Computational and Experimental Evidence of Emergent Equilibrium Isotope Effects in Anion Receptor Complexes

Article abstract of DOI:10.1021/jacs.7b00612

The measurement of a deuterium equilibrium isotope effect (EIE) for the aryl CH···Cl^- interaction of anion receptor 1H/1D is reported. Computations corroborate the results of solution measurements for a small, normal EIE in the full complex (K_a^H/K_a^D = 1.019 ± 0.010). Interestingly, isotope effects involving fragments of the anion receptor (urea, aryl ring, etc.) are predicted to produce an inverse effect. This points to an unusual and subtle structural organization effect of the anion receptor complex that changes the nature of the combined interactions to a normal isotope effect. The reversal of EIE values suggests that overall architecture of the anion receptor can dramatically impact the nature of bonding in these complexes.

Full text of DOI:10.1021/jacs.7b00612

This is an open access article published under an ACS AuthorChoice License, which permits
copying and redistribution of the article or any adaptations for non-commercial purposes.
Communication
pubs.acs.org/JACS
Computational and Experimental Evidence of Emergent Equilibrium
Isotope Effects in Anion Receptor Complexes
Blakely W. Tresca,†,# Alexander C. Brueckner,‡ Michael M. Haley,*,† Paul H.-Y. Cheong,*,‡
and Darren W. Johnson*,†
†Department of Chemistry & Biochemistry and the Materials Science Institute, University of Oregon, Eugene, Oregon 97403-1253,
United States
‡Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, Oregon 97331, United States
S
* Supporting Information
were also dependent on the location of the deuteration (KHa /KaD
ABSTRACT: The measurement of a deuterium equili-
brium isotope effect (EIE) for the aryl CH···Cl−
interaction of anion receptor 1H/1D is reported.
Computations corroborate the results of solution measure-
ments for a small, normal EIE in the full complex (KHa /KaD
= 1.019 0.010). Interestingly, isotope effects involving
fragments of the anion receptor (urea, aryl ring, etc.) are
predicted to produce an inverse effect. This points to an
unusual and subtle structural organization effect of the
anion receptor complex that changes the nature of the
combined interactions to a normal isotope effect. The
reversal of EIE values suggests that overall architecture of
the anion receptor can dramatically impact the nature of
bonding in these complexes.
range from 1.0139 to 1.0828). This example highlights the
hypothesis that aggregate zero-point energy (ZPE) vibration
effects are responsible for changes in DEIE rather than
inductive effects. These two studies highlight the power of
EIE studies in fundamentally understanding reversible inter-
actions.
The Perrin method for competitive titration requires only a
fast exchanging system with resolved resonances for the precise
measurement of relative association constants by NMR
spectroscopy. This method has previously been applied to
guest binding in supramolecular cage complexes.7,8 We have
adapted this method to the study of DEIEs on CH···Cl−
hydrogen bonding by both solution measurements and
computations. The synthesis of a selectively deuterated
arylethynyl bis-urea anion receptor 1D enables competitive
titration DEIE measurements in DMSO-d6. The observed
DEIE of the singly deuterated compound 1D and quantum
mechanical computations corroborate the importance of
vibrational changes on the observed DEIE. We elucidate
specific structural origins for DEIEs in anion receptor
complexes and have discovered an emergent behavior: the
DEIE of the receptor complex is uniquely different than DEIEs
of various fragments of the complex. In other words, the nature
of multiple hydrogen bonding in the anion receptor is uniquely
different from simpler hydrogen bonding complexes.
he ability of isotopes to alter the strength and selectivity of
T
binding in noncovalent interactions is well-documented
for biological and nonbiological systems.1,2 Numerous methods
have been developed to utilize selective labeling experiments to
differentiate the importance of individual interactions on the
complete symphony of binding, including relative chromato-
graphic retention times,3 binding studies by mass spectrometry
and spectroscopic titrations,4 and enzyme kinetic studies.5,6
Deuterium equilibrium isotope effect (DEIE) measurements in
biological systems highlight the complex effects of deuterium
labeling on noncovalent interactions.5 Recent studies combin-
ing experiments and computations provide insight into the
mechanisms of DEIEs in supramolecular systems that
ultimately facilitate the development of models for ab initio
sensor and drug design.4,7−13
1H has been previously shown to act as a strong CH donor
with log Ka for Cl− of 4.39 0.62 in CHCl3.16 Isotopologue 1D
is prepared by methods similar to those reported in previous
papers (Scheme 1).16−18 Visible-light catalyzed deamination in
DMF-d7 allows for the selective synthesis of monodeuterated
intermediate 2.19 The final receptor 1D and key intermediates
were characterized by 1H, 2H, and 13C NMR spectroscopy, and
high-resolution mass spectrometry (see Supporting Informa-
tion).
Two classic examples of such a combined experiment-and-
computation approach are the acidities of deuterium-labeled
pyridines and glucose binding to human brain hexokinase
(HBH). Lewis and Schramm have extensively studied the EIE
of tritiated glucose binding to HBH.14,15 Tritium EIEs for
binding to HBH range from 0.927 to 1.027 depending on the
site of CH substitution. Proximal hydrogen bonding groups in
the binding site consistently explained normal EIEs, and steric
interactions caused the large inverse isotope effect. In an
approach that inspires our current experiments, competitive pH
titrations by 13C NMR spectroscopy of 1:1 mixed H/D-
pyridines allowed for the direct measurement of KHa /KaD as
small as 10−4.11 DEIEs favored dx-pyridine protonation and
As shown by Perrin et al., 13C NMR spectroscopy is
exceptionally sensitive to chemical shifts from isotopic
labeling.11,20 Figure 1a illustrates the similarity between 1H/
1D. The two key differences are the loss of the Ha peak in 1D
and small shifts in 13C peaks. Competitive titrations were
performed in DMSO-d6 with ∼1:1 mixture of 1H and 1D
Received: January 18, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.7b00612
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
The remaining 1H and 13C NMR peaks produce simpler and
consistent binding curves, which can be split into two
categories (Figure 1b). First, several peaks reach a saturation
point near 15 equiv. Cl− and remain mostly flat for the
remainder of the titration (Ha, Ce, and Cab). The second
category includes peaks that never appear to reach saturation in
the range studied but continue to increase linearly after 15
equiv. (Hg, Hf, Cx, and Cd). The second case is consistent with
a weak 1:2 complex, whereas the first case is more indicative of
a 1:1 binding event. An estimated value for the overall Ka (log
β) is obtained from the fitting of the 1H resonances and Cab to
a 1:2 1:Cl− model using the combined [1H+1D]. The binding
in DMSO-d6 is significantly weaker (log β ∼ 1.8) than in
CHCl3 (log Ka = 4.39 0.62), which is expected for a solvent
system with high polarity and strong hydrogen bond acceptors.
As well, the second Cl− association is likely very weak with K1a:2
< 5. Based on the curve shape and position on 1 of atoms for
the two binding isotherm categories, it is clear that the first Cl−
binds in the expected pocket to CHa with both ureas, and then
a second Cl− weakly binds to one of the ureas at high [Cl−],
forcing the alkyne to rotate open to accommodate it.22,23 The
complex binding isotherm, however, does not prevent the use
of a linearized plot to determine the relative Ka values for 1H
and 1D with greater precision than possible by direct analytical
titration and nonlinear regression.
Scheme 1. Selective Deuterium Labeling of Anion Receptor
1H/1D, Summary of Equilibrium Isotope Effect, Anion
Binding Energy and Wiberg Bond Index
receptors at a combined concentration of 6.1 mM, and Bu4NCl
was added in 5 μL aliquots as a 1.8 M solution.21 Many 1H and
13C signals shift over the course of the titration, and four 13C
peaks are resolved by isotope shifts between 1H and 1D to
differentiate the two species. The labeled 13C signal (Ca) is too
weak for accurate measurement of δ during the titration, which
leaves three signals (alkyne C1, alkyne C2, and Cab) for possible
fitting.
The binding isotherm for mixed 1H:1D is plotted in Figure
1b. Cab is the closest carbon to the labeled CH hydrogen bond
donor and, along with the alkyne peaks, is well resolved
between 1H and 1D with greater intensity than the labeled
peak. The Cab peak and alkyne C2 are complex curves indicative
of possible multistep equilibria. Alkyne C1 presents a large
downfield shift and the isotherm for this carbon approximates a
binding curve for a 1:1 association. A large shift in the alkyne
carbons is surprising due to the large distance from the nearest
hydrogen bonding site. One explanation is the through space
effect of an approaching anion that shields the alkyne. An
alternative effect is rotation about the alkyne upon binding of
the anion, a conformational change that has also been observed
in similar alkyne receptors.22,23
The linearized plot of KaH/KDa , eq 1, provides an accurate
measure of the DEIE from a competitive titration with errors as
small as 0.0004 reported.8,11 The method requires free and
bound species to be in fast equilibrium with resolved peaks but
is independent of receptor concentration. Typical methods of
Ka determination rely on nonlinear regression, and errors are
restricted to ∼5−10% by the accuracy in concentration. Least
squares linear regression of the 13C NMR chemical shifts of 1H
and 1D, eq 1, provides the relative KHa /KaD with greater
precision.20 The chemical shifts for alkyne C1 13C peaks were fit
with an R2 = 0.996 shown in Figure 1c. The weighted average
KHa /KDa was 1.019 0.010 for two independent titrations.
(δHo − δH)(δD − δDf ) = (KaH/KaD)(δH − δHf )(δDo − δD)
(1)
Quantum mechanical computations are effective at predicting
kinetic and equilibrium isotope effects.10,11,24 Structures were
optimized using PBE/6-31G(d) level of theory with the
1
Figure 1. (a) H (top) and 13C (bottom) NMR spectra of 1H (blue) and 1D (red) in DMSO-d6. Cab is the aryl carbon between Ha and Hb. (b)
Binding isotherm with Cl− for 1:1 1H:1D in DMSO-d6 by 1H (solid lines) and 13C (dashed lines) NMR spectroscopy; lines added as a guide for the
eye. Isotherm shape is consistent with weak 1:2 binding where K1a:1 ≫ Ka1:2 13C NMR peaks are used for linear EIE fitting; the isotherms overlap,
.
although the peaks are resolved. (c) Linearized plot of KHa /KaD (eq 1) for 1H:1D 13C NMR peak corresponding to alkyne C1.
B
DOI: 10.1021/jacs.7b00612
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
polarizable continuum model (PCM) for DMSO as imple-
mented in Gaussian 09.25−27 Exhaustive conformational
searches led to structures of anion receptor complexes and
free receptors that corroborate known crystal structures of
related compounds (see SI). The computed EIE for Cl−
binding (complex [1H/1D···Cl−]) was 1.020, in good agree-
ment with experimental values (1.019 0.010). Moreover, the
binding energy between the receptor and Cl− was computed to
be −7.57 kcal mol−1, in good agreement with experiments
(−5.99 kcal mol−1).16,28
0.979. These results are surprising because no data from the
fragments of the anion receptor would suggest that a normal
EIE is possible (nonadditive), yet the experimentally measured
and computed EIE is 1.020. This indicates that the isotope
effect for [1H/D···Cl−] is more than the sum of its parts.
The computation of the fragment complexes revealed
structural details relevant to elucidating the origin of this
unexpected EIE of 1H/1D. In 3, the CH···Cl− geometry is bent
(161°) with Cl− still within the plane of the aryl group. In 4 and
6, this geometry is completely linear, just as in 1H/1D.
Although repulsions between the chloride anion and the alkyne
groups may be responsible for this geometric change, the CH···
Cl− distance in the monoalkynylated complex 4 is shorter,
whereas the distance in the bis-alkynylated complex 6 is even
shorter (2.66 Å vs 2.57 Å vs 2.54 Å, respectively). These data
are consistent with an inductive enhancement of hydrogen
bonding by the electronegative alkynes. Similar trends can be
observed in complexes 5 and 7. The computed natural bond
orbital (NBO) analysis revealed that the Wiberg bond index of
the hydrogen bond in 3 is 0.029, whereas it is 0.036 in 4 and
0.039 in 6.29 Similarly, the bond index for complex 5 and 7 are
0.025 and 0.028, respectively. The bond index for the
corresponding interaction in 1H/1D is 0.035, matching closely
the alkynylated complexes 4 and 6. This would suggest the EIE
does not originate from the central aryl CH hydrogen bonding.
Complexes 8 and 9 reveal that the geometry of hydrogen
bonding in 1H/1D is significantly distorted (Chart 1). The
The computed 1H/1D structure is shown in Figure 2. The
chloride anion is quintuply hydrogen bonded by the central aryl
Chart 1
Figure 2. Quantum mechanically optimized structure of anion
receptor 1H/1D with chloride anion (PBE/6-31G*/PCM(DMSO)).
CH and the four urea protons. The two substituted urea arms
of the anion receptor are not planar in the presence of the
anion, and the terminal urea aryl rings rest on top of each other.
This structure forces an asymmetric hydrogen bond between
the chloride anion and the two protons of each urea, the proton
that is distal from the CH site is closer (2.19 Å) than the proton
proximal to the CH (2.87 Å).
The normal EIE favors association of Cl− with the unlabeled
receptor 1H. We fragmented the receptor into key components
to explore the isotope effects of the basic receptor segments
(Figure 3, S20). All fragments led to more inverse EIE values.
For example, the addition of an alkyne to 4 (i.e., 6) lowered the
DEIE from 1.000 to 0.989. Removing half of the receptor (5)
also led to an inverse DEIE (0.993), and the addition of
another alkyne (7) results in an even more inverse DEIE of
ideal urea NH···Cl− distance is 2.38−2.43 Å, which deviates
significantly from 1H/1D. NBO analysis also shows that the
bond indices for hydrogen bonds in 8 and 9 are 0.057 and
0.044, respectively; significantly different from those of 1H/1D
(0.015 and 0.087). These observations are consistent with the
hypothesis that the unusual arrangement of urea NH and aryl-
CH hydrogen bonds is responsible for the unexpected EIE of
1H/1D.
Figure 3. Computed EIE values involving chloride complexes with fragments of the anion receptor 1H/D.
C
DOI: 10.1021/jacs.7b00612
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
(3) Tran, C. D.; Mejac, I.; Rebek, J.; Hooley, R. J. Anal. Chem. 2009,
In summary, the synthesis of a selectively labeled receptor
has allowed for the first accurate measurement of an EIE in an
aryl CH···X− hydrogen bond. The combined evidence
presented here is consistent with the categorization of certain
aryl CH donors as hydrogen bond donors. The DEIE
measurements from competitive titrations and computations
agree on a small, normal DEIE that favors the formation of a
CH hydrogen bond over a CD hydrogen bond. This study,
more importantly, shows that supramolecular binding is a
complex phenomenon that cannot be reduced to the sum of its
parts. The reversal of EIE values between the complete anion
receptor complex versus the fragments suggest that isotope
effects are extremely sensitive to the overall architecture of the
anion receptor. This discovery is highly relevant to recent
applications of isotope labeling in pharmaceuticals, where the
introduction of isotopes may improve the strength and
selectivity of protein-drug interactions.
81, 1244−1254.
(4) Laughrey, Z. R.; Upton, T. G.; Gibb, B. C. Chem. Commun. 2006,
970−972.
́
(5) Swiderek, K.; Paneth, P. Chem. Rev. 2013, 113, 7851−7879.
(6) Wang, Z.; Singh, P.; Czekster, C. M.; Kohen, A.; Schramm, V. L.
J. Am. Chem. Soc. 2014, 136, 8333−8341.
(7) Mugridge, J. S.; Bergman, R. G.; Raymond, K. N. Angew. Chem.,
Int. Ed. 2010, 49, 3635−3637.
(8) A similar method was applied to the supramolecular complex of
benzyltrimethylphosphonium with a tetrahedral cage. The 31P NMR
resonance was used to measure small normal DEIEs for association to
the exterior and interior of the cage: (a) Mugridge, J. S.; Bergman, R.
G.; Raymond, K. N. J. Am. Chem. Soc. 2012, 134, 2057−2066.
(b) Mugridge, J. S.; Bergman, R. G.; Raymond, K. N. J. Am. Chem. Soc.
2010, 132, 1182−1183.
(9) Balevicius, V.; Aidas, K.; Svoboda, I.; Fuess, H. J. Phys. Chem. A
2012, 116, 8753−8761.
(10) Zhao, C.; Parrish, R. M.; Smith, M. D.; Pellechia, P. J.; Sherrill,
C. D.; Shimizu, K. D. J. Am. Chem. Soc. 2012, 134, 14306−14309.
(11) Perrin, C. L.; Karri, P. J. Am. Chem. Soc. 2010, 132, 12145−
12149.
ASSOCIATED CONTENT
■
S
* Supporting Information
(12) Rechavi, D.; Scarso, A.; Rebek, J. J. Am. Chem. Soc. 2004, 126,
7738−7739.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b00612.
(13) Zhao, Y. L.; Houk, K. N.; Rechavi, D.; Scarso, A.; Rebek, J. J.
Am. Chem. Soc. 2004, 126, 11428−11429.
Complete synthesis and characterization for all com-
pounds, titration data, coordinates, energies, and
frequencies for all computed structures (PDF)
(14) Lewis, B. E.; Schramm, V. L. J. Am. Chem. Soc. 2001, 123, 1327−
1336.
(15) Lewis, B. E.; Schramm, V. L. J. Am. Chem. Soc. 2003, 125, 4785−
4798.
(16) Tresca, B. W.; Hansen, R. J.; Chau, C. V.; Hay, B. P.; Zakharov,
L. N.; Haley, M. M.; Johnson, D. W. J. Am. Chem. Soc. 2015, 137,
14959−14967.
AUTHOR INFORMATION
■
Corresponding Authors
*haley@uoregon.edu
*paulc@science.oregonstate.edu
*dwj@uoregon.edu
(17) Carroll, C. N.; Berryman, O. B.; Johnson, C. A.; Zakharov, L.
N.; Haley, M. M.; Johnson, D. W. Chem. Commun. 2009, 2520−2522.
(18) Kimball, D. B.; Weakley, T. J. R.; Haley, M. M. J. Org. Chem.
2002, 67, 6395−6405.
ORCID
(19) Majek, M.; Filace, F.; Jacobi von Wangelin, A. Chem. - Eur. J.
2015, 21, 4518−4522. The low yield of 2 results from a side product
produced by kinetic isotope effects in DMF-d7.
Michael M. Haley: 0000-0002-7027-4141
Darren W. Johnson: 0000-0001-5967-5115
(20) Perrin, C. L.; Fabian, M. A. Anal. Chem. 1996, 68, 2127−2134.
(21) Titrations with pure 1D were also performed by UV−vis and 1H
NMR; however, these are limited because differences in the raw data
and Ka values are not significant enough to distinguish 1H and 1D.
(22) Gavette, J. V.; Evoniuk, C. J.; Zakharov, L. N.; Carnes, M. E.;
Haley, M. M.; Johnson, D. W. Chem. Sci. 2014, 5, 2899−2905.
(23) Gavette, J. V.; Mills, N. S.; Zakharov, L. N.; Johnson, C. A.;
Johnson, D. W.; Haley, M. M. Angew. Chem., Int. Ed. 2013, 52, 10270−
10274.
Present Address
#Molecular Foundry, Lawrence Berkeley National Laboratory,
Berkeley, CA 94720
Notes
The authors declare the following competing financial
interest(s): NIH grant R01-GM087398 funded early-stage
intellectual property that was licensed by SupraSensor
Technologies, a company co-founded by M.M.H. and D.W.J.
(24) Singh, A.; Gangopadhyay, D.; Popp, J.; Singh, R. K. Spectrochim.
Acta, Part A 2012, 99, 136−143.
ACKNOWLEDGMENTS
■
(25) (a) Adamo, C.; Barone, V. J. Chem. Phys. 1999, 110, 6158−
6169. (b) Hehre, W. J.; Ditchfield, R.; Pople, J. A. J. Chem. Phys. 1972,
56, 2257−2261.
This work was supported by the NIH (R01-GM087398 to
D.W.J./M.M.H.) and NSF (CHE-1352663 to P.H.-Y.C.). We
thank the NSF for an NMR spectrometer grant (CHE-
1427987) and for computing infrastructure in part provided by
the NSF Phase-2 CCI, Center for Sustainable Materials
Chemistry (CHE-1102637). P.H.-Y.C. is the Bert and Emelyn
Christensen professor. A.C.B. acknowledges financial support
by the Barnes Fellowship. The authors acknowledge the
Biomolecular Mass Spectrometry Core of the Environmental
Health Sciences Core Center at Oregon State University (NIH
P30ES000210).
(26) Miertus, S.; Scrocco, E.; Tomasi, J. Chem. Phys. 1981, 55, 117−
̌
129.
(27) (For complete author list, see SI). Frisch, M. J.; Trucks, G. W.;
Schlegel, H. B.; et al. Gaussian 09; Gaussian Inc.: Wallingford, CT,
2009.
(28) Computations showed that the receptor-anion-TBA complex
(1H/D···Cl−···TBA+) was endergonic by 7.0 kcal mol−1, and
moreover, the computed EIE of 1.005 also did not match experiments.
Taken together, these suggest that the anion receptor solely binds to
the Cl− anion rather than the ion pair (Cl−···TBA+).
(29) Foster, J. P.; Weinhold, F. J. Am. Chem. Soc. 1980, 102, 7211−
7218.
REFERENCES
■
(1) Wade, D. Chem.-Biol. Interact. 1999, 117, 191−217.
(2) Kohen, A.; Limbach, H.-H. Isotope Effects In Chemistry and
Biology; CRC Press: Boca Raton, FL, 2005.
D
DOI: 10.1021/jacs.7b00612
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX