Stabilization of Near Identical Hydrogen Bonded Octameric Water Clusters in Crystal Structures of Three Distinct Non-Charged Polyamide Macrocyclic Host Molecules

In this paper, we present a comparative analysis of the solid state structures of three well-resolved hydrates of macrocyclic host molecules 1a, 1b, and 2 containing an intrannular amide-aryl substituent (lariat arm) connected to a fixed 26-membered ring in a normal (-NHCOAr, hosts 1a and 1b) or reverse manner (-CONHAr, host 2). Despite different chemical structures, these hosts crystallize as isostructural tetrahydrates in the same P-1 space group. Moreover, their crystals exhibit identical hydrogen bond motifs resulting in a stabilization of an almost identical unusual octameric water cluster built from the cyclic tetramer core and four water molecules, attached sequentially in an “up-and-down” manner. Further analysis reveals that, among the series, the structure of host 2 provides the most suitable environment for the accommodation of this type of water cluster.


Introduction
Crystal engineering relies on understanding how multiple weak intermolecular interactions influence the organization and packing of molecules in the solid state, and the application of such knowledge in the development of new solids characterized by specific physical and chemical properties [1]. One of its main tasks is the answer to the problem of what is the dependence between the molecular structure and crystal form. Isostructurality is defined as a phenomenon describing different systems exhibiting similarity in their structures [2]. Isostructurality in single-component organic crystals appears for compounds of closely related molecular structures. There are several reports where the exchange of functional groups does not alter the packing in the crystal structure. Functional groups best known to exhibit such behavior are halogen groups [3]. Additionally, the exchange of Me/Cl [4], Me/Br [5], as well as CH 2 /NH substitution [6], are also well known to result in isostructurality of the structures. Occurrence of 3D isostructurality in the presence of strong hydrogen bonds, namely N-H· · · N with the presence of weak C-H· · · N [7], C-H· · · F, C-H· · · π [8] interactions and π-π stacking was also reported. Bucar et al. [9] showed that isostructural crystals can be obtained by an exchange of a > C=O to > C=S, but only if the studied oxygen/sulfur atom is not involved in hydrogen bonding. Isostructurality induced by a change of the substituent in para-position (-NO 2 /-OMe) of a lariat arm in macrocyclic pentaamides was also reported [10]. Isostructurality is more likely to occur for multicomponent crystals [11], where only one of the components is changed. It can be seen for two-component host-guest inclusion compounds [12], as well as for three-component crystals [13]. Isostructurality induced by the solvent is also observed [14,15]. It is a phenomenon often found in pharmaceutical molecular salts [16], and cocrystals [17][18][19]. There are not many examples involving multi-component macrocyclic isostructural crystals, they include solvates of tetrahaloethynyl cavitands [20] and complexes of cucurbit [6] uril with three glycine containing dipeptides [21].
Unclosed cryptands (UCs) [22][23][24][25][26][27] belong to the class of fairly new macrocyclic receptors, structurally related to well-established cryptands [28][29][30][31][32]. They are, however, much more flexible than their rigid analogs, as they are built from the macroring of various sizes to which is connected a labile intrannular substituent-lariat arm. Due to their inherent flexibility, they are able to adjust their shape to highly demanding guests, in particular anions. Lately, we showed that 26-membered pentamide unclosed cryptands are able to accommodate octameric water clusters in their structure [10,33], proving that such molecules are a good choice for studying the hydration phenomena of small organic compounds, as well as serving as a model for more complicated structures of biological importance. It may prove very useful as studying the hydration behavior of biological molecules still presents a challenge for modern science [34,35].
We are focused on investigating the crystal structures of closely related macrocyclic compounds, which allows us to examine how incremental structural changes affect the crystal packing [10,36,37]. During our study, we proved that the presence of para-substituent in the lariat arm of 26-membered unclosed cryptand is essential for the stabilization of octameric water cluster [10]. It is worth mentioning that such stabilization occurs for UCs possessing amido-phenyl group in lariat arm substituted with electron-withdrawing (-NO2, as in host 1a), as well as electron-donating group (-OMe, as in host 1b). On the contrary, structural studies of a macrocycle equipped with an unsubstituted amido-phenyl substituent revealed the occurrence of a water chain stabilized by two unclosed cryptand molecules.
In the present paper, we investigate how the attachment of an intraannular amido-pnitrophenyl substituent (lariat arm) on the 26-membered unclosed cryptand of type 1 influences the molecular packing and possible stabilization of water clusters in so-designed host 2 (see the red-colored amide groups in Figure 1). Previous solution studies reveal that such structural isomerism of the amide group has a beneficial impact on the molecular recognition of anions [22,24]. The host 2 has been synthetized and its crystal structure was determined by X-ray crystallography. It was found, unexpectedly, that host 2 contains near the same octameric water cluster as previously found in the crystal structures of analogs 1a [33] and 1b [10]. A comparative crystal structure analysis has been carried out to identify the role of macrocyclic skeleton and lariat arm on the formation of water clusters in the crystal lattice of hosts 1a-b and 2.

Results and Discussion
The target macrocyclic hosts 1a and 1b were obtained in very high yields using a "one-pot" post-macrocyclisation procedure involving deprotection and functionalization of Boc-protected macrocyclic amine 3 (Scheme 1a) [27], whereas the novel host 2 was prepared by a direct MeONa-assisted macrocyclization [10] between α,ω-diester 4 and hydrochloric salt of α,ω-diamine 5 (Scheme 1b). Previous solution studies reveal that such structural isomerism of the amide group has a beneficial impact on the molecular recognition of anions [22,24]. The host 2 has been synthetized and its crystal structure was determined by X-ray crystallography. It was found, unexpectedly, that host 2 contains near the same octameric water cluster as previously found in the crystal structures of analogs 1a [33] and 1b [10]. A comparative crystal structure analysis has been carried out to identify the role of macrocyclic skeleton and lariat arm on the formation of water clusters in the crystal lattice of hosts 1a-b and 2.

Results and Discussion
The target macrocyclic hosts 1a and 1b were obtained in very high yields using a "one-pot" post-macrocyclisation procedure involving deprotection and functionalization of Boc-protected macrocyclic amine 3 (Scheme 1a) [27], whereas the novel host 2 was prepared by a direct MeONa-assisted macrocyclization [10] between α,ω-diester 4 and hydrochloric salt of α,ω-diamine 5 (Scheme 1b). Similar to the previously described unclosed cryptands 1a and 1b, macrocycle 2 crys tallizes as a pair of enantiomers in a triclinic space group P-1. In the solid state, unclosed cryptands in general may adopt three different conformations: T-, C-, and S-shaped [37] All three macrocycles studied here, 1a, 1b, and 2, adopt C-shaped conformation. The C shaped conformation of macrocycle 2 is stabilized by a net of 8 inter-and intramolecula hydrogen bonds (Figure 2a).  Molecules of 2 form dimers, where nitro-substituted aromatic rings from the laria arms of adjacent host molecules are arranged in an antiparallel mode and are connected via CH … O, as well as π-stacking interactions ( Figure 2b). In addition, the lariat arm is held in its position via H-bond between N7 nitrogen atom of the main macroring and the car bonyl atom O3 in the lariat arm, which is further connected with O1W molecule. The fur ther stabilization is realized through interactions with water molecules O2W, O3W, and O4W (for details describing hydrogen bonds, see Table 1). Similar to the previously described unclosed cryptands 1a and 1b, macrocycle 2 crystallizes as a pair of enantiomers in a triclinic space group P-1. In the solid state, unclosed cryptands in general may adopt three different conformations: T-, C-, and S-shaped [37]. All three macrocycles studied here, 1a, 1b, and 2, adopt C-shaped conformation. The C-shaped conformation of macrocycle 2 is stabilized by a net of 8 inter-and intramolecular hydrogen bonds (Figure 2a). Similar to the previously described unclosed cryptands 1a and 1b, macrocycle 2 crystallizes as a pair of enantiomers in a triclinic space group P-1. In the solid state, unclosed cryptands in general may adopt three different conformations: T-, C-, and S-shaped [37]. All three macrocycles studied here, 1a, 1b, and 2, adopt C-shaped conformation. The Cshaped conformation of macrocycle 2 is stabilized by a net of 8 inter-and intramolecular hydrogen bonds (Figure 2a). Molecules of 2 form dimers, where nitro-substituted aromatic rings from the lariat arms of adjacent host molecules are arranged in an antiparallel mode and are connected via CH … O, as well as π-stacking interactions ( Figure 2b). In addition, the lariat arm is held in its position via H-bond between N7 nitrogen atom of the main macroring and the carbonyl atom O3 in the lariat arm, which is further connected with O1W molecule. The further stabilization is realized through interactions with water molecules O2W, O3W, and O4W (for details describing hydrogen bonds, see Table 1). Molecules of 2 form dimers, where nitro-substituted aromatic rings from the lariat arms of adjacent host molecules are arranged in an antiparallel mode and are connected via CH· · · O, as well as π-stacking interactions ( Figure 2b). In addition, the lariat arm is held in its position via H-bond between N7 nitrogen atom of the main macroring and the carbonyl atom O3 in the lariat arm, which is further connected with O1W molecule. The further stabilization is realized through interactions with water molecules O2W, O3W, and O4W (for details describing hydrogen bonds, see Table 1). Table 1. Analysis of hydrogen bonds stabilizing one molecule of host 2 (entries 1-9) and analysis of hydrogen bonds stabilizing water octamer in the structure of 2 (entries 10-13).

Entry
Interaction

Stabilization of Discrete Octameric Water Cluster
Further crystal analysis reveals that the confined space of unclosed cryptand 2, similarly to the previous cases of hosts 1a and 1b, stabilizes a discrete water octamer built from a tetrameric cyclic water cluster (for selected examples of discrete water tetramers, see references: [38][39][40][41][42]) with four water molecules attached in an "up-and-down" manner to each corner of the tetramer (O1W, O2W, O3W, and O4W; Figure 3).  Table 1. Analysis of hydrogen bonds stabilizing one molecule of host 2 (entries 1-9) and analysis of hydrogen bonds stabilizing water octamer in the structure of 2 (entries 10-13).

Entry
Interaction

Stabilization of Discrete Octameric Water Cluster
Further crystal analysis reveals that the confined space of unclosed cryptand 2, similarly to the previous cases of hosts 1a and 1b, stabilizes a discrete water octamer built from a tetrameric cyclic water cluster (for selected examples of discrete water tetramers, see references: [38][39][40][41][42]) with four water molecules attached in an "up-and-down" manner to each corner of the tetramer (O1W, O2W, O3W, and O4W; Figure 3).  The average distance between water molecules in cyclic tetramer (O2W-O4W) equals to 2.853 Å, while average distance between water molecules from the tetramer and dangling water molecule O1W and O3W equals to 2.768 Å, which gives an overall average of 2.811 Å (all H-bonds in the octameric water cluster are summarized in Table 1, entries 10-13). The core cyclic water tetramer is higher in energy by at least 35.3 kcal•mol −1 than the global minimum with the cage symmetry [33,43]. This indicates a large energetic compensation of this water assembly by the crystal lattice. The average distance between water molecules in cyclic tetramer (O2W-O4W) equals to 2.853 Å, while average distance between water molecules from the tetramer and dangling water molecule O1W and O3W equals to 2.768 Å, which gives an overall average of 2.811 Å (all H-bonds in the octameric water cluster are summarized in Table 1, entries 10-13). The core cyclic water tetramer is higher in energy by at least 35.3 kcal·mol −1 than the global minimum with the cage symmetry [33,43]. This indicates a large energetic compensation of this water assembly by the crystal lattice.

Comparative Analysis of Crystals
To elucidate the role of a macrocyclic skeleton and the lariat arm on the formation and stability of the discrete water cluster, we conducted a comparative crystal structure analysis of tetrahydrates of hosts 1a-b and 2. The three studied compounds (1a, 1b, and 2) Molecules 2021, 26, 2787 5 of 12 crystallize in the triclinic space group P-1 and their unit cell parameters and cell volumes exhibit very similar values (Table 2).  In addition to having very similar cell parameters, the crystal structures of hosts 1a, 1b, and 2 also display similar crystal packing features. All hosts crystallize as a pair of enantiomers and adopt C-shaped conformation (the overlay of three molecules of unclosed cryptands 1a, 1b, and, 2 is presented in Figure 4a). It can be seen from the superposition of 1a, 1b, and 2 that, although the lariat arms of 1a and 1b, which differ in the p-substituent in the terminal phenyl group overlay perfectly, the lariat arm of 2 is slightly shifted. It is due to the reversed position of the amide group in the lariat arm which bonds with O1W molecule via O-H· · · O interactions (Figure 4b).  Despite the obvious differences in the structures of unclosed cryptands 1a, 1b, and 2, all of them exhibit very similar hydrogen bond motifs, which results in a stabilization of similar octameric water clusters ( Figure 5, Table 3 and Table S1). Despite the obvious differences in the structures of unclosed cryptands 1a, 1b, and 2, all of them exhibit very similar hydrogen bond motifs, which results in a stabilization of similar octameric water clusters ( Figure 5, Table 3 and Table S1). The average distance between the oxygen atoms of water molecules forming water cluster is 2.841 Å, 2.925 Å, and 2.811 Å in the crystal structures of hosts of 1a, 1b, and 2, respectively. This parameter is related to the relative stability of the water assembly in a confined environment. It can be therefore concluded that, among the studied macrocyclic compounds, host 2 provides the most suitable molecular architecture for the formation of this type of water cluster.
To get a better insight into the role of the attachment of lariat' amide group (i.e., "normal" vs. "reverse" manner), we further closely inspected the molecular environment around the water cluster, as well as the geometrical parameters describing the water cluster, with particular emphasis on closely related isomeric hosts 1a and 2 (Figures 6 and 7, Table S1).  [a] For host 1b see Table S1. [b] Difference between parameter from the X-ray structure of host 2 and host 1.
The average distance between the oxygen atoms of water molecules forming water cluster is 2.841 Å, 2.925 Å, and 2.811 Å in the crystal structures of hosts of 1a, 1b, and 2, respectively. This parameter is related to the relative stability of the water assembly in a confined environment. It can be therefore concluded that, among the studied macrocyclic compounds, host 2 provides the most suitable molecular architecture for the formation of this type of water cluster.
To get a better insight into the role of the attachment of lariat' amide group (i.e., "normal" vs. "reverse" manner), we further closely inspected the molecular environment around the water cluster, as well as the geometrical parameters describing the water cluster, with particular emphasis on closely related isomeric hosts 1a and 2 (Figures 6 and 7, Table S1).  [a] For host 1b see Table S1. [b] Difference between parameter from the X-ray structure of host 2 and host 1.
At first sight, the analysis of the molecular environment reveals a close similarity between the crystal structures of hosts 1a and 2, however, closer inspection reveals some important differences, in particular in the arrangements of host molecules and the conformation of aliphatic butylene linkers. These differences are confirmed by the Hirshfeld fingerprint plots (Figures 7a,c and d) and a very comparable share of intermolecular interactions (Figure 7e).
The weak and nondirectional van der Waals interactions account for nearly 56% of the total surface area, in which H•••H dominates over C-H•••C π contacts (45% vs. 11-13%). The short and highly directional hydrogen bonds (mainly between water molecules and with water molecules hydrogen bonded to oxygen carbonyl atoms and NH amide protons) account for 33-34% of the total surface area. In contrast, the share of intermolecular interactions for hosts 1a and 2 vs. 1b highlights much larger differences, plausibly related to the presence of different para-substituent (-NO2 for 1a and 2 vs. -OMe for 1b). Although all these three hosts 1a, 1b, and 2 crystallize as isostructural tetrahydrates, it is evident that replacement of the para-substituent from the nitro-to methoxy group markedly increases the share of H•••H and C-H•••H interactions (56.1%, 57.6% vs. 66.6% for 1a and 2 vs 1b, respectively), whereas the share of At first sight, the analysis of the molecular environment reveals a close similarity between the crystal structures of hosts 1a and 2, however, closer inspection reveals some important differences, in particular in the arrangements of host molecules and the conformation of aliphatic butylene linkers. These differences are confirmed by the Hirshfeld fingerprint plots (Figure 7a,c,d) and a very comparable share of intermolecular interactions (Figure 7e).
The weak and nondirectional van der Waals interactions account for nearly 56% of the total surface area, in which H· · · H dominates over C-H· · · C π contacts (45% vs. 11-13%). The short and highly directional hydrogen bonds (mainly between water molecules and with water molecules hydrogen bonded to oxygen carbonyl atoms and NH amide protons) account for 33-34% of the total surface area.
In contrast, the share of intermolecular interactions for hosts 1a and 2 vs. 1b highlights much larger differences, plausibly related to the presence of different para-substituent (-NO 2 for 1a and 2 vs. -OMe for 1b). Although all these three hosts 1a, 1b, and 2 crystallize as isostructural tetrahydrates, it is evident that replacement of the para-substituent from the nitro-to methoxy group markedly increases the share of H· · · H and C-H· · · H interactions (56.1%, 57.6% vs. 66.6% for 1a and 2 vs. 1b, respectively), whereas the share of O· · · H interactions becomes smaller (33.7%, 32.7% vs. 26.2% for 1a and 2 vs. 1b, respectively).
More importantly, analysis of the lengths and angles of molecular interactions in the water clusters reveals that water cluster is more stable in the crystal lattice of host 2 than in host 1a. In particular, the hydrogen bonds are considerably shorter (up to 0.06 Å), as well as the angles between water molecules are more directional (e.g., angle (c) is 171.6 • vs. 176.6 • in 2 and 1a, respectively). This clearly highlights that the molecular environment of host 2 is much more suitable for the formation of confined water octamer.

Reagents and General Methods
All reagents were used as received. The solvents were dried by distillation over the appropriate drying agents. All solvents were obtained from common suppliers and used as received. TLC was carried out on Merck Kieselgel F254 plates (Merck, Germany). Melting points were determined using a hot-stage apparatus and were uncorrected. The NMR spectra were recorded on a Varian Mercury 600 (Varian, Palo Alto, CA, USA) instrument. Chemical shifts are reported in ppm (δ) and are set to the solvent residue peak. J coupling constants values are reported in Hz. Mass spectral analyses were performed with the ESI-TOF technique on a Mariner mass spectrometer from PerSeptive Biosystem (Waltham, MA, USA).

Synthesis of Molecular Host 2
To a stirred solution of diamine dihydrochloride 4 (380 mg, 1.0 mmol) and diester 5 (420 mg, 1.0 mmol) and in anhydrous methanol (100 mL, c = 0.01 M), a freshly prepared solution of sodium methoxide (6.0 mmol, 6 equivalents) in anhydrous methanol (5 mL) was added. The resulting mixture was stirred at rt for 7 days and then silica gel (~3 g) was added. Solvent was evaporated yielding a yellowish residue which was purified by column chromatography using a gradient of methanol in CH 2 Cl 2 [99:1→95:5 v/v] as an eluent. Evaporation of the solvent and recrystallization from MeOH yielded the target product 2 (210 mg, 32%) in the form of yellow crystals (mp 169-171 • C). 1

Crystallography
The measurement of 2 crystals was performed on a KM4CCD κ-axis diffractometer with graphite-monochromated MoK α radiation. The crystal was positioned at 50 mm from the CCD camera. A total of 637 frames were measured at 1 • intervals with a counting time of 20 s. The data were corrected for Lorentz and polarization effects. Multi-scan absorption correction has been applied. Data reduction and analysis were carried out with the Agilent program [44]. The structure was solved by direct methods and refined using SHELXL-2014/7 [45] [49]. The crystal packing coefficients (CP k ), defined as: where Z is the number of molecules in the unit cell, V cell is the volume of the cell taken from the X-ray structure, and V mol is the volume of geometryoptimized molecule (only hydrogen positions were refined) calculated at DFT/B3LYP-D3/6-31G(d) level of theory using Spartan'18 program [50]. Hirshfeld calculations were performed using the default parameters of the Crystal Explorer 17.5 program [51][52][53][54].
Single monocrystals of compound 2 suitable for X-ray crystallographic analysis were obtained by diffusion of water vapor into DMSO solution of a mixture of 2 and an excess amount of TBAH 2 PO 4 salt (~10 equivalents). Crystal data for 2·(H 2 O) 4

Conclusions
In conclusion, we present a comparative analysis of the single-crystal X-ray structures of three hydrates of macrocyclic host molecules 1a, 1b, and 2 bearing various intrannular substituent (lariat arm) connected to a fixed 26-membered ring in a normal (-NHCO-p-C 6 H 4 X, 1a: X = NO 2 , 1b: X = OMe) or reverse manner (2, -CONH-p-C 6 H 4 NO 2 ) fashion. Despite structural differences, all hosts crystallize in P-1 space group as isostructural tetrahydrates crystals containing an unusual and energetically disfavored [24] octameric water cluster. The water assembly is built from the cyclic tetramer and four dangling water molecules that are placed in alternate "up-and-down" positions. Further examination of crystals, reveals that reversing the mode of attachment of amide group in 2 (relative to compounds 1a and 1b), changing the binding mode of relocation induces the conformation of the molecule by the formation of hydrogen bonds via the carbonyl O3 atom (N7-H7· · · O3 and O1W-H1V· · · O3) and amidic N2 atoms (N2-H2· · · O3W). Lariat arms of hosts 1a and 1b overlay perfectly, while the lariat arm of host 2, due to the reversed position of amide group is slightly shifted. This structural difference, however, does not influence significantly the general conformation of the macrocyclic skeleton nor its ability to stabilize water cluster. In fact, this inversion was found to markedly increase the stability of water cluster by host 2 (avg. d O· · · O = 2.811 Å) compared to corresponding water assemblies in crystal structures of hosts 1a (avg. d O· · · O = 2.841 Å) and 1b (avg. d O· · · O = 2.925 Å).

Supplementary Materials:
The following are available online, Table S1: Selected geometrical parameters describing water clusters stabilized in the structures of macrocyclic host molecules 1a, 1b, and 2; Figure S1: 1 H NMR (600 MHz) and 13