Supramolecular Pathway Complexity of a Host‐Guest System Revealed in the Single‐Crystal Structural Snapshots

ABSTRACT

The host‐guest aqueous complexation gives rise to a library of crystal structures of distinct inclusion modes and solvation states, all assembled from p‐sulfonato‐calix[4]arene as a host and p‐amino‐benzamidine as a guest. Benefitting from single‐crystal X‐ray diffraction analysis and microscopic observation, we show that these host‐guest structures emerge progressively from kinetically trapped highly solvated assemblies to thermodynamically favored inclusion complexes as a function of time and concentration. The host‐guest system is also highly sensitive to solvent composition, as reflected in the alteration of ¹H NMR spectra and crystallization behavior between water‐alcohol and purely aqueous solutions. The work illustrates how crystallization can serve as a structural probe of pathway‐dependent supramolecular maturation, enabling direct observation of kinetically and thermodynamically accessible assembly states in aqueous host‐guest systems.

The host‐guest structures of variable solvation and inclusion states emerge within supramolecular landscape as a function of time, concentration, and solvent. The work illustrates how crystallization can serve as a structural probe of pathway‐dependent supramolecular evolution from kinetically trapped highly solvated assemblies to thermodynamically favored inclusion complexes.

1. Introduction

Recent works have shown that pathway complexity may emerge even in simple supramolecular systems [1, 2, 3]. This phenomenon implies accessibility of different supramolecular outcomes from the same molecular components depending on the specific route the assembly follows. Usually, the diversification of these routes stems from the delicate balance of kinetic and thermodynamic factors, as well as reversible and weak nature of noncovalent forces bringing molecular components together. The pathway‐dependent molecular self‐assembly may arise from the interplay of sequential binding and conformational interconversion [4], kinetic trapping of metastable structures [5, 6], or from the consumption of chemical “fuel” directing the system to operate under out‐of‐equilibrium conditions [7, 8]. Capture and structural characterization of transient (kinetic) ensembles that undergo further evolution into stable (thermodynamic) supramolecular structures, particularly in aqueous environment, present a significant challenge [9, 10].

At the same time, the solution‐based crystallizing systems occasionally display multi‐phase pathway complexity revealed as the initial crystallization of metastable phase(s) followed by step‐wise transformation to the most stable crystal form under the given conditions. This empirical observation long known as the Ostwald's rule of stages traditionally refers to the solution crystallization of different polymorphic forms [11]. In such scenario, the firstly formed least‐stable form resembles closely a solution state and has the lowest activation energy barrier. Its crystallization is kinetically favored. This initial crystal form can transform through dissolution and recrystallization into the more stable phase via cascade route until the thermodynamically stable phase is reached. Recently, similar multi‐phase step‐like transformations have been noticed in other supramolecular materials, such as gels [12, 13], supramolecular polymers [14], or supramolecular nanoaggregates [15]. Especially in the supramolecular polymerization area, pathway complexity has become an important research direction, opening up possibilities to isolate kinetically trapped species, operate interconversion between different states, or gain precise control over size and structure of supramolecular aggregates [16, 17, 18].

In the present study, we explore host‐guest supramolecular self‐assembly manifested as time‐differentiated step‐wise crystallization as a result of pathway complexity in the host‐guest system. The macrocyclic host in this system is p‐sulfonato‐calix[4]arene (C4S), one of the most widely studied macrocycles of excellent water solubility and biocompatibility [19, 20]. It is known to form supramolecular architectures and superstructures coassembling with small guest molecules [21, 22], metal ions [23], drugs [24, 25], and even proteins [26]. The supramolecular assembly is driven by its versatile inclusion/complexation properties, metal coordination, electrostatics, and hydrogen bonding. The guest is p‐amino‐benzamidine (ABZ) bearing cationic hydrogen bond donors at two opposite ends of the molecule. Building on our previous work [27, 28], we harness the interplay of host‐guest interactions and crystallization events to capture, structurally identify, and monitor the slow progression of kinetically trapped highly solvated host‐guest structures towards stable inclusion complexes under varied conditions of time, concentration, and solvent. All cocrystallization experiments have been conducted employing two commercially available forms of the macrocyclic host, C4S sulfonic acid and C4S sodium salt. Using single‐crystal X‐ray diffraction and optical microscopy, we identified and structurally characterized nine C4S‐ABZ host‐guest complexes along with a new crystal form of the C4S tetrasodium salt. Although these host‐guest crystal forms consist of the same host and guest components, they are not strictly polymorphs because they are not chemically identical. The host‐guest structures of variable solvation/hydration and inclusion states emerging within supramolecular landscape unveil rich pathway complexity and provide crystallographic snapshots of the gradual evolution from kinetically trapped solvated assemblies to thermodynamically favored inclusion complexes in aqueous media. This study affords direct structural insight into pathway‐dependent host‐guest self‐assembly and highlights how subtle variation in solvation, concentration and competitive binding events reshape supramolecular organization in aqueous media.

2. Results and Discussion

The cocrystallization of C4S sulfonic acid with p‐amino‐benzamidine dihydrochloride in 0.5 mL of 1:1 water‐ethanol solution (10 mg of C4S and 5.6 mg of ABZ) affords two crystal complexes emerging on different time scale, Figure 1. Within several hours of setting up crystallization experiment, we noticed the appearance of very large crystals of prismatic shape (upper micrograph, Figure 1). Single‐crystal X‐ray analysis of this freshly crystallized Form I revealed the host‐guest structure of the exclusion binding of p‐amino‐benzamidine and large amount of incorporated solvent molecules. Within the next days we observed the appearance of new form firstly as thin needles and later as rod‐shaped crystals (middle micrograph, Figure 1). The crystal growth of Form II has been accompanied by the slow dissolution of large crystals of Form I. The solution‐mediated transformation was slow enough to be monitored through the time span of several days. Two crystal forms I and II coexisted in the mother solution within this time frame. After app. 6 days only crystals of Form II have remained in the solution (lower micrograph, Figure 1). Single crystal X‐ray analysis showed the new crystals to be of different structure exhibiting inclusion type complexation between C4S and ABZ. As the kinetically favored complex I crystallizes rapidly but then gradually dissolves, we assumed that its formation/crystallization may depend on the concentration. Indeed, simple dilution of the initial mother solution (using 1 mL of the solvent instead of 0.5 mL in the case of two‐step crystallization) results in the exclusive crystallization of Form II, Figure 1. Under lower concentration conditions the first crystals appear only after several days.

FIGURE 1.

Schematic illustration of the host‐guest cocrystallization pathways of p‐sulfonato‐calix[4]arene C4S (sulfonic acid) and p‐amino‐benzamidine ABZ under high and low concentration conditions. At high concentration rapid cocrystallization affords Form I as very large prismatic crystals (upper photo micrograph) that over time (several days) transform spontaneously in solution into rod‐like crystals Form II (lower photo micrograph). Form II crystallizes directly under low concentration conditions. Crystal structure of Form I contains much more water and ethanol solvent molecules (crystal packing shown along b direction) than Form II of more compact crystal packing (shown along b direction). The scale bars are 500 µm.

Crystallographic characterization of Forms I and II showed remarkable differences in the host‐guest structure and solvation, Table S1. The asymmetric unit of Form I (triclinic, P‐1 space group) comprises two C4S macrocycles, four ABZ dications positioned outside of the hosts, 4.5 of ethanol and 32 water molecules, Figure S1. C4S macrocycles filled with ethanol molecules are in the roughly cone conformation, the dihedral angles between the aryl rings and the mean plane of the C4S molecules defined by four methylene carbon atoms are provided in Table S3. The included ethanol molecules do not form hydrogen bonds with upper rim sulfonate groups of C4S, interacting instead with water molecules residing above the cavity, Figure 2A. The inclusion of ethanol molecule is sustained by C─H⋯π interactions between its methylene/methyl groups and aromatic subunits of C4S, the short C─H⋯π distances (toward centroids of the aromatic rings) are in the range of 3.64–3.98 Å. ABZ dications are embedded vertically between C4S bilayers connecting them by amidinium‐sulfonate and ammonium‐sulfonate hydrogen bonding. Overall structure is highly solvated with multiple water (app. 16 H₂O molecules per one C4S) and some ethanol molecules. In Form II (also triclinic, P‐1 space group) asymmetric unit comprises two crystallographically distinct C4S molecules of different inclusion mode and conformation, four ABZ dications, 0.5 of ethanol and app. 15 water molecules (disordered), Figure S2. One of the C4S holds ABZ in roughly longitudinal orientation, residing above the pinched cone of the macrocycle, Figure 2B. The included ABZ molecule forms three amidinium‐sulfonate and one ammonium‐sulfonate hydrogen bonds. Due to such multipoint hydrogen bonding between guest and host the overall inclusion mode is shallow as the guest prefers to stay near the anionic sulfonate rim of the macrocycle. The macrocycle is strongly pinched (Table S3) to serve the optimal interaction with ABZ. The shallow inclusion mode is similar to the previously described structure of C4S with doubly charged bis‐amidinium guest [29]. The filling of another C4S cavity in cone conformation (Table S3) is prone to considerable structural disorder modelled as two positions of ABZ (Figure 2C, in blue and pink) and two positions of ethanol molecule. ABZ molecule of major occupancy (in blue, Figure S3) perches above the C4S upper rim forming two ammonium‐sulfonate and one amidinium‐sulfonate hydrogen bonds. ABZ of minor occupancy (in pink, Figure. S3) pivots deeper into the cavity through its ammonium group pointing to one of C4S aromatic rings (N⋯centroid distance of 3.39 Å suggests N‐H⋯π interaction). This ABZ is in the slantways orientation with respect to C4S cavity with only one amidinium‐sulfonate hydrogen bond present. Such inclusion mode is also typical for unsubstituted benzamidine guest with the same host, as previously reported by our group [27]. The notable feature of Form II is high degree of overall structural disorder required to be introduced in the model despite collecting good quality diffraction data. Majority of C4S sulfonate groups, ABZ and water molecules have been treated as disordered. The structure is less solvated (only app. 7 H₂O molecules per one C4S) and denser than Form I, as can be seen from the comparison of two crystal packings, Figure 1. Form II is further stable in the solution upon its slow evaporation. In the dry sample these crystals became opaque and loose diffraction quality. Additionally, some plate‐shaped crystals of p‐amino‐benzamidine dichloride (refcode WOBCEW) [30] together with small prisms of new host‐guest Form III (monoclinic, space group P2₁/c) can be identified in the dry sample. Its asymmetric unit consists of one C4S with one included ABZ dication, and seven water molecules, of which two are in the form of hydroxonium H₃O⁺ cations, Figure S4. The C4S molecule is even more pinched (Table S3) than in complex II and host‐guest fit is pretty optimal, as all four amidinium donors are engaged in the hydrogen bonding with sulfonate groups, ammonium‐sulfonate hydrogen bond is conserved, Figure 2D,E. It is evident that the inclusion of ABZ requires distortion (pinching) of the macrocycle compared to the inclusion of ethanol molecule happening in app. cone conformation. An interesting feature of Form III is the presence of two hydroxonium cations that can be considered as Zundel type species (H₅O₂)⁺ of very short O···O distances of 2.46 and 2.48 Å representing strong hydrogen bonds. Such supramolecular stabilization of Zundel type cations was previously observed in the crystalline complexes of C4S with phenanthroline [31] and benzamidine [27] guests.

FIGURE 2.

Inclusion modes in Forms I‐III: (A) ethanol molecule residing in the cone shaped cavity in Form I; (B) ABZ inclusion in the pinched cone in Form II; (C) structural disorder of ABZ (in blue and pink) in Form II; (D) macrocycle is strongly pinched upon ABZ inclusion in Form III; (E) amidinium‐sulfonate and ammonium‐sulfonate hydrogen bonds between guest and host in Form III. (B) and (C) represent two crystallographically independent C4S molecules of different conformation and inclusion mode in Form II.

The crystallization of kinetic Form I under high concentration conditions reflects pathway complexity of host‐guest molecular self‐assembly arising from competing host‐ethanol interactions. Solvent molecules are certainly noninnocent components in the host‐guest aqueous systems. They can compete for the place in the macrocyclic cavity of the host molecule, or form hydrogen bonds with host or guest molecules. Such competition is noticeable as the conformational deformation (pinching) of the calix[4]arene macrocycle is required to fit ABZ guest in the cavity. In the first step of host‐guest molecular encounter ABZ molecules cling to C4S macrocycles externally through ion‐ion and hydrogen bonding interactions realized via positively charged amidinium and ammonium functional groups, while cavity is still filled with ethanol molecules. Under high concentration conditions such kinetic associate crystallizes rapidly (Form I) reflecting early molecular events in the solution state. Eventually, the kinetically formed complex I progresses towards thermodynamic inclusion complex II through dissolution‐recrystallization transformation as a function of time. In fact, the ¹H NMR spectra show the influence of solvent media on the host‐guest complexation. We have compared two solvent systems, namely D₂O/CD₃OD (v/v = 1/1, pH 2) mixture and D₂O (unbuffered, pH 2), Figure 3A,B. In both solvent systems aromatic proton resonances of ABZ are strongly shifted toward the upfield region in the presence of C4S, indicating insertion of the guest in the host macrocyclic cavity. In aqueous solution, the chemical shifts of ABZ proton signals are larger (Δδ of 1.26 and 0.87) than in water‐methanol mixture (Δδ of 0.90 and 0.31), suggesting stronger host‐guest interactions in the absence of competing alcohol solvent. The cocrystallization in 0.5 mL of water (10 mg of C4S and 5.6 mg of ABZ) results in the rapid formation of microprecipitate of fiber‐like morphology, Figure 3C. This initial phase undergoes spontaneous structural evolution into rod‐shaped crystals appearing overnight within still present fibers, Figure 3D. Over the next several days only single crystals remain in the mother solution, Figure 3E. These crystals are of the same unit cell parameters and structure as Form II obtained from water‐ethanol mixture, differing somewhat in the disorder modelling of one of included ABZ molecules (Form II _H2O , Supporting Information). The dilution of the initial mother solution to 1 mL results in the formation of large flower‐like single crystal aggregates of Form II _H2O bypassing fiber‐like phase, Figure 3F. The crystals appear in the shorter timespan (overnight) compared to the crystallization from water‐ethanol mixture under identical starting conditions (several days).

FIGURE 3.

Partial ¹H NMR spectra of C4S sulfonic acid, p‐amino‐benzamidine dihydrochloride, and their host‐guest complex recorded on an Agilent 400 MHz instrument at room temperature in (A) D₂O/CD₃OD (v/v = 1/1) and (B) D₂O; C(C4S) = 5 mM, C(ABZ) = 5 mM, pH = 2.0; (C) fiber‐like morphology of microprecipitate formed upon mixing of aqueous solutions of C4S and ABZ; (D) first crystals of Form II _H2O appear within precipitate overnight; (E) only crystals Form II _H2O remain in the solution after several days; (F) in the diluted solution the crystallization occurs directly omitting fiber‐like phase. All scale bars are 500 µm.

The same set of experiments has been performed using C4S sodium salt instead of C4S sulfonic acid. Two additional factors should be considered. First is the presence of sodium cations that can either compete with the target guest [32, 33] or assist in the formation of hetero‐ternary complexes comprising C4S, organic guest and metal ion [34]. In fact, many examples of such ternary structures can be found in the Cambridge Structural Database. Secondly, aqueous solutions of C4S sodium salt are neutral to slightly basic, in contrast to C4S sulfonic acid affording acidic (pH 1.5‐2) solutions. The dissolving of C4S sodium salt with p‐amino‐benzamidine dihydrochloride (acidic compound) affords solutions of pH ∼ 4. We recorded relevant ¹H NMR spectra in D₂O/CD₃OD (v/v = 1/1, pH 4) and D₂O (unbuffered, pH 3.8) solvents. In both solvent systems aromatic proton resonances of ABZ are shifted toward the upfield region in the presence of C4S, confirming inclusion nature of the host‐guest complex despite potential competition from sodium cations. In water‐methanol mixture the signals of the ABZ protons in the presence of C4S sodium salt show much smaller upfield shifts (Δδ of only 0.19 and 0.11) compared to C4S sulfonic acid described above, Figure S12. The significant difference is most probably related to the presence of methanol molecules and sodium cations, both as competitors to ABZ. In water, the same signals of ABZ protons show again pronounced upfield shifts (Δδ of 1.17 and 0.82), regaining strong binding in the host‐guest system, Figure S13.

The cocrystallization of C4S sodium salt with p‐amino‐benzamidine dihydrochloride in 0.5 mL of water‐ethanol solution (10 mg of C4S and 5.6 mg of ABZ) affords different crystal complexes than in the case of C4S sulfonic acid, Figure 4. The next day from starting crystallization experiment (app. 20 h) the plate‐like crystals of square shape emerged (upper micrograph, Figure 4) that appear to be of orthorhombic Pbcn space group. This crystal form IV comprises C4S complexed with p‐amino‐benzamidine and sodium cations. Within the next several hours after examination of the sample under the microscope the novel crystals appear among square plates, firstly as small prisms growing over next several days into larger prisms, (middle micrograph, Figure 4). The yellowish prismatic crystals of Form V are of monoclinic C2/c space group. Despite some disintegration of plate‐like initial crystals, two forms coexist further, also under the conditions of the slow evaporation of mother solution. Crystals of Form V can be obtained exclusively (lower micrograph, Figure 4) under lower concentration conditions, for example diluting the initial mother solution from 0.5 to 1 mL, or using less ABZ dihydrochloride (2.8 instead of 5.6 mg) in the same solvent volume. The crystallization occurs after several days.

FIGURE 4.

Schematic illustration of the host‐guest cocrystallization pathways of p‐sulfonato‐calix[4]arene C4S (sodium salt) and p‐amino‐benzamidine ABZ under high and low concentration conditions. At high concentration rapid cocrystallization affords Form IV as square plate crystals (upper photo micrograph) that over time (several days) transform spontaneously in solution into Form V (lower photo micrograph). Form V crystallizes directly under low concentration conditions. Crystal structure of Form IV contains both sodium and ABZ cations leading to coordination polymeric structure, crystal packing of the metallo‐supramolecular assembly is shown along b direction. Form V is devoid of sodium cations, its crystal packing is shown along [1 0 1] direction. The scale bars are 500 µm.

Single crystal X‐ray analysis of square‐plate crystal of orthorhombic Form IV revealed that only half of C4S macrocycles is filled with ABZ molecules, while another half is occupied with disordered solvent molecules, Figure S5. Two crystallographically distinct C4S molecules take different shapes, one holding ABZ in the longitudinal orientation is strongly pinched, another one with disordered solvent is of more symmetrical cone shape (Table S3). The inclusion of ABZ in the pinched C4S is assisted by four amidinium‐sulfonate and one ammonium‐sulfonate hydrogen bonds, similarly as in Form III, Figure S6. Figure 5A shows these two C4S molecules as a pseudo‐capsular ensemble. Both C4S are coordinated to sodium cations at the sulfonate rims. Altogether, the asymmetric unit of IV comprises two C4S, two and a half ABZ molecules as dications, three sodium cations (of which two are disordered), and 13.5 water molecules, Table S2. Overall, 2‐D coordination polymeric structure of corrugated layers is formed in the ab plane. Monoclinic Form V is devoid of sodium cations. Its asymmetric unit consists of one C4S, three and a half ABZ molecules, 7.75 of water and 0.25 ethanol molecules, Figure S7. The structure model is highly disordered despite nice crystals and good quality diffraction data. The interior of the macrocyclic cavity was modelled as a disorder of ABZ molecule over two positions. First one pivoting into the cavity via amino group in almost vertical orientation (in blue, Figure 5B), two N⋯centroid distances of 3.60 and 3.94 Å suggest potential N─H⋯π interaction with aromatic walls of C4S. In such inclusion geometry amidinium group of ABZ is distant from C4S upper rim, preventing possibility of host‐guest hydrogen bonding. Instead, amidinium‐water and amidinium‐sulfonate hydrogen bonds are realized with adjacent C4S molecules externally. The second position of ABZ is in the slantways orientation (in pink, Figure 5C) assisted with one amidinium‐sulfonate hydrogen bond, while amino group points to one of C4S aromatic rings, again N⋯centroid distance of 3.28 Å suggests N‐H⋯π interaction. To account for the electroneutrality of the complex the ABZ molecules have been introduced in the model as monocations with positive charge on the amidine groups only. ABZ molecule has two functional groups of different pKa values. While amidine group remains cationic over wide pH range (its pKa is 12–13), the pKa value of anilinium (4‐amino) group is ∼4.6 meaning that both protonated and neutral forms are present at pH 4.

FIGURE 5.

(A‐E) Inclusion variants in Forms IV‐VII; (F) microprecipitate of dendritic aggregates forms rapidly upon mixing of aqueous solutions of C4S sodium salt and ABZ; after 1 day first crystals of Form VI appear within precipitate, after 2 days the initial microprecipitate dissolves and crystals of VI and VII remain in solution; (G) in the diluted solution the crystallization of very large prisms of Form VII takes place, later minor presence of rod‐like crystals of Form VI were noticed. All scale bars are 500 µm.

The same cocrystallization experiments in water give different outcomes than in water‐ethanol mixtures. The rapid microprecipitation occurs in 0.5 mL of water (10 mg of C4S sodium salt and 5.6 mg of ABZ). The dendritic aggregates can be observed under the microscope, Figure 5F. The next day after microprecipitation the rod‐shaped crystals appeared in the vial. Single crystal X‐ray diffraction analysis revealed the new host‐guest complex Form VI in the triclinic P‐1 space group, Figure S8. Its asymmetric unit consists of one C4S, two and a half of ABZ in mixed protonation state and 7.5 water molecules. The structure is mostly ordered and hydrogen atoms of ammonium and amino groups of fully occupied ABZ have been found on the difference maps and refined. ABZ molecule residing above the pinched macrocycle is a dication (Figure 5D), it is held by three amidinium‐sulfonate and one ammonium‐sulfonate hydrogen bonds. There is also half of ABZ dication disordered by symmetry outside the cavity. One ABZ is monocationic, and the charge is balanced in the crystal complex. The mixed protonation state of ABZ amino groups agrees with the hydrogen bonding pattern. Over the next days, the initial dendritic microprecipitate dissolved completely and many smaller rod‐like crystals of Form VI together with some prism‐shaped crystals appeared. This new Form VII (monoclinic, P2₁/n space group) can be obtained directly from the less concentrated initial solution, namely using 1 mL of water instead of 0.5 mL. No precipitation has been observed, while very large prismatic crystals emerged after several days Figure 5G. Interestingly, after one week, several small rod‐shaped crystals of Form VI can be noticed between predominant large prisms of Form VII. Asymmetric unit of Form VII comprises one C4S, three ABZ in mixed protonation state, and five water molecules, Figure S9. ABZ residing above C4S macrocycle is dicationic (Figure 5E), while the remaining two are monocationic. The inclusion/perching mode and hydrogen bonding pattern is similar to Form VI.

Two additional host‐guest crystal forms and novel structure of C4S sodium complex have been identified in the dry samples after complete evaporation of mother solutions. Form VIII (monoclinic, P2₁/c, Table S2) originates from the cocrystallization experiment in 0.5 mL of water‐ethanol solution (10 mg C4S sodium salt and 5.6 mg ABZ) initially affording crystals of Forms IV and V. In the dry sample small prismatic crystals of Form VIII were present together with some remnants of square plate like crystals of Form IV. Asymmetric unit is composed of one C4S, three ABZ (one as dication and two as monocations) and only one water molecule, Figure S10. The structure is well‐ordered and even hydrogen atoms of ammonium and amino groups of ABZ molecules could be identified from the difference map and refined. Form IX (triclinic, P‐1 space group, Table S2) originates from the cocrystallization in 0.5 mL of water‐ethanol solution (10 mg C4S sodium salt and 2.8 mg ABZ) initially affording crystals of Form V only. In the dry sample crystals of Form IX were present concomitantly with crystals of new C4S sodium complex, Form C4S_Na. Asymmetric unit of Form IX comprises one C4S, four ABZ (all as monocations, two disordered by symmetry), and 2.75 water molecules, Figure S11. The ABZ inclusion/perching mode and hydrogen bonding pattern in forms VIII and IX are similar to the majority of previous complexes (forms II, III, IV, VI and VII).

Form C4S_Na is a tetra‐sodium complex in the P2₁/c space group crystallized as a nonmerohedral twin with 9.5 water molecules per formula unit, Figure 6. There are two water molecules shallowly included in the cavity, one of full occupancy O9W coordinated to sodium cation, and another noncoordinated O10W refined with 50 % occupancy. Each of these water molecules shows one close distance to the plane of neighboring aryl ring suitable for O─H···π hydrogen bonding, 3.20 Å for O9W and 3.26 Å for O10W. All water molecules besides O10W are coordinated to sodium cations. This structure differs from all tetra‐sodium and penta‐sodium C4S crystal complexes known from the previous studies. The first crystallographic characterization of the tetra‐sodium C4S complex showing inclusion of single water molecule into aromatic cavity traces back to 1991 [35]. However, no coordinates were given and the structure is not deposited in the Cambridge Structural Database. Later, Fucke et al. redetermined this structure using both X‐Ray (refcode REBYEE03) and neutron (refcode REBYEE04) diffraction data, and reported two new polymorphic modifications (refcodes REBYEE01 and REBYEE) of tetra‐sodium C4S hydrate with app. 14 water molecules [36]. All these polymorphs contain one intracavity water molecule engaged in two O‐H···π hydrogen bonds with the interior of the macrocycle. For penta‐sodium C4S complexes the coordination is realized both at the sulfonate and hydroxyl rims, the macrocyclic cavity is filled either with ethanol molecule (refcode QADYOK) [37] or two water molecules (refcode SAFJEO) [38]. The most recent C4S sodium complex of triclinic symmetry (refcode AMIQAS) comprises 2.5 sodium cations (disordered) per macrocycle implying mixed protonation states of calix[4]arene molecules within the crystal [39].

FIGURE 6.

Crystal structure of novel form of C4S tetra‐sodium complex – Form C4S_Na; two water molecules O9W and O10W are shallowly included in the macrocyclic cavity (pinched), four sodium cations are coordinated to sulfonate groups and water molecules (aqua ligands shown as red balls). The crystal packing (along b direction) is of classical bilayer character.

3. Conclusion

The aqueous complexation between macrocyclic host p‐sulfonato‐calix[4]arene C4S and guest p‐amino‐benzamidine ABZ gives rise to nine crystalline supramolecular assemblies of distinct inclusion modes and solvation states emerging from pathway complexity in host‐guest self‐assembly. Under high concentration conditions the kinetically trapped Form I of the exclusion complexation mode between host C4S sulfonic acid and guest ABZ crystallizes rapidly. This structure features ethanol inclusion in the macrocyclic cavity of calix[4]arene and large content of solvent (water and ethanol) in the crystal representing early transient stages of the host and guest molecular encounter. Over time the crystals of Form I progress towards inclusion complex Form II via the route of spontaneous dissolution‐recrystallization transformation. Under low concentration conditions the host‐guest inclusion Form II crystallizes directly. The replacement of the macrocyclic host with C4S sodium salt results in the drastic change of the complexation/crystallization outcome. Under high concentration conditions Form IV of metallo‐supramolecular structure forms rapidly, with half of the calix[4]arene molecules occupied by ABZ guests and another half taken by solvent molecules. Under lower concentration conditions cocrystallization of C4S sodium salt and ABZ gives directly inclusion Form V devoid of sodium cations. Additional host‐guest crystal forms emerge under slow evaporation of mother solution conditions, typically comprising less water molecules per host‐guest pair than earlier crystallizing crystal complexes. These late‐appearing structures (forms III, VIII and IX) are not only less‐solvated, but also less‐disordered and structurally better adapted in terms of host‐guest complementarity. The host‐guest system is also highly sensitive to solvent media, as reflected in the alteration of ¹H NMR spectra and crystallization behavior between water‐alcohol and purely aqueous solutions. Overall, the system represents a unique example of host‐guest pathway complexity manifested in multiple crystalline supramolecular structures assembled from the same host and guest molecular components depending on time, concentration, and solvent conditions. More broadly, the work illustrates how crystallization can serve as a structural probe of pathway‐dependent supramolecular maturation, enabling direct observation of kinetically and thermodynamically accessible assembly states in aqueous host‐guest systems.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

The authors have cited additional references within the Supporting Information [41, 42, 43].

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Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article. CCDC 2555344‐2555353 (for Forms I‐IX) and 2555343 (for C4S_Na) contain the supplementary crystallographic data for this paper [40].