As the foundation elements of natural world,anions play important roles in a range of chemical,biological and environmental processes ^{[1, 2, 3]}. In the past a few decades,supramolecular chemists have made significant advances toward anion recognition ^[4].

Until recent five years,the property of neutral C-H site(s) has proved to effectively bind with anionviaintermolecular hydrogen bond(s) both in experimental and computational investigations ^{[5, 6]}. The finding attracts the interests both in chemistry and biology fields because C-H bond is exist in the majority (97%) of chemical compounds ^[7]. On the other hand,the nitrogen atom with sp²hybridization (N(sp²)) also received significant attention of chemists and biologists for so long time with its metal binding and intermolecular hydrogen acceptor properties,but have rarely been evaluated in anion complexation ^{[8, 9]}.

Recently,we reported the facile synthesis of two novel tetraimidazolium macrocycles,cyclo^[2](2,6-bis-(1H-imidazol-1-yl)pyridine)^[2](1,3-dimethylenebenzene) ( 1⁴⁺),and cyclo^[2] (2,6-bis-(1H-imidazol-1-yl)pyridine)^[2](2,6-dimethylenepyridine) ( 2⁴⁺),with the names as ‘‘Texas-sized’’ molecular boxes ^[10]. The small differences between them locate on two sites of the bridged groups toward the center of the macrocycle (X sites in Fig. 1).

Fig. 1

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Fig. 1. Structures of ‘‘Texas-sized’’ molecular box 14+and 24+.

More specifically,macrocycle 1⁴⁺has neutral C-H bonds whereas 2⁴⁺instead with N(sp²) groups. These two structures provide a model that evaluate the effect of neutral C-H bondvs.N(sp²)for anion binding in similar chemical environment. For the purpose, the interactions between host 1⁴⁺or 2⁴⁺and four anion guests with different shapes (i.e.Cl^-,N₃^-,NO₃^-and H₂PO₄^-)were studied in detail to investigate the influence of neutral C-H bond or N(sp²) group in anion complexations. As shown below,the supramolecular complexes formed between macrocycle 1⁴⁺or 2⁴⁺and anions were distinct in stoichiometries,association constants,and binding modes. The results develop the knowledge of the different effects between two wide-spread active sites, namely neutral C-Hvs.N(sp²),and their roles in anion binding even in biology. 2. Experimental

2.1. Instruments and reagents

Deuterated dimethyl sulfoxide (DMSO-d₆) was purchased from Cambridge Isotope Laboratory (Andover,MA). All the other solvents and chemicals were purchased commercially (Aldrich, Acros,or Fisher) and used without further purification. All the guest anions were studied as their tetrabutylammonium (TBA⁺) salts (i.e. TBA⁺.Cl^-,TBA⁺.N₃^-,TBA⁺.NO₃^-and TBA⁺.H₂PO₄^-). ¹H NMR spectra were recorded in DMSO-d₆on a Bruker Advanced Instrument (600 MHz). The chemical shifts of the proton signals are reported relative to tetramethylsilane (TMS). For all the anion binding studies involving 1⁴⁺or 2⁴⁺,the starting counter anions were always hexafluorophosphate (i.e.PF₆^-),which is obtained as reference ^[10].

The crystallographic data were collected on a Rigaku Mercury2 (2×2 bin mode) or a Saturn724 + (2×2 bin mode) CCD diffractormeter using a graphite monochromator with Mo Ka radiation (l= 0.71073 Å ). The data were collected using w-scans with a scan range of 1° at low temperature using a Rigaku Tek50 low-temperature device on the Rigaku Mercury2 (2×2 bin mode), or a Rigaku XStream low-temperature device on the Saturn724 + (2×2 bin mode) diffractometer.

The compound for electrospray ionization mass spectrometry (ESI-MS) analysis were obtained with anion exchange processvia adding 10 molar equiv. of TBA⁺.A^-(A^-=Cl^-,N₃^-,NO₃^-and H₂PO₄^-) in the acetonitrile solution of 1⁴⁺.4PF₆^-or 2⁴⁺.4PF₆^-with concentration as 5.00×10^-3mol/L. White precipitate products were collected and washed with acetonitrile for ESI-MS spectroscopy study. The ESI-MS data were collectedviainfusion on a liquid chromatograph mass spectrometer (LC-MS-2010) operating in positive ion model. 2.2. Experimental procedure

The Job-plots experiment: As described in Ref. ^{[11, 12]},the whole concentration of host and guest remain constant during the process as below.

The titration experiment: Following the procedure shown in Ref. ^[12],in the process of every titration,the concentration of host remains stable as 5.00×10^-4mol/L. 2.3. The single crystal X-ray structure study 2.3.1. Crystal cultivation

Diffraction grade crystals were obtained by slow evaporation from solution in the mixture of water andN,N-dimethylformamide (DMF) as described below. 2.3.2. Data refinement

Data reduction was performed using CrystalClear ^[13]. The structures were solved by direct methods using SIR97 ^[14] and refined by full-matrix least-squares on F2 with anisotropic displacement parameters for the non-H atoms using SHELXL-97 ^[15]. The hydrogen atoms were calculated in ideal positions with isotropic displacement parameters set to 1.2×Uequiv. of the attached atom (1.5×Uequiv. for methyl hydrogen atoms). The utility ROTAX ^[16] in the program WinGX ^[17] was used to look for possible twins. The function,w(|Fo|2-|Fc|2),was minimized. Neutral atom scattering factors and values used to calculate the linear absorption coefficient are from the International Tables for X-ray Crystallography (1992). All ellipsoid figures were generated using SHELXTL/PC ^[18]. Tables of positional and thermal parameters,bond lengths and angles,torsion angles,figures and lists of observed and calculated structure factors are located in the cif documents available from the Cambridge Crystallographic Center viaquoting reference numbers 842438,979059 and 979060. 3. Results and discussion 3.1.¹H NMR Job-plots

To confirm the binding stoichiometry between macrocycle host 1⁴⁺or 2⁴⁺and anion guests with different shapes (namely Cl^-(ball),N₃^-(linear),NO₃^-(triangle) or H₂PO₄^-(tetrahedron)),Jobplots were performed for every host/guest pair respectively. The Job-plots of 1⁴⁺and Cl^-or N₃^-displayed maximum values at 0.5, which are consistent with a 1:1 (host: guest) binding stoichiometry; meanwhile the data of 1⁴⁺and guest anion NO₃^-or H₂PO₄^-peaked at 0.67,indicating the complexation stoichiometry both are 1:2 (host: guest) (Fig. 2a). Differently,host 2⁴⁺bind every kind of guest (i.e. Cl^-,N₃^-,NO₃^-or H₂PO₄^-) with the same 2:3 (host:guest) stoichiometry,which were characterized with all the maximum values at 0.6 in their Job-plots (Fig. 2b). Obviously, the small difference between two macrocycle anion receptors (neutral C-H on 1⁴⁺vs.N(sp²)on 2⁴⁺) with analogous skeleton causes their distinct binding modes with same anion,resulting in different equilibrium. It is noted that one macrocyclic host has the potential to bind more than one small inorganic anionic guest in the solution. The multi-anion binding property was also supported viathe additional evidences from electrospray ionization mass spectrometry (ESI-MS) study in gas phase and single crystal X-ray crystallography study shown below. There are two possible reasons of the fact: (1) One host with big cavity can bind small anionic guests both inside and outside of its cavity; (2) Macrocycle hosts contains tetracationic charge so that it could combine with multiple small single charge anion.

Fig. 2

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Fig. 2. Job-plots corresponding to the binding between host 14+([host] + [guest] = 5.00×10-4mol/L) (a), or 24+([host] + [guest] = 1.00×10-3mol/L) (b) and anion guests with different representation symbol constructed from 1H NMR (600 MHz) spectral data.

In order to complete the binding study in solution besides stoichiometry and obtain the associate constants between 1⁴⁺or 2⁴⁺and every anion guest (namely Cl^-,N₃^-,NO₃^-or H₂PO₄^-), corresponding titration experiments were carried out respectively. As Fig. 3a shows below,the titration results between macrocycle host 1⁴⁺or 2⁴⁺and Cl^-have been discussed in detail to explain the different effect of neutral C-H or the electron pair of N(sp²) on analogue anion receptor backbones. The large low field chemical shift change of H(8) on 1⁴⁺with increasing Cl^-suggested that the neutral C-H...Cl intermolecular hydrogen bond(s) play an important role in the complexation. It is observed that the signal of H(1) on 1⁴⁺shows less low field chemical shift,even the protons has more acidity than H(8) (Fig. 3a). The possible reason of the phenomenon is the influence of intermolecular anion-π interactions. Further supporting evidence of the deduction came from the signal of H(3) on 1⁴⁺moving to the high field with additional Cl^-. Meanwhile the signal of H(6) on 1⁴⁺shifts to low field,which implied that anion-pinteractions is mainly supplied viapyridinyl moieties other than bridged benzene rings on 1⁴⁺(Figs. 3b and 4a).

Fig. 3

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Fig. 3. 1H NMR (600 MHz) binding isotherms of H(1), H(8) on 14+, H(1')on 24+(a), or H(3), H(6) on 14+, H(3'), H(7')on 24+(b) with additional Cl-in DMSO-d6at 300 K. The red dash lines show the non-linear curve fit of the experiment data to the appropriate equation.

As the titration process between 1⁴⁺and Cl^-,the low field chemical shift change of H(1')on 2⁴⁺with increasing Cl^-gave out the evidence of intermolecular hydrogen bonds (Fig. 4b). Differently, when bridged benzene rings of 1⁴⁺were replaced with pyridine moieties (i.e. 2⁴⁺),the chemical shift of H(3'),H(7')on 2⁴⁺took an updown change tendency relative to 1⁴⁺. More specifically,the low field chemical shift change of H(3') and the high field chemical shift change of H(7')on 2⁴⁺co-supported the suggestion that the anion-p interaction between 2⁴⁺and Cl^-was primarily providedviabridged pyridine ring (the pyridinyl fragments with proton H(7’)),instead of pyridine plane on 2,6-bis-(1H-imidazol-1-yl) pyridine with proton H(3'),which efficiently support the anion-π interactions between 1⁴⁺and Cl^-as discussed before. Besides Cl^-(as the representative of anion with ball shape),what were explored in the study included the interactions between macrocycle anion receptor 1⁴⁺or 2⁴⁺and the anion guests with different shapes (i.e.N₃^-(linear),NO₃^-(triangle), or H₂PO₄^-(tetrahedron)). Surprisingly,the chemical shift change tendencies of the proton signals on 1⁴⁺or 2⁴⁺were similar as they did with additional Cl^-,no matter what shape the anion guest was introduced. The results suggested that for all the studied small inorganic anion guests,the macrocycle anion receptor 1⁴⁺or 2⁴⁺adopted analogical binding mode as it does for Cl^-.

Fig. 4

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Fig. 4. 1H NMR spectroscopic titration of 14+(5.00×10-4mol/L) (a), and 24+(5.00×10-4mol/L) (b) with increasing Cl-in DMSO-d6at 300 K (600 MHz).

Based on every binding stoichiometry between each host-guest pair detectedviaJob-plots,the functional relationship between the chemical shift change of proton signals on the host and the corresponding concentration of anion guest was investigatedvia Hyperquad2003 program. Finally,the values ofK1and/orK2of anion complexation were obtained by non-linear curve fit ^{[19, 20]}. The results of ¹H NMR Job-plot and titration study has been summed up in Table 1. Herein,K1of macrocycle 1⁴⁺is much larger than 2⁴⁺for every anion guest binding,which also further support that neutral C-H on benzene rings of 1⁴⁺stabilize the anion complexationvia intermolecular hydrogen bond formation. On the other hand,the N(sp²) electron pair of bridged pyridinyl moieties with H(7) on 2⁴⁺tends to form possible intramolecular hydrogen bond(s). Both factors were suggested to cause the largerK1values of 1⁴⁺than 2⁴⁺for the same anion complexation as shown in Table 1.

Table 1

Table 1 Summary of equilibrium and the calculated association constantsKa.

Table 1 Summary of equilibrium and the calculated association constantsKa.

ESI-MS experiments were also carried out to detect the host/ guest interactions. The results listed in Table 2 lead us to suggest that the host/guest complexes may also exist in the gas phase.

Table 2

Table 2 Summary of ESI-MS results.

Table 2 Summary of ESI-MS results.

The data collection and structure refinement statistics of single crystal X-ray structures,namely [ 1⁴⁺4Cl^-2DMF.4H₂O] and [2⁴⁺2PF₆^-.2NO₃^-] involving into this work are summarized in Table 3. The single crystal structure of [ 1⁴⁺4NO₃^-.4H₂O] reported before ^[10] was displayed here for the following discussion.

Table 3

Table 3 X-ray crystallographic data of [ 14+.4NO3-4H2O], [ 14+.4Cl-2DMF 4H2O] and [24+.2PF6-2NO3-].

Table 3 X-ray crystallographic data of [14+.4NO3-4H2O], [14+.4Cl-2DMF 4H2O] and [24+.2PF6-2NO3-].

The single crystal X-ray structure of [ 1⁴⁺4NO₃^-.4H₂O] has been studied to show the flexibility of 1^4+[10]. But the effect of neutral C-H bond for anion bindingviaintermolecular hydrogen bonds has not been discussed. In this structure, 1⁴⁺contains one NO₃^-anion in its cavity. The ‘‘clip’’ conformation of macrocycle with NO₃^-result in a molecular ‘‘sandwich’’ structure,which is stabilizedvia intermolecular hydrogen bond between NO₃^-and neutral C-H on C(18) (characterized with short distance between O(4) on NO₃^-and C(18) less than 3.4 Å ),as well as anion-pinteractions between the plane of NO₃^-anion and the neighbor pyridinyl moieties on 1⁴⁺with distance less than 3.3 Å (Fig. 5). Select bond distances are as follows: selected interatomic hydrogen bonding distances [Å]: O(4)...C(18) 3.393,O(6)...C(37) 3.730. Selected interatomic angles: C(18)-H(18A)...O(4) 141.98,C(37)-H(37A)...O(6) 131.08. Selected anion...pdistances [Å]: O(5)...N(3) 3.757,O(5)...N(8) 3.294,O(5)...C(23) 3.250.

Fig. 5

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Fig. 5. Top view with the ellipsoid form (a), as well as the side view (b), top view (c) and front view (d) with the stick form of the ‘‘clip like’’ conformation of14+.with a insert NO3-anion present in14+.4NO3-4H2O. Displacement ellipsoids are scaled to the 25% probability level. All the other molecules have been omitted for clarity.

Meanwhile,it is noted that two other neighbor NO₃^-anion bind with 1⁴⁺mainlyviaanion-pinteractions with ‘‘outside’’ mode, which is characterized with the short distances (less than 3.0 Å) between the plane of NO₃^-anion and the neighbor pyridinyl moieties of 2,6-bis-(1H-imidazol-1-yl)pyridine on 1⁴⁺,as the solution study implied before (Fig. 6). Select bond distances are as follows: selected anion...pdistances [Å]: N(11)...C(27) 3.285, N(11)...N(8) 3.763,N(11)...N(9) 3.502,N(11)...C(26) 3.416, O(1)...C(26) 3.618,O(1)...C(27) 3.618,O(1)...N(9) 3.522, O(1)...C(29) 3.491,O(2)...C(26) 3.638,O(3)...N(8) 3.140, O(3)...N(9) 3.071,O(3)...C(27) 2.993,O(3)...C(28) 3.223; N(13)...C(8) 3.374,N(13)...N(3) 3.741,N(13)...N(4) 3.399, N(13)...C(7) 3.754,O(7)...C(9) 3.652,O(7)...C(10) 3.653, O(7)...N(4) 3.452,O(8)...C(9) 3.020,O(8)...N(4) 3.153, O(8)...C(8) 3.141,O(8)...N(3) 3.116,O(9)...C(7) 3.758, O(9)...C(8) 3.748.

Fig. 6

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Fig. 6. Top view with ellipsoid form (a), as well as the top view (b), front view (c) and side view (d) showing14+.in stick form, along with the two co-bound NO3-anions (in space filling form) present in 14+.4NO3-.4H2O. Displacement ellipsoids are scaled to the 25% probability level. All the other molecules have been omitted for clarity.

A new single crystal X-ray structure,the pure chloride salt of 1⁴⁺was obtained by slow evaporation from solution using mixtures of water and DMF. The structure of [1⁴⁺4Cl^-2DMF.4H₂O],shows the neutral C-H on aromatic ring (e.g. benzene) can act as strong intermolecular hydrogen bond donor for not only anionic species, but also neutral molecules binding (e.g.DMF and H₂O). As listed below, 1⁴⁺bind with DMF and H₂Omainlyvia intermolecular hydrogen bond between O(1) and C(32) with distance less than 3.6 Å ,and between O(6WA) and C(32) with distance less than 3.7 Å.

p-pdonor-acceptor interactions also appeared between the plane of DMF molecules and the neighbor 2,6-bis-(1H-imidazol-1-yl) pyridinyl moieties on 1⁴⁺with distance less than 3.5 Å (Fig. 7). Select bond distances are as follows: selected interatomic hydrogen bonding distances [Å ]: O(1)...C(32) 3.522,O(6WA)...C(32) 3.603. Selected interatomic angles: C(32)-H(32A)...O(1) 113.58,C(32)-H(32A)...O(6WA) 169.28. Selected p...pdistances [Å ]: O(1)...C(1) 3.042,O(1)...C(28) 3.060,O(2)...C(9) 3.365,O(2)...C(20) 3.365, C(39)...C(1) 3.558,C(39)...C(27) 3.268,C(42)...N(4) 3.314, C(42)...N(7) 3.363,N(11)...C(27) 3.475,N(11)...C(4) 3.557, N(12)...C(10) 3.549,N(12)...C(22) 3.519.

Fig. 7

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Fig. 7. Top view with ellipsoid form (a), as well as the side view (b), top view (c) and front view (d) showing the ‘‘clip like’’ conformation of14+.in stick form, along with the two co-bound DMF and one water molecules (in space filling form) present in [14+.4Cl-2DMF.4H2O]. Displacement ellipsoids are scaled to the 25% probability level. All the other molecules have been omitted for clarity.

With the similar procedure to obtain [1⁴⁺4NO₃^-.4H₂O],but instead 1⁴⁺4PF₆^-with 2⁴⁺4PF₆^-,only half PF₆^-in 2⁴⁺4PF₆^-was substituted by NO₃^-and gave out the single crystal structure of [2⁴⁺2PF₆^-.2NO₃^-]. The possible reason is the weaker interaction of 2⁴⁺than 1⁴⁺for NO₃^-binding. It is suggested that the intramolecular hydrogen bond formation between hydrogen bond-acceptor (N(sp²)) on bridged pyridine and its neighboring cationic C-H on imidazolium ring. Herein, 2⁴⁺bind with NO₃^-mainlyvia anion-pinteraction between NO₃^-and neighbor imidazolium ring with N(1),which is characterized with the short distance (less than 3.4 Å ) (Fig. 8). Select bond distances are as follows: selected anion...pdistances [Å ]: O(1)...C(2) 3.249, O(1)...N(1) 3.329,O(2)...N(1) 3.629,O(2)...N(2) 3.279, O(2)...C(2) 3.337,O(2)...C(4) 3.577,O(2)...C(5) 3.599; N(1B)...N(1) 3.410,N(1B)...N(2) 3.336,N(1B)...C(2) 2.963, O(3)...N(2) 3.353,O(8)...C(2) 3.034,O(3)...C(5) 3.309, O(3)...C(6) 3.234. These findings in single crystal analysis also support the conclusion from studies in solution and gas phase described above.

Fig. 8

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Fig. 8. Top view with ellipsoid form (a), as well as the top view (b), side view (c) and front view (d) showing the host 24+.in stick form, along with the two co-bound NO3-anions (in space filling form) present in [24+.2PF6-.2NO3-]. Displacement ellipsoids are scaled to the 25% probability level. All the other molecules have been omitted for clarity.

In summary,the interactions between different artificial macrocycle anion receptor ( 1⁴⁺or 2⁴⁺) and four anion guests with various shapes (i.e.Cl^-,N₃^-,NO₃^-,H₂PO₄^-) were studied in detail via ¹H NMR spectroscopy studies in DMSO-d₆,ESI-MS studies,and single crystal X-ray diffraction analyses. Neutral C-Hvs.N(sp²) present in the similar chemical environment for anion binding property comparison. The results have shown that neutral C-H bonds on 1⁴⁺effectively stabilize anion combinationviastrong C-H...Cl hydrogen bond(s). Differently,N(sp²) sites on 2⁴⁺with intramolecular hydrogen bond potential weaken the interaction with anion species. The present findings may not only provide the guidance for design of artificial anion receptor,but also help to improve understanding of anion binding mode in biology system. Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. 21202199 to Han-Yuan Gong, and No. 21372258 to Li-Jin Xu),The Young One-Thousand-Talents Scheme,and Beijing Normal University. Thanks also go to Dr. JunFeng Xiang for his assistance with the NMR spectroscopic analyses.