Dimeric molecular clips based on glycoluril

Glycoluril dimers 4a and 4b were prepared and successfully transformed to glycoluril dimeric clips 7a and 7b. H NMR spectroscopy was used to reveal that the dimeric clip 7b with methyl substituents in its structure exists as a mixture of two distinct conformers in D2O at ambient temperature, while 7a possesses only one stable conformer under the same conditions due to its bulky phenyl substituents. The coalescence temperatures for 7b and its precursor 5b were determined. Both dimeric clips 7a and 7b formed complexes with methylviologen in D2O in which each of two clip-binding sites was occupied by one molecule of the guest. Thus, this study uncovers possible use of glycoluril dimers 4a and 4b as general building blocks for glycoluril-based supramolecular host molecules with two binding sites in their structure.


Introduction
Cucurbiturils are an important class of supramolecular host molecules that are prized for their ability to strongly and selectively bind organic molecules in water. [1][2][3][4][5][6] These macrocycles consist of glycoluril units connected by methylene bridges. Recent progress in the chemistry of cucurbiturils enables the preparation of their functionalized derivatives, [7][8][9][10][11][12][13] which can be covalently attached to various substrates and thus can be used for real applications. Along with the synthesis of cucurbituril derivatives, other supramolecular hosts based on glycoluril have been prepared, including anionic receptorsbambusurils [14][15][16] -and acyclic glycoluril oligomers. [17][18][19] The latter group of compounds features high affinities toward anaesthetics, nitrosamines and various drugs. [20][21][22] Despite recent progress in cucurbituril synthesis, there have been only two reported approaches dealing with the preparation of cucurbituril dimers. The first is based on the condensation of acyclic glycoluril hexamers with tetraaldehydes; 23,24 the second relies on the conjugation of two monofunctionalized cucurbiturils. 25 These cucurbituril dimers have been used as building blocks for the construction of supramolecular polymers and oligomers.
We decided to investigate a different approach for the preparation of dimeric cucurbiturils and their acyclic analogs. Our work was inspired by compound 4a (Scheme 1) previously reported in the literature. 26,27 This compound contains two glycoluril units connected through phenylene links. Therefore, we wanted to investigate whether this structural motif could be suitable for the construction of cucurbituril-based compounds. As a first approximation of a cucurbituril derivative, we selected to prepare dimeric clip 7a. The successful preparation of this compound should uncover the possibility of attaching methylene bridges to nitrogen atoms of sterically hindered glycoluril units of 4a.

Results and discussion
The starting building block 4a for this dimeric clip has already been reported. 26,27 According to the literature, compound 4a can be prepared by condensation of urea or urea derivative 8 ( Fig. 1) with compound 3a in refluxing formic or acetic acid with a catalytic amount of sulfuric acid. However, when the synthesis was repeated in our laboratory under these conditions, only compound 9 with one glycoluril moiety was formed.
Fortunately, the reaction of urea with 3a in toluene in the presence of TFA provided dimer 4a in 95% yield. Compound 4a was then transformed to cyclic ether 5a by its reaction with paraformaldehyde in aqueous HCl (Scheme 1). Compound 5a was further reacted with hydroquinone to provide dimeric molecular clip 6 bearing OH groups on its terminal aromatic walls in 69% yield. Clip 6 was alkylated using 1,3-propanesultone to give water-soluble clip 7a in 68% yield.
The phenyl substituent present in glycoluril dimer 4a can be a source of low reactivity and low solubility of this compound, as previously reported on the corresponding glycolurils for cucurbituril syntheses. 8 Thus, we decided to prepare dimer 4b in which two phenyls are replaced by methyl substituents. The synthesis of 4b required precursor 3b, which was not commercially available (unlike 3a) and had to be prepared from 1,4-diethynylbenzene (1) (Scheme 1). Alkyne 1 was deprotonated with n-butyllithium and subsequently treated with methyl iodide to give 2. Compound 2 was oxidized to 3b using an RuCl 3 /NaIO 4 mixture. Having compound 3b in our hands, we performed its reaction with urea in an aqueous HCl/CH 2 Cl 2 emulsion to obtain glycoluril dimer 4b, which was transformed into cyclic ether 5b by its reaction with paraformaldehyde. Each of the glycoluril units of 5b bears methyl groups in the methine position, which are significantly smaller compared to phenyl groups in 5a. Cyclic ether 5b can react directly with compound 10 yielding molecular clip 7b in 59% yield. This approach was significantly less efficient in the case of 5a as bulky phenyl groups lower the reactivity of the ether groups towards the aromatic ring. This observation is in agreement with previously reported synthesis. 16

Conformational properties
When recording the 1 H NMR spectra of compounds 5b and 7b at 30 1C in DMSO, we observed two signals instead of the expected one signal for each of their protons. These two sets of signals can be attributed to two distinct conformers, which were likely enabled by the rotation of two single C-C bonds connecting the central phenylene group to the glycoluril moieties. The rotation was slow on the NMR timescale at 30 1C; thus, the conformers were distinguished by their unique NMR spectra. Steric hindrance between methyl groups (attached to the methine carbon atoms of the glycoluril units) and the side walls of glycoluril units were likely the reason why the rotation was restricted. We decided to label the first conformer syn (the methyl groups located on the same side of the central phenylene group) and second anti (the methyl groups on opposite sides) (Fig. 2). Unfortunately, we could not distinguish which set of signals in the NMR spectra belonged to which conformer. Conformers of 5b, as well as of 7b, are present in a ratio of 58 : 42, which is based on the integration of the corresponding signals in the NMR spectra. The ratio corresponds to a 0.2 kcal mol À1 energy difference (DE)  between the two conformers. The situation is different in the case of glycoluril dimer 4b, which shows only one set of signals under the same conditions. This indicates that the rotation is fast on the NMR timescale as it is not restricted, due to the absence of the methylene side-groups featuring in 5b and 7b. Glycoluril dimer 4a and its derivatives 5a and 7a also showed only one set of signals in their NMR spectra. However, these compounds distinguish themselves from 4b, 5b, and 7b by the presence of phenyl instead of methyl groups in the two methine positions of the glycoluril dimer core. We assume that two phenyl groups are too bulky to be fitted in the syn conformation. This is why 4a and its derivatives adopt only the sterically favorable anti conformation.
In order to gain information on the energy characteristics of the conformational changes in 5b, the 1 H NMR spectra in DMSO-d 6 with temperature rising to 130 1C were recorded (Fig. 3). It was discovered that some signals in the NMR spectra show coalescence at a different temperature from the others. The coalescence temperature of signals 2b and 4 was 90 1C, while 1, 2a and 3b had already coalesced at 60 1C. The reason why there are two coalescence temperatures observed instead of one may be that compound 5b possesses two C-C bonds around which the rotation occurs. Due to this, there is a possibility of two different kinds of rotational movements: (i) rotation of phenylene with respect to the glycoluril units and (ii) rotation of one glycoluril unit with respect to the other. The signals of protons 1 and 4 were used for the calculation of Gibbs activation energies of both rotations (DG ‡ ) using the Eyring equation. The resulting values of DG ‡ are shown in Table 1. The whole calculation is described in detail in the ESI. † In contrast to 5b, dimeric molecular clip 7b showed only a single coalescence temperature, 110 1C. The values of DG ‡ of 7b calculated using singlets 1 and 4 were greater than those of 5b (Table 1). This means that the xylylene side walls of the clip 7b are a source of more significant steric hindrance than the cyclic ether groups of 5b. In the case of compound 4b, the DG ‡ might be determined by measurement of the NMR spectra at low temperature. However, compound 4b was insoluble in common organic solvents including DMF. The only suitable NMR solvent for this compound was DCOOD. The melting point of formic acid is 8.4 1C and therefore we could not measure the lowtemperature spectra and determine the DG ‡ of compound 4b.

Supramolecular properties
The ability of dimeric clips 7a and 7b to act as supramolecular hosts was investigated by 1 H NMR spectroscopy. Inclusion complexes between viologens with cucurbiturils as well as their acyclic versions have been previously described. However, interactions of molecular clips resembling those present in dimeric clips 7a and 7b with viologen have not been reported. Thus, methylviologen 11 was selected as a suitable guest for supramolecular studies with dimeric clips 7a and 7b and their interactions in D 2 O (Fig. 4).
The aromatic signals of 11 underwent an upfield shift after addition of clips 7a and 7b to the guest solution. This indicates the formation of an inclusion complex between the clips and methylviologen. Since compounds 7a and 7b have two sites that could encapsulate guest 11, it was expected that these compounds would form 1: 2 complexes, in which each clip arm holds one guest molecule 11. To prove the expected binding mode, the stoichiometry of the complex was determined by the continuous variation method (Job's plot). The maximum of Job's curve appeared approximately at x-coordinate = 0.66 which proves that 1 : 2 complexation is predominant at equilibrium (Fig. S19 and S20, ESI †). This binding mode was further visualized by a model obtained by quantum chemical calculations (Fig. 5).
A plot of the chemical shifts of signals 1 and 2 of the guest was fitted to the binding isotherm of the 1 : 2 binding model to   obtain the corresponding association constants (Table 2). Unlike clip 7a, the situation in the case of clip 7b was complicated by its ability to adopt two different conformers. In aqueous solution, the conformers were always in a ratio of 58 : 42; the equilibrium was not affected by the presence of guest 11. We were not able to determine the association constants for separate conformers due to the presence of four binding sites (each conformer has two indistinguishable binding sites) during titration with 11. However, if the concentration of 11 was kept constant during the titration and the concentration of 7b was varied, it was possible to use the signals of guest 11 to establish the association constants describing the affinity of 11 towards an equilibrium mixture of both conformers of 7b. The association constants determined for complexes 7aÁ11 2 and 7bÁ11 2 are summarized in Table 2.
Hosts 7a and 7b bind methylviologen 11 with a very similar overall affinity and in both cases the binding is associated with a negative cooperativity. The observed negative cooperativity can be rationalized by the inclusion of the guest molecule in the first binding side of the dimeric clip which causes conformational changes at the second binding site of the host. However, in our case the binding sites are separated by a rigid phenylene moiety. Therefore, we do not expect that the negative cooperativity is a result of conformational changes. We presume that binding of the first cation 11 affects the distribution of electron density in the host molecule, which makes it less attractive for another guest 11. Thus, electronic effects, rather than conformational changes, are the reason for the negative cooperativity. Finally, we determined the binding between 11 and model compound 12 to be (8.82 AE 0.77) Â 10 3 M À1 which is in good agreement with the values obtained for binding sites in dimeric clips.

Conclusion
We prepared compounds 7a and 7b as the first examples of glycoluril-based dimeric molecular clips. Clip 7b, as well as other compounds derived from glycoluril dimer 4b, can adopt two different conformations, which can be distinguished by means of NMR. On the other hand, free rotation is forbidden in compounds based on 4a due to the presence of the bulky phenyl substituent. Both dimeric clips 7a and 7b form complexes with methylviologen in a 1 : 2 ratio and with submillimolar stability in water. Our work demonstrated that structural motifs represented by compounds 4a and 4b can be converted to supramolecular hosts with two binding sites. Thus, a similar strategy should be applicable to the synthesis of dimers of acyclic glycoluril oligomers or cucurbituril dimers. The syntheses of these compounds are under development in our laboratory.

General methods
Starting materials were purchased from commercial suppliers and used without further purification. NMR spectra were recorded using a Bruker Avance 500 spectrometer operating at frequencies of 500.13 MHz ( 1 H) and 125.77 MHz ( 13 C), and a Bruker Avance 300 spectrometer operating at frequencies of 300.13 MHz ( 1 H) and 75.48 MHz ( 13 C). Both spectrometers were equipped with a BBFO probe. High-resolution mass spectra were recorded on an Agilent 6224 Accurate-Mass TOF LC-MS spectrometer using a multimode ESI/APCI ion source and a KDS Model 100 Series manual pump for sampling.

Synthetic procedures and characterization
Glycoluril dimer (4a). Compound 3a (2.480 g, 7.43 mmol) was dissolved in toluene (150 mL); urea (8.91 g, 0.149 mol) and trifluoroacetic acid (7.5 mL) were added and the mixture was heated to reflux in a flask equipped with a Dean-Stark trap for 12 h. A precipitate was formed at the bottom of the flask during the reaction. Toluene was then removed by decantation and the precipitate was treated with methanol (200 mL) and sonicated for 30 min. The suspension was filtered and the solid was washed with acetone and dried in vacuo to give 4a as a white powder (3.605 g, 95%). 1 H NMR (300 MHz, 96% D 2 SO 4 , DMSO-d 6 as external reference) d 6.72 (t, J = 7.2 Hz, 2H), 6.59 (t, J = 7.2 Hz, 4H), 6.53 (s, 4 H), 6.24 (d, J = 7.8 Hz, 4H). 13  Cyclic ether (5a). Compound 4a (3.01 g, 5.89 mmol) was mixed with paraformaldehyde (5.30 g, 0.177 mol); water (48 mL) and 35% HCl (72 mL) were added and the suspension was heated to reflux for 16 h. The suspension was cooled to RT and filtered. The solid material was washed with water and dried in vacuo to give 5a as a white powder (3.64, 91%). 1  Dimeric clip (6). Compound 5a (1.20 g, 1.77 mmol) was dissolved in TFA (15 ml); hydroquinone was added (4.21 g, 38 mmol) and the mixture was heated to reflux for 6 h and then allowed to cool to RT. The product was precipitated with methanol (100 ml) and the precipitate was collected by filtration. The crude product was dissolved in DMSO (15 ml) at 80 1C. Methanol (80 ml) was added to the solution and the mixture was cooled to RT. The precipitate was collected by filtration and washed with methanol and diethyl ether to give 6 as a white powder (1.29 g, 69%). 1  Dimeric clip (7a). Compound 6 (0.510 g, 0.49 mmol) was dissolved in 2.5 M NaOH (3.5 mL). The solution was treated with 1,3-propanesultone (0.911 g, 7.46 mmol) in dioxane (3.5 mL). The reaction mixture was stirred at RT for 8 h. The resulting precipitate was collected by filtration, washed with methanol and suspended in 2.5 M NaOH (7 mL). The suspension was again treated with 1,3-propanesultone (2.139 g, 17.51 mmol) in dioxane (9 mL). The reaction mixture was stirred at RT for 8 h. The resulting precipitate was collected by filtration and washed with methanol. The crude product was dissolved in water (20 mL) and precipitated with acetone (120 mL). The precipitate was collected by filtration, washed with methanol and diethyl ether and dried in vacuo to afford 7a as a yellowish powder (0.728 g, 68%). 1,4-Bis(1,2-dioxopropane-1-yl)benzene (3b). Compound 2 28 (0.771 g, 5.00 mmol) was dissolved in a mixture of CCl 4 (15.5 mL) and CH 3 CN (15.5 mL). A mixture of NaIO 4 (6.42 g, 30.02 mmol), NaHCO 3 (67.3 mg, 0.80 mmol) and MgSO 4 (308.0 mg, 2.56 mmol) in water (20 mL) was added to the solution. Aqueous 0.01 M RuCl 3 (4 mL, 0.04 mmol) was added and the reaction mixture was stirred at RT for 1.5 h. The mixture was diluted with ethyl acetate (100 ml) and the organic phase was separated from the precipitated salts by decantation. The organic phase was dried over MgSO 4 and the solvents were removed by rotary evaporation. The crude product was dissolved in ethyl acetate and filtered through a plug of silica gel. The solvent was removed by rotary evaporation to give compound 3b as a yellow solid (0.605 g, 55% Cyclic ether (5b). Paraformaldehyde (1.60 g, 53.3 mmol) was dissolved in 9 M HCl (20 mL) at 60 1C, the solution was cooled to RT and compound 4b (0.690 g, 1.78 mmol) was added and the solution was stirred for 24 h. Water was added to the mixture (11 mL) and stirred for another 12 h. The resulting precipitate was filtered, washed with water and dried in vacuo to give ether 5b as a white powder (0.465 g, 47%). Compound 5b was obtained as a mixture of two conformers in a 58 : 42 ratio.  13  Dimeric clip (7b). Compound 5b (0.460 g, 0.83 mmol) and compound 10 (2.31 g, 5.83 mmol) were dissolved in a mixture of TFA (12 mL) and acetic anhydride (12 mL). The solution was heated to 95 1C for 4 h. The solvents were removed by rotary evaporation and the resulting solid was washed with methanol (200 mL). The crude product was dissolved in water (15 mL) and precipitated with acetone (120 mL). The precipitate was filtered and dissolved in water (20 mL); the pH of the solution was adjusted to 7 using 1 M NaOH and the water was removed by rotary evaporation. The resulting solid was washed with methanol and diethyl ether and dried in vacuo to afford 7b as a yellowish solid (1.01 g, 59%). 1 H NMR (500 MHz, D 2 O) major conformer: d 7.71 (s, 4H), 7.03