Designing Synthetic Receptors for Shape-Selective Hydrophobic

Chapter 19

Designing Synthetic Receptors for Shape -Selective Hydrophobic Binding

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 2, 2016 | http://pubs.acs.org Publication Date: September 29, 1994 | doi: 10.1021/bk-1994-0568.ch019

Craig S. Wilcox, Neil M . Glagovich, and Thomas H. Webb Department of Chemistry, University of Pittsburgh, Pittsburgh, P A 15260

It has been 20 years since the discovery that simple water soluble cyclophanes can be effective synthetic receptors for hydrophobic binding of lipophilic substrates. These synthetic receptors are small enough that quite accurate structural and thermodynamic binding data are obtainable and yet are large enough to offer the computational chemist most of the challenges found in biological receptors. The course of development of water soluble cyclophanes has been notably conservative. Here we review some data from our own work and from other laboratories and attempt to identify results and observations that will interest the computational chemist. More intense use of calculations and computer aided design methods will benefit the experimentalist and the theoretical chemist. Predictions of the behaviour of these synthetic systems can provide a valuable test for the validity of contemporary quantitative theories and as confidence in the calculations grows, rapid and more innovative progress in receptor design will follow. In the mid-1970's Tabushi initiated work on macrocyclic polyammonium ions 1 composed of alternating aromatic rings and aliphatic spacers (1-2). These molecules are structurally related to important ansate cyclophanes that Murakami was investigating at that time (5), and to the smaller water insoluble cyclophanes first prepared (and christened) by Cram (4,5). Tabushi pointed out that the "water soluble cyclophanes" contain lipophilic cavities and would provide a valuable model of enzyme-substrate and receptor-effector complexation (6). Since that time numerous +4

0097-6156/94/0568-0282$08.00/0 © 1994 American Chemical Society

In Structure and Reactivity in Aqueous Solution; Cramer, Christopher J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

19. WILCOX ET AL.

Receptors for Shape-Selective Hydrophobic Binding

descendants of Tabushi's synthetic receptor have appeared. Representative examples (Figure 1) include macrocyclic receptors reported by Koga (who provided the first crystallographic evidence of inclusion complexation, the first examples of chiral recognition, and the first asymmetric syntheses using these molecules), Diederich, Dougherty, and Wilcox (7-10).


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Figure 1. The descendants of Tabushi's receptor. These four hosts illustrate how the field has evolved as chemists have pursued receptors that have greater substrate binding affinity and selectivity. Water soluble cyclophanes today are often chiral and enantiomerically pure. This allows binding affinities for enantiomeric substrates to be determined. In addition, in three of these hosts, the polar groups that are required for water solubility are placed at locations more remote from the interior pocket than the polar groups in Tabushi's host. This relocation was first pursued in the belief that binding would be increased because desolvation of the ammonium groups, proposed to occur upon guest binding in Koga's host, would be avoided. However, Schneider has recently shown that with electron rich aromatic guests, binding to a host related to 2 is in fact assisted by the positively charged group close to the guest (11). Finally, these hosts illustrate the trend toward increasingly rigid hosts. Restricting Conformational Freedom. Our initial goal in this area was to find synthetic methods for making hosts less flexible than the hosts reported by Tabushi and Koga (12). Such preorganization of In Structure and Reactivity in Aqueous Solution; Cramer, Christopher J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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the host was desirable on theoretical grounds: it should lead to enhanced binding affinities because the entropy change on binding (AS) would be less negative (13, 14). Diminishing conformational flexibility would also offer a chance for greater selectivity in binding. To achieve this goal we have used dibenzodiazocine substructures, and two early hosts we prepared incorporating this structural unit are illustrated in Figure 2 (3,13,14). Host 6 has more conformational freedom than host 7 and is the less effective receptor. One important difference between these receptors was revealed by studying the temperature dependence of binding. The entropy change for association of host (6) with 2,4,6-trimethylphenol is -15 cal moHK~*. Usually, hydrophobic association of non-polar solutes is characterized by positive entropy changes, but this negative AS is typical for the less organized synthetic receptors and suggests that the conformational freedom of the unbound receptor is substantially restricted when the guest arrives. In contrast, the entropy change for binding of host 7 to the same guest is +7 cal m o H K ~ l (15). Better organization of the receptor prior to association leads to diminished loss of conformational freedom and stronger binding.

Figure 2. The more rigid cyclophane 7 excludes aliphatic cyclohexanoid substrates. A n x-ray diffraction study verified that the shape and size of host 7 is perfectly suited to aromatic guests (Figure 3). Because the available space is complementary to aromatic guests but is too narrow for an aliphatic guest, cyclohexanoid substrates will not bind to host 7 and binding of aromatic substrates is not inhibited in the presence of menthol or cyclohexanol.

Figure 3. (a) Crystal structure of 7 bound to p-xylene. (b) Packing diagram. In Structure and Reactivity in Aqueous Solution; Cramer, Christopher J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

19. WILCOX ET AL.


To further pursue the rigidification and shape-optimization of these cyclophanes, we developed receptor 5 (Figure 1). It was created based upon a non-macrocyclic receptor that we first reported in 1988 (16,17). Receptor 5 binds cyclohexanoid substrates strongly in aqueous media and reverses the selectivity observed with receptor 7 in that it binds cyclohexanoid substrates better than aromatic substrates. Furthermore, this receptor is diastereoselective. Cyclohexane derivatives that have axial substituents are usually bound less well than diastereomeric derivatives with equatorial substituents. For example (+)-menthol ( K = 3 900 M~l) binds almost 3 times more strongly than (+)-isomenthol (Ka = 1 400 M * 1). Equatorial hydroxyl groups are in general better solvated than axial hydroxyl groups and therefore i f relative K ' s depended only on guest solvation forces then cw-4-tertbutylcyclohexanol with an axial hydroxyl group would bind more strongly than its equatorial diastereomer. However the shape selectivity of receptor 5 overcomes this solvation factor and rran,s-4-tert-butylcyclohexanol binds seven times more strongly ( K = 40 000 M" ) than the cis isomer ( K = 5 900 M " ) . Molecular modeling using Amber and M M 3 force fields allows the possible receptor-substrate interactions to be visualized (18-20). The qualitative results are consistent with the binding studies and indicate that the distance between the sides of receptor 5 is very well suited to forming a sandwich type complex with cyclohexane rings. These calculations also leave no doubt that an axial substituent will not be accommodated in this receptor (Figure 4).


a

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Figure 4. (a) Optimized geometry of menthol bound in receptor 5. (b) Structure of the tetramethyl ester of receptor 5 as determined by x-ray diffraction methods. The effects of temperature on binding of menthol to receptor 5 are particularly interesting because here, for the first time, a synthetic receptor exhibited temperature dependent association consistent with classical hydrophobic binding. As the temperature increased, there was a slight increase in association constant. A van't Hoff plot for the data indicates ΔΗ = +1.5 kcal mol" and AS = +22 cal mol" Κ* . The heats of hydration of alkanes such as menthol are negative while the entropy of hydration is also negative. Positive heats of association (an event leading to dehydration of some hydrophobic surface area) are typical of true hydrophobic binding. 1


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Variations on Receptor 5.


We decided to vary the structure of receptor 5 to examine the effects of systematic structural variation. Three new receptors, 8, 9, and 10, were prepared by using structural components that were regioisomers of the components incorporated in receptor 5. Our interest was in comparing binding affinity and selectivity among these four receptors and in developing computational methods to allow predictions of the relative binding affinities. (See Figure 5.)

Figure 5. Structural isomers of receptor 5. Evaluation of binding. Receptors 8, 9,10, and 5 are structural isomers. N M R titration studies were used to evaluate binding (21,22). A summary of the reported association constants is given in Table I, where data for binding of all four hosts to menthol enantiomers can be compared. Table I. Association constants for cyclophane hosts (8), (9), (10), and (5) Substrate Receptor 8 9 10 5 (-)-menthol 72±7 M " 4300±640 M " 335±6 M " 3±1 M " (+)-menthol a 3900±480 M " l 293177 M " Experiment not performed. 1

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Receptors 8 and 10 exhibit extremely poor complexation with the enantiomers of menthol. In contrast, the association constants ( K ) of receptor 5 with menthol enantiomers are over 3500 M ~ l . The data reveal receptor 9 is not much better, and a



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287 Receptors for Shape-Selective Hydrophobic Binding

binds only modestly with these guests (Kg ~ 300 M~*). What is the reason for these failures? Are accessible computational methods capable of predicting such failures? What went wrong. One important piece of evidence is provided by the *H-NMR data for receptors 8 and 10. In aqueous buffer these receptors gave unusual spectra. The signals expected from the bridging methylene groups of the dianiline unit were shifted far upfield. Normally this signal occurs at about 3.5 ppm. But in aqueous solution the signals for 8 and 10 arose at approximately 1.8 and 0.87 ppm, respectively. This would happen if the protons of the methylene bridge were in the shielding region of one or more aromatic rings. The reason receptors 8 and 10 are so ineffective as receptors in aqueous media is that they collapse ("deflate") to form crescent shaped molecules that have no binding site available. The methylene protons of the bridge are placed directly within the shielding region of the aryl rings of the receptor and large upfield shifts result. We say the hosts "sickle" because the shapes of the receptors are reminiscent of sickled erythrocytes. Figure 6 show receptors 8 and 10 in two possible "sickled" conformations.

(a)

(b) Figure 6. Stereoviews of receptors 8 (a) and 10 (b) in sickled or closed forms. We find that sickling must be a solvation driven phenomenon because it is unique to aqueous solutions. The tetramethyl esters of the macrocycles do not sickle in chloroform. The sickling process involves motions that are too rapid to resolve by N M R . Only a weighted average of the sickled and unsickled receptor resonances could be observed. The barrier to sickling must therefore be less than 18 kcal m o H . Given a crude estimate that the upfield shift of a proton in this situation would be about 2.5 ppm, we can estimate that the host 10 is about 95% in the sickled form, while host 8 may be only about 60% in the sickled form. However, modeling results (discussed below) show that these hosts have alternative ways to eliminate molecular voids and can fold in a manner that would not place the bridging methylene in a strongly shielding environment (Figure 6b). So such very simple interpretations, though tempting, may be quite erroneous. In Structure and Reactivity in Aqueous Solution; Cramer, Christopher J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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Receptor 9 is also inferior to 5, but we propose this is not due to a propensity to invert like receptor 8. N M R data indicate that the bridging diphenylmethane unit is exterior to the cavity and its protons resonate at unexceptional field strengths. We think that in 9, where the 2,7-disubstituted ethenoanthracene unit was used, the distance across the cavity is too small to allow cyclohexanoid rings to easily enter. Conformational analysis of this receptor indicates the cleft is squeezed shut, and has a cleft more narrow than receptor 5 and too small to easily encapsulate a cyclohexane ring. Are such results predictable? These data showed that solvent was an important factor in the sickling process. We carried out Monte Carlo conformational searches for each of the four macrocycles using Still's Generalized Born equation and surface area model, GB/SA (23). The results for receptor 5 calculations using a water model: Of 37 structures located, 14 were sickled, and they ranged in calculated energy up to 16 kcal m o l above the lowest energy structure. The remaining minima had open cavities, ranging in energy from 2.5-7 kcal mol" above the lowest energy conformer. In listing of all conformers in order of conformational energy, there was very little overlap between the domains of sickled and open conformers. In contrast, using a chloroform solvation model we located 49 conformational minima and the differences between sickled and open conformers were much diminished. The most stable open conformer was only 0.4 kcal mol" above the overall minimum, and although the 7 lowest energy conformers were sickled, the preference for sickled conformers was not as strong as it was in water. For receptor 8 the first open conformation was calculated to lie 5 kcal mol" above the overall minimum so these very crude calculations do confirm that receptor 8 is more prone to sickling than receptor 5. N M R data indicate, however, that receptor 5 is not so strongly sickled as these energy calculations suggest. The open form is probably favored for this receptor. Many improvements are required in these calculations. For example it is important to accurately assign atomic charges for all atoms in the receptor. The solvation calculations are very sensitive to atomic charge (23). Most importantly, we need a good way to use these energies to calculate relative free energies of the conformers taking into account the effects of entropy in stabilizing open and sickled forms. The preference for the open form in receptor 5 might be due to the greater internal entropy available to that family of conformational substates. - 1

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Concluding Comments. Systematic variations of this model receptor demonstrated that overall binding energies are very sensitive to small structural modifications. Molecular sub-units which are overly flexible, like the substituted diphenylmethane units, can lead to undesirable diminishment in association energy. They do so because they allow the receptors to deflate or "sickle" by folding in upon themselves. This folding process, like the binding event itself, is driven by hydrophobic forces. Recently we prepared receptor 11 (Figure 7). This receptor eliminates the diphenylmethane unit and cannot sickle. Even though this receptor has rather poor complementarity with menthol, it binds strongly (Ka = 45 000 M " ) . Its free energy of association with menthol is thus about 1.4 kcal/mol greater than that measured for the more flexible receptor 5 - a 25% improvement. Complementarity without rigidity can give poor results (if sickling occurs) or good results (when sickling is not favored). Increasing rigidity, as in 11, when achieved at the expense of complementary structure does not eliminate binding. On the contrary, receptor 11 is far better than receptor 5. Our next goal will be to prepare a receptor that has good shape complementarity (Figure 4) but is more preorganized than receptor 5 and cannot sickle. 1



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Figure 7. A receptor that cannot sickle, but does fold. For confident planning and efficient progress in this field, the molecular engineer requires accurate and conveniently accessible computational tools. The computational chemist requires precise data for testing new methods of calculation. The data obtained for these model receptors can provide a useful challenge to theorists and computational chemists. The uncertainties of measuring enthalpies and entropies of association between proteins and their substrates are large compared to the uncertainties that attend observations on model receptors. The computational chemist can quickly assess the success of an approach by testing it against binding affinities measured for synthetic receptors of the type described here. Predictions made on the basis of calculations can be quickly verified and additional data to test hypotheses arising from the calculations can be obtained at a cost below that required to prepare and characterize mutant proteins. Closer cooperation between computational chemists and synthetic chemists involved in preparing model receptors will accelerate the pace of progress in both fields. Acknowledgment Our work in this area has been supported by a grant from the National Institute of General Medical Sciences. Literature Cited 1. 2. 3.

Tabushi, I.; Kuroda, Y.; Kimura, Y. Tetrahedron Lett. 1976, 8827. Tabushi, I.; Kimura, Y.; Yamamura, K.J.Am. Chem. Soc. 1978, 100, 1304. Murakami, Y.; Aoyama, Y.; Ohno, K.; Dobashi, K.; Nakagawa, T.; Sunamoto, J. J. Chem. Soc. Perkin Trans.I1976, 1320-1326. 4. Cram, D. J.; Steinberg, H. J. Am. Chem. Soc. 1951, 73, 5691-5704. 5. Cram, D. J. in Cyclophanes; Kheen, P. M.; Rosenfeld, S. M., Eds.; Academic Press: New York, NY, 1983; pp 1-21. 6. Tabushi, I.; Yamamura, K. Topics Curr. Chem. 1983, 113, 145-182. 7. Odashima, K.; Itai, Α.; Iitaka, Y.; Koga, K.J.Am. Chem. Soc. 1980, 102, 25042505. 8. Diederich, F. in Cyclophanes; Stoddart, J. F., Ed.; Royal Society: Cambridge, 1991. 9. Petti, Μ. Α.; Sheppodd, T. J.; Barrans, R. E.; Dougherty, D. A.J.Am. Chem. Soc. 1988, 110, 6825-6840. 10. Cowart, M. D.; Sucholeiki, I.; Bukownik, R. R.; Wilcox, C. S. J. Am. Chem. Soc. 1988, 110, 6204-6210. In Structure and Reactivity in Aqueous Solution; Cramer, Christopher J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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11. Schneider, H.-J.; Blatter, T.; Zimmerman, P. Angew. Chem. Int. Ed. Engl. 1990, 29, 1161-1162. 12. Wilcox, C. S. Tetrahedron Lett. 1985, 26, 5749-5752. 13. Wilcox, C. S.; Cowart, M . D. Tetrahedron Lett. 1986, 27, 5563-5566. 14. Wilcox, C. S. in Inclusion Phenomena and Molecular Recognition; Atwood, J. Α., Ed.; Plenum: New York, NY, 1990. 15. Zawacki, F. PhD Dissertation, Univ. of Pittsburgh, 1993. 16. Wilcox, C. S.; Greer, L. M.; Lynch, V. J. Am. Chem. Soc. 1987, 109, 18651867. 17. Webb, T. H.; Suh, H.; Wilcox, C. S. J. Am. Chem. Soc. 1991, 113, 8554-8555. 18. Weiner, S. J.; Kollman, P. Α.; Nguyen, D. T.; Case, D. A. J. Comput. Chem. 1986, 7, 230. 19. Allinger, N. L.; Zhi-qiang, S. Z.; Chen, K. J. Am. Chem. Soc. 1992, 114 61206133. 20. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440. 21. Wilcox, C. S. in Frontiers in Supramolecular Organic Chemistry and Photochemistry; Schneider, H.-J.; Dürr, H., Eds.; VCH:Weinheim, 1991. 22. Glagovich, Ν. M.; Webb, T. H.; Suh, H. Geib, S.; Wilcox, C. S. Proc. Indian Acad. Sci. 1994, (in press). 23. Still, W. C.; Tempczyk, Α.; Hawley, R. C.; Hendrickson, T. J. Am. Chem. Soc. 1990, 112, 6127-6129. RECEIVED April 5, 1994


Designing Synthetic Receptors for Shape-Selective Hydrophobic

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