Enhanced Chiral Recognition by Cyclodextrin Dimers

In this article we investigate the effect of multivalency in chiral recognition. To this end, we measured the host-guest interaction of a β-cyclodextrin dimer with divalent chiral guests. We report the synthesis of carbohydrate-based water soluble chiral guests functionalized with two borneol, menthol, or isopinocampheol units in either (+) or (−) configuration. We determined the interaction of these divalent guests with a β-cyclodextrin dimer using isothermal titration calorimetry. It was found that—in spite of a highly unfavorable conformation—the cyclodextrin dimer binds to guest dimers with an increased enantioselectivity, which clearly reflects the effect of multivalency.


Introduction
Cyclodextrins (CDs) are artificial chiral receptors that bind numerous hydrophobic chiral molecules and are well known as stationary phases in liquid chromatography and capillary electrophoresis [1]. Most research efforts to investigate the interaction of enantiomeric guests with the chiral cavity of CDs have focused on monovalent inclusion complexes with only a single interaction between a hydrophobic guest and a CD host. However, the binding constants of enantiomeric guest molecules usually show only marginal differences and in consequence a very modest degree of chiral recognition is observed [2][3][4][5][6][7]. The introduction of substituents such as nucleobases to the primary face of β-CD leads to an increase in chiral recognition of natural compounds such as borneol and camphor [8]. This effect OPEN ACCESS is based on an additional stabilization induced by secondary interactions such as hydrogen bonding and electrostatics.
It is our hypothesis that multivalent complexes involving multiple host-guest interactions will not only result in an increased overall binding affinity (expressed as ΔG or K a ) but also induce a significant increase in enantioselectivity (expressed as ΔΔG). Multivalency plays a key role in cell-cell and cell-matrix recognition processes in biological systems [9]. In recent years, a variety of model systems to study multivalency were investigated. Such model systems include dendrimers [10], polymers [11], nanoparticles [12], vesicles [13,14] and self-assembled monolayers [15]. In the majority of cases, multivalent interactions are additive-not cooperative-and lead to higher overall binding affinities (K a ), lower dissociation rates (k d ), and higher selectivities.
As a proof-of-principle, we investigated the divalent interaction of dimers of chiral guests with β-CD dimers. CD dimers with various linkers are readily available and their interaction with monovalent as well as divalent guests has been investigated in detail, although mainly with achiral hydrophobic guests [16][17][18][19][20][21][22][23][24][25][26][27]. The interaction of CD dimers with divalent guests can either lead to divalent 2:2 macrocyclic complexes or extended supramolecular polymers, depending on the geometry and the concentration of host and guest [28][29][30]. In some cases it is possible to switch between monovalent, divalent and multivalent complexes by external stimulation [31,32].
In this contribution, we report the interaction of chiral divalent guest molecules based on borneol, isopinocampheol and menthol. We investigated whether a change from a monovalent (1:1) to a divalent (2:2) complexation leads to a significant increase in binding affinity and/or selectivity. To this end, water soluble dimers of borneol, isopinocampheol and menthol in (+) and (-) configuration as well as a β-CD dimer were synthesized. The thermodynamic parameters of the interaction between the dimers and β-CD and the β-CD dimer, respectively, were measured by isothermal titration calorimetry (ITC).

Results and Discussion
To investigate the chiral recognition between guest and host dimers, a set of water soluble molecules based on menthol, borneol and isopinocampheol either in (+) and (-) configuration were synthesized. The water solubility was improved by conjugation of a maltose moiety to the spacer that links the two hydrophobic guests. The synthesis is outlined in Scheme 1. The secondary chiral alcohols (either in (+) or (-) configuration) were converted into the carboxylic acid derivatives 1a-1f via Williamson ether synthesis by reaction with bromoacetic acid using sodium hydride as a base. Maltose was peracetylated and converted into azide 2. Synthesis of the dimers started out from propargylamine which was converted into the corresponding dimethylester 3 via Michael addition. By reacting 3 with an excess of ethylenediamine, alkyne functionalized diamine 4 was obtained in quantitative yield. The hydrophobic guest units 1a-f were coupled to 4 under peptide coupling conditions with EDCI and NMM in DMF to provide 5a-f. Azide 2 was conjugated to 5a-f by a Cu(I) catalyzed click reaction to give 6a-f. Dimers 7a-f were obtained by deprotection of the acetyl functions using Zemplèn conditions. Experimental details and analytical data are provided as supporting information. A selection of 1 H and 13 C NMR spectra is also provided. The NMR and MS data are consistent with the molecular structures. The dimeric CD host was synthesized according to a known literature procedure [33]. However, in contrast to the literature procedure, the peracetylated monoazide of β-CD was used. The synthesis is outlined in Scheme 2. The β-CD was converted to the monoazide 10 by reaction of monotosylate 9 with sodium azide. After peracetylation of 10, the protected monazide CD 11 was obtained in good yield. After Cu(I) catalyzed click reaction of CD 11 with dialkyne linker 12 followed by deprotection of the acetyl functions under Zemplèn conditions, the CD dimer 14 was obtained in good yields. Experimental details and analytical data are provided as supporting information. The NMR and MS data are consistent with the molecular structures.
The chiral recognition of the guest molecules 7a-f with β-CD as well as with CD dimer 14 as host molecules was investigated using ITC. In all ITC experiments, guests 7a-f were titrated into host 14 (or β-CD) in water at 25 °C. Deionized water (rather than buffer solutions) was used for all ITC experiments, since both hosts and guests are non-ionic and buffers might complicate the ITC analysis due to salt effects. In order to compare the ITC data for the monovalent interaction with β-CD and the divalent interaction with dimer 14, the concentration of guests and hosts were set as follows: [1a-f] = 5 mM, [β-CD] = 1 mM, [14] = 0.5 mM. Two representative titrations are shown in Figure 1. Additional titrations are provided in the supporting information. The ITC data are summarized in Table 1    It was o binding con K a = 0.83and entropic of the free se The inte K a = 3.14-However, as han anticip binding proc binding even concentratio observed th nstants wit -2.00 × 10 4 cally favour econdary al eraction of -11.21 × 1 s will be sh pated on the cess can be nt, which c on C eff [25].
Stepwi molecule.  The first binding step can be interpreted as an intermolecular binding event with binding constant K 1 , which is directly correlated to the intrinsic binding constant K i . The factor 4 in Equation 1 is based on the four degenerate binding modes for the first complexation step.

Overview
The second binding step with binding constant K 2 is an intramolecular process which can be expressed by the intrinsic binding constant K i and the effective concentration C eff . C eff describes the concentration of host available to the second guest unit after the first guest unit is bound to the dimer. This expression is used to differ between inter-and intramolecular complexation. The factor ½ in Equation 2 is due to the fact that the concentration of free host halves after the first complexation step.
Since the overall binding constant K a is the product of K 1 and K 2 , it is possible to calculate C eff from K a and K i .
Alternatively, a theoretical approximation of C eff can be obtained from Equation 5.
Equation 5 describes the possibility of two linked chain ends to react in a defined volume. In Equation 5, N Av is the Avogadro constant and r 0 is the distance between the two guest moieties of 7a-f. For these guest dimers r 0 was estimated to be around 2 nm. According to Equation 5, a C eff ≈ 50 mM would thus be expected for a divalent interaction of guests 7a-f with host 14.
However, the ITC data indicate an interaction that is far from optimal. K a can be directly obtained from the ITC titration of 7a-f with 14, while K i can be obtained from the titrations with β-CD. Equation 4 then provides the values for C eff . It can be seen from Table 1 that the observed C eff is in the range 0.126-0.268 mM which is two orders of magnitude lower than the value calculated from Equation 5! In other words, the interaction of 7a-f is only partially divalent, and a substantial fraction of the divalent guest is bound in a monovalent mode only. An explanation for this unexpected finding was reported by Monflier and coworkers during the course of our investigation [34]. This group showed that a single glucose unit of the β-CD in dimer 14 forms a rotational isomer which leads to a pseudorotaxane-like structure (Figure 3). This preferred conformation leads to an effective blocking of one cavity, which strongly diminishes the binding behavior towards the chiral dimers 7a-f. Indeed, as shown in Table 1, the binding stoichiometry of 14 and 7a-f is not 1:1 as expected but significantly lower, indicating that not all CD cavities are available for complexation. This behavior is in good agreement with the 1 H-NMR spectra of 14 which shows doubled signals for the aromatic protons [33]. The triazole protons give three particularly well-separated signals: two for the unsymmetric pseudorotaxane like structure and one for the free symmetric CD dimer. In the study by Monflier and coworkers, the amount of the symmetric species in aqueous media was determined to be as little as 20%. It was also shown that a monovalent interaction with adamantyl carboxylate does not lead to a

Conclusions
A new class of water soluble chiral guest dimers based on the (+) and (-) enantiomers of borneol, menthol and isopinocampheol was synthesized. In addition, a CD dimer was prepared. The formation of divalent inclusion complexes between the guest dimers and the CD dimer was measured by using ITC. The data obtained were compared with the monovalent interaction of the guest dimers with unmodified β-CD. It was found that the dimer largely isomerizes into a pseudorotaxane that preferentially binds in a monovalent rather than a divalent fashion. Nevertheless, it was shown that chiral recognition is amplified even if a substantial fraction of the cavities of the CD dimer are not available for complexation. In retrospect, it is obvious that chiral recognition can be significantly improved by a better choice of the CD dimer, for example by using a hydrophilic linker. Furthermore, it is anticipated that higher order multivalent interactions (e.g., of trimers and polymers) will show much higher enantioselectivities.