Carboxylated Pillar[5]arene Meets Medicinal Biguanides: Host − Guest Complexes with Alexidine and Phenformin in the Crystal and Solution/Gas Phase

: Here, we discuss crystal and solution/gas-phase complexes of carboxylated pillar[5]arene with two cationic guests, alexidine and phenformin, revealing host − guest and assembly curiosities, the role of hydrogen bonding, and cavity inclusion versus exo -mode binding. We show that the combination of carboxylated pillar[5]arene with bis(biguanidinium) guest alexidine results in the crystallization of open-type supramolecular architecture. This is also the first crystal structure of alexidine ever reported. The crystallization of pillar[5]arene with biguani-dinium drug phenformin affects a rare solid-state complex comprising two cavity inclusion modes within the same crystal lattice. The winner in the competition between ethanol molecules and an organic cation (phenformin) for access to the cavity of pillar[5]arene is undecided, visualized as a “snapshot” of these two inclusion possibilities in one crystal structure. Our results demonstrate that carboxylated pillar[ n ]arenes can be a useful addition to the macrocyclic toolkit for the facilitation of the crystallization of bio(macro)molecules. Moreover, the IM-MS analysis of the precrystallization solutions of pillar[5]arene host and biguanide guests has shown the presence of structures and conformations closely related to those observed in the crystal forms. The most intriguing results obtained for a pillar[5]arene − alexidine complex imply a conformational evolution of the complex over 24 h. The IM-MS analysis complemented by theoretical calculations may be applied to predict and examine the crystallization process of host − guest systems, complementing crystallographic studies.


■ INTRODUCTION
Carboxylated pillar [n]arenes are water-soluble macrocyclic hosts of remarkable molecular recognition properties. 1 Although their repeating units framing the macrorings are similar to those of more mature calix[n]arenes, the pillar architecture of unique curvature with two entrances to the inner space differentiates them from the bowl-shaped calix-type receptors.First reported by Ogoshi in 2010, 2 carboxylated pillar[n]arenes have received a lot of attention in aqueous supramolecular chemistry. 3For example, they have been used in various host−guest assemblies, 4,5 as sensors, 6 drug delivery systems, 7,8 in supramolecular chemotherapy, 9,10 for cell imaging, 11,12 or as scaffolds for supramolecular polymers. 13owever, their solid-state assemblies and structures have barely been touched in comparison to flourishing solution host−guest chemistry.One can imagine employing the confined space and carboxylato-rimmed platforms of pillar[n]arenes in terms of solid-state design toward various architectures based on inclusion properties, hydrogen bonding, π−π, and charge-transfer donor−acceptor interactions.We decided to fill this gap by directing our attention to the crystallization and structure elucidation of carboxylated pillar[n]arenes and their supramolecular assemblies.We managed for the first time to trap in the crystal state both pillar [5]arene 14 and pillar [6]arene 15 in the form of host−guest complexes.We have shown that carboxylated pillar [5]arene (CPA5) forms inclusion and exclusion complexes with electron-deficient viologen guests 16 and coordination complexes with metal cations. 17In the current study, we have chosen two biguanide cationic guests, alexidine (ALEX) and phenformin (PFM), as partner species to crystallize with CPA5, as shown in Figure 1.Biguanides are important pharmaceutical molecules of the rich palette of activity; they have been used inter alia as antidiabetic, antiseptic, or antimalaria drugs. 18lexidine is a well-known biocidal and antiseptic commonly used in oral hygiene products and in contact lens disinfecting solutions.In terms of the chemical structure, ALEX is a bis(biguanide) with the hexamethylene chain linking two biguanides attached to ethyl−hexyl end groups.It is of high interest as a small-molecule ligand that specifically binds to bacterial targets.Although activity of ALEX toward bacteria is biologically known, 19 little information exists on the mechanism of action and its binding stoichiometry. 20Recently, the interaction between ALEX and lipopolysaccharides (LPS, commonly known as endotoxins), the major constituents of the Gram-negative bacteria outer cell wall, have been studied. 21he spectroscopic methods and MD simulation suggested mid-to-high binding of ALEX with lipopolysaccharides due to electrostatic interactions between positively charged guanidinium groups on alexidine and negatively charged phosphate groups present in LPS.From the crystal engineering viewpoint, ALEX is a completely unexplored drug molecule.No crystal structure comprising ALEX can be found in either the CSD or PDB.It is not surprising considering its high conformational flexibility due to prevalence of alkyl chains.
Phenformin is a biguanide antidiabetic drug of a mode of action similar to metformin, the most prescribed drug for type II diabetes.PFM was withdrawn from clinical use in many countries due to its toxicity because of the high risk of lactic acidosis.However, the past decade features tremendous interest in biguanides in line with the repurposing of FDAapproved drugs for cancer treament and prevention. 22reclinical studies suggest potential of PFM as an antitumor agent, particularly in combination with immunotherapy. 23,24FM has shown even more metabolic and pharmacologic potential than metformin, while its toxicity due to side effects is lower than that of some current cancer treatments. 25he objective of this work was to elucidate the complexation modes of alexidine and phenformin with carboxylated pillar [5]arene, gaining insights from crystal structure analysis.Due to the multitopic nature of both host (pillar [5]arene) and guests (biguanidinium), several interaction and association scenarios can be considered.The biguanidinium cations are potential donors of multiple hydrogen bonds that are compatible with carboxyl rims of CPA5 in terms of guanidinium−carboxylate supramolecular synthons.The hydrogen bonding network can potentially be sustained in either the inclusion or exclusion host−guest complexes due to the proximity of biguanidinium moieties to carboxylated rims of the macrocycle.Also, favorable ion pairing between cationic sites on guests and (partially) ionized carboxylated rims of pillar [5]arene should be taken into account.It can be hypothesized that the pillar [5]arene cavity can potentially include either a hydrophobic moiety of the drug molecule due to hydrophobic effect or a positively charged biguanidinium group due to cation−π interactions with the cavity interior.
We show that the combination of CPA5 with supposedly "uncrystallizable" bis(biguanide) guest alexidine results in the crystallization of an exclusion complex of an open-type supramolecular architecture.This is also the first crystal structure of alexidine (ALEX) ever reported.The crystallization of CPA5 with the biguanide drug phenformin (PFM) results in a rare solid-state complex comprising two cavity inclusion modes within the same crystal lattice.
Along with crystallographic studies, precrystallization solutions containing CPA5 and ALEX or PFM were subjected to analysis by ion mobility mass spectrometry (IM-MS), complemented by theoretical calculations.The main objective of this part of the study was the detailed structural characterization of the noncovalent complexes in the mother solutions from which the crystals grew, whereby identifying similarities and the differences between solid phase and solution/gas-phase chemistry. 26This unique insight into the precrystallization process using the IM-MS approach has been successfully applied previously to characterize several host− guest complexes with CPA5 27 and CPA6, 15 providing evidence of their distinct primary conformational ordering in mother solutions.

Crystal Structure of the CPA5−Alexidine Complex.
The crystallization experiments of CPA5 with alexidine dihydrochloride in a water/ethanol solution proved successful and provided us with suitable crystals for single-crystal X-ray diffraction analysis.We were excited to solve the crystal structure of the CPA5 complex with ALEX in a P4 2 /nbc space group, which is the first crystal structure of ALEX reported so far.The asymmetric unit comprises half of a CPA5 molecule positioned on the 2-fold rotation axis (perpendicular to the molecular axis of macrocycle), half of ALEX on the 2-fold rotation axis, and disordered water and ethanol molecules, as shown in Figure 2A,B.The ALEX lies outside of the macrocyclic cavity of CPA5, instead wrapping its external walls, interacting with both carboxylated rims of the macrocycle.Pillar [5]arene is in the undistorted prismatic conformation, with its cavity being filled with disordered ethanol/ water molecules.
ALEX adopts an S-like conformation and is positioned on the 2-fold rotation axis, as shown in Figure 2C.Thus, the two halves of the molecule are symmetry-equivalent.Its hexamethylene and ethyl−hexyl aliphatic chains have been treated as disordered in the structure model.The only ordered regions of the molecule are two biguanidinium groups richly participating in hydrogen bonding.The biguanidinium groups are extensively delocalized, as evidenced by the similarity of the C−N distances (1.321(6)−1.352(5)Å).It appears from the hydrogen bonding network around biguanidinium groups that the central nitrogen atom is not protonated�the absence of the HB donor in its vicinity and steric blockade of these atoms by crooked aliphatic chains.Such a protonation scheme of the biguanide group is in agreement with the previous structural works on bis(biguanidinium) salts. 28Each biguanidinium group has six donors of hydrogen bonds.Five HB are formed toward oxygen atoms of CPA5 molecules, and one HB is donated to water molecule.In total, one ALEX interacts with five adjacent CPA5 molecules in the crystal lattice.Two symmetry-equivalent biguanidinium groups form hydrogen bonds toward two rims of the parent CPA5 macrocycle (from the asymmetric unit, Figure 3A).Then, each of biguanidinium group donates HB toward two additional CPA5 molecules in the crystal lattice, as shown in Figure 3B.One guanidine subunits forms HB with carboxyl and phenoxy oxygen atoms of one CPA5, and the second guanidine subunit forms bifurcated HB with the carboxyl oxygen atom of another CPA5 molecule.
The similar S-shaped conformation has previously been observed for chlorhexidine, another wide spectrum bis-(biguanide) antiseptic.In terms of the chemical structure, chlorhexidine shares with ALEX two biguanidinium groups connected by a hexamethylene linker, but in chlorhexidine, biguanidinium groups are further attached to chlorophenyl moieties.These p-chlorophenyl groups (in place of ethyl− hexyl aliphatic chains in ALEX) can take part in additional stabilizing interactions (like π−π, C−H•••π, and C−H•••Cl), thus facilitating self-assembly and crystallization.In fact, the first crystal structures of chlorhexidine have been obtained from its cocrystallization studies with a set of anionic calix [4]arenes, as reported by Tony Coleman et al. 29 In one of these crystal complexes, namely, in the case of calix [4]arene disubstituted at the lower rim by methoxy−carboxylate groups, chlorhexidine adopts an S-shaped conformation (refcode WODGIG), which is very similar to the conformation of ALEX in the complex with CPA5.Thus, alexidine and chlorhexidine share first crystallization success stories benefiting from the macrocycles as generic cocrystal formers. 30Later, chlorhexidine has been crystallized in the form of its sulfate and carbonate salts, 31 and as cyclohexylsulfamate salt. 32Very recently, the crystal structure of the chlorhexidine complex with acetyltransferase Eis from Mycobacterium tuberculosis has been determined as part of the study of FDA-approved drug repositioning to fight drug-resistant tuberculosis. 33Chlorhexidine has been shown to enter the hydrophobic substrate binding cavity of the Eis enzyme (PDB entry 8F4A) and form a strong salt bridge between its positively charged biguanidinium groups and the carboxyl groups of Asp, acting as an Eis inhibitor.
The bent S-shaped conformation of ALEX is beneficial in terms of maximization its HB propensity toward macrocyclic rims.The extensive HB network generated by two biguanidinium groups results in the completely different mutual organization of CPA5 molecules in the crystal lattice compared to the previously studied solid-state complexes of CPA5.Although in all known crystal structures of CPA5, macrocyclic molecules are assembled in parallel back-to-back orientation into layers, here, adjacent CPA5 is rotated by 90°i n respect to each other, as shown in Figure 4A.Four adjacent CPA5 molecules lie in the ab plane, with their rims being perpendicular to each other defining the central hole of such tetrameric assembly.There are four symmetry-equivalent O−   16 These are the only HB taking place between adjacent CPA5, each macrocycle participating in four such symmetryequivalent HB�in two as a donor and in two as an acceptor.The occurrence of short HB confirms the deprotonation of two carboxylic substituents of the macrocycle.The anionic groups have been assigned as those labeled C9C−O2C(O3C) at one portal and the symmetry-related group at another portal.The deprotonation of two carboxyl groups per macrocycle agrees well with dicationic nature of ALEX and a 1:1 stoichiometry of the complex.While rims outline a tetragonal opening filled with disordered water molecules, the external walls of each four macrocycles define rectangular space occupied by ALEX molecules.The biguanidinium groups of ALEX are crowded near the vertices of the rectangle in the proximity to macrocyclic rims, and aliphatic chains (colored in cyan) are directed toward the hydrophobic center of the rectangle.The alexidine molecules staple the next CPA5 layers in the c direction by guanidinium−carboxylate hydrogen bonding, leading to the pillar [5]arene stacking with macrocyclic rims aligned and thereby forming an open-type three-dimensional HB network, as shown in Figure 4B.The resulting network has large water-filled channels that are parallel to the c axis.The walls of the channels are framed with hydrophilic carboxyl/ate rims of the CPA5 molecules.The diameter of the channels is app. 10 Å, which corresponds to the distance between CPA5 rims framing the tetragonal hole.If we consider that the inner space of the macrocycle is also filled with solvent molecules and both rims are free to access, the structure can be described as a 3D network with interconnected channels.Altogether, these channels occupy appr.25.3% of the crystal volume as calculated by the PLATON procedure for solvent-accessible voids (probe radius 1.2 Å). 34 Unfortunately, the crystals lose their diffraction ability when out from the mother solution.Still, the new assembly pattern of CPA5 molecules in a channel-type HB network reveals an interesting possibility to build open-type architectures with proper partner molecules.
IM-MS Studies of the CPA5−Alexidine Complex.IM-MS combines the merits of using mass spectrometry to identify and determine the mass-to-charge (m/z) value of analyte ions and the ion mobility technique to further detail their threedimensional structural shape.The inference of the structural properties of ions by IM-MS relies on both experimental and theoretical studies of the motion of ions in a buffer gas, more precisely, on the determination of the crucial value of the collision cross section (CCS).When a soft ionization method such as electrospray is used to ionize and transfer an analyte from solution to the gas phase, then IM-MS offers unique insights into the structural properties of noncovalent aggregates, adding to our growing understanding of their structural properties in solution.Hence, this approach is well suited for structural analysis of supramolecular systems, providing insights into their preorganization in the mother solution, one step prior to the crystallization process.Recent advances in instrumentation and the development of computational strategies for IM-MS have enabled its use for tremendously challenging structural studies, including macrocycle−anion complexes, supramolecules with confined spaces and interlocked structures, 35−38 and self-assembled systems such as metallosupramolecular complexes. 39he mass spectrum recorded for the CPA5 and ALEX mixture has flourished with 1:1 and higher-order aggregates from singly to triply charged ions.Also present on the spectrum are peaks corresponding to the uncomplexed host molecule as singly, doubly, triply, and even quadruply charged ions (Figure S1).The structural analysis based on IM-MS studies complemented by theoretical calculations was performed for singly and doubly deprotonated CPA5−ALEX complexes.
The singly deprotonated complex of ALEX−CPA5 shows a single, Gaussian-shaped AT distribution (Figure 5A) with a mean arrival time value AT = 16.2 ms (for a given reported IM conditions).With AT significantly shifted toward a higher value, it also displays a broader AT distribution than the CPA5 anion, suggesting the presence of multiple indistinguishable conformations of the complex involved in the ion population.When the analyte solution was examined after 24 h, a shift in the AT distribution corresponding to the anion of the complex is observed (marked as a dotted line), indicating the evolution of the complex's conformation over time toward more extended conformations.The crystal-like conformation (Figure 6A) described earlier as S-shaped conformation (Figure 3A) and additional conformation generated by an extensive conformational search in the gas phase (Figure 6B, for details on the calculations, see the SI material) were considered to be present in the gas-phase population of a singly deprotonated CPA5−ALEX ion.The CCS value calculated for the conformation resulting from the relaxation of the crystal-like structure in the semiempirical MOPAC PM7 force field 41 is 21 Å 2 higher than the experimental value.This discrepancy between theoretical and experimental CCS values suggests an extensive conformational shrinkage of the complex ion compared to its crystalline counterpart.
Another conformation (Figure 6B) obtained through an exhaustive theoretical conformational search is significantly more compact than the crystal-like structure due to extensive optimization of the prevailing gas-phase self-solvation interactions.In this conformation, ALEX solvates the outer part of CPA5, similar to the crystal conformation; only here, ALEX is more compressed.The observation of the AT shift in Figure 5A can be explained by the initial formation of a more compact conformer (Figure 6B) observed at AT = 16.2 ms, as shown in Figure 5A (ΔCCS t-TWIM = 10 Å 2 = 2.5%), which evolves over time (24 h) to a more extended conformation toward a crystallike structure.
Slightly different structural features from those of the singly deprotonated complex were revealed for its doubly deproto-nated form.In this case, two distinct Gaussian-shaped distributions (Figure 5B) were observed in IM-MS experiments with mean AT values of 4.1 and 4.3 ms.The determined TWIM CCS N2 values for these two distinct AT distributions are 406 ± 2 and 418 ± 5 Å 2 ; hence, two different conformation families varying by 12 Å 2 in the CCS value are anticipated for the doubly deprotonated form of CPA5−ALEX.Analysis of the analyte solution over time revealed that the population of the less extended isomer with AT = 4.1 ms decreases (dotted line in Figure 5B), and the more extended form becomes dominant after 24 h.Similar to a single deprotonated complex, the t CCS value calculated for crystal-like conformation obtained by relaxation of a doubly deprotonated complex in the semiempirical MOPAC PM7 force field is higher than the experimental values by 30 and 18 Å 2 for the less and more extended forms, respectively.Therefore, one can expect significant conformational changes of the complex compared to its crystal form.An additional structure that was considered represents a conformation, which is similar to the one where singly charged complex ALEX is wrapped around CPA5 more tightly than that for the crystal-like form to ensure the effective interactions between biguanidine cations and carboxyl/ate on both rims of CPA5 (Figure 7B).This conformation was calculated to have a lower t CCS value, which matches well the experimental value (ΔCCS t-TWIM = 6 Å 2 = 1.5%).
A joint IM-MS experimental and theoretical study clearly shows that the conformations of the exclusion complex of ALEX and CPA5 change over time.Initially observed at lower AT values, compact structures of both singly and doubly deprotonated CPA5−ALEX complexes reorganize in a precrystallization solution into more extended forms.Although in this initial study we focused on the short-time results (24 h), we were able to catch the conformational evolution of the CPA5−ALEX complex.Further ongoing experiments focus on a more detailed study of the precrystallization solution over a  Crystal Growth & Design longer period of time and for less flexible ligands in order to increase the simplicity and accuracy of the system under study, which, in turn, is important for obtaining highly reliable experimental as well as theoretical results.
Crystal Structure of the CPA5−Phenformin Complex.CPA5 crystallizes in the presence of phenformin hydrochloride from a water/ethanol solution to afford prismatic crystals of the host−guest complex.The crystal structure was determined and refined in the monoclinic P2 1 /c space group.The asymmetric unit comprises two CPA5 molecules, three phenformin guests, together with ethanol and water molecules, as shown in Figure 8A.We were quite surprised to find out that the "filling" of two crystallographically unique macrocycles in the solid-state complex was different.One of the CPA5 includes a PFM molecule, while another has two ethanol molecules in its cavity.Additional two PFM molecules reside outside of cavities positioning their biguanidinium groups in the proximity of carboxyl rims of the macrocycles, as shown in Figure 8B.The structure is a rare example showing two different host−guest inclusion scenarios trapped in one crystalline phase.It reflects the competition between solvent molecules (ethanol) and organic cations (phenformin) for access to the macrocyclic cavity of CPA5.The winner in this competition is undecided; obviously, the energetic gap between the inclusion of two molecules of ethanol and the inclusion of cationic phenformin must be really small, visualized as a "snapshot" of these two inclusion possibilities in one crystal structure, as shown in Figure 9. From our previous work, we know that the addition of ethanol as a cosolvent is necessary to solubilize the carboxylic form of CPA5 in an aqueous solution.It also provides a means to crystallize carboxylated pillar [5]arene and its host−guest complexes.The first crystal structure of CPA5 reported was an actual inclusion complex with ethanol. 14The similar capture of two different inclusion modes by per-ethylated pillar [5]arene within one crystal form has recently been described. 43wo host−guest substructures correspond to the cavity inclusion of a tetracyanobenzene guest and THF solvent molecules in one crystal lattice.Earlier Atwood et al. reported host−guest cocrystals of pyrogallol [4]arenes of dual inclusion mode. 44The bowl-shaped macrocycles include pyrene guest   and solvent (ethanol or 2-propanol) molecules within one crystal form.Vilela and Dalgarno observed unusual cocrystallization of two different calix [4]diquinone conformations (partial cone and 1,3-alternate) within a single crystal. 45hese complex crystal structures give us better insight into nuances of the host−guest molecular recognition and crystallization as second-order supramolecular process showcasing various scenarios from the host−guest structural landscape.
We modeled all three phenformine guests as dications in the structure model.The reasons we considered were as follows: (1) each of biguanidinium group is likely to donate eight hydrogen bonds as appears from their chemical surroundings and (2) there are numerous short HB between adjacent CPA5 molecules characteristic for charge-assisted carboxylic−carboxylate supramolecular synthons, witnessing deprotonation of multiple substituents.Two crystallographically unique CPA5 molecules have been modeled as either a dianion�macrocycle penetrated by PFM, or a teraanion�filled with ethanol and interacting with external PFM via macrocyclic rims.Thus, the charge balance in the structure is satisfied.The crystal structure of the dicationic form of PFM in the host−guest complex with anionic p-sulfonatocalix [4]arene (refcode VAPQUA) has previously been reported by Liu et al. 46 This structure shows the inclusion of PFM in the folded U-shaped conformation inside the bowl cavity of calix [4]arene.The complex is the sole example of the solid-state host−guest system of PFM reported so far.Also, the crystal structure of the diprotonated PFM in the form of its metal salt with tetrachlorozincate has been described. 47Besides these examples, there are two crystal structures of phenformin chloride deposited in the CSD�the first one determined in 1979, 48 and later redetermined in 1998 to find positions of all H atoms and the protonation scheme of the biguanidinium fragment. 49Considering biomolecules, the crystal structures of the PFM and the NADP (nicotinamide adenine dinucleotide phosphate) ternary complex with E. coli dihydrofolate reductase have been determined in the initial study on the interaction of antidiabetic biguanidine drugs with folate-dependent enzymes. 50The crystal structure of the ternary complex (PDB entry 5UIH) shows that the biguanidine forms a salt bridge with the Asp "folate hook" residue and additional H-bonds with the carbonyl groups of isoleucine and tyrosine.The PFM molecule in the binding site of the enzyme is in the bent nonlinear conformation intermediate between the extended one observed in our inclusion complex with the prismatic cavity of CPA5 and the U-shaped one as a result of its inclusion into the p-sulfonatocalix [4]arene bowl cavity.
The phenformin (labeled X, colored in blue, Figure 9A) inside CPA5 is completely shrouded by the macrocycle.The macroring encircles the ethylene linker and part of the cationic guanidinium group of the guest featuring C−H•••π and N + − H•••π interactions between PFM and the host interior.The aromatic group and the rest of biguanidinium reside in cavity "extensions" encircled by −CH 2 −COOH substituents at both rims of the macrocycle.The included PFM molecule adopts a nearly elongated conformation to fit into the rigid prismatic cavity of pillar [5]arene.Another CPA5 molecule has two ethanol molecules inside the cavity (colored in yellow, Figure 9B) and two water molecules in the plane of one rim.The hydroxyl groups of both ethanols are positioned in the proximity of the carboxyl groups of the macrocyclic rims and are hydrogen-bonded to water molecules.
Two remaining PFM molecules are complexed exo with respect to the macrocyclic cavities, forming a rich HB network between cationic biguanidinium groups and carboxyl rims of pillar [5]arenes.PFM Y (colored in sea green, Figure 10A  phenformin Y (in sea green) forms five HBs with carboxyl oxygen atoms of pillar [5]arenes and three HBs with water molecules.(B) Phenformin Z (in pink) forms seven HBs with carboxyl oxygen atoms of pillar [5]arenes and one HB with a water molecule.

Crystal Growth & Design
HB dimers is (2.66 ± 0.05)Å.For carboxylic−carboxylate synthons, shorter distances are typical of (2.54 ± 0.06) Å.Thus, HB between adjacent CPA5 molecules of •••O distances less than 2.6 Å is statistically more likely to be formed between a carboxylic oxygen donor and a carboxylate oxygen acceptor.This confirms the deprotonation (ionization) of multiple substituents on the macrocycles and fulfils the charge neutrality of the complex with cationic PFM.The CPA5 molecules are assembled in a back-to-back orientation into parallel layers perforated by PFM Z with HB connecting macrocycles within the layer, as shown in Figure 11.CPA5 molecules from adjacent layers are in a rim-to-rim orientation with additional HB stapling layers.PFM Y (see green)and the biguanidinium group of PFM Z (pink) reside between the layers in a more hydrophilic region surrounded by carboxyl groups of the macrocycles and water molecules.The layer-type packing of parallel CPA5 molecules connected by the carboxylic− carboxyl/ate HB network has been previously observed in the crystal structures of the CPA5 complexes with viologen derivatives 16 and pentamidine. 20M-MS Studies of the CPA5−Phenformin Complex.The mass spectrum recorded for an electrosprayed solution containing CPA5 and PFM has revealed the formation of a 1:1 complex, observed as monoanionic species [PFM + CPA5 -H]̅ at m/z = 1394.3(Figure S2, SI).No higher-order aggregates were observed in the mass spectrum.The peaks corresponding to the uncomplexed host molecule as singly, doubly, triply, and even quadruply charged ions accompany the CPA5−PFM complex in the spectrum.The CPA5−PFM complex exhibits a single, Gaussian-shaped AT distribution in the ion mobility spectrum (Figure 12A).The AT of the complex is shifted toward higher values compared to the bare host.The experimental collision cross section ( TWIM CCS N2 ) of the complex determined by the calibration approach 36 is 27 Å 2 higher than that of the bare host.
Further conclusions on the structural properties of the CPA5−PFM complex were obtained by comparing its experimental collision cross section with the theoretical cross section obtained for model structures.The first model structure considered here was the crystal-like conformation of the CPA5−PFM complex (Figure 12B), obtained by relaxing its crystal-derived conformation in the MOPAC PM7 force field. 37The collision cross section calculated for this crystal-like conformation is consistent with the experimental values, thus indicating the structural congruence between the solid and the solution/gas phases.In this conformation, a doubly protonated PFM molecule is trapped and symmetry positioned in the interior of the triply negatively charged CPA5 host.It represents a classical inclusion associate, similar to that earlier reported for CPA5−PTM 20 and CPA6−dMV 15 complexes.A further extensive conformational search performed in the gas phase (for details, please see SI) has led to the additional type of conformation that was also considered.This conformation, shown in Figure 12C, is 78.9 kJ/mol, more stable than the crystal-like conformation.It represents a similar type of inclusion to the crystal-like   38 structure with PFM slightly shifted in the CPA5 cavity to increase the efficiency of multiple and cooperative intermolecular hydrogen bonding between carboxyl(ate)−biguanide cation.This conformation holds a slightly lower CCS value but is still in excellent agreement with the experimental value.Other types of conformations in which the PFM is partially or fully extended from the host cavity were excluded based on their substantially higher CCS values.
The conformation of the CPA5−PFM complex in the precrystallization solution revealed by IM-MS studies agrees well with that observed in the confined space.The absence of the second exo-associate, which, in turn, was additionally found in the crystal complex, can be explained by its low stability, which prevents its observation under increased pressure during the IM-MS experiment.

■ CONCLUSIONS
The anchoring of cationic biguanidinium groups at the carboxylated pillar [5]arene rims by charge-assisted hydrogen bonding is sufficient to reduce the degree of freedom of flexible alexidine molecules and their integration into the crystalline complex.The ability to trap alexidine within a solid-state assembly offers a unique possibility to explore its conformational properties and interaction modes in a confined space, mimicking those of active sites of natural macromolecules.The described complex is the first case of crystallographic authentication of alexidine benefiting from its host−guest cocrystallization with pillar [5]arene.Our results demonstrate that carboxylated pillar[n]arenes can be a useful addition to the macrocyclic toolkit for the facilitation of the crystallization of bio(macro)molecules. 52 Excellent examples have been provided by the Peter Crowley group on the use of anionic calix[n]arenes 53,54 and cucurbit [7]uril 55 as mediators of protein assembly and crystallization.Employing alexidine and CPA5 as cocrystallizing partners affects an open-type assembly of a perpendicular pattern of macrocyclic molecules.This is a sole example of a hydrogen-bonded three-dimensional network build from CPA5 featuring open packing with large solvent channels.
Both inclusion and exclusion modes of the host−guest complexation have been revealed for phenformin with a carboxylated pillar [5]arene.The complexes are based on the guanidinium−carboxyl/ate hydrogen bonds and cation−π interactions between the included phenformin cation and the cavity interior of the pillar [5]arene macroring.The presence of two inclusion scenarios for the pillar [5]arene host in the crystal complex, one with a phenformin cation and one with ethanol molecules, showcases an evenly matched race of different guest species for the confined space of the macrocyclic cavity.
The IM-MS analysis of the precrystallization solutions of the CPA5 host and ALEX or PFM has shown the presence of structures and conformations closely related to that observed in the crystal lattice.The most intriguing results obtained for the CPA5−ALEX complex imply a conformational reordering of the complex over 24 h.The ability of IM-MS to capture the dynamics and directionality in crystal lattice formation, to some extent shown for the CPA5−ALEX complex, provides evidence that this approach may be applied to predict and examine the crystallization process of host−guest systems, complementing crystallographic studies.
Experimental details, photomicrographs of crystals, single-crystal X-ray diffraction, and ion mobility mass spectrometry experimental details and theoretical calculations (PDF)

Figure 2 .
Figure 2. Expanded asymmetric unit of the exclusion host−guest complex between carboxylated pillar[5]arene and alexidine: (A) side view and (B) top view.The macrocyclic cavity is filled with disordered ethanol (yellow) and water molecules.The water molecules in the crystal lattice and hydrogen atoms are not shown for clarity.(C) S-shaped conformation of the alexidine molecule in the crystal complex; the molecular surface is shown in cyan color.Figure 3. Hydrogen bonding between alexidine biguanidinium groups as donors and oxygen atoms of pillar[5]arene as acceptors.(A) Two biguanidinium groups form hydrogen bonds toward two rims of one macrocycle.(B) Each of the biguanidinium group is hydrogen bonded with two additional pillar[5]arene molecules in the crystal lattice.

Figure 3 .
Figure 2. Expanded asymmetric unit of the exclusion host−guest complex between carboxylated pillar[5]arene and alexidine: (A) side view and (B) top view.The macrocyclic cavity is filled with disordered ethanol (yellow) and water molecules.The water molecules in the crystal lattice and hydrogen atoms are not shown for clarity.(C) S-shaped conformation of the alexidine molecule in the crystal complex; the molecular surface is shown in cyan color.Figure 3. Hydrogen bonding between alexidine biguanidinium groups as donors and oxygen atoms of pillar[5]arene as acceptors.(A) Two biguanidinium groups form hydrogen bonds toward two rims of one macrocycle.(B) Each of the biguanidinium group is hydrogen bonded with two additional pillar[5]arene molecules in the crystal lattice.

Figure 4 .
Figure 4. (A) Part of the tetragonal supramolecular assembly sustained by hydrogen bonding between adjacent pillar[5]arene rims.(B) Open-type tetragonal packing of pillar[5]arene−alexidine assembly viewed along the c direction; alexidine molecules are colored in cyan.Tetragonal channels framed by carboxylated rims of macrocycles are filled with disordered water molecules (not shown).

Figure 5 .
Figure 5. Gaussian-fitted AT distributions of selected and extracted (A) singly and (B) doubly deprotonated ions corresponding to the ALEX−CPA5 complex and CPA5 recorded at wave velocity (WV) = 500 m/s, wave high (WH) = 32 V along with the determined experimental CCS values ( TWIM CCSN2�collision cross section in nitrogen drift gas determined by traveling wave ion mobility). 40Dotted lines represent AT distributions of the complex recorded after 24 h.

Figure 7 .
Figure7.PM7-optimized structures of the doubly deprotonated CPA5−ALEX complex obtained from theoretical studies and calculated collision cross-section values using MobCal-MPI.38

Figure 8 .
Figure 8. Asymmetric unit of the host−guest complex between carboxylated pillar[5]arene and phenformin.One of the two crystallographically independent macrocycles includes a phenformin guest molecule (colored in blue), while another includes two ethanol molecules (colored in yellow) in the cavity.Two additional phenformines reside outside (colored pink and green), positioning biguanidinium groups in the proximity of macrocyclic rims.(A) side view and (B)top view with pillar[5]arenes molecular surface colored in gray.

Figure 9 .
Figure 9. Two host−guest inclusion modes trapped within one crystal lattice: (A) Phenformin X (colored in blue) and (B) ethanol; water molecules taking part in the hydrogen bond network are shown as cyan balls.
) is positioned roughly perpendicular to the macrocycles.There are five N + −H•••O HB between the biguanidinium group and oxygen atoms from four adjacent macrocycles and three HB toward water molecules.PFM Z (colored in pink, Figure 10B) is also complexed outside, forming seven HB with oxygen atoms from five adjacent CPA5 molecules and one HB with a water molecule.Despite multiple biguanidinium−carboxyl/ate HB networks, there are still plentiful HB interactions between adjacent CPA5 molecules.In total, there are 18 O−H•••O hydrogen bonds per two nonequivalent macrocycles, with carboxyl(ate) groups acting as donors and acceptors of hydrogen bonds of the O•••O distance range 2.521(8)− 2.746(17) Å.Many of these HB are shorter than 2.6 Å, which is characteristic of a carboxylic−carboxylate supramolecular synthon.From statistical data extracted from CSD on O•••O distance distribution for intermolecular carboxylic−carboxylic and carboxylic−carboxylate synthons, it appears that chargeassisted carboxylic−carboxylate HBs are statistically shorter than neutral carboxylic−carboxylic HBs. 51According to this analysis performed on high-resolution nondisordered structures, the •••O distance distribution for carboxylic−carboxylic

Figure 10 .
Figure 10.Hydrogen bonds formed by biguanidinium groups of two phenformin guests complexed outside of macrocyclic cavities: (A) phenformin Y (in sea green) forms five HBs with carboxyl oxygen atoms of pillar[5]arenes and three HBs with water molecules.(B) Phenformin Z (in pink) forms seven HBs with carboxyl oxygen atoms of pillar[5]arenes and one HB with a water molecule.

Figure 11 .
Figure 11.Crystal packing of the carboxylated pillar[6]arene− phenformin complex viewed along a direction.Included phenformin molecules are colored blue, and phenformin complexed outside the cavities is colored sea green and pink.

Figure 12 .
Figure 12. (A) Gaussian-fitted AT distributions and determined experimental collision cross-section values ( TWIM CCS N2 �collision cross section in nitrogen drift gas determined by traveling wave ion mobility) of the selected and extracted singly charged ions of the CPA5−PFM complex and the host recorded at wave velocity (WV) = 500 m/s, wave high (WH) = 32 V. (B) Structure of the crystal-like complex and (C) gas-phase conformation obtained from theoretical studies.t CCS values of discussed structures were obtained using MobCal-MPI.38