Supramolecular Assembly of Uridine Monophosphate (UMP) and Thymidine Monophosphate (TMP) with a Dinuclear Copper(II) Receptor.

Understanding the intermolecular interactions between nucleotides and artificial receptors is crucial to understanding the role of nucleic acids in living systems. However, direct structural evidence showing precise interactions and bonding features of a nucleoside monophosphate (NMP) with a macrocycle-based synthetic molecule has not been provided so far. Herein, we present two novel crystal structures of uridine monophosphate (UMP) and thymidine monophosphate (TMP) complexes with a macrocycle-based dinuclear receptor. Structural characterization of these complexes reveals that the receptor recognizes UMP through coordinate-covalent interactions with phosphates and π-π stackings with nucleobases and TMP through coordinate-covalent interactions with phosphate groups. Furthermore, the receptor has been shown to effectively bind nucleoside monophosphates in the order of GMP > AMP > UMP > TMP > CMP in water at physiological pH, as investigated by an indicator displacement assay.

Introduction

In recent years, there has been a growing interest in designing artificial receptors that recognize nucleotides as target substrates,[1−27] with the goal to mimic their biological interactions with enzymes.[25,28] Nucleotides are the fundamental building blocks of DNA and RNA, playing many critical roles in biology. For instance, n class="Chemical">adenosine triphosphate (ATP) is used in generating universal chemical energy for cellular functions, guanosine triphosphate (GTP) is involved in the synthesis of DNA and proteins, and uridine monophosphate (UMP) is the central building block in the synthesis of RNA.[29,30] Although an abundance of information is available on solution binding studies and theoretical calculations predicting the binding modes of nucleotides,[4−26] structural reports of nucleotide complexes with synthetic receptors are indeed limited.[31−40] In 2008, Bianchi and co-workers reported the first structure of a nucleoside triphosphate complex with a synthetic receptor, showing hydrogen bonding and π–π stacking interactions between a thymidine 5′-triphosphate and a polyamine-based receptor.[31] Several structures of nucleotides, nucleosides, or nucleobases as pure or mixed ligands with transition metal ions were also reported.[32−40] In particular, the central role played by nucleoside monophosphates (NMPs) in many biological events makes them attractive targets for mimicking their biological interactions.[41−46] For instance, as characterized by X-ray analysis, UMP was found within the cavity of a Bacillus subtilis orotidine 5′-monophosphate decarboxylase via hydrogen bonds and salt bridges between the nucleotide and the protein.[46] However, to the best of our knowledge, a crystallographically characterized NMP complex with a macrocycle-based synthetic receptor has not been reported yet.

Most of the synthetic receptors for nucleotides are mainly based on the construction of recognition sites with positive charges from polyazamacrocycles that have been shown to be capable of binding nucleotides[19−21,47−52] and of numerous catalytic activities, including hydrolase, phosphatase, and kinase activity.[53−57] Their interactions, however, ocn class="Chemical">cur primarily through hydrogen bonding or electrostatic interactions between the charged groups and the phosphate moieties of nucleotides, which often depend on the pH of the solution. On the other hand, macrocycle-based dinuclear metal complexes are attractive hosts that have shown to bind nucleotides through Lewis acid/base (coordinate–covalent) interactions in water at physiological pH.[58−64] Fabbrizzi and co-workers synthesized an expanded dinuclear cryptate that contains two copper(II) ions as binding sites to selectively recognize GMP with respect to other NMPs in MeOH/water solution.[63] It was suggested that in addition to phosphate groups, enolate oxygen atoms of GMP formed bonds with the metal centers. Hydrazone-based dinuclear copper(II) complexes recently synthesized by Dharmaraj and co-workers have been shown to bind DNA via an intercalative mode.[64]

Even though several transition metal complexes have been employed for nucleotide complexation, the design of artificial receptors that effectively recognize nucleotides in n class="Chemical">water at neutral pH still remains a challenge. Recently, we have been interested in simple and easily synthesizable macrocycle-based dinuclear complexes as probes for anion recognition, which are expected to be more flexible than their bicyclic analogues, thereby allowing them to interact with anions of variable shapes and sizes.[65−68] As learned from nature,[42−46] an ideal receptor for nucleotide binding should be designed on a multipoint recognition strategy to bind phosphate and aromatic base simultaneously. Herein, we describe a macrocyclic dinuclear copper(II) receptor 1 that possesses two metal centers and two aromatic groups, providing effective binding sites for multipoint recognition of NMPs (Chart ). We have structurally characterized two NMP complexes: one with UMP and the other with thymidine monophosphate (TMP), showing multipoint recognition with a dinuclear copper(II) receptor 1. The receptor and NMP alternatively assemble into a one-dimensional chain through bonds between the phosphates and the metal ions.

Chart 1

Chemical Structures of the Free Macrocycle L, Receptor 1 and Eosin Y (EY)

Results and Discussion

Synthesis

The receptor 1 was synthesized from the reaction of L with 2 equiv of CuBr2 in n class="Chemical">H2O, as described previously.[65] The molecule was characterized by single crystal X-ray diffraction analysis, confirming the formation of a dinuclear complex, as shown in Figure . Numerous attempts to obtain crystals of 1 with different nucleotides provided two X-ray quality crystals of UMP and TMP complexes that were grown from an aqueous solution of 1 containing 2 equiv of UMP and TMP, respectively. The isolated crystals were found to be stable at room temperature and characterized by single crystal X-ray diffraction analysis.

Figure 1

Crystal structure of [1·Br2]2+ (where 1 = [CuII2(L)]4+) (see Chart ).

Crystal Structure Analysis

Receptor 1

Structural analysis of the dinuclear n class="Chemical">copper complex (1) reveals that it crystallizes in the triclinic space group P1, with two CuII ions at both ends of the macrocycle, forming a dinuclear complex. As shown in Figure , the macrocycle is folded, where each CuII is coordinated with three macrocyclic nitrogens and one bromide in a square planar coordination environment. The two square planes remaining on the same side are approximately parallel with respect to each other, thus allowing the macrocycle to adopt a rigid boat-like structure. The Cu–Br distances are almost equal, with Cu–Br distances of 2.5705(17)–2.5582(17) Å. The bonded bromides are in equatorial positions, as opposed to the previously reported complex [CuII2(L)Br3(H2O)]+ in which one bromide coordinated to a copper ion in a square pyramidal geometry with an elongated Cu–Br bond (Cu–Br = 2.959(4)).[65] The remaining two bromides (Br3 and Br4) are outside the cavity interacting with the secondary nitrogen atoms (N4 and N19) via NH···Br interactions. Thus, the receptor incorporates positive charges and planar hydrophobic groups as potential binding sites.

Figure 2

Crystal structures of UMP complex with 1: (a) asymmetric unit of [1·(UMP)2]2, (b) single macrocyclic unit showing [1·(UMP)2] motif, and (c) assembled UMP complex showing a chain along the c axis. Hydrogen atoms and external solvent molecules are omitted for clarity.

UMP Complex of 1

The treatment of the receptor 1 with 2 equiv of Na2UMP in n class="Chemical">H2O/dimethyl sulfoxide (DMSO) (2:1, v/v) yielded suitable crystals for X-ray analysis. Structural analysis by single crystal X-ray diffraction reveals that the asymmetric unit of the complex contains two macrocycles, four CuII ions, and four UMP groups, providing two nearly identical UMP-bound units connected through phosphate groups (Figure a). The macrocycles are faced in opposite directions, whereas both UMP groups remain on the same side of each macrcocycle. Clearly, two coordinating bromides in [1·Br2]2+ (Figure ) are exchanged with two UMP guests, forming the receptor–UMP complex. As shown in Figure b, two UMP units in their anionic forms are coordinated to the macrocyclic host through metal–ligand bonds, showing a folded conformation with aromatic spacers between the copper binding sites. The noteworthy feature of this structure is that each UMP binds to the receptor via two types of intermolecular interactions: (i) traditional metal–ligand (coordinate–covalent) interaction between CuII and phosphate and (ii) face-to-face π–π stacking (centroid–centroid distances = 3.61–3.63 Å)[69] between one aromatic ring of 1 and the uracil base (pyrimidine-2,4-dione) of UMP. The two aromatic rings are slightly shifted with respect to each other with a dihedral angle of 4.97 (1)°. Such an assembled complex of NMP with a synthetic receptor exhibiting multipoint recognition has not been structurally characterized before. The X-ray structure of the protein–DNA complex reported earlier showed similar types of interactions in which four positively charged groups on the protein (Arg 16, 21, 80 and Lys 46) exhibited strong electrostatic interactions with the DNA phosphates.[70] Four aromatic residues (Tyr 26, 34, 41 and Phe 73) interacted via π–π stackings with each of the five nucleotide bases.

Crystal structures of UMP complex with 1: (a) asymmetric unit of [1·(n class="Chemical">UMP)2]2, (b) single macrocyclic unit showing [1·(UMP)2] motif, and (c) assembled UMP complex showing a chain along the c axis. Hydrogen atoms and external solvent molecules are omitted for clarity.

In the complex [1·(UMP)2]2, each n class="Chemical">CuII is coordinated with three macrocyclic N atoms and two anionic O atoms in a square pyramidal coordination environment. The distances of Cu···O bonds are 1.921(9) (Cu1···OIC) and 1.915(9) Å (Cu2···O1D), which are comparable to the corresponding distances reported for a copper complex with 5′-UMP (1.92(1) and 1.93(1) Å).[32] Two uracil bases of the coordinated nucleotides in the complex are stacked with the aromatic rings of the receptor in a sandwich fashion (Figure c). Thus, this particular geometrical conformation of the receptor makes it ideal to recognize two UMPs, providing an example of a structurally characterized macrocycle-based nucleoside complex.

TMP Complex of 1

We were also successful in growing X-ray quality crystals of 1 with TMP from the treatment of 1 with 2 equiv of n class="Chemical">Na2TMP in water. As shown in Figure a, the structural aspects of this crystal are nearly similar to that of the UMP complex, containing two macrocycles, four CuII ions, and four TMP, whereas two nearly identical TMP-bound units are connected through phosphate groups of TMP in an asymmetric unit. Figure b shows the structural motif of a single macrocyclic unit as [1·(TMP)2], with two coordinating TMP groups on the same side of the folded macrocycle. However, as opposed to the 1-UMP complex exhibiting strong π–π interactions, there are no such interactions between the receptor’s rings and the thymine base (5-thethylpyrimidine-2,4-dione) in the 1-TMP complex. This could be possibly due to the steric effect of the methyl group that is present on the thymine base. A similar steric effect of methyl groups was also predicted theoretically for the binding of thymine and uracil with a cholesterol-armed triazacyclononane.[71] In crystals, each TMP serves as a linker of two macrocyclic units through phosphate and copper bonds. Notably, in both cases, the receptor and the NMP alternatively assemble into a one-dimensional supramolecular polymer, forming a 1-D chain through bonds between phosphates and metal ions (Figures c and 3c).

Figure 3

Crystal structure of TMP complex of 1: (a) asymmetric unit of [1·(TMP)2]2, (b) single macrocyclic unit showing [1·(TMP)2] motif, and (c) assembled TMP complex showing a chain along the a axis. Hydrogen atoms and external solvent molecules are omitted for clarity.

Crystal structure of TMP complex of 1: (a) asymmetric unit of [1·n class="Chemical">(TMP)2]2, (b) single macrocyclic unit showing [1·(TMP)2] motif, and (c) assembled TMP complex showing a chain along the a axis. Hydrogen atoms and external solvent molecules are omitted for clarity.

Fluorescence Binding Studies Based on IDA Mechanism

An indicator displacement assay that was first employed by Anslyn in anion binding chemistry[72] was used to examine the binding ability of the receptor 1 (in the form of [n class="Chemical">Cu2LBr4]) for nucleoside monophosphates (NMPs) in solution. As shown in Figure , upon the increasing addition of 1 (2.0 × 10–4 M) to EY (eosin Y, 2.0 × 10–6 M) in water buffered with 20 mM HEPES at pH 7.4, the fluorescence intensity (I) gradually decreased possibly due to the Lewis acid–base interactions between the host and the negatively charged EY. The addition of 5 equiv of 1 to EY resulted in an almost complete quenching of the emission (fluorescent OFF). This phenomenon is attributed to the formation of a receptor–indicator complex (1·EY) that quenches the excited state of EY through an energy-transfer process.[66] The change in the fluorescence intensity (I/I0) was analyzed by a nonlinear regression method, which gave the best fit with a 1:1 binding model,[73] yielding the association constant of 5.17 (in log K).

Figure 4

Decrease of fluorescence intensity of EY upon the gradual addition of 1 in water at pH 7.4. λex = 470 nm, λem = 537 nm. The inset shows the titration plot of I/I0 against [1]0/[EY]0.

Decrease of fluorescence intensity of EY upon the gradual addition of 1 in n class="Chemical">water at pH 7.4. λex = 470 nm, λem = 537 nm. The inset shows the titration plot of I/I0 against [1]0/[EY]0.

The complex 1·EY was then investigated for its potential to bind several n class="Chemical">NMPs including UMP, GMP, AMP, TMP, and CMP in water at pH = 7.4. As depicted in Figure , the addition of UMP (2.0 × 10–3 M) to 1·EY (1/EY = 2:1, [EY]0 =2.0 × 10–6 M) led to a complete restoration of fluorescence intensity of EY. This observation suggests the formation of a receptor–UMP complex while the bound dye EY releases from 1·EY to the solution, as signaled by the fluorescence enhancement (fluorescent ON). Similar fluorescence enhancement was also observed on the addition of other analytes to 1·EY (Supporting Information). Analysis of the titration data from the fitting of I/I0 with [anion]0/[1·EY]0 suggested the formation of a 1:1 complex. The calculated conditional constants (Kcon) for [1·EY + anion = 1·anion + EY], which are dependent on the experimental conditions, are listed in Table , showing the binding strength in the order of GMP > AMP > UMP > TMP > CMP. For comparison, we also investigated the binding interactions of 1·EY for ADP (as diphosphate), ATP (as triphosphate), and inorganic phosphate. The chemosensor showed a strong binding affinity (log Kcon) in the range from 5.12 (3) to 5.23 (3) toward GMP, UMP, and AMP, as compared to that of other anions included. However, no interactions were observed for nucleosides, such as adenosine, uridine, guanosine, cytidine, and thymidine, suggesting that the binding of NMP to 1·EY occurs primarily through the interactions of phosphate groups with 1·EY. Several dimetallic complexes have been previously reported by others with NMPs[63] or nucleoside polyphosphate[64] in solution. For example, Hamachi and co-workers reported a xanthene-based Zn(II) complex that was shown to strongly bind several polyphosphate derivatives in the range from (log Kcon) 5.69 to 6.23; however, it showed moderate affinity for cAMP (3.14) and no affinity for AMP or GMP.[62] The observed binding trend, as listed in Table , roughly suggests that in addition to the metal–ligand interactions between the metal centers and the phosphate groups, the higher conjugated π-rings in nucleobases (e.g., GMP and AMP) contribute enhanced interactions possibly due to the π–π interactions between the nucleobases and the receptor’s aromatic groups. The appreciably higher binding strength of the ensemble 1·EY for UMP (log Kcon = 5.1) with respect to that for TMP (log Kcon = 4.8) is consistent with the crystal structure of [1·(UMP)2]2 exhibiting π–π interactions (Figure a), whereas such interactions are absent in [1·(TMP)2]2 due to the steric effect of the methyl group present on the thymine base (Figure a). However, the solution binding model is in contrast to that observed in the solid states for UMP or TMP, where suitable crystals of the NMP complexes were obtained under different conditions. Such discrepancy in anion binding modes in the solid state and solution was reported in the literature.[74,75] It is important to note that at this pH, an NMP may be present as different species (e.g., diprotonated or monoprotonated); thus, this value may not account for the ionic state used for the titration or present in crystals. The results indicate that the ensemble 1·EY shows potential to act as a “fluorescent OFF–ON” probe for the fluorescence sensing of biologically relevant anions. A similar ensemble of [Ni2L(H2O)6]4+ with EY was previously reported that selectively binds biologically relevant oxalate anions, displaying the sharp color change in water at physiological pH.[67]

Figure 5

Enhancement of fluorescence intensity of 1·EY upon the addition of UMP (a) and TMP (b) in water at pH 7.4. λex = 470 nm, λem = 537 nm. The insets show the titration plot of I/I0 against [NMP]0/[1·EY]0.

Table 1

Conditional Constants (Kcon) for Various Anions for the Equilibrium: 1·EY + Anion = 1·Anion + EY, As Measured by the Indicator Displacement Assay in Water at pH 7.4a

anionb	log Kcon
GMP	5.23(3)
AMP	5.18(2)
UMP	5.12(3)
TMP	4.78(3)
CMP	4.71(3)
ADP	5.00(2)
ATP	4.92(3)
HPO42–	4.10(2)

Error limit in Kcon was less that 15%, which was based on the standard deviation from the fit of experimental values.

GMP = guanosine monophosphate, AMP = adenosine monophosphate, UMP = uridine triphosphate, TMP = thymidine Monophosphate, CMP = cytidine monophosphate, ADP = adenosine diphosphate, and ATP = adenosine triphosphate.

Enhancement of fluorescence intensity of 1·EY upon the addition of n class="Chemical">UMP (a) and TMP (b) in water at pH 7.4. λex = 470 nm, λem = 537 nm. The insets show the titration plot of I/I0 against [NMP]0/[1·EY]0.

GMP = n class="Chemical">guanosine monophosphate, AMP = adenosine monophosphate, UMP = uridine triphosphate, TMP = thymidine Monophosphate, CMP = cytidine monophosphate, ADP = adenosine diphosphate, and ATP = adenosine triphosphate.

Conclusions

We have shown that a dinuclear receptor possessing multipoint binding sites recognizes UMP via coordinate–covalent interactions for n class="Chemical">phosphates and hydrophobic π–π stackings for aromatic bases and TMP via coordinate–covalent interactions for phosphates, resulting in the formation of two rare examples of NMP complexes with a macrocycle-based artificial receptor. The structures clearly show the intermolecular bonding features between two discrete molecules, mimicking nucleotide–protein interactions, for example, in B. subtilis orotidine 5′-monophosphate decarboxylase,[46] in ribonucleotide reductases of Salmonella typhimurium,[42] and in single-stranded DNA with G5BP of bacteriophage fd.[70] In addition, the receptor has been successfully employed in an indicator displacement approach[72] for fluorescence sensing of nucleoside monophosphates in water at physiological pH. Understanding the interactions between nucleotides and synthetic receptors is a key step toward understanding the functions of nucleic acids in life. Taken together, these findings may be useful in developing highly sensitive molecular sensors for living cells[19,20] and in drug design for biomedical applications.[64,76,77]

Experimental Section

General

All reagents and solvents were purchased as reagent grades and were used without further purification. NMR spectra were recorded on a Varian Unity INOVA 500 FT-NMR. Chemical shifts for samples were measured in n class="Chemical">CDCl3 and calibrated with TMS as an internal reference. The elemental analysis was carried out using an ECS 4010 Analytical Platform (Costech Instrument). Fluorescence titration studies were performed using a Fluoromax-4 spectrofluorometer (HORIBA Scientific) in water at pH 7.4. The pH was adjusted by using 0.02 M HEPES purchased from Sigma-Aldrich Chemical Company. Experimental conditions were used as follows: λex = 470 nm, λem = 537 nm, dex = 2, and dem = 3 for eosin Y dye.

L

The free macrocycle was synthesized following the procedure as described before.[65] In a typical reaction, N-methyl-2,2′-diaminodiethylamine (1.1719 g, 10.0 × 10–3 mol) in CH3OH (350 mL) and n class="Chemical">terephthalaldehyde (1.3413 g, 10.0 × 10–3 mol) in CH3OH (350 mL) were added slowly to a three-neck round-bottom flask containing 500 mL of CH3OH over 6 h under stirring at 0 °C. The reaction mixture was further stirred overnight at room temperature. The solvent was evaporated, and the imine thus obtained was reduced with NaBH4 (1.56 g) at room temperature for 8 h. The solvent was removed under reduced pressure, and the resulting reaction product was dispersed in water (100 mL). The macrocycle was extracted from the aqueous phase to organic phase by CH2Cl2 (3 × 100 mL). The organic portions were collected and dried by anhydrous MgSO4 (2 g). The solid was filtered off and the solvent was evaporated to dryness. The crude product was purified by column chromatography (neutral alumina, 2% CH3OH in CH2Cl2). Yield: 1.6342 g, (74%). 1H NMR (500 MHz, CDCl3, TMS): δ 7.19 (s, 8H, ArH), 3.75 (s, 8H, ArCH2), 2.78 (t, 8H, J = 5 Hz, NCH2CH2), 2.54 (t, 8H, J = 5 Hz, NCH2CH2), 2.16 (s, 6H, NCH3), 13C NMR (125 MHz, CDCl3): δ 138.92 (Ar-C), 128.00 (Ar-CH), 56.54 (ArCH2), 53.92 (NCH2CH2), 47.00 (NCH2CH2), 42.21 (NCH3). Anal. Calcd for C26H42N6: C, 71.19; H, 9.65; N, 19.16. Found: C, 71.27; H, 9.68; N, 19.22.

[Cu2II(L)Br4]·H2O, 1

The free amine L (146 mg, 0.332 mmol) was mixed with 2 equiv of n class="Chemical">CuBr2 (148 mg, 0.662 mmol) in 5 mL of H2O. The solution was stirred at 60 °C for 1 h. After the solvent was evaporated under reduced pressure, the solid product was suspended in 2 mL of diethyl ether and collected by filtration. The blue microcrystals were dissolved in 2 mL of water–methanol mixture (1:1, v/v) and recrystallized by slow evaporation of the solution, providing air stable X-ray quality crystals after 3 days. Yield: 247 mg, 84%. Anal. Calcd for C26H44Br4Cu2N6O: C, 34.57; H, 4.91; N, 9.30. Found: C, 34.48; H, 4.94; N, 9.27. The compound was further characterized by X-ray diffraction analysis.

[Cu2II(L)(UMP)2]2·2(C2H6OS)·24H2O

The metal complex (1) (88 mg, 0.1 mmol) was dissolved in 10 mL of n class="Chemical">water–DMSO (5:1, v/v) with 2 equiv of UMP (uridine 5′-momophosphate disodium) (74 mg, 0.2 mmol). After the solution was stirred for 20 min at room temperature, the mixture was kept for crystallization at room temperature, yielding X-ray quality crystals after one month. The compound was characterized by X-ray diffraction analysis.

[Cu2II(L)(TMP)2]2·14H2O

The metal complex (1) (88 mg, 0.1 mmol) was dissolved in 10 mL of n class="Chemical">water–DMSO (5:1, v/v) with 2 equiv of TMP (thymidine 5′-momophosphate disodium) (73 mg, 0.2 mmol). After the solution was stirred for 20 min at room temperature, the mixture was kept for crystallization at room temperature, yielding X-ray quality crystals after 1 month. The compound was characterized by X-ray diffraction analysis.

Fluorescence Binding Studies

Binding Constant of 1 for EY

The binding affinity of 1 for EY was determined from the titration of n class="Chemical">EY with 1 in water at pH 7.4. The pH of the solution was maintained using 20 mM HEPES in water. In this case, the quenching of fluorescence was observed due to the addition of 1 to EY. Initial concentrations of EY and 1 were 2.0 × 10–6 and 2.0 × 10–4 M, respectively. Each titration was performed by 20 measurements varying [1]0/[dye]0 = 0–20, and the conditional constant K was calculated by fitting the ratio of I/I0 (Φ =I/I0, where I0 = initial fluorescence intensity of EY, I = fluorescence intensity after the addition of 1) with a 1:1 association model[73] using the equation (the error limit in K was less than 15%).

Binding Constant of 1·EY for NMP

The binding affinity of 1·EY for different n class="Chemical">NMPs was examined from the competition reaction, and binding constants were calculated from the titration of 1·EY with anions in water under the same experimental conditions described in the preceding section. Initial concentrations of 1·EY and an NMP were 2.0 × 10–6 and 2.0 × 10–3 M, respectively. In this case, EY was displaced from 1·EY by the addition of an NMP, resulting in an enhancement of the fluorescence intensity. Each titration was performed by 20 measurements varying the [anion]0/[1·EY]0 = 0–200, and the association constant K was calculated by fitting the ratio of I/I0 (Φ = I/I0, where I0 = initial fluorescence intensity of 1·EY, I = fluorescence intensity after the addition of an anion) with a 1:1 association model[73] using the equation (the error limit in K was less than 15%).

X-ray Crystallography

The crystallographic data and details of data collection for the crystals (1, [1·(UMP)2]2 and [1·n class="Chemical">(TMP)2]2) are given in Tables S1–S3. Intensity data for 1 and [1·(UMP)2]2 were collected using a diffractometer with a Bruker APEX ccd area detector and graphite-monochromated Mo Kα radiation (λ = 0.71073 Å),[78,79] whereas those for [1·(TMP)2]2, with a Rigaku diffractometer, with a Pilatus CMOS area detector and Cu Kα radiation (λ = 1.54178 Å),[80] as described before.[81] The structures were refined by SHELXL2013 program.[82]

There were two macrocycles, four Cu(II) ions, and four n class="Chemical">UMP groups in the asymmetric unit of the cell for [1·(UMP)2]2. The asymmetric unit contains 24 water molecules and two DMSO molecules (not shown in figures for clarity). One of the two DMSO molecules was shown to be disordered. The occupancies of the T-labeled DMSO refined to 0.665(10) and 0.335(10) for the unprimed and primed atoms, respectively. Restraints on the positional parameters of the disordered DMSO, the positional parameters of hydrogens bonded to N and O atoms of the complex, and the displacement parameters of two water oxygens were required. In the case of [1·(TMP)2]2, there were also two macrocycles, four Cu(II) ions, and four TMP groups per asymmetric unit. Several waters were so disordered that they were best fit using the Squeeze program.[83] Restraints on the positional parameters of the disordered atoms and the displacement parameters of all atoms were required. CCDC 1015181, 1019485, and 1536988.