corresponding Δδ values for the reference complexes
suggests that both OH groups are fully H-bonded in the 1:1
The titration data for the AA•DD complexes fit well to a
simple 1:1 binding isotherm in all cases, and the apparent
association constant, K1, is an order of magnitude larger than
the values of Kref in Table 2. This suggests cooperative formation
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Table 4. Association Constants and Complexation-Induced
Changes in Chemical Shifts (ppm) Obtained by Fitting the
Data from 1H NMR Titrations in CCl4 at 298 K to a 2:1
Binding Isotherma
Δδ1 (ppm)
H2 H3 H1
Δδ2 (ppm)
complex K1 (Mꢀ1
)
K2 (Mꢀ2
)
H1
H2 H3
3a•4a
3a•4b 260 ( 80
3a•4c 310 ( 30
230 ( 70
1200
1200
1200
1200
1400
1700
1700
1700
1400
1700
1700
1700
ꢀ0.2 ꢀ0.3 2.1 ꢀ0.2 ꢀ0.3 2.6
ꢀ0.1 ꢀ0.4 2.0 ꢀ0.3 ꢀ0.2 3.0
ꢀ0.2 ꢀ0.4 2.2 ꢀ0.3 ꢀ0.3 2.8
ꢀ0.2 ꢀ0.3 2.0 ꢀ0.2 ꢀ0.3 2.6
ꢀ0.2 ꢀ0.4 2.1 ꢀ0.2 ꢀ0.3 3.2
3a•4d 160 ( 10
3b•4a 410 ( 40
3b•4b 910 ( 310
3b•4c 650 ( 340
3b•4d 240 ( 20
ꢀ0.2 ꢀ0.5 2.4
0.0 ꢀ0.2 2.7
Figure 4. Association constant, K1, obtained by fitting the titration data
to a 1:1 binding isotherm compared with the value of K1 obtained by
fitting the data to a 2:1 binding isotherm.
ꢀ0.2 ꢀ0.4 2.3 ꢀ0.1 ꢀ0.2 2.6
ꢀ0.2 ꢀ0.3 2.1 ꢀ0.2 ꢀ0.3 2.8
ꢀ0.2 ꢀ0.4 2.7 ꢀ0.2 ꢀ0.3 3.5
3c•4a
3c•4b 540 ( 50
3c•4c 290 ( 90
3c•4d 200 ( 10
310 ( 90
ꢀ0.3 ꢀ0.5 2.3
0.1 ꢀ0.2 2.3
ꢀ0.2 ꢀ0.4 2.2 ꢀ0.1 ꢀ0.2 2.4
ꢀ0.4 ꢀ0.2 2.1 ꢀ0.3 ꢀ0.2 2.5
by as much as a factor of 10 in some titrations, indicating a
significantly better fit to the 2:1 isotherm. Analysis of the
speciation indicates that the population of the 2:1 complex does
not exceed 30% in any of the titrations, and this has a small but
detectable impact on the shape of the binding isotherm. Table 4
gives the association constants K1 and the limiting complexation-
induced changes in chemical shift obtained by fitting the titration
data to a 2:1 binding isotherm. The values of K1 reported in
Table 4 are similar to those listed in Table 3 for the simple 1:1
isotherm (Figure 4).
The values of K1 from the 2:1 binding isotherm were used in
eq 6 to determine the values of EM and hence estimate the
populations of the open and closed 1:1 complexes, o-AA•DD and
c-AA•DD (eqs 7and8). The results are listed in Table 5.
a See Figure 1 for proton labeling scheme. K1 and K2 are the macroscopic
association constants. The value of K2 was fixed at 4Kref2 using the values
in Table 2. Δδ1 and Δδ2 are the limiting complexation-induced changes
in chemical shift for the 1:1 and the 2:1 complexes, respectively.
complexes. The values of Δδ for the phenol CH protons (H1 and
H2) are also very similar to those observed for the reference
complexes, indicating that the recognition units are in similar
environments in all cases.
Although there is no clear evidence for the presence of the o-
AA•DD or (AA)2•DD complexes in the titration data presented
above, the speciation of these additional complexes can be
estimated based on the value of Kref.
2
EM ¼ ðK1 ꢀ 4Kref Þ=2Kref
ð6Þ
ð7Þ
population of o-AA•DD ¼ 4Kref =K1
Kðo-AA•DDÞ ¼ 4Kref
ð2Þ
population of c-AA•DD ¼ ðK1 ꢀ 4Kref Þ=K1
ð8Þ
2
K½ðAAÞ2•DDꢁ ¼ 4Kref
ð3Þ
In all cases, the value of 1/2KrefEM is greater than 1 (Table 5),
which means that the closed complex, c-AA•DD, is more stable
than the open partially bound 1:1 complex, o-AA•DD. However,
the population of the partially bound open complex is significant.
In the complex with the highest EM (3b•4b), 9% of the 1:1
complex is in the partially bound open form, but when the linker
groups are long and flexible (e.g., 3c•4d), the open partially
bound intermediate constitutes 42% of the 1:1 stoichiometry
species. The most striking feature of the results in Table 5 is that
the value of EM does not depend very strongly on the nature of
the linkers. Table 5 includes an estimate of the total number of
rotors connecting the two H-bonding sites (r), and although this
varies from 5 to 20, the range of EM values is rather narrow
(0.1ꢀ0.9 M).
Using these relationships, it is possible to refit the titration data
to a 2:1 binding isotherm that allows for the presence of all three
complexes.
K1 ¼ Kðc-AA•DDÞ þ Kðo-AA•DDÞ
¼ 2Kref 2EM þ 4Kref
ð4Þ
ð5Þ
2
K2 ¼ K½ðAAÞ2•DDꢁ ¼ 4Kref
When the data were reanalyzed using a 2:1 isotherm, the
quality of the fit to the experimental data improved in every case.
It is important to note that the value of K1 was fitted as a variable
in this analysis, but the value of K2 was not: K2 was fixed using
eq 5 and the value of Kref from Table 2. However, the limiting
changes in chemical shift for the 1:1 and 2:1 species, Δδ1 and
Δδ2, were fit as independent variables, because the value of Δδ1
depends on the relative populations of the open and closed 1:1
complexes. Thus, fitting to the 2:1 isotherm requires an extra
variable compared with fitting to a 1:1 isotherm, and the quality
of the fit to the experimental data is expected to improve
automatically. Nevertheless, the root-mean-square difference
between the calculated and experimental chemical shifts changed
’ DISCUSSION
The results in Table 5 allow us to explore the relationship
between the structure of the linker groups, L1 and L2, and the
effective molarity, EM, which quantifies the chelate cooperativity
associated with formation of intramolecular H-bonds. Based on
studies of EM values for the rates of covalent cyclization
reactions, the two key parameters that are expected to affect
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Table 5. Effective Molarities (M) Associated with Formation
of the Intramolecular H-Bond in c-AA•DD Complexes at 298
reality, it is the difference in conformational mobility between
the free and bound states that we are interested in, but the crudest
method for calibrating the system is simply to count rotors in the
linkers.47 Some assumptions must be applied in counting what is
and is not a rotor, and the approach we have adopted is illustrated
in Figure 5: the carbamate and the phenol OH groups are
considered to have fixed conformations in conjugation with the
aromatic rings; rotation around the arylꢀoxygen bonds is
equivalent to rotation around the arylꢀcarbon bonds, because
spinning of the intermediate phenylene units does not affect the
geometry of the complex; and the first intermolecular H-bond is
considered to be a rotor (labeled 3 in Figure 5). For the 3a•4a
complex, where both L1 and L2 are CH2, there are only five
rotors, as indicated in Figure 5. Increasing the length of the
linkers increases the number of rotors (r) up to 20 as detailed in
Table 5. This definition of r implies that the minimum value is r =
1, which is the rotor associated with the formation of the first
intermolecular H-bond, labeled 3 in Figure 5.
Figure 6 shows the relationship between the number of rotors
and EM for the complexes described in Table 5 (black dots). A
clear trend is difficult to identify, but the results are more
interesting when compared with data for other noncovalent
cyclization processes. Figure 6 includes EM values for more rigid
H-bonded complexes that we have reported previously, where r =
1 (Figure 7a,b, open circles in Figure 6),31 and data from the
folding of sarcosine oligomers, where r is very large (r = 12ꢀ50)
(Figure 7c, gray dots in Figure 6).48 As might be expected, the
more rigid complexes have higher EM values, and the very
flexible systems have lower EMs. However, these results are
particularly interesting when compared with the corresponding
EM data for covalent cyclization reactions. Ring strain compli-
cates the analysis of covalent cyclization processes, but Mando-
lini has collated literature data to estimate the EM for the rate of a
strain-free covalent cyclization reaction as a function of the
number of rotors in the linker.35 This relationship is plotted as
the gray line in Figure 6. For large numbers of rotors, the
behavior of the covalent and noncovalent systems is practically
identical. In this region, the relationship between log EM and log
r is linear with a slope of ꢀ1.55. This is very close to the
theoretical value of ꢀ1.5 that is expected for random walk
encounters between end groups on a flexible chain.38 However,
as the number of rotors decreases, the very large EM values that
are found for covalent cyclization of small rings are not observed
for the noncovalent systems. Instead, the noncovalent EM values
continue along the random walk line right up to the intersection
with the log EM axis at EM ≈ 10 M. Thus, it appears that there is
a relatively simple relationship between EM and the number of
rotors (eq 9). This is consistent with the observations on
ringꢀchain equilibria in supramolecular polymerization
processes.49
a
K in CCl4
population (%)
complex
rotors (r)
EM (M)
1/2KEM
c-AA•DD
o-AA•DD
3a•4a
3a•4b
3a•4c
3a•4d
3b•4a
3b•4b
3b•4c
3b•4d
3c•4a
3c•4b
3c•4c
3c•4d
5
7
0.3
0.3
0.4
0.2
0.5
0.9
0.6
0.2
0.3
0.5
0.2
0.1
2.4
2.8
3.6
1.4
4.4
9.8
6.7
1.9
3.1
5.4
2.5
1.4
70
74
78
57
81
91
87
65
75
84
71
58
30
26
22
43
19
9
9
16
7
9
11
18
9
13
35
25
16
29
42
11
13
20
a The average error in EM is (50%.
Figure 5. Approach used to count the number of rotors (r) that are
conformationally restricted on formation of an intramolecular H-bond.
When L1 and L2 are rigid, i.e., CH2, there are five independent rotors
which are labeled 1ꢀ5.
EM ꢂ 10rꢀ3=2
ð9Þ
EM are ring strain and conformational flexibility.35,36 In the
complexes described here, the inherent flexibility of the linker
groups together with the relatively weak geometric constraints
imposed by H-bonding should allow the formation of c-AA•DD
complexes that are relatively strain-free, so we will focus on the
conformational flexibility of the linker chains. Formation of an
intramolecular H-bond leads to cyclization of the complex and
restriction in the conformational mobility of the linkers. In
For systems of this type, where there is one rotational degree
of freedom associated with formation of the first intermolecular
H-bond (Figure 5), the maximum value of EM is on the order of
10 M, the intercept of 1.0 on the log EM axis in Figure 6. The
value of EM is expected to be somewhat higher for perfectly rigid
systems where there is no conformational mobility, and the Page
and Jencks upper limit of 100 M is consistent with the data in
Figure 6.37
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ARTICLE
Theory suggests that 100 M might represent an upper limit for
the value of EM for a noncovalent intramolecular interaction.37
The analysis presented here supports this idea, and indicates that
introduction of rotors reduces the value of EM from this
maximum in accord with a random walk analysis of the encounter
probability of the chain ends. There are two important implica-
tions. Noncovalent EMs never reach the very high values
observed for covalent processes, which places limitations on
the magnitudes of the effects that one is likely to achieve through
the use of chelate cooperativity in supramolecular assembly and
catalysis. Second, the decrease in EM due to the introduction of
conformational flexibility is less dramatic than one might expect
on the basis of the behavior of covalent systems, which implies
that the losses in binding affinity caused by poor preorganization
are limited. Figure 6 shows that the EM falls by 2 orders of
magnitude when the number of rotors is increased from 1 to 20.
This implies that floppy design rather than preorganization could
prove a fruitful strategy for supramolecular chemists. The EM
does not fall below 0.1 M for any of the systems described here,
and this means that the key chelate cooperativity parameter KEM
will always be greater than 1, provided relatively high affinity
interactions are used (Kref > 10 Mꢀ1). Under these conditions,
there will always be a thermodynamic benefit from chelate
cooperativity, even for loosely linked multivalent systems.12
Figure 6. Relationship between the effective molarity (EM/M) at 298
K for covalent (gray line) and noncovalent (data points) cyclization
processes and the number of rotors in the linking chain (r). The
noncovalent EM values come from this work (black dots), related but
more rigid H-bonded complexes (open circles) and folding of sarcosine
oligomers (gray dots). The best fit straight line for the noncovalent data
(black line) corresponds to log EM = 1.08 ꢀ 1.55 log r (r2 = 0.91).
’ EXPERIMENTAL SECTION
Synthesis Compound 2a. A mixture of 1a (0.579 g, 5.35 mmol),
4-nitrophenyl chloroformate (1.10 g, 5.35 mmol), and triethylamine
(0.75 mL, 5.35 mmol) in dichloromethane (50 mL) was stirred under
nitrogen atmosphere at room temperature for 4 days. Bis(2-ethylhex-
yl)amine (1.61 mL, 5.35 mmol) and triethylamine (0.75 mL, 5.35
mmol) were added, and the solution was stirred under nitrogen atmo-
sphere at room temperature for an additional 5 days. The solution was
diluted with dichloromethane (50 mL), washed with water (30 mL) and
brine (30 mL), dried with sodium sulfate, and condensed under reduced
pressure. The resulting oil was purified by column chromatography
eluting with a dichloromethane and hexane. The product was isolated as
a clear oil: yield 1.27 g (63%). 1H NMR (250 MHz, CDCl3) δH = 7.15
(d, 2H, J = 8), 6.98 (d, 2H, J = 8), 3.28 (m, 4H), 2.34 (s, 3H), 1.75 (m,
2H), 1.35 (m, 16H), 0.92 (t, 12H, J = 7). 13C NMR (62.8 MHz, CDCl3)
δC = 155.5, 149.4, 134.5, 129.7, 121.4, 51.1, 38.1, 37.4, 30.6, 28.7, 23.9,
23.1, 20.8, 14.1, 10.7. MS (EIþ) m/z (rel intens) = 268 (65), 375 (10)
[Mþ]. HRMS (EIþ) calcd for C24H41NO2 375.3134, found 375.3134.
FT-IR (thin film) νmax/cmꢀ1 2958, 2928, 2872, 1713, 1512, 1462, 1417,
1379, 1214, 1199.
Compound 2b. A mixture of 1b (0.5 g, 2.5 mmol), 4-nitrophenyl
chloroformate (0.5 g, 2.5 mmmol), and triethylamine (0.35 mL, 2.5
mmol) in dry dichloromethane (50 mL) was stirred under nitrogen
atmosphere at room temperature for 2 days. The solution was concen-
trated under reduced pressure. The crude product was dissolved in
dichloromethane (15 mL), and bis(2-ethylhexyl)amine (0.97 mL, 2.7
mmol) and triethylamine (0.37 mL, 2.7 mmol) were added to the
solution that was stirred overnight under nitrogen atmosphere at room
temperature. The resulting slurry was dissolved in dichloromethane
(50 mL), washed with hydrochloric acid 1 M (50 mL), sodium
hydroxide 1 M (50 mL), water (50 mL), and brine (50 mL), dried with
sodium sulfate, and then condensed under reduced pressure. The
obtained oil was purified on silica eluting with a dichloromethane and
hexane. The product was isolated as a clear oil: yield 0.3164 g (27%). 1H
NMR (250 MHz, CDCl3) δH = 7.39 (m, 5H), 6.98 (m, 4H), 5.06 (s,
2H), 3.27 (m, 4H), 1.75 (m, 2H), 1.34 (m, 16H), 0.92 (t, 12H, J = 6).
13C NMR (62.9 MHz, CDCl3) δC = 156.0, 155.6, 145.4, 128.6, 127.9,
Figure 7. Experimental noncovalent EM data in Figure 6 is plotted
(a, b) for H-bonded complexes with limited conformational mobility
(open circles in Figure 6), and (c) for the folding of flexible sarcosine
oligomers (gray dots in Figure 6).
’ CONCLUSIONS
We have developed a simple supramolecular model system for
investigating the relationship between covalent structure and
chelate cooperativity in the formation of intramolecular
H-bonds. A chemical double mutant cycle analysis was used to
confirm that the molecular design successfully avoids any com-
plications due to secondary electrostatic interactions or allosteric
cooperativity. However, there is a significant population partially
bound states in some of the systems studied, and quantitative
analysis of the titration experiments required consideration of
partially bound complexes that contain only one H-bond, as well
as fully bound 1:1 complexes that contain two H-bonds, and 2:1
complexes. Effective molarities (EMs) were measured for 12
different systems, where the number of rotors in the chains
connecting the two H-bond sites was varied from 5 to 20. The
association constants vary by less than an order of magnitude for
all 12 complexes, and the variation in EM is remarkably small
(0.1ꢀ0.9 M). The results were combined with data from the
literature to come up with a general relationship for estimating
the relationship between EM and the number of rotors in the
connecting chains (r): EM ≈ 10rꢀ3/2. The behavior is very
different from that observed for covalent processes, where there
is an increase in EM of several orders of magnitude for the
cyclization of small rings, and the maximum value is on the order
of 107 M.
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The Journal of Organic Chemistry
ARTICLE
127.4, 122.5, 115.4, 70.5, 51.1, 50.9, 38.1, 37.4, 30.6, 28.7, 23.8, 23.1,
14.1, 10.7. MS (ESþ) m/z (rel intens) = 468 (100) [M þ Hþ], 485
(40), 490 (10) [M þ Naþ]. HRMS (ESþ) calcd for C30H45NO3Na
490.3297, found 490.3274. FT-IR (thin film) νmax/cmꢀ1 2957, 2928,
2859, 1714, 1507, 1463, 1418, 1379, 1194, 1152, 1104, 1025, 863, 819,
734, 696, 522.
2932, 2917, 2853, 1608, 1509, 1451, 1473, 1395, 1369, 1298, 1229, 1169,
1105, 1048, 1022, 983, 823, 802, 768, 556.
Compound 4a. A mixture of 3a (0.620 g, 3.1 mmol), 4-nitrophenyl
chloroformate (1.245 g, 6.2 mmol), and triethylamine (0.86 mL, 6.2
mmol) in dichloromethane (50 mL) was stirred under nitrogen atmo-
sphere at room temperature for 48 h. Bis(2-ethylhexyl)amine (1.90 mL,
6.3 mmol) and triethylamine (0.86 mL, 6.2 mmol) were added, and the
solution was stirred overnight under nitrogen atmosphere at room
temperature. The solution was diluted with dichloromethane
(50 mL), washed with water (30 mL) and brine (30 mL), dried with
sodium sulfate, and condensed under reduced pressure. The resulting oil
was purified by column chromatography eluting with a mixture of ethyl
acetate and hexane. The product was isolated as a clear oil: yield 0.894 g
(76.8%). 1H NMR (250 MHz, CDCl3) δH = 7.16 (d, 4H, J = 8), 7.01 (d,
4H, J = 8), 3.95 (s, 2H), 3.28 (m, 8H), 1.74 (m, 4H), 1.33 (m, 32H), 0.91
(m, 24H). 13C NMR (249.9 MHz, CDCl3) δC = 155.4, 150.0, 137.7,
129.7, 121.6, 51.1, 50.9, 40.6, 38.1, 37.4, 30.6, 28.7, 23.9, 23.8, 23.1, 14.1,
10.7. MS (ESþ) m/z (rel intens) = 736 (70) [M þ Hþ], 758 (100) [M
þ Naþ]. HRMS (ESþ) calcd for C47H78N2O4Na 757.5859, found
757.5855. FT-IR (thin film) νmax/cmꢀ1 2957, 2927, 2859, 1717, 1508,
1464, 1417, 1379, 1198, 1165, 1053, 1018, 753, 508.
Compound 4b. A mixture of 3b (6 g, 25.8 mmol), 4-nitrophenyl
chloroformate (10.4 g, 51.6 mmol), and triethylamine (7.2 mL, 51.6
mmol) in dichloromethane (300 mL) was stirred under nitrogen
atmosphere at room temperature for 48 h. Bis(2-ethylhexyl)amine
(37.1 mL, 123.4 mmol) and triethylamine (14.4 mL, 103 mmol) were
added, and the solution was stirred overnight under nitrogen atmo-
sphere at room temperature. The solution was washed with 1 M
hydrochloric acid, 1 M sodium hydroxide, water, and brine, dried with
sodium sulfate, and condensed under reduced pressure. The resulting oil
was purified by column chromatography eluting with a mixture of
dichloromethane and hexane. The product was isolated as a clear oil:
yield 4.0 g (20%). 1H NMR (400 MHz, CDCl3) δH = 7.09 (d, 4H, J = 8),
7.03 (d, 4H, J = 8), 5.67 (s, 2H), 3.25 (m, 8H), 1.75 (m, 4H), 1.35 (m,
32H), 0.92 (m, 24H). 13C NMR (100.6 MHz, CDCl3) δC = 155.5,
154.1, 146.7, 122.6, 117.3, 92.1, 51.1, 50.9, 38.1, 37.4, 30.6, 28.7, 23.9,
23.8, 23.1, 14.1, 10.7. MS (ESþ) m/z (rel intens) = 768 (80) [M þ Hþ],
790 (100) [M þ Naþ], 806 (10) [M þ Kþ]. HRMS (ESþ) calcd for
C47H79N2O6 767.5938, found 767.5928. FT-IR (thin film) νmax/cmꢀ1
2957, 2927, 2859, 1717, 1505, 1464, 1418, 1379, 1185, 1139, 1014, 864,
818, 752, 521.
Compound 3b50. Compound 1b (20.02 g, 99.98 mmol) was
partially dissolved in dichloromethane (150 mL). To this solution were
added solid potassium hydroxide (16.96 g, 302.26 mmol) and tetra-N-
butylammonium bromide (3.83 g, 11.88 mmol). The mixture was stirred
at room temperature for 40 h and then filtered over silica gel. Evapora-
tion of the solvent gave a white solid (8.25 g, 20 mmol) that was
dissolved in ethyl acetate (275 mL) and hydrogenated with palladium-
on-charcoal (10% Pd, 0.4 g) and hydrogen gas at atmospheric pressure
and room temperature. After removal of the catalyst via filtration, the
solvent was evaporated and the resulting oil was recrystallized from ethyl
1
acetate as a white solid: yield 4.24 g (91%). H NMR (400 MHz,
DMSO) δH = 9.09 (s, 2H), 6.88 (d, 4H, J = 8), 6.69 (d, 4H, J = 8), 5.57
(s, 2H). 13C NMR (62.8 MHz, DMSO) δC = 152.5, 149.2, 117.7, 115.7,
92.2. Mp =121ꢀ123 °C. MS (ESꢀ) m/z (rel intens) = 231 (100) [M ꢀ
Hþ]. HRMS (ESꢀ) calcd for C13H11O4 231.0657, found 231.0660. FT-
IR (thin film) νmax/cmꢀ1 3393, 3034, 2040, 1867, 1609, 1503, 1452,
1407, 1365, 1317, 1241, 1196, 1169, 1145, 1111, 1100, 1038,1003, 821,
809, 723, 639.
Compound 3c51. Compound 1b (20.00 g, 99.90 mmol) was
dissolved in acetone (125 mL). To this solution were added anhydrous
potassium carbonate (20.50 g, 148.33 mmol) and 1,3-dibromopropane
(5.07 mL, 49.90 mmol), and the mixture was refluxed for 3 days. The
resulting solution was allowed to cool down and then filtered. The
solvent was evaporated, and the crude product was dissolved in toluene
(100 mL), washed with sodium hydroxide 1 M, and dried with sodium
sulfate. Evaporation of the solvent gave a white solid, which was purified
by recrystallization from acetone. After filtration, the crystals were dried
(8.62 g, 19.57 mmol), dissolved in a mixture of tetrahydrofuran
(220 mL) and methanol (58 mL), and hydrogenated with palladium-
on-charcoal (10% Pd, 2 g) and hydrogen gas at atmospheric pressure and
room temperature. After removal of the catalyst via filtration through
celite, the solvent was evaporated and the product was crystallized from
ethyl acetate as a yellow solid: yield 3.00 g (59%). 1H NMR (400 MHz,
DMSO) δH = 8.93 (s, 2H), 6.76 (d, 4H, J = 8), 6.66 (d, 4H, J = 8), 4.00
(t, 4H, J = 6), 2.07 (m, 2H). 13C NMR (62.9 MHz, DMSO) δC = 151.3,
151.2, 115.7, 115.4, 64.7, 28.9. Mp = 142ꢀ143 °C. MS (ESꢀ) m/z (rel
intens) = 259 (100) [M ꢀ Hþ]. HRMS (ESꢀ) calcd for C15H15O4
259.0970, found 259.0980. FT-IR (thin film) νmax/cmꢀ1 3394, 3035,
2953, 2040, 1609, 1504, 1452, 1407, 1364, 1317, 1293, 1240, 1197, 1169,
1145, 1100, 1038, 995, 974, 820, 809, 724, 639.
Compound 4c. A mixture of 3c (4.6 g, 17.7 mmol), 4-nitrophenyl
chloroformate (8.5 g, 42.2 mmol), and triethylamine (5.9 mL, 42.3
mmol) in dichloromethane (300 mL) was stirred under nitrogen
atmosphere at room temperature for 48 h. Bis(2-ethylhexyl)amine
(35.1 mL, 116.9 mmol) and triethylamine (16.3 mL, 116.9 mmol) were
added, and the solution was stirred overnight under nitrogen atmo-
sphere at room temperature. The solution was washed with 1 M
hydrochloric acid, 1 M sodium hydroxide, water, and brine, dried with
sodium sulfate, and condensed under reduced pressure. The resulting oil
was purified by column chromatography eluting with dichloromethane
and methanol. After removal of the solvent under reduced pressure, the
Compound 3d51. Compound 1b (13.34 g, 66.60 mmol) was
dissolved in acetone (83.33 mL). To this solution were added anhydrous
potassium carbonate (13.66 g, 98.87 mmol) and 1,10-dibromodecane
(9.98 g, 33.26 mmol), and the mixture was refluxed for 3 days. The
product precipitated as crystals that were washed with acetone, 1 M
sodium hydroxide, and water (16.28 g, 30.24 mmol). After filtration, the
crystals were dried (1 g, 1.86 mmol), partially dissolved in a mixture of
chloroform and methanol (250 mL), and hydrogenated with palladium-
on-charcoal (10% Pd, 0.23 g) and hydrogen gas at atmospheric pressure
and room temperature. After removal of the catalyst via filtration
through celite, the solvent was evaporated and the product was crystal-
lized from a mixture of chloroform and methanol as a white solid: yield
0.22 g (33%). 1H NMR (400 MHz, DMSO) δH = 8.88 (s, 2H), 6.72 (d,
4H, J = 9), 6.65 (d, 4H, J = 9), 3.83 (t, 4H, J = 6), 1.65 (m, 4H), 1.34 (m,
12H). 13C NMR (62.9 MHz, DMSO) δC = 151.5, 151.0, 115.7, 115.3,
67.9, 28.9, 28.8, 28.7, 25.5. Mp =161ꢀ163 °C. MS (ESꢀ) m/z (rel
intens) = 357 (100) [M ꢀ Hþ]. HRMS (ESꢀ) calcd for C22H29O4
357.2066, found 357.2070. FT-IR (thin film) νmax/cmꢀ1 3352, 3038,
1
product was isolated as a clear oil: yield 4.8 g (35%). H NMR (250
MHz, CDCl3) δH = 7.01 (d, 4H, J = 8), 6.89 (d, 4H, J = 8), 4.14 (t, 4H, J
= 6), 3.27 (m, 8H), 2.25 (m, 2H), 1.74 (m, 4H), 1.34 (m, 32H), 0.92 (m,
24H). 13C NMR (62.9 MHz, CDCl3) δC = 156.0, 155.6, 145.3, 122.5,
115.1, 65.0, 51.1, 37.4, 30.6, 29.3, 28.7, 23.9, 23.1, 14.1, 10.7. MS (ESþ)
m/z (rel intens) = 796 (30) [M þ Hþ], 818 (100) [M þ Naþ], 834 (50)
[M þ Kþ]. HRMS (ESþ) calcd for C49H83N2O6 795.6251, found
795.6229. FT-IR (thin film) νmax/cmꢀ1 2957, 2927, 2859, 1715, 1507,
1464, 1418, 1379, 1294, 1193, 1141, 1105, 1060, 1010, 862, 819,
753, 520.
Compound 4d. A mixture of 3d (1.0 g, 2.79 mmol), 4-nitrophenyl
chloroformate (1.1 g, 5.56 mmol), and triethylamine (0.8 mL, 5.52
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ARTICLE
mmol) in dichoromethane (50 mL) was stirred under nitrogen atmo-
sphere at room temperature for 48 h. Bis(2-ethylhexyl)amine (1.7 mL,
5.60 mmol) and triethylamine (0.8 mL, 5.52 mmol) were added, and the
solution was stirred overnight under nitrogen atmosphere at room
temperature. After evaporation of the solvent, the solution was diluted
again with dichloromethane (50 mL), washed with water (30 mL) and
brine (30 mL), dried with sodium sulfate, and condensed under reduced
pressure. The resulting oil was purified on column chromatography
eluting with ethyl acetate and hexane. The product was isolated as a clear
oil: yield 0.8 g (32.1%). 1H NMR (250 MHz, CDCl3) δH = 7.00 (d, 4H, J
= 8), 6.87 (d, 4H, J = 8), 3.94 (t, 4H, J = 6), 3.28 (m, 8H), 1.76 (m, 8H),
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1.32 (m, 44H), 0.92 (m, 24H). 13C NMR (62.9 MHz, CDCl3) δC
=
156.3, 155.7, 145.0, 122.4, 114.9, 68.4, 51.0, 50.8, 38.1, 37.4, 30.6, 29.5,
29.4, 29.3, 28.7, 26.0, 23.9, 23.8, 23.1, 14.1, 10.7. MS (ESþ) m/z (rel
intens) = 916 (100) [M þ Naþ]. HRMS (ESþ) calcd for C56H96N2O6-
Na 915.7166, found 915.7164. FT-IR (thin film) νmax/cmꢀ1 2956, 2926,
2857, 1717, 1508, 1464, 1418, 1380, 1295, 1197, 1152, 1104, 861, 818,
753, 521.
1H NMR Titrations. A 10 mL sample of host dissolved in CCl4 was
prepared at known concentration (0.1ꢀ0.6 mM). A 0.6 mL fraction of
this solution was used to record a 1H NMR spectrum using a capillary of
D2O as a lock signal. The host stock solution was used to prepare a 2 mL
solution of guest at known concentration (40ꢀ400 mM), so that the
concentration of the host remained constant throughout the titration.
Aliquots of the guest solution were successively added to the NMR tube
1
containing the host solution, and a H NMR spectrum was recorded
after each addition. Changes in chemical shift for the host aromatic and
phenolic protons signals were fit to 1:1 and 2:1 binding isotherms using
purpose written software. Each titration was repeated at least twice, and
the error is quoted as twice the standard deviation.
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’ ASSOCIATED CONTENT
S
Supporting Information. Calculation of EM for the
b
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data for all compounds. This material is available free of charge
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Corresponding Author
c.hunter@sheffield.ac.uk
Notes
Phone: þ44 (0)114 2229476. Fax: þ44 (0)114 2229436.
’ ACKNOWLEDGMENT
We thank the EPSRC for funding.
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