The Recognition of Viologen Derivatives in Water by N-Alkyl Ammonium Resorcinarene Chlorides

Three water-soluble N-alkyl ammonium resorcinarene chlorides decorated with terminal hydroxyl groups at the lower rims were synthesized and characterized. The receptors were decorated at the upper rim with either terminal hydroxyl, rigid cyclohexyl or flexible benzyl groups. The binding affinities of these receptors towards three viologen derivatives, two of which possess an acetylmethyl group attached to one of the pyridine nitrogens, in water were investigated via H NMR spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry (ITC). ITC quantification of the binding process gave association constants of up to 10 M. Analyses reveal a spontaneous binding process which are all exothermic, and are both enthalpy and entropy driven.

CH-π interactions and hydrogen bonds. 28,29 We recently reported the binding of small neutral molecules such as amides and diamides in organic media with cooperativity by NARX receptors. 30 The four spatially fixed anions act as halogen bond acceptors leading to a variety of complex architectures, such as deep-cavity cavitands, pseudo-capsular, and capsular assemblies. 31,32 The NARXs are generally soluble in alcoholic and non-polar solvents. Recently we synthesized the first water soluble NARX receptors by attaching terminal hydroxyl groups at the upper rim. 33 Therein, we showed that the water soluble NARXs exist in C 4v crown conformation and bind a variety of aliphatic, aromatic and halogenated alkanes and arenes in aqueous media. 33 In this study, three new water-soluble NARCl receptors decorated with four terminal hydroxyl groups at the lower rims were synthesized and characterized ( Figure 1). The first NARCl receptor (5), is also functionalized at the upper rim with four terminal hydroxyl groups, making it extremely water soluble (35 mg/mL). The receptor (7) is functionalized at the upper rim with four rigid cyclohexyl groups and the third receptor (9) possesses four flexible benzyl groups at the upper rim. Mono-methyl 4,4'-bipyridine (11), 34 mono-acetylmethyl 4,4'-bipyridine (12), and hetero methyl-acetylmethyl 4,4'bipyridine (13) molecules were also synthesized as potential guests. The recognition of these guests (11)(12)(13) by the NARX receptors (5, 7 and 9) were investigated in water via 1 H NMR spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry (ITC) analyses.

RESULTS AND DISCUSSION
Synthesis of the receptors and the viologen guests. The synthesis of 5, 7 and 9 starts with resorcinarene 3, which was synthesized through reported procedures. 35 Ethanolamine, cyclohexyl amine and benzyl amine in the presence of excess formaldehyde participate in a Mannich condensation with 3 to form tetrabenzoxazines 4, 6 and 8, respectively. 36,37 The reaction with ethanolamine leads to a mixture of the five and six membered azoxazine rings 4a and 4b, respectively. The un-isolated crude product containing the five-and six-membered ring compounds in the presence of concentrated HCl under refluxing conditions, lead to the same final product 5 ( Figure 1). Cleavage of the pure tetrabenzoxazines 6 and 8 under similar conditions give NARCls 7 and 9. The detailed synthetic procedures of the NARCl receptors are reported in the Supporting Information (Schemes S1-S3; Figures S1-S5).  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59   The 4,4'-bipyridine guest 11 was synthesized according to a reported procedure. 34 The other bipyridine guests, 12 and 13, were synthesized by reacting chloroacetone with 4,4'-bipyridine or guest 11, respectively (Schemes S4, S5; Figures S6-S8). Suitable single crystals of mono-methyl 4,4'-bipyridine (11) and hetero-methyl-acetylmethyl 4,4-bipyridine (13) were obtained and analyzed (Figures S9-S11). Structural analysis verified the bipyridine was successfully substituted. In 11, the N-methyl 4,4'-bipyridyl cation is paired with the iodide anion with the anion close to the cationic nitrogen of the viologen molecule. While in 13, although the dicationic viologen is as expected, the original counter anions are replaced by 1.5 Iand 0.5 I 3 during the crystallization. Electrostatic forces contribute to the arrangement of the ion pairs. A mechanism for formation of the triiodide from iodide was proposed in a recent crystallographic study. experiments also showed the increased stability of keto tautomer as compared to possible enol form. This is likely due to existence of two resonance structures of keto tautomer (see SI, Figure S12).
Quantification of the Binding Process via Isothermal Titration Calorimetry (ITC) Studies. The interaction between the hosts and guests was quantified through a series of ITC experiments in H 2 O (Figure 4, Figures S23, S24). The thermodynamic parameters of host-guest binding (K, ∆H, ∆S, and ∆G) were extracted from fitting to a single binding site model (Table 1). When comparing the ITC titrations of the three NARCl receptors (5, 7 and 9) with the guests (11)(12)(13), several considerations can be made. The ∆H and ∆G values indicate the binding process to be exothermic and spontaneous at 298 K. ∆H and T∆S results also indicate that complexation of guests 11-13 by the receptors to be both enthalpy and entropy driven in most cases. In three cases (11@5, 13@5, 13@9), negative ∆S values indicate these process to be enthalpy driven.

Complexation Studies via Fluorescence Spectroscopic Analysis.
We also used optical titrations to analyze the binding processes. Fluorescence enhancement and/or quenching were observed from titration experiments conducted at 298 K with solutions of the hosts 5, 7 and the guests 11-13. A solution of the guests (0.1 M) at 298 K was titrated into a solution of the hosts 5 and 7 (2 mL, 125 µM). In all cases, fluorescence spectra were collected after thermal equilibration at 298 K ( Figure 5, Figure S25-S29).

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The Journal of Organic Chemistry   1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57 58 59 The red shift in fluorescence emission spectra of the guest 13, registering two maximum (458 nm and 545 nm), was observed. The lack of a clear isosbestic point indicates that there are more than two species in the system, which is hypothesized to be the formation of aggregates due to the high concentration of guests used in the system. Fluorescence enhancement was observed for the guests during the titration with the hosts 5 and 7. The excited-state vibrational dynamics of the viologen guests 11-13 appears to play the key role in the observed enhancement, as well as the red shift of the emission when the viologens concentration is increased. Studies by Galoppini and coworkers, 39 and Pal and coworkers, 40 show similar behavior of a viologen and the dye Brilliant Green, a triphenylmethane derivative, where restriction of intramolecular bond rotations between the aryl rings induced by complexation/encapsulation with cucurbiturils resulted in enhanced fluorescence.

CONCLUSION
In conclusion, we synthesized three water-soluble NARCl receptors (5, 7 and 9) with varying hydrophilicity of the upper rim substituents. These receptors exists in the C 4v bowl-shaped conformation as observed from their relatively simple 1 H NMR spectra. The binding properties of the NARCl receptors and three water-soluble viologen derivatives (11)(12)(13) were investigated in water via NMR, ITC, and fluorescence studies. Binding constants of 10 3 M -1 were observed via ITC analyses. The hosts show higher affinity towards the acetylmethyl-derived viologen guests 12 and 13 over the methyl viologen derivative 11. The higher affinity can be attributed to hydrogen bond interactions between the host cation-anion seam and the guest carbonyl groups. This study illustrates the versatility of the NARXs, which in water possesses hydrophobic cavities and hydrophilic cation-anion seam. The ease of functionalization of resorcinarene type receptors into water soluble NARCl receptors make resorcinarenes a very interesting class of receptor compounds. This versatility renders the NARXs as suitable receptors for a variety of guests in water.
General procedure for the synthesis of tetrabenzoxazines from the resorcinarene 3. To a solution of the resorcinarene 3 (5.5 mmols) and excess formaldehyde (6 mL) in ethanol (40 mL), the amine (23.3 mmols) in ethanol (15 mL) is added slowly and stirred at room temperature for 24 h. The precipitate that separated is filtered, recrystallized in a methanol/n-hexane mixture and dried.
General procedure for the synthesis of the N-alkyl ammonium resorcinarene chlorides from the tetrabenzoxazines. Into a solution of the tetrabenzoxazine (0.82 mmol), 3 mL concentrated HCl (37%) and 4 ml H 2 O in 50 ml isopropanol is heated under reflux. Water and formaldehyde are removed by azeotropic distillation with chloroform. The remaining isopropanol is evaporated and the crude product triturated with diethyl ether to give the N-alkyl ammonium resorcinarene chloride.