& Host–Guest Systems

Allosteric Binding of Capsaicin by a Bis(b-cyclodextrin)-2,2’-bipyridine Receptor

Christopher Kremer and Arne L�tzen*[a]

Dedicated to Prof. Dr. Julius Rebek, Jr. on the occasion of his 70th birthday

Abstract: A new b-cyclodextrin-based receptor thatshowed allosteric binding behavior towards capsaicin inaqueous solution was prepared. By NMR titration and non-linear regression, we obtained binding constants, whichincreased more than fivefold when an effector (Zn2+) wasbound to a central 2,2’-bipyridine that acts as the alloste-ric center.

Allosteric regulation is one of the most important mechanismsin nature to control functions of certain proteins and enzymes.Therefore, it is hardly surprising that in the years after 1961,when this mechanism was first discovered by Monod andJacob, allosteric recognition has gained wide interest in thefields of medicine, as well as in chemistry.[1] In the last years,great progress has been made to implement this principle intosupramolecular host–guest systems to control the binding af-finity of receptors towards their guest molecules.[2]

The term allosteric was derived from the Greek words allw&

(allos, the other) and ste1e�& (stere�s, solid (object)) andstands for cooperative effects in the selective binding of morethan one substrate to different binding sites of a receptor.These effects result in a conformational change of the hostafter the binding of the first sub-strate (also called the effector),which influences its binding af-finity towards the other sub-strate(s) in a positive (positive al-losteric regulation) or a negativemanner (negative allosteric regu-lation).

2,2’-Bipyridine showed to beideally suited as allosteric centerdue to its ability to switch be-tween its anti- and syn-confor-mations upon binding to suita-

ble transition-metal ions as effectors,[3] and thus, it is a widelyused motif ever since Rebek and co-workers created the firstallosteric system based on 2,2’-bipyridines in 1979.[4] However,only a handful of the receptor systems based on this motif re-ported to date are able to bind neutral molecules.[5]

In our group, we synthesized several systems based on 2,2’-bipyridine as allosteric center and two resorcin[4]arenes to actas artificial receptors for nonpolar guest substrates.[6] However,the cavities provided by those proved to be rather small forour purposes.

In a second approach, we utilized b-cyclodextrins due totheir known ability to bind a great variety of nonpolar sub-strates[7] and their tunable solubility, which we connected tothe bipyridines by thiourea moieties.[8] Herein, we report thenew, water-soluble derivative 1 of these systems and its allos-terically controlled binding behavior towards capsaicin.

The synthesis of 1 was achieved by linking 4,4’-diisothiocya-nato-2,2’-bipyridine 3[9] to peracetylated 6A-amino-6A-deoxy-b-cyclodextrin 4, gaining the fully acetyl protected compound 2,which was deacetylated by using sodium methoxide andsodium hydroxide (Scheme 1).

Compound 1 formed a [Zn(1)2](OTf)2 complex upon additionof Zn(OTf)2. In principle, this is fine, binding to one effectorcauses the formation of two cavities each consisting of two cy-clodextrin moieties that can both bind one guest molecule.

However, a 1:2 metal-to-ligand stoichiometry is not very con-venient for obtaining quantitative information about the bind-ing behavior, because this could lead to an equilibrium of sev-eral possible host-to-guest stoichiometries (1:0, 1:1, 1:2), whichis difficult to analyze. To circumvent this problem and quantifythe potentially positive allosteric effect, we used stericallycrowded phenanthroline ligand 5 in combination with zinc(II)

Figure 1. Structure of capsaicin and phenanthroline ligand 5.

[a] C. Kremer, Prof. Dr. A. L�tzenKekul�-Institut f�r Organische Chemie und BiochemieRheinische Friedrich-Wilhelms-Universit�t BonnGerhard-Domagk-Strasse 1, 53121 Bonn (Germany)Fax: (+ 49) 228-73-9608E-mail : arne.luetzen@uni-bonn.de

Supporting information for this article is available on the WWW underhttp ://dx.doi.org/10.1002/chem.201403503.

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CommunicationDOI: 10.1002/chem.201403503

ions (Figure 1).[6b] According to the general concept of hetero-leptic phenanthroline complexes (“HetPhen”) that was estab-lished in 1997 by Schmittel and L�ning,[10] this phenanthrolineligand prefers to form heteroleptic complexes with suitabletransition-metal ions and sterically noncrowded phenanthro-line or 2,2’-bipyridine ligands. The ethylene glycol units make 5fairish soluble in aqueous solutions. By using this concept, wecould synthesize a 1:1:1-complex of 1, 5 and Zn(OTf)2, whichcould be characterized by 1H NMR and mass spectrometryanalyses (see the Supporting Information).

In the next step, we quantitatively analyzed the bindingproperties of 1 and its complex [Zn(5)(1)](OTf)2 towards capsai-cin (Figure 1). Capsaicin is known as the “hot” ingredient ofpeppers, but has also antimicrobial, as well as anti-insect, prop-erties and is thus very interesting for agricultural purposes.[11]

Furthermore, it is known to have antihypertensive[12] and possi-bly antitumor qualities.[13] There is one example for a 1:1 cap-saicin-b-cyclodextrin complex, having a binding constant of1187 m

�1.[14]

First, we examined the binding stoichiometry of 1 or itscomplex with 5 and Zn2 + , respectively, on the one hand, andcapsaicin on the other hand, by recording the 1H NMR signalsof the terminal methyl groups of capsaicin in different solu-tions, varying the concentrations according to the method ofcontinuous variation (also known as Job’s plot).[15] We foundthe stoichiometry to be 1:1 in both cases. Figure 2 shows onepossible conformation of the ternary effector host–guest com-plex that we obtained by semi-empirical calculations (PM6).[16]

To obtain the binding constants, we performed NMR-titra-tion experiments, varying the concentration of the host, andmeasuring once again the shifts of the terminal methyl groups

of capsaicin. Because the exchange of capsaicin between thecavity of 1 and the solution is fast on the NMR time scale, weused nonlinear regression methods for obtaining the binding

Scheme 1. Synthesis of 1.

Figure 2. Model of one conformer of the complex 1 + 5 + Zn2+ with capsai-cin based on semi-empirical PM6-calculations. For more clarity, capsaicin isdisplayed in the blue space-filling presentation, carbon atoms of 5 are inblack, and carbon atoms of 1 in grey. Hydrogen atoms are omitted for clari-ty. Further colors : zinc(II) green, nitrogen blue, sulfur yellow, oxygen red.

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constants from the observed dif-ferences in chemical shifts. Forsystem 1 in the “off” state (notcoordinated to a metal ion), wecalculated a binding constant of1380�40 m

�1, which is well inthe order of the results of Shenet al.[14] However, for the systemin the “on” state (complex of 1,5 and Zn2+), we obtained7420�720 m

�1, which is morethan five times higher than inthe “off” state. This result nicelyshows that the concept of allo-steric regulation fits perfectly forthis system. These values couldfurthermore be verified by ana-lyzing the binding behavior ofzinc(II) triflate (5) or the complexof 5 and zinc(II) triflate alone to-wards capsaicin, which wereeither very small (�50 m

�1) oreven too small to be measured,and hence, negligible(Scheme 2).

In a second step, we wantedto examine if the step from the“off” state to the “on” statecould be reversed again by theaddition of ethylenediamine-tetraacid (EDTA) disodium salt.EDTA is a far better ligand forZn2+ than 1 or 5. We obtaineda binding constant of 210�20 m

�1. This value, which is considerably smaller than the onereceived from pure 1, can be explained by a competition ofseveral possible guest molecules that fit into the host’s cavityafter this operation: not only capsaicin can be taken up, butalso phenanthroline 5 and potentially even the Zn–EDTA com-plex.[17]

In summary, we were able to synthesize and evaluate b-cy-clodextrin-based receptor 1, which showed positive allostericbinding behavior towards capsaicin: binding in the “on” stateobtained upon coordination of a [Zn(5)]2 + complex fragmentas an effector is more than five times better than in the “off”state, in which 1 is not coordinated to a metal ion. Unfortu-nately, the use of [Zn(5)]2 + as the effector did not allow multi-ple switching between the “off” and the “on” states, because 5and/or the Zn2+–EDTA complex became competitive sub-strates for the cyclodextrin cavities. Hence, future work will befocused on the search and use of other effectors that do notcause such problems. Nevertheless, these results pave theavenue for the development of powerful functional supra-molecular receptors, functions of which can be regulated by al-losteric effects.

Acknowledgements

Financial support from the DFG (SFB 624) is gratefully acknowl-edged. C.K. thanks the Studienstiftung des deutschen Volkesfor a doctoral scholarship.

Keywords: allosteric regulations · bipyridines · capsaicin ·cyclodextrins · host–guest systems

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Scheme 2. Binding model of the positive allosteric binding behavior of 1 towards capsaicin regulated by thezinc(II) complex of phenanthroline ligand 5. [a] Smaller binding constant compared to the binding of capsaicin by1 alone due to the presence of competing guests (EDTA–Zn2 + complex, phenanthroline 5).

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Chem. 2000, 112, 975 – 977; Angew. Chem. Int. Ed. 2000, 39, 945 – 947;c) Y. Tomohiro, A. Satake, Y. Kobuke, J. Org. Chem. 2001, 66, 8442 – 8446;d) T. Haino, Y. Yamanaka, H. Araki, Y. Fukazawa, Chem. Commun. 2002,402 – 403; e) T. Nabeshima, A. Hashiguchi, Tetrahedron Lett. 2002, 43,1457 – 1459; f) M. H. Al-Sayah, R. McDonald, N. R. Branda, Eur. J. Org.Chem. 2004, 173 – 182; g) F. Durola, J. Rebek, Jr. , Angew. Chem. 2010,122, 3257 – 3259; Angew. Chem. Int. Ed. 2010, 49, 3189 – 3191.

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[17] Please note that adding an excess of zinc(II) to the mixture of capsaicin,1, 5, Zn2 + , and EDTA did not allow to reestablish the formation of the[Zn(5)(1)]2 + complex. However, one might be able to overcome thisproblem by just using zinc(II) ions as an effector, because this might bemuch easier to remove from the [Zn(1)2]2 + complex that we have ob-served. Although one might not be able to determine accurate bindingconstants for this systems, it might still be very useful from a functionalpoint of view, for example, for the extraction or transport of nonpolarsubstrates, such as capsaicin especially, because it can bind up to twoguest molecules.

Received: May 13, 2014

Published online on June 20, 2014

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