Conformationally Mobile Acyclic Cucurbit[n]uril-Type Receptors Derived from an S-shaped Methylene Bridged Glycoluril Pentamer
We report the synthesis of the conformationally mobile S-shaped glycoluril pentamer building block 3a and two new acyclic CB[n]-type receptors P1 and P2. P1 (9 mM) and P2 (11 mM) have moderate aqueous solubility but their host\xe2\x80\xa2guest complexes are poorly soluble. Host P1 does not undergo intermolecular self-association whereas P2 does (Ks = 189\xc2\xb127 M\xe2\x88\x921). 1H NMR titrations show that P1 and P2 are poor hosts toward hydrophobic (di)cations 6 \xe2\x80\x93 11 (P1: Ka = 375\xe2\x80\x931400 M\xe2\x88\x921; P2: Ka = 1950\xe2\x80\x9319800 M\xe2\x88\x921) compared to Tet1 and Tet2 (Tet1: Ka = 3.09 \xc3\x97 106 to 4.69 \xc3\x97 108 M\xe2\x88\x921; Tet2: Ka = 4.59 \xc3\x97 108 to 1.30 \xc3\x97 1010 M\xe2\x88\x921). Molecular modelling shows that P1 and P2 exist as a mixture of three different conformers due to the two S-shaped methylene bridged glycoluril dimer subunits that each possess two different conformations. The lowest energy conformers of P1 and P2 do not feature a well-defined central cavity. In the presence of guests, P2 adapts its conformation to form 1:1 P2\xe2\x80\xa2guest complexes; the binding free energy pays the energetic price of conformer selection. This energetically unfavorable conformer selection results in significantly decreased Ka values of P1 and P2 compared to Tet1 and Tet2.
Introduction.<!>Results and Discussion.<!>Goal of the Study.<!>Synthesis and Characterization of Pentamer Bis(Cyclic Ether) 3a and Hosts P1 and P2.<!>Conformational Properties of Pentamer Derived Hosts P1 and P2.<!>Self-Association Studies Performed for P1 and P2.<!>Attempted Use of P1 and P2 as Solubilizing Excipients for Insoluble Drugs.<!>Qualitative 1H NMR Investigations of Host\xe2\x80\xa2Guest Recognition.<!>Measurement of the Host\xe2\x80\xa2Guest Binding Constants.<!>Modelling of the Conformations of Host-Guest Complexes.<!>Discussion of the Trends in the Binding Constants.<!>Conclusions.<!>Computational Details.<!>Experimental Details.<!>Compound 3.<!>Host P1.<!>Host P2.
<p>Over the past two decades, there have been great advances in the preparation of members of the cucurbit[n]uril (n = 5, 6, 7, 8, 10, 13–15, Figure 1) family of molecular container compounds.1 The defining features of CB[n] molecular containers are their two symmetry equivalent ureidyl carbonyl portals which are highly electrostatically negative and their central hydrophobic cavity.2,3 Given these structural features, CB[n] are excellent hosts for hydrophobic (di)ammonium ions which often bind with Ka values in the 106 – 109 M−1 range and in select cases with Ka values exceeding 1012 M−1 in aqueous solution.3,4 The high affinity of macrocyclic CB[n]•guest complexes has been traced to the presence of high energy waters in the cavity of CB[n] that are released upon complexation.5 CB[n] hosts are also quite selective and large differences in Ka values are seen upon application of suitable stimuli (e.g. pH, electrochemical, photochemical).6,7 Accordingly, CB[n] have emerged as an outstanding platform for the development of functional supramolecular systems including chemical sensors, molecular machines, supramolecular polymers and materials, and drug delivery systems.7,8 In recent years, the development of methods to prepare per- and mono-functionalized CB[n] hosts have allowed their strategic merger with polymers, solid phases, surfaces, nanoparticles, targeting ligands, antibodies, and fluorophores which has further extended their applicability in the chemical, biological, and biomedical arenas.9</p><p>Over the years, the Isaacs and Šindelář groups have been very interested in the mechanism of CB[n] formation, especially the formation of the S-shaped and C-shaped diastereomeric methylene bridged glycoluril dimers.10,11–13 The S-shaped forms are kinetic products whereas the C-shaped forms are the thermodynamic products which eventually lead to macrocyclic CB[n] by cyclooligomerization.14 Inverted CB[n] (i-CB[n], Figure 1) have also been isolated;15
i-CB[n] feature a pair of methine H-atoms pointing into the CB[n] cavity and possess a pair of adjacent S-shaped units. The binding affinity of i-CB[n] (n = 6, 7) toward typical hydrophobic ammonium ions are weaker than the corresponding diastereomeric CB[n].15 More recently, we have described the preparation of a class of receptors that feature a central glycoluril oligomer that is capped with two terminal aromatic walls (e.g. Tet1 and Tet2, Figure 1).16,17 By virtue of their glycoluril oligomer backbone, these receptors are preorganized into a C-shape and retain the essential binding features of the CB[n] family (e.g. tight and selective recognition of hydrophobic (di)cations). Accordingly, these hosts are referred to as acyclic CB[n]-type receptors. Numerous variants have been created, by us and others, that differ in the nature of the central glycoluril oligomer, the terminal aromatic walls, and the appended solubilizing groups.16,18 Of the acyclic CB[n] based on glycoluril tetramer prepared to date, Tet1 and Tet2 have been used extensively because of their high binding affinity which has enabled their function as solubilizing excipients for insoluble drugs and as in vivo sequestration agents for neuromuscular blockers (e.g. rocuronium and vecuronium)19,20 and drugs of abuse such as methamphetamine.21 Host Tet1 has displayed excellent biocompatibility according to the usual in vitro (e.g. cell death and metabolic activity, mutagenicity, lack of hERG ion channel inhibition) and in vivo (e.g. maximum tolerated dose, blood gases, blood pH, mean arterial pressure) assays.16 The group of Prof. Ruibing Wang has used macrocyclic CB[7] as a sequestering agent in related applications.22 Previously, we have prepared and studied the molecular recognition properties of analogues of Tet1 and Tet2 based on central glycoluril monomer, dimer, and trimer (Mon1 – Tri1 and Mon2 – Tri2).23 We found that Mon1 – Tri1 and Mon2 – Tri2 do not function well as solubilizing agents for insoluble drugs due to their smaller cavities which result in lower binding constants. Accordingly, we wondered whether acyclic CB[n]-type receptors that feature an extended glycoluril oligomer (e.g. pentamer) might display higher binding affinity toward their guests than Tet1 and Tet2 and therefore function as superior sequestration agents. In this paper, we describe the synthesis of pentamer derived acyclic CB[n] hosts P1 and P2 and investigations of their molecular recognition properties.</p><!><p>This results and discussion section is organized as follows. First, we describe the design, synthesis, and characterization of hosts P1 and P2. Second, we measure the solubility of P1 and P2 in water. Third, we performed 1H NMR titrations to determine the Ka values of P1 and P2 toward guests 6 – 11. Finally, we discuss the trends in Ka values of P1 and P2 toward their guests using Tet1 and Tet2 as comparators and rationalize the observed changes based on molecular modelling studies.</p><!><p>The initial goal of this study was to determine the impact of the extension of the glycoluril oligomer backbone from tetramer (e.g. Tet1 and Tet2) to pentamer on the molecular recognition properties toward typical cationic guests. In the process, however, we uncovered that the synthesized pentamer hosts P1 and P2 feature two S-shaped units that endow them with conformational flexibility. Accordingly, we expanded our study to include the influence of conformational isomerism on guest binding.</p><!><p>To prepare acyclic CB[n]-type hosts derived from glycoluril pentamer, we took advantage of a building block approach that relies on the double electrophilic aromatic substitution reaction between a central glycoluril oligomer and a dialkoxy aromatic wall. The condensation of glycoluril (1) with dimethylglycoluril bis(cyclic ether) (2) which was conducted at lower temperature (90% aq. MeSO3H, 8–12 °C) in order to control the oligomerization process gave a complex crude reaction mixture from which we could isolate a single methylene bridged glycoluril pentamer (3) in gram scale batches (Scheme 1). The ES-MS spectrum of 3 confirms its molecular formula (C38H46N20O12) and that it is composed of two equivalents of 1 and three equivalents of 2. Because the substituents on the convex face of adjacent glycoluril rings may point in the same or opposite directions (e.g. C-shaped or S-shaped units), there are 10 possible diastereomers of 3. The four diastereomers (3a – 3d) depicted in Scheme 1 are C2v-symmetric whereas the six diastereomers that are not shown are Cs-symmetric. The 1H NMR spectrum of 3 recorded in DMSO-d6 shows three distinct resonances for the methyl groups (Hk – Hm), three pairs of doublets for the diastereotopic methylene bridges (Hc – Hh), and a pair of doublets for the glycoluril methines (Hi and Hj). Similarly, the 13C NMR spectrum of 3 in DMSO-d6 displays a total of 14 resonances which is only consistent with a C2v-symmetric structure. Compound 3c corresponds to the desired glycoluril pentamer that consists of all C-shaped subunits. In contrast, 3a and 3b contain 2 S-shaped and 2 C-shaped segments whereas 3d possesses 4 S-shaped segments. Given the known thermodynamic preference for the C-shaped diastereomers,10,13 we initially presumed that we had isolated 3c and subsequently proceeded to create the pentamer derived hosts. It was only later, after observing the poor molecular recognition properties of P1 and P2 that we discovered that in reality we had isolated 3a. The relative stereochemistry of 3a, P1, and P2 were fully assigned by a combination of 1H, 13C, selective 1D NOE, and 2D NMR experiments (Supporting Information). In brief, once the 1H NMR has been fully assigned, we can use the selective 1D NOE experiments to step from the cyclic ether termini of 3a toward the center determining relative stereochemistry at each step along the way. As shown in Figure 2, Hc, Hd, and (CH3)l show NOEs when (CH3)k is irradiated. The resonance for (CH3)l shows NOEs to He, Hf, and most importantly Hi which establishes the C-shaped relative stereochemistry of the terminal pairs of glycolurils. Proton Hi is coupled to and shows an NOE to the adjacent Hj. Irradiation of Hj shows a main NOE to Hj and very small NOEs to Hg and Hh but does not show an NOE to (CH3)m which establishes that the central glycoluril is connected to its neighbors by S-shaped stereochemistry. The identity of Hg and Hh as adjacent to the central glycoluril was confirmed by HMBC cross peaks from Hg and Hh to the central O=Cy which is a half-intensity resonance. From our previous studies of methylene bridged glycoluril dimers we know that the CH2-bridges involved in S-shaped connections have smaller 3JHCH coupling constants that those involved in C-shaped connections (≈13.6 Hz vs. ≈16.0 Hz).10,11,13 The observed coupling constant between Hg and Hh (3JHCH = 13.7 Hz) of 3a further supports the depicted diastereomer.</p><p>The sulfonated dialkoxy benzene and dialkoxy naphthalene sidewalls (4 and 5) required to synthesize hosts P1 and P2 were available from previous studies.24 As shown in Scheme 2, the attachment of walls 4 and 5 to pentamer 3a was conducted under acidic conditions (TFA, 75 °C) in the presence of Ac2O to increase reactivity25 to deliver P1 (22%) and P2 (27%) after purification by trituration with hot water (P1) and by precipitation from water (P2). Hosts P1 and P2 were characterized by spectroscopic means. The high resolution electrospray ionization mass spectra for P1 displayed an ion at m/z 547.8031 ([M-4Na+H]3-, calculated for C62H75N20O26S4 547.8020) whereas P2 displayed an ion at m/z 872.2222 ([M-4Na+2H]2-, calculated for C70H80N20O26S4 872.2223) which establish the molecular formulas required of P1 and P2. Figure 3 shows the 1H NMR spectra recorded for P1 and P2 in DMSO-d6. As expected, host P1 exhibits a singlet for the four symmetry equivalent Ha protons, three resonances for the methyl groups (Hk, Hl, Hm), a pair of doublets for the equatorial methine protons (Hi and Hj), three pairs of doublets for the bridging methylene groups (Hc – Hh) in the required 4:4:4:4:4:4 ratio, and three resonances for the O(CH2)3SO3Na sidearms expected for a C2v-symmetric structure (Figure 3a). Figure 3b shows the fully assigned 1H NMR spectrum for P2 which displays a similar pattern of resonances in accord with C2v-symmetry. The 13C NMR spectra recorded for P1 (P2) display 20 (22) resonances (Supporting Information, Figures S10 and S21) in accord with the 20 (22) resonances expected based on C2v-symmetry. As described above for 3a, the relative stereochemistry of the glycoluril units of P1 and P2 was established based on the combined inference of 1H, 13C, COSY, HSQC, HMBC, and NOE experiments (Supporting Information). After having firmly established the constitutions and relative stereochemistry of P1 and P2 we moved on to determine their inherent solubility in aqueous solution. For this purpose, samples of P1 and P2 were weighed on a microbalance and then dissolved in at room temperature in the smallest amount of D2O possible with the aid of sonication; the obtained solubility of P1 (≈ 9 mM) and P2 (≈ 11 mM) was then calculated in the standard way using the known mass, molecular weight, and volume.</p><!><p>The chemical structures of 3a and hosts P1 and P2 contain two S-shaped connections between adjacent glycolurils (e.g. the substituents at the equator of the glycoluril units are on opposite sides of the oligomer chain). From previous work, we know that each S-shaped segment can adopt two different conformations where the substituents on the convex face of one glycoluril point toward the concavity of the other glycoluril and vice versa.10,11,13 Accordingly, there are three distinct conformations for compounds 3a, P1, and P2; Figure 4 depicts the three conformers (folds) of P1 which we refer to as P1-F1, P1-F2, and P1-F3. From previous work, we also know that symmetrical S-shaped methylene bridged glycoluril dimers bearing H-atoms13 or CO2Et groups10,11 on their convex face undergo fast conformational exchange processes between the two chemically equivalent and isoenergetic S-shaped conformations such that the H-atoms on the bridging CH2-groups are rendered chemically equivalent and appear as a singlet in the 1H NMR spectrum. Finally, the sharp 1H NMR spectra observed (Figure 3) for P1 and P2 display the number of resonances expected for C2v-symmetric P1-F1 or P1-F3 but not for Cs-symmetric P1-F2. This result indicates that P1 (P2) is either fixed in the P1-F1 or P1-F3 (P2-F1 or P2-F3) folded form or is undergoing fast conversion between all three conformers on the chemical shift timescale. Experimentally, no significant changes in the 1H NMR of P2 were observed upon cooling to 10 °C in D2O. It should be noted that P1-F1 features two terminal molecular clip-like clefts26 shaped by one aromatic wall, whereas P1-F3 possesses a potential cavity that is reminiscent of i-CB[n]. The flexibility of sidearms in P1-F3 does not forbid induced cavity formation upon guest binding.</p><p>To gain insights into the relative populations of the different conformational states, computational methods using DFT were employed. To decipher the structural features of the methylene bridged glycoluril pentamer containing an inverted glycoluril unit, model systems comprising the glycoluril trimers TriMe and TriH (Figure 5) were investigated. Based on our benchmarking of computational methods (Supporting Information, Tables S7, S8, S9, S10, S11), we employed the low-cost method B97–3c for geometry optimization and the accurate hybrid DFT functional PBE0 corrected for dispersion interactions (D3BJ) for final energy consideration. Both geometry and final energy calculations were performed in an implicit water environment provided by the SMD model. This model offers both polar and non-polar contributions to the solvation energies, and it is thus suitable for consideration of thermochemistry. The relative conformer stabilities obtained at the PBE0-D3BJ-SMD/def2-TZVPP//B97-3c-SMD level of theory are given in Table 1 (Supporting Information, Tables S12 and S13 contain detailed data). For TriH, the three forms were of nearly equal relative energy (TriH-F3: 0.0 kcal mol−1; TriH-F2: 0.2 kcal mol−1; TriH-F1: 0.6 kcal mol−1) as expected based on literature precedent for the corresponding glycoluril dimers. In sharp contrast, for TriMe, the three folded forms are predicted to be of very different energies (TriMe-F3: 5.1 kcal mol−1; TriMe-F2: 3.5 kcal mol−1; TriMe-F1: 0.0 kcal mol−1). The lower stabilities of the F2 and F3 conformers of TriMe is caused by solvent and internal contributions, but the data obtained in vacuum (Supporting Information) indicate that steric factors also make a significant contribution. We believe that the steric bulk of the methyl groups on the central glycoluril of TriMe effectively dictates that the molecule folds into the TriMe-F1 form to avoid placing the Me-groups into the concavity of the adjacent glycoluril rings. Several aspects of the geometries of the different folded structures of TriMe and TriH are noteworthy. For example, a comparison of the terminal H3C•••CH3 distance dMM for TriH-F1 is 9.231 Å, whereas the corresponding distance for TriMe-F1 is 8.978 Å. The shorter dMM distance in TriMe arises from the increased curvature of the glycoluril trimer due to the presence of the methyl groups on the concave face. In contrast, the H3C•••CH3 distance for TriH-F3 is 9.724 Å, whereas the corresponding length for TriMe-F3 is increased to 13.841 Å. Rather than deforming by changing the curvature of the glycoluril trimer as observed for F1, the F3 folded form of TriMe releases the tension by an end-to-end twisting of the glycoluril trimer unit. This twisting was observed computationally for all structures. We quantified the twist by two dihedral angles, τ1 and τ2, describing the local (on the inverted glycoluril) and the global twist of glycoluril ribbon (Supporting Information, Tables S11 and S13; τ2 is defined in Figure 5a). Due to the aforementioned steric factors, the global twist was found to be more pronounced for TriMe-F3 (τ2 = 57.7°) than for TriH-F3 (τ2 = 0.2°).</p><p>The use of glycoluril substituents as a conformational control element in this context is new.27 Our intention is to use P1 or P2 as a host for alkylammonium ions via its F3 folded form, which will require us to pay an energetic penalty to bias the conformational ensemble toward the P1-F3 or P2-F3 folded forms. Of course, the energetics of the F1 – F3 folded forms of P1 and P2 will be modified by the presence of the additional glycolurils and terminal aromatic rings which may bias the conformational ensemble toward the F3 form due to π−π interactions. To get at these questions, computational methods were employed. We used simplified models of P1 and P2 with removed solubilizing groups; these models are labeled as P1' and P2'. By this simplification, we tried to avoid possible problems with a not well-defined conformational preference of the flanking O(CH2)3SO3− groups and the presence of negative charge (−4), which could be problematic for reliable DFT quantum chemical calculations. The computations of P1' and P2' were performed with the same methodology used for TriMe and TriH and the calculated relative conformational stabilities of P1' and P2' are provided in Table 1. In the case of P1', calculations revealed conformational preferences very similar to TriMe. This indicates that the conformational preferences are mainly dictated by the presence of the double S-shaped central glycoluril rather than the aromatic sidewalls. Interactions between the aromatic side walls of P1' and the central methyl groups (P1'-F2) or with the second aromatic sidewall (π−π stacking, P1'-F3) were observed, but these interactions are not large enough to counterbalance other effects such as solvation (see Table S15 for energy decomposition). In the case of P2', the difference between the F1 and F2 (+1.8 kcal mol−1) forms is similar to that of P1' (+3.1 kcal mol−1) or TriMe (+3.5 kcal mol−1), but the P2'-F3 form is the lowest energy conformational form.</p><p>Figure 6 shows the minimized geometries of the P2'-F1, P2'-F2, and P2'-F3 conformations of host P2'; an analogous figure is given for P1' in the Supporting Information (Figure S103). In contrast to the idealized C2v-symmetric line bond structures shown above in Figure 4, we observe more compact conformations for P2'-F2 and P2'-F3, presumably due to the van der Waals interactions between the central Me-groups of P2' and the aromatic sidewall(s) in these conformations. For P2'-F3 (Figure 6a), we additionally observe offset π−π interactions between the faces of the naphthalene sidewalls. These interactions are responsible for the overall preference for F3 (0.0 kcal mol−1) over F2 (4.1 kcal mol−1) and F1 (2.3 kcal mol−1). For guest binding to occur within P2'-F3, the disruption of these intramolecular non-covalent interactions must be counterbalanced by stronger host•guest non-covalent interactions. For critical assessment of the obtained results two additional contributions must be mentioned which are not available in our calculations. The first is the absence of thermal motions (mainly entropy) in the calculated energies. It can be expected that F3 will have lower entropy than F1 because its compact structure will limit movements of its aromatic walls (Figure S107 shows the dynamic behavior of aromatic walls in P2•P2 dimer). This effect will decrease the stability of the F3 conformer relative to F1. Second, destabilizing electrostatic interactions between the negatively charged solubilizing groups would be expected to be larger for P2-F3 than for P2-F1.</p><!><p>As a prelude to the planned host•guest binding studies, we investigated the self-association properties of P1 and P2 to ensure that the measured Ka values would not be influenced by host self-association.28 Accordingly, we prepared solutions of P1 and P2 at their maximal solubility in D2O and measured their 1H NMR spectra as a function of [P1] or [P2] down to 0.12 mM. We did not observe any significant changes in the chemical shifts for P1 over the 9 mM – 0.12 mM concentration range which indicates that P1 does not undergo significant self-association (Supporting Information, Figure S24). Figure 7a shows the chemical shift of Hm as a function of [P2]. We fitted the change in chemical shift to a two-fold self-association model29 which allowed us to extract the self-association constant of P2 (Ks = 189 ± 27 M−1). Because chemical exchange is fast on the NMR time scale, it is not possible to obtain precise information about the geometry of P2•P2 from the NMR experiments. Accordingly, we performed molecular modelling; Figure 7b shows a representative snapshot of the P2-F1•P2-F1 dimer from a 1 μs long molecular dynamics simulation which is consistent with the observed upfield shifting of the Ha and Hb resonances of the aromatic sidewall and the resonance for (CH3)m upon dimerization. The geometry of P2•P2 depicted in Figure 7b is reminiscent of the geometry of dimeric molecular clips prepared by the Nolte and Isaacs groups which feature the aromatic sidewall of one molecule penetrating into the cleft of the opposing molecule and vice versa.26 We also attempted to model the dimer from the P2-F2 form. However, soon after the start, both flanking side arms underwent conformation change into F1 (see Figure S108).</p><!><p>Given our previous work on the use of acyclic CB[n]-type receptors as solubilizing excipients for insoluble drugs,30 we initially tested the solubilization abilities of P1 and P2 toward a small panel of insoluble drugs (paclitaxel, fenofibrate, itraconazole, tamoxifen and ethynylestradiol, Figure 8). For this purpose, we separately prepared 7 mM solutions of P1 and P2 in 20 mM sodium phosphate buffered D2O and dispensed the solution into a series of vials to which an excess of insoluble drug was added. After mixing overnight (16 h), the insolubles were removed by filtration through a 0.45 μm polyethersulfone membrane filter and the solution and a known volume of a solution of trimesic acid (1 mM) was transferred to an NMR tube for analysis. No drug solubilization was detected by 1H NMR indicating that P1 and P2 are not promising candidates as solubilizing excipients for insoluble drugs.</p><!><p>In order to understand the poor solubilizing ability of P1 and P2 we decided to perform qualitative host•guest binding studies at 1:1 and 1:2 host:guest ratios. Initially, we attempted to prepare solutions of host P1 (1 mM) and guests 7, 9, and 10 (≥ 2 mM) and observed the formation of precipitates indicating the poor solubility of the complexes. Similar observations were made for solutions of host P2 (2 mM) and guests 7 – 9. These problems can be avoided by working at lower concentrations of hosts P1 and P2 (e.g. 0.3 mM). It is not possible to reach saturation due to the low binding affinity for host•guest complexes of P1 and P2 (vide infra) and therefore experimentally observable complexation induced changes in chemical shift are small particularly for P1. For example, Figures 9a–c show the 1H NMR spectra recorded for 8 (0.3 mM) and 1:1 and 1:2 mixtures of P1 and 8 which exhibit upfield shifts of ≤ 0.2 ppm under these conditions. In contrast, Figure 10a–c shows the 1H NMR spectra recorded for 8 (1.0 mM) and 1:1 and 1:2 mixtures of P2 and 8. Clear upfield shifting of the aromatic H-atoms (Hq and Hr) of 8 upon complexation suggest the formation of a complex where the aromatic rings of guest 8 are located inside rather than on the exterior of host P2. The guest exchange processes were fast on the 1H NMR chemical shift timescale which is expected when the host•guest complexes are relatively weak. The Supporting Information (Figures S26 – S43) shows the analogous 1H NMR spectra recorded for guests 7 – 11 and hosts P1 and P2 which suggests the complexation of the central hydrophobic regions of guests 7 – 11 inside hosts P1 and P2 potentially in their P1-F3 and P2-F3 conformations.</p><!><p>Initially, we attempted to measure the Ka values for the host•guest complexes by isothermal titration calorimetry (ITC). Unfortunately, under our usual conditions (20 mM sodium phosphate buffer, pH 7.4) very little heat was evolved and the data could not be fitted to a standard 1:1 binding model. Accordingly, we turned to 1H NMR titrations. The titration of 8 (0.06 mM) with P2 (0 – 0.96 mM) conducted in 20 mM sodium phosphate buffer (pH 7.4) again resulted in only very small changes in chemical shift of 8 which made clear that P1 and P2 were poor hosts. Therefore, we changed the medium to the less competitive unbuffered D2O for determination of Ka values for P1 and P2. Figure 9d shows the change in chemical shift of (Hq, Hr, and Hs) of a fixed concentration of guest 8 (0.3 mM) upon titration with host P1 (0 – 2.01 mM); the solid line represents the best fitting of the data to a 1:1 binding model implemented within Scientist™ (Supporting Information) with Ka = 1100 ± 50 M−1. Similarly, Figure 10d shows the change in chemical shifts (Hq, Hs) of a fixed concentration of guest 8 (0.04 mM) recorded during the titration with host P2 (0 – 541 μM). The solid line in Figure 10d represents the best non-linear least squares fit of the data to a binding model that takes into account the self-association of P2 along with 1:1 host:guest binding (Supporting Information, page S70) with Ka = 19800 ± 400 M−1. Related titrations were performed for hosts P1 and P2 with guests 7 – 11 and are presented in the Supporting Information. The Ka values are collected in Table 2. From the fitting of 1H NMR titrations data curves (Supporting Information) we were also able to extract the limiting chemical shifts of the P1•guest and P2•guest complexes and calculate the complexation induced changes in chemical shift (Δδ, Table 3). A perusal of Table 3 reveals that for the naphthalene walled hosts, the complexation induced changes in chemical shifts (Δδ) of guests are significantly larger for Tet2 than for P2. Similarly, between the benzene walled hosts, the Δδ values are larger for Tet1 than for P1. These disparities suggest that the geometry of the P1•guest and P2•guest complexes are not directly analogous to those of Tet1 and Tet2. Accordingly, we wondered whether these weak binding processes might simply reflect electrostatic and hydrophobic interaction between the guest and the outside of the aromatic walls of the host or potentially to one of the clip-like cavities of P1-F1 or P2-F1. To test this possibility, we performed titrations between guests 6 – 11 and aromatic sidewalls 4 and 5 (Supporting Information). No changes in chemical shift were observed for mixtures of benzene derived wall 4 and guests 6 – 11; accordingly no Ka values or Δδ values are reported in Tables 2 and 3 for wall 4. For naphthalene wall 5 we did observe changes in chemical shift upon titration with guests 6 – 11; we fitted those changes to a 1:1 binding model to obtain Ka values and Δδ values (Tables 2 and 3). The Ka values for the complexation between wall 5 and 6 – 11 are 6.7 – 23.6-fold weaker than between host P2 and 6 – 11 and the Δδ values (Table 3) are much smaller for 5 than for P2. Based on this data we exclude the possibility that guests 6 – 11 simply bind to the exterior face of the aromatic sidewalls of host P2. The 1:1 stoichiometry of the P2•guest complexes was confirmed for guests 6, 7, and 10 by constructing Job plots (Supporting Information). The 1:1 stoichiometry suggests that the guest•P2 complexes exist as the guest•P2-F3 conformer. For the very weak complexes between P1 and guests 6 – 11 the Job plots were inconclusive with no clear maxima. The utility of Job plots has been called into question, especially for weak complexes studied under dilute conditions.31 In addition to the Δδ values for the guest upon complexation, we also monitored the changes in P2 chemical shift upon complexation with 6 – 11 (Supporting Information) and generally observe upfield shifts for Hg (≈ 0.3 ppm) and Hh (≈ 0.1 ppm) and a slight downfield shift (≤ 0.1 ppm) for He upon complexation. Hg and Hh are the diastereotopic protons on the methylene bridges involved in the S-shaped connections at the center of P2. The chemical shifts of the diastereotopic methylene bridges is CB[n] type hosts resonate at quite different chemical shifts due to the anisotropic effects of the ureidyl C=O group with the H-atoms nearer the lone pairs on oxygen appearing substantially downfield of those pointing away from the C=O groups. Accordingly, the significant upfield movement of Hg and Hh upon binding provides additional support for our conclusion that P2 undergoes conformation change upon binding to yield the P2-F3•guest complexes.</p><p>Table 2 also presents the Ka values for complexes of Tet1 with guests 6 – 11 measured previously by ITC in 20 mM sodium phosphate buffer (pH 7.4, RT) as a comparator for P1.32 The logical comparator for P2 is Tet2, but unfortunately, the Ka values for Tet2 complexes with 6 – 11 were unknown. Accordingly, we measured the Ka values by isothermal titration calorimetry. We attempted direct ITC titrations but quickly found that the Ka values exceeded the dynamic range of the measurements (c-value > 300).33 Accordingly, we decided to perform competition ITC.34 In competition ITC, a host and an excess of a weak binding guest of known Ka and ΔH is titrated with an excess of a stronger binding guest; fitting of the data to a competition binding model then allows extraction of the Ka and ΔH values for the tighter binding complex. Figure 11a shows the thermogram for the titration of a mixture of host Tet2 (105 μM) and trans-1,4-diamino cyclohexane dihydrochloride (9b, 750 μM) as competitor in the cell with guest 9 (1.0 mM) in the syringe. Figure 11b shows a plot of the integrated heats versus the Tet2:9 molar ratio fitted to a competition binding model in the PEAQ-ITC data analysis software which allowed the determination of the strength of the Tet2•9 complex (Ka = 2.76 × 109 M−1; ΔH = −13.3 kcal mol−1). Table 2 reports the binding constants for the Tet2•guest complexes by competition ITC and the data is given in the Supporting Information.</p><!><p>The optimized structures of the pentamers P1' and P2' (Figure 6) were used to model the complexes with guest 8. In the case of F3, we inserted the guest 8 into an artificially created cavity. The structures obtained for P2'•8 fully optimized in implicit water are summarized in Figure 6 whereas the analogously obtained structures of P1'•8 are shown in Figure S105. In the F1 and F2 forms, guest 8 binds into the clip-like cavity. In the case of F3, the aromatic walls undergo an out-of-plane twisting and reorganization to maximize contact with the guest, and as a result, they no longer π−π stack with each other anymore. For P1 and P2 which bear O(CH2)3SO3− solubilizing groups, there may be steric interactions or electrostatic interactions between solubilizing groups, and it can be expected that the computationally obtained complexes with the P1' and P2' hosts in the F3 conformational state may not fully represent the real situation. The computed relative stabilities of the complexes of 8 with P1' and P2' (Table 1) revealed that the most stable conformer is F3 for P1'•8 and F1 for P2'•8. This indicates that the preference for conformational states can change during the binding. Similar to the free hosts, two contributions were not included in our analysis, the thermal motions (entropy) and impact solubilizing groups, which may have a destabilizing effect on the complexes in the F3 conformational state as discussed above.</p><!><p>An examination of the Ka values in Table 2 reveal a number of significant trends. First, both P1 and P2 are relatively poor hosts with the Ka values for the series of (di)ammonium ions (6 – 11) – generally excellent guests for CB[n]-type receptors – ranging from 375 to 1400 M−1 for P1 and from 1950 to 19800 M−1 for P2. Amongst guests 6 – 11, guests 7 and 8 which contain aromatic rings bind most tightly to P1 and P2, presumably due to π−π interactions in the complexes. Second, P2 is always a better host than P1 toward 6 – 11 with ratios of Ka values as follows: 6 (19.9-fold), 7 (12.6-fold), 8 (18-fold), 9 (4.6-fold), 10 (4.7-fold), 11 (5.2-fold). We believe that P2 is a slightly better host than P1 due to either a larger population of the F3 conformer or the larger π-surfaces of P2 which form stronger non-covalent interactions with guests, or a combination of the two. Similarly, a comparison of the Ka values of Tet2 and Tet1 toward 6 – 11 shows that Tet2 is uniformly the superior host (6 (5.1-fold), 7 (15-fold), 8 (4.5-fold), 9 (123-fold), 10 (4200-fold), 11 (42-fold)). Related trends have been seen previously for the complexes of Tet1 and Tet2 toward insoluble drugs and neuromuscular blocking agents.19,30 We attributed these trends to the potential for augmented π−π interactions with the naphthalene walled hosts Tet2 and P2. For hosts Tet1 and Tet2 the selectivity is largest for the bulkier alicyclic guests 9 – 11 and smallest for the narrow aliphatic guest 6 which suggests that smaller host Tet1 must undergo energetically costly cavity expansion to accommodate the larger guests. The narrow dynamic range of Ka values for hosts P1 and P2 does not allow us to draw any firm conclusions regarding guest size preference. Previously, we have observed that Tet1 is a more potent host than Tri1 and that Tet2 is a more potent host than Tri2.23 The data in Table 2 allows an analogous comparison of P1 with Tet1 and P2 with Tet2. We find that Tet1 is a substantially better host than P1 toward guests 6 – 11 (6: 2.3 × 105-fold, 7: 1.3 × 105-fold, 8: 4.3 × 105-fold, 9: 2.5 × 104-fold, 10: 2.9 × 103-fold, 11: 4.5 × 104-fold) and that Tet2 is far superior than P2 (6: 6.0 × 104-fold, 7: 1.5 × 105-fold, 8: 1.1 × 105-fold, 9: 6.6 × 105-fold, 10: 2.5 × 106-fold, 11: 3.6 × 105-fold). Overall, the binding data shows that P1 and P2 are relatively poor hosts toward hydrophobic (di)cations 6 – 11 which are generally excellent guests for CB[n]-type hosts. We surmise that the poor performance of P1 and P2 is because the uncomplexed hosts must undergo an energetically costly folding process to populate the P1-F3 and P2-F3 conformation before guest binding (Figure 6).</p><!><p>In summary, we have described the synthesis of an important new glycoluril oligomer building block (S-shaped pentamer 3) and its transformation into two new acyclic CB[n]-type receptors P1 and P2. Hosts P1 and P2 have moderate solubility in water (≈ 9 and ≈ 11 mM); P1 does not self-associate whereas P2 undergoes only weak intermolecular self-association (Ks = 189 ± 27 M−1). P1 and P2 are relatively poor hosts toward (di)cationic guests 6 – 11 as established by 1H NMR titrations (P1: 375 to 1400 M−1; P2: 1950 to 19800 M−1). Host P2 with its larger naphthalene rings is the more potent host in all cases relative to P1. In sharp contrast, guests 6 – 11 form much tighter complexes (103 – 106 fold) with acyclic CB[n] based on glycoluril tetramers (Tet1 and Tet2). The relatively poor recognition abilities of P1 and P2 are traced to their ability to adopt three conformational isomers around their S-shaped segments (F1 – F3). For P1 there is a computational derived preference for the F1-fold (TriMe-F3: +5.1 kcal mol−1; P1'-F3: +4.0 kcal mol−1) whereas for P2 there is a smaller preference for the F3-fold (P2'-F1: +2.3 kcal mol−1). The need to pay the energetic cost to shift the conformational equilibrium toward the F3-folded forms of P1 and induce cavity formation in the F3-folded forms of P1 and P2 required for 1:1 host:guest cavity binding results in the observed low Ka values. Interestingly, calculations performed for TriH show no preference for the F1-fold which suggests that the Me-substituents play a major steric role in biasing the F1 – F3 equilibrium. In turn, this observation opens up the use of substituted glycolurils as building blocks to rationally design S-shaped glycoluril oligomers with well-defined conformational ensembles. In conclusion, these results highlight the importance of controlling the ensemble of conformations open to a host (e.g. maximizing host pre-organization) and minimizing host self-complexation when attempting to maximize host•guest binding affinity.</p><!><p>The relative stabilities of the F1, F2, and F3 conformers were investigated on simplified models employing density functional theory (DFT) approach. These models included TriMe and TriH containing two S-shaped connections between adjacent glycolurils, models P1' and P2' with solubilizing groups (O(CH2)3SO3Na) absent representing simplified versions of P1 and P2, and complexes of P1' and P2' with guest 8. Initial structures were built in silico in the three conformational states and then their geometries were optimized employing B97–335 method in an implicit water described by the SMD36 model. Finally, the relative stabilities were evaluated at the PBE0-D3BJ/def2-TZVPP level of theory37 in the SMD implicit water on the optimized geometries. The employed computational methodology was thoroughly tested and showed similar accuracy as MP2/CBS. Further computational details, optimized geometries, and their absolute energies are available in the Supporting Information. All quantum chemical calculations presented in the main text were performed in Orca 4.2.1.38</p><p>A possible structure of the P2•P2 dimer was investigated by molecular dynamics simulations performed in the Amber 16 package.39 The P2 host was considered in the F1 and F2 folds, which resulted in two possible dimeric structures (P2-F1•P2-F1 and P2-F2•P2-F2). These dimers were built in silico and described by the GAFF force field in MD simulations.40 Each dimer was immersed into a box filled by an explicit water solvent described the TIP3P model with electroneutrality maintained by 8 sodium cations. In total, each system was simulated for 1 μs at a temperature of 300 K and a pressure of 100 kPa.</p><!><p>Compounds 4 and 5 were prepared according to the literature procedures.24 NMR spectra were measured on 400 MHz, 500 MHz, 600, and 800 MHz spectrometers (400, 500, 600, 800 MHz for 1H NMR; 126 MHz for 13C NMR) at room temperature in the stated deuterated solvents.</p><!><p>Glycoluril 1 (4.78 g, 33.6 mmol) was dissolved in 90% aq. methanesulfonic acid (80 mL). Then the solution was cooled to 8–12 °C using an ice bath within 20 min. and 2 (15.97 g, 62.8 mmol) was added in one portion and the reaction was stirred at 8–12 °C for 2 h and then 2 h at room temperature. The reaction mixture was poured into acetone (1.4 L) that had been cooled in ice for 30 min. to give a precipitate which was obtained by filtration. The crude solid was washed with ethanol. The crude solid was then dissolved in acetonitrile/water (1:1 v:v, 200 mL) and stored in the refrigerator for 2–3 days. The resulting precipitate was isolated by filtration and then purified by successive cycles of stirring and centrifuging using the following solvent series: DMSO (6 mL), H2O (12 mL), CH3CN/H2O 1:1 (20 mL), acetone (30 mL), Et2O (30 mL). The final off-white residue was dried under high vacuum to give 3 (847 mg, 5%). M.p. > 300 °C. IR (ATR, cm−1): 2917w, 2849w, 1711s, 1452s, 1371m, 1308m, 1250m, 1224m, 1187m, 1079m, 1012w, 958w, 918w, 862w, 770m. 1H NMR (DMSO-d6, 800 MHz, 30 °C): 5.54 (d, J = 15.2, 4H), 5.51 (d, J = 8.6, 2H), 5.21 (d, J = 13.7, 4H), 5.16 (d, J = 11.0, 4H), 5.09 (d, J = 8.6, 2H), 4.86 (d, J = 11.0, 4H), 4.71 (d, J = 13.7, 4H), 4.27 (d, J = 15.2, 4H), 1.82 (s, 6H), 1.70 (s, 6H), 1.64 (s, 6H).13C NMR (DMSO- d6, 126 MHz, 30 °C): 154.7, 154.7, 153.8, 78.00, 77.0, 72.4, 70.6, 69.5, 62.4, 48.2, 47.5, 17.8, 16.6, 15.8. HR-MS (ESI): m/z 1091.4953 ([M + hexanediamine + H]+), C44H63N22O12, calculated 1091.4996; 546.2520 ([M + hexanediammonium]2+), C44H64N22O12, calculated 546.2532.</p><!><p>Compound 3 (1.57 g, 1.60 mmol) was charged to a round bottomed flask followed by trifluoroacetic acid (5.1 mL), Ac2O (5.1 mL), and then finally 4 (1.47 g, 3.68 mmol) was added. The reaction mixture was stirred and heated at 75 °C for 3 h. The reaction mixture was poured into MeOH (65 mL) and the resulting precipitate was isolated by filtration. The crude solid was triturated with boiling water (30 mL) and then cooled in the refrigerator. The resulting solid was collected by centrifugation, dissolved in water and adjusted to pH 7 with 1 M aqueous NaOH. The solution was filtered to remove dust and then concentrated to dryness by rotary evaporation to afford host P1 as an off-white solid (597 mg, 22%). M.p. > 300 °C. IR (ATR, cm−1): 3427m, 2944w, 1703s, 1460s, 1374m, 1311m, 1182s, 1081m, 1036s, 844w, 796w, 786w. 1H NMR (DMSO-d6, 400 MHz, 30 °C): 6.84 (s, 4H), 5.50 (d, J = 15.9 Hz, 4H), 5.41 (d, J = 8.6 Hz, 2H), 5.28 (d, J = 16.2 Hz, 4H), 5.13 (d, J = 13.8 Hz, 4H), 5.05 (d, J = 8.6 Hz, 2H), 4.59 (d, J = 13.8 Hz, 4H), 4.17 (d, J = 15.9 Hz, 4H), 4.13 (d, J = 16.2 Hz, 4H), 4.00 – 3.93 (m, 8H), 2.69 – 2.56 (m, 8H), 2.03 – 1.97 (m, 8H), 1.73 (s, 6H), 1.67 (s, 6H), 1.60 (s, 6H). 13C NMR (D2O, 126 MHz, 30°C, dioxane as internal reference): 157.3, 157.1, 156.5, 150.7, 128.6, 115.6, 80.0, 79.7, 78.3, 71.6, 69.4, 64.4, 49.4, 48.7, 48.3, 35.6, 25.2, 16.5, 16.2, 15.4. HR-MS (ESI): m/z 547.8031 ([M-H]3-), C62H75N20O26S4, calculated 547.8020; 410.5995 ([M-H]4-), C62H74N20O26S4, calculated 410.5997.</p><!><p>A round bottomed flask was charged with 3 (976 mg, 1.00 mmol), trifluoroacetic acid (3.2 mL), Ac2O (3.2 mL), and then finally 5 (1.01 g, 2.3 mmol). The reaction mixture was stirred and heated at 75 °C for 3 h. The reaction mixture was poured into MeOH (50 mL) and the precipitate was isolated by filtration. The crude solid was dissolved in water (25 mL) and precipitated by the addition of KCl (900 mg, 12.0 mmol). The precipitate was isolated by centrifugation and then dissolved in water and adjusted to pH 7 with 1 M aqueous NaOH. The solution was filtered to remove dust and then concentrated to dryness by rotary evaporation to afford P2 as a pale yellow solid (492 mg, 27%). M.p. > 300 °C. IR (ATR, cm−1): 3421w, 2999w, 2979w, 2944w, 1707s, 1458s, 1373m, 1311m, 1225m, 1183s, 1079m, 1034m, 950w, 785w, 757w. 1H NMR (DMSO-d6, 400 MHz, 30 °C): 7.99 (m, 4H), 7.56 (m, 4H), 5.51 (d, J = 15.5 Hz, 4H), 5.38 (d, J = 8.7 Hz, 2 H), 5.34 (d, J = 15.8 Hz, 4H), 5.06 (d, J = 13.9 Hz, 4H), 4.97 (d, J = 8.7 Hz, 2H), 4.51 (d, J = 13.9 Hz, 4H), 4.41 (d, J = 15.8, 4H), 4.22 – 4.18 (m, 4H), 4.16 (d, J = 15.5 Hz, 4H), 3.93– 3.91 (m, 4H), 2.77 – 2.73 (m, 8H), 2.18 – 2.15 (m, 8H), 1.76 (s, 12H), 1.63 (s, 6H). 13C NMR (D2O, 126 MHz, 30 °C, dioxane as internal reference): 157.0, 156.7, 156.1, 148.9, 128.1, 127.3, 127.0, 122.9, 79.7, 79.3, 78.1, 74.8, 71.4, 64.2, 49.2, 48.6, 48.2, 36.8, 25.8, 16.4, 16.1, 15.9. HR-MS (ESI): m/z 872.2222 ([M-H]2-), C70H80N20O26S4, calculated 872.2223; 581.1456 ([M-H]3-), C70H79N20O26S4, calculated 581.1458; 435.6078 ([M-H]4-), C70H78N20O26S4, calculated 435.6075.</p>