Thermodynamic Analysis of Allosteric and Chelate Cooperativity in Di- and Trivalent Ammonium/Crown-Ether Pseudorotaxanes

Article abstract of DOI:10.1021/acs.orglett.5b02592

A detailed thermodynamic analysis of the axle-wheel binding in di- and trivalent secondary ammonium/[24]crown-8 pseudorotaxanes is presented. Isothermal titration calorimetry (ITC) data and double mutant cycle analyses reveal an interesting interplay of p

Full text of DOI:10.1021/acs.orglett.5b02592

Letter
pubs.acs.org/OrgLett
Thermodynamic Analysis of Allosteric and Chelate Cooperativity in
Di- and Trivalent Ammonium/Crown-Ether Pseudorotaxanes
Karol Nowosinski, Larissa K. S. von Krbek, Nora L. Traulsen, and Christoph A. Schalley*
Institut fur Chemie und Biochemie, Freie Universitat Berlin, Takustraße 3, 14195 Berlin, Germany
̈
̈
S
* Supporting Information
ABSTRACT: A detailed thermodynamic analysis of the axle-wheel binding
in di- and trivalent secondary ammonium/[24]crown-8 pseudorotaxanes is
presented. Isothermal titration calorimetry (ITC) data and double mutant
cycle analyses reveal an interesting interplay of positive as well as negative
allosteric and positive chelate cooperativity thus providing profound insight
into the effects governing multivalent binding in these pseudorotaxanes.
ultivalency1 is fundamental for many biological
Mprocesses such as virus-docking to cell surfaces. Strong,
yet reversible Velcro-like binding is achieved through multiple
interactions. Binding strength gains depend not only on the
number of binding sites and their preorganized arrangement
but also on allosteric and chelate cooperativity.2 The number of
binding sites needs to be known for an analysis of cooperativity.
In biological systems, this is often not the case and precise
thermodynamic analyses are challenging. The systematic
variability of binding site number and spacer design is an
advantage of synthetic complexes.3 Intriguing examples are the
“molecular elevators”, triply threaded ammonium/crown-ether
pseudorotaxanes which can be switched between two stations
along their axles.4 Here, multivalent binding not only causes
higher binding strengths but also controls the relative positions
of the subunits and thus function. While kinetic effectsa slow
third threading step following two much faster oneshave
been investigated,5 a thorough thermodynamic analysis is not
Figure 1. Pseudorotaxane axles and wheels.
yet available.
Among the reports on di- and trivalent pseudorotaxanes,4−6
only three analyze cooperativity effects in detail.7 Attractive
spacer−spacer interactions are an important factor for positive
chelate cooperativity in divalent ammonium/crown-ether
pseudorotaxanes 1@9−4@9 (Figure 1).7a,c In this work, we
use isothermal titration calorimetry (ITC) to determine
binding parameters of di- and trivalent ammonium/crown-
ether pseudorotaxanes to assess allosteric and chelate
cooperativity effects which govern multiple threading. We use
the term “allosteric” in a broader sense. It comprises not only
structural changes but also, for example, electronic effects that
the first binding event has on the next as they occur in
complexes that contain either monovalent axles and di- or
trivalent wheels or, vice versa, monovalent wheels with di- or
trivalent axles. Beyond this, the analysis of chelate cooperativity
is performed by double mutant cycles that permit determi-
nation of effective molarities EMa measure for chelate
cooperativityfrom only four titration experiments.8
The synthesis of the compounds under study is exemplarily
shown in Scheme 1 for the two trivalent ones (for details, see
Supporting Information). Briefly, the axles were prepared by
ether synthesis followed by reductive amination. The free
amines were then protonated with HCl followed by an anion
exchange of Cl− against PF6 to increase solubility and reduce
−
competition of the crown and anion for the binding sites in
organic solvents. Trivalent crown ether 10 was made by
oxidative cyclotrimerization.
All pseudorotaxanes form spontaneously when axles and
wheels are combined in CHCl3/CH3CN mixtures. The solvent
ratio can be adjusted to achieve sufficient solubility of both
1
components. Typical complexation-induced H NMR signal
shifts demonstrate a quantitative formation of the pseudo-
Received: September 8, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b02592
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Letter
rotaxanes (Figure 2). Particularly pronounced upfield shifts are
observed for spacer protons Hg and Hh (6@9) as well as H6,
H7, and H9 (7@10), which experience the anisotropy of the
anthracene spacers. In contrast, the benzylic protons Hd, He
(6@9) and H4, H5 (7@10) significantly shift downfield caused
by the formation of C−H···O hydrogen bonds between the
polarized C−H bonds next to the charges and crown ether
oxygen atoms. Threading of nonsymmetric axles renders the
protons of all crown ether CH2 groups diastereotopic and cause
two sets of signals with complex coupling patterns for these
protons in the pseudorotaxane, another clear piece of evidence
for pseudorotaxane formation. Pseudorotaxane formation is
also confirmed by ESI mass spectrometry (Supporting
Information).
Scheme 1. Syntheses of Trivalent Axle 7 and Wheel 10
After establishing pseudorotaxane formation, isothermal
titration calorimetry has been used to determine standard
Gibbs binding energies, binding enthalpies and binding
entropies. The data obtained for all mono-, di-, and trivalent
pseudorotaxanes are summarized in Table 1. The monovalent
pseudorotaxane was measured twice, as solubility required two
different CHCl3/CH3CN solvent mixtures: 2.2:1 for the di- and
1:1 for the trivalent case.
Four titration experiments suffice for a detailed analysis of
allosteric and chelate cooperativity. By determining the
monovalent binding constant as a reference and by titrating
the di- or trivalent axle with monovalent crown ethers and vice
versa the di- or trivalent crown ether with monovalent axles,
one can determine allosteric cooperativity, which describes any
steric or electronic effect that one binding event has on the
following ones and which are not related to the presence of
both spacers. In the two latter titration experiments, the
statistical factors9 need to be taken into account for the two or
three binding steps (Supporting Information). Normalizing the
experimentally determined values K1, K2, and K3 with these
int
statistical factors provides the intrinsic binding constants Kn
that equal Kmono in the absence of allosteric cooperativity. An
allosteric cooperativity factor αallo can thus easily be defined (eq
1) for the n-th binding step (divalent pseudorotaxane: n = 2,
trivalent pseudorotaxane n = 2, 3). A value of αnallo > 1 indicates
positive allosteric cooperativity, and αnallo < 1 indicates negative
Figure 2. 1H NMR spectra (500 MHz, 298 K, 2.5 mM) of (a) axle 6,
(b) pseudorotaxane 6@9, and (c) crown ether dimer 9 in CDCl3/
CD3CN 2:1 and (d) axle 7, (e) pseudorotaxane 7@10, and (f) crown
ether trimer 10 in CDCl3/CD3CN 1:1.
Table 1. Thermodynamic Data Obtained for Mono-, Di-, and Trivalent Pseudorotaxanes from ITC Experiments
int
pseudorotaxane
K [M−1
]
ΔG [kJ mol−1
]
ΔH [kJ mol−1
]
TΔS [kJ mol−1
]
Kσ
Kn [M−1
]
αallo
EM [mM] EM Kmono
779 164
a
6@9
K
67,200 6,800
720 80
−27.6 0.3
−16.3 0.4
−14.2 0.3
−16.4 0.3
−12.3 0.4
−7.5 0.2
−29.9 0.3
−18.3 0.2
−15.9 0.2
−14.4 0.2
−18.2 0.2
−13.3 0.2
−10.6 0.2
−7.8 0.2
−64.1
−54.0
−15.0
−48.0
−75.2
−30.7
−67.4
−32.3
−32.0
−38.7
−24.6
−25.8
−22.1
−15.5
−36.5
−37.7
−0.8
a
6@82
K1
K2
K1
K2
4
180
310
185
145
310 40
1
4
1
2
1.5
0.7
a
52@9
735 90
−31.6
−62.9
−23.2
−37.5
−14.0
−16.1
−24.3
−6.4
145 20
a
c
c
c
5@8
K
420 50
210
b
7@10
K
173,000 18,000
1,640 170
600 60
47
12
b
7@83
K1
K2
K3
K1
K2
K3
6
2
275
300
510
250
110
105
1.2
2.0
340 40
2/3
6
b
53@10
1,510 160
220 30
−12.5
−11.5
−8.7
2
0.4
0.4
70 10
2/3
b
c
c
c
5@8
K
520 50
2
260
a
b
c
Titration in CHCl3/CH3CN 2.2:1. Titration in CHCl3/CH3CN 1:1. Our statistical factors take into account that the monovalent axle can be
threaded into the monovalent crown ether in two different orientations and thus account for the symmetry of the monovalent axle that the other
axles do not have. Kmono thus equals half the measured binding constant for the monovalent pseudorotaxanes (Kmono = 210 M−1 in CHCl3/CH3CN
2.2:1 and Kmono = 260 M−1 in CHCl3/CH3CN 1:1).
B
DOI: 10.1021/acs.orglett.5b02592
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allosteric cooperativity (Kσ is the corresponding symmetry
factor).
(Figure 3). The positive charges are electrostatically connected
by three interdigitating PF6 counterions. After binding of the
−
1
Kσ
Knint
Kmono
Kn
αnallo
=
=
Kmono
(1)
Ammonium ion binding to crown ethers in low dielectric
constant solvents is known to be strongly affected by ion
pairing effects.10 As the analysis of the concentration depend-
ence of NMR shifts10a,d revealed, these effects affect the binding
constants of the crown ether/ammonium complexes so
strongly, because they are large for the free ammonium salts,
while they are negligible for the crown ether complexes, in
which the two charges are quite remote from each other as
evidenced also by crystal structure analyses.10d,11 Besides steric
and electronic effects that a binding event has on the
subsequent binding step, the allosteric cooperativity factor
Figure 3. MM2 force-field-optimized structures12 of the di- and
trivalent axles 6 and 7 folded through ion-pairing effects.
first crown ether, two such ion pairs have been broken up, so
that ion pairing is partially destroyed and the competition of
the second crown with the counterions is diminished. After
binding of the second crown, also the last such interaction is
lost and the crown/anion competition for the ammonium
group is even lower, thus resulting in an even larger positive
allosteric effect.
In marked contrast, the two pseudorotaxanes 52@9 and 53@
10 exhibit negative allosteric cooperativity that is more
pronounced in the trivalent case. This effect can be rationalized
by a polarization of the aromatic π-systems of the spacers after
binding of a positively charged axle. The first binding event
causes a polarization of the spacer toward the positive charge
and thus reduces electron density at the remaining free binding
sites and the following binding interaction. The finding that the
second and third binding steps in 53@10 exhibit similar αallo
factors of 0.4 is well in agreement with this hypothesis.
In the di- and trivalent pseudorotaxanes 6@9 and 7@10,
both allosteric effects will at least in part counterbalance each
other. For the divalent pseudorotaxane 6@9, one would expect
no significant overall effect, as the product of both allosteric
effects is essentially one. For 7@10, one would expect an
overall negative allosteric effect. These arguments demonstrate
how a detailed analysis of allosteric cooperativity can be very
fruitful even in the absence of a substantial net effect, because
the separation of these counterbalancing effects helps in
analyzing which factors affect binding strongly and should
thus be optimized in the design of supramolecular complexes.
A look at the effective molarities and the EM Kmono values for
6@9 and 7@10 clearly reveals positive chelate cooperativity
effects for both pseudorotaxanes. With EM Kmono = 164, the
divalent pseudorotaxane exhibits by far the larger effect
compared to EM Kmono = 12 for the trivalent one. A direct
comparison is, however, not easily possible, as the spacer
structures are not directly related to each other and spacer−
spacer interactions are thus not comparable. As the double
mutant cycles waive all other effects except for those caused by
the spacers incorporated in the pseudorotaxanes, we can
conclude that a good geometric fit of axle and wheel and in
addition favorable spacer−spacer interactions such as π−π
stacking between the two spacers are the origins of positive
chelate cooperativity, which is important for the optimal design
and high-yield syntheses of the above-mentioned “molecular
elevators” and other multiply threaded, functional molecular
machines. Furthermore, spacer−spacer interactions may alter
the binding properties of different stations along the axles by
favoring the closest one above the more remote one. An
analysis of spacer−spacer interactions is therefore also very
allo
αn also comprises those (and only those) changes in ion
pairing effects that depend on valence, while it excludes
differences between ion pairing of free axle and pseudorotaxane
that analogously occur for the monovalent pseudorotaxane.
A fourth titration, i.e. that of the di/trivalent crown ether
with the di/trivalent axle, completes the data required for the
evaluation of chelate cooperativity, which describes all
cooperativity effects that result from tethering both binding
partners. With the data of all four experiments available, the
effective molarities EM for the di- and trivalent pseudorotax-
anes can be determined from double mutant cycles as detailed
in the Supporting Information. EM is a measure of the
preference of intramolecular ring closure over the formation of
supramolecular polymers. Note that a trivalent complex
undergoes two cyclization steps, each one requiring an EM
value. Without a divalent reference available, we use an
apparent EM here as defined in the Supporting Information.
The product EM Kmono is a dimensionless number, which
provides a measure for chelate cooperativity: If EM Kmono ≫ 1,
binding occurs with positive chelate cooperativity and the
formation of closed cyclic complexes is preferred. If EM Kmono
<
1, negative chelate cooperativity is encountered resulting in
open complexes that can oligomerize. The double mutant
cycles are constructed in such a way that that effects that are
not related to chelate cooperativity are warranted to cancel.
This also includes the allosteric effects. Furthermore, the ion
pairing effects hardly affect chelate cooperativity, as the double
mutant cycle does not contain any free ammonium ions and is
exclusively based on crown ether complexes, which are not
affected by ion pairing effects significantly (also, see Supporting
Information).
Binding in our ammonium/crown pseudorotaxanes is
enthalpy-driven (Table 1). All binding entropies are negative,
indicating higher order upon pseudorotaxane formation. The
liberation of solvent during binding thus does not over-
compensate for the entropic effects related to the particle
number reduction and conformational fixation of spacers.
These unfavorable entropic effects are nevertheless more than
counterbalanced by the quite strongly negative binding
enthalpies.
Positive allosteric cooperativity is found for pseudorotaxanes
6@82 and 7@83. In 7@83, this effect is even more pronounced
in the last binding step. We attribute this positive allosteric
effect to the formation of ion pairs within the di- and trivalent
axles in the rather unpolar solvents used. Taking 7@83 as an
example, free axle 7 can fold into a cyclic array of the three arms
C
DOI: 10.1021/acs.orglett.5b02592
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
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helpful in the design of the best spacers for a desired molecular
machine.
In conclusion, this study analyzes in detail the thermody-
namics of the formation of one di- and one trivalent
ammonium/crown-ether pseudorotaxane. Based on data
obtained by ITC, double mutant cycles were used to provide
insight into chelate cooperativity. The same binding data also
revealed interesting positive as well as negative allosteric
cooperativity effects. They allow us to obtain insight into the
details of multivalent binding and will thus help in designing
and synthesizing other optimized pseudorotaxanes and multiply
threaded molecular machines.
ASSOCIATED CONTENT
* Supporting Information
■
(10) (a) Jones, J. W.; Gibson, H. W. J. Am. Chem. Soc. 2003, 125,
7001−7004. (b) Huang, F.; Jones, J. W.; Slebodnick, C.; Gibson, H.
W. J. Am. Chem. Soc. 2003, 125, 14458−14464. (c) Gasa, T. B.;
Spruell, J. M.; Dichtel, W. R.; Sørensen, T. J.; Philp, D.; Stoddart, J. F.;
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.5b02592.
̌
Kuzmic, P. Chem. - Eur. J. 2009, 15, 106−116. (d) Gibson, H. W.;
Jones, J. W.; Zakharov, L. N.; Rheingold, A. L.; Slebodnik, C. Chem. -
Eur. J. 2011, 17, 3192−3206.
Experimental procedures, characterization data, original
spectra, ITC titrations and double mutant cycle analyses
(PDF)
(11) (a) Davlieva, M. G.; Lu, J.-M.; Lindeman, S. V.; Kochi, J. J. Am.
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Chem. Soc. 2004, 126, 4557−4565. (b) Fallon, G. D.; Lau, V. L.;
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o321−o323. (c) Ashton, P. R.; Campbell, P. J.; Chrystal, E. J. T.;
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P. A.; Williams, D. J. Angew. Chem., Int. Ed. Engl. 1995, 34, 1865−1869.
(d) Gibson, H. W.; Wang, H.; Bonrad, K.; Jones, J. W.; Slebodnick, C.;
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(12) CaChe 5.0 program package, Fujitsu, Krakow/Poland.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: c.schalley@fu-berlin.de.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for financial support from the Deutsche
Forschungsgemeinschaft (SFB 765). L.K.S.v.K. thanks the
Studienstiftung des Deutschen Volkes for a Ph.D. fellowship.
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