Solution and structural binding studies of phosphate with thiophene-based azamacrocycles

Abstract

Two thiophene-based monocyclic receptors L1 and L2 have been studied for phosphate binding in solutions (D₂O and DMSO-d₆) by ¹H NMR and ³¹P NMR titrations, and in the solid state by single crystal X-ray analysis. Results from ¹H NMR titrations suggest that the ligands bind phosphate anions in a 1:2 binding mode in DMSO-d₆, with the binding constants of 5.25 and 4.20 (in log K), respectively. The binding of phosphate to L1 and L2 was further supported by ³¹P NMR in D₂O at pH = 5.2. The crystal structure of the phosphate complex of L1 reveals unambiguous proof for the formation of a ditopic complex via multiple hydrogen bonds from NH···O and CH···O interactions.

Phosphate is a key building block of nucleic acids, playing critical roles in many biochemical processes [1]. The translocation of phosphate between DNA and proteins in living cells is an essential step in the regulation of metabolic processes [2]. Energy production and storage processes within the body are regulated by phosphorylated compounds, such as adenosine triphosphate [3]. It is known that the phosphate level in serum and saliva is linked to several diseases including hyperparathyroidism, vitamin D deficiency and Fanconi syndrome [4]. It is also widely used in fertilizer and drug-related industries [5]. Because of its significant roles in environment, healthcare and biochemical applications, molecular recognition of phosphate by synthetic molecules is a growing area of current research in supramolecular chemistry [6]. However, phosphate binding in water is unfavorable due to its high free energy of hydration (ΔG⁰ = −465 J/mol) [7]. Polyamine-based receptors that are water soluble, are often used to bind phosphates in water over a wide range of pH [8]. For examples, Martell and coworkers synthesized hexaazamacrocyclic ligands and used them to encapsulate a pyrophosphate via four hydrogen bonds [9]. A hexaprotonated 26-membered polyammonium macrocycle reported by Bowman-James and coworkers was shown to form a complex with six di-hydrogen phosphate anions and two neutral phosphoric acid molecules, illustrating dipotic behavior of the receptor [10]. Bianchi, García-España, Paoletti and coworkers reported a tetraprotonated macrocycle [18]aneN₆ that binds two pyrophosphate anions via NH···O and CH···O bonds [11]. Lu and coworkers showed that an octa-protonated p-xylyl-based cryptand encapsulated a phosphate anion via multiple hydrogen bonds [12]. Other reported receptors that are neutral molecules including amides [13], thioamides [14], ureas [15], thioureas [16], pyrroles [17] and indoles [18] were shown to bind phosphate in organic solvents. Our recent studies on various macrocycle-based receptors indicated that hydrogen bonding and electrostatic interactions are major binding forces in stabilizing complexes; thus providing insight into the binding modes of anions and conformations of host molecules [19-21]. Herein, we report the binding aspects of two thiophene-based macrocycles L1 and L2 via ¹H NMR and ³¹P NMR in solutions. We also report the structural characterization of a phosphate complex with L1, forming a ditopic complex with one dihydrogen phosphate and one monohydrogen phosphate.

The hexamine ligands L1 and L2 were synthesized through Schiff-base condensation reaction (Scheme 1) of the corresponding dipodal amine with two equivalents of 2,5-thiophendicarboxaldehyde followed by the reduction with NaBH₄, as reported earlier [20-22]. The tosylate salts were prepared by reacting the free ligands with p-toluenesulfonic acid in methanol. The analysis of ¹H NMR data suggested the formation of an adduct with six tosylate groups providing six positive charges on the macrocyclic moiety. The phosphate complex was synthesized by the addition of a few drops of aqueous phosphoric acid to L1 dissolved in methanol. Crystals suitable for X-ray analysis were grown from the slow evaporation of the solution at room temperature.

Scheme 1.

Synthetic scheme for the macrocycles L1 and L2: Reaction conditions: (i) methanol, 0 °C, 24 hr stirring; (ii) NaBH₄, methanol, overnight stirring.

¹H NMR titrations of [H₆L1]⁶⁺ and [H₆L2]⁶⁺ were performed to study the binding interactions with phosphate using n-Bu₄N⁺H₂PO₄⁻ (TBAH₂PO₄) in D₂O as well as in DMSO-d₆. The incremental addition of the anion (20 mM) to [H₆L1]⁶⁺ (2 mM) in D₂O (pH = 5.2) resulted in a downfield shift of aliphatic protons. The non-linear regression analysis of the shift change of several independent protons of [H₆L1]⁶⁺ showed moderate binding for phosphate (log K = 2.0 M⁻¹), providing a good fit of a 1:1 binding model [23]. However, under identical conditions, there was no significant change in any protons of [H₆L2]⁶⁺ upon the addition of TBAH₂PO₄ to the host solution, indicating weak host-guest interactions in D₂O. We, therefore, proceeded to investigate the binding properties of [H₆L1]⁶⁺ and [H₆L2]⁶⁺ in DMSO-d₆. Figure 1 displays the stacking of ¹H NMR titration spectra of [H₆L1]⁶⁺ obtained after the increasing amount of phosphate anion (ranging 0 to 10 equivalents), showing a gradual upfield shift of both the aromatic and the aliphatic protons of the macrocyclic moiety at room temperature. The changes in the chemical shift of the aromatic proton (ArH) as a function of the anion concentration are displayed in Figure 1. The non-linear regression analysis of the changes in the chemical shift of the ligand as recorded with an increasing amount of anionic solution provided the best fit for a 1:2 binding model [24]. The ligand [H₆L2]⁶⁺ also shows a similar binding trend with phosphate in DMSO-d₆. Association constants presented in Table 1 suggest that the binding process of each ligand involves the formation of both a 1:1 (ligand:anion) complex and a 1:2 (ligand:anion) complex. However, a 1:2 complex is stronger than a 1:1 complex in DMSO-d₆, supporting the X-ray structure (discussed later). A similar binding mode was previously reported for phosphate with [26]aneN₆C₆ [10]. As shown in Table 1, the overall binding constant of L1 for phosphate in DMSO-d₆ is slightly higher than that of L2 which could be due to the reduced hydrogen bonding ability of the methylated compound.

Figure 1.

(a) ¹H NMR spectra of [H₆L1] (Ts)₆ (2 mM) with an increasing amount of TBAH₂PO₄ (20 mM) in DMSO-d₆. (b) Titration curve of [H₆L1] (Ts)₆ with TBAH₂PO₄ showing the change in the chemical shift of Ar-H (H1) against an increasing concentration of phosphate in DMSO-d₆.

Table 1.

Association constants of the ligands for phosphate as determined by ¹H NMR titrations.^a

Ligand	Log K
	D2O	DMSO-d6
L1	2.0 (1:1)	1.54 (1:1) 5.25 (1:2)
L2	b	1.92 (1:1) 4.20 (1:2)

^aEstimated error was less than 15%

^bNo significant NMR shift was observed.

The interaction of [H₆L1]⁶⁺ and [H₆L2]⁶⁺ with TBAH₂PO₄ in D₂O was also investigated by phosphorus ³¹P NMR at room temperature. Because of the lower sensitivity of ³¹P NMR compared to ¹H NMR, a higher concentration of TBAH₂PO₄ (10 mM) was loaded to an NMR tube, and a host solution (25 mM) was prepared as a titrant. The ³¹P resonance was calibrated against an aqueous phosphoric acid used in a sealed capillary tube. Figure 2 shows the ³¹P NMR spectra of TBAH₂PO₄ (10 mM) after the addition of two equivalents of [H₆L1]⁶⁺ and [H₆L2]⁶⁺ in D₂O. As clearly shown in Figure 2a, the signal at δ_P = 0.218 ppm for the free TBAH₂PO₄ shifts upfield to δ_P = −1.635 ppm (Δδ_P = 1.853 ppm) after the addition of [H₆L1]⁶⁺ (2 equivalents). The other ligand [H₆L2]⁶⁺ also shows similar shifting to δ_P = −1.404 ppm but to a lesser extent (Δδ_P = 1.622 ppm), indicating a lower affinity for the phosphate anion than that with [H₆L1]⁶⁺. This upfield shift suggest the formation of the phosphate complex due to the strong electrostatic interactions, where the bound phosphate is shielded by the interacting macrocycle. Similar upfield shifts of phosphorus resonance were reported due to the complexation by polyamine-based hosts [12,25].

Figure 2.

³¹P NMR spectra of n-Bu₄N⁺H₂PO₄⁻ in D₂O recorded at room temperature (a) free TBAH₂PO₄ (δ_P = 0.218 ppm) (10 mM) (b) TBAH₂PO₄ + 2 equivalents of [H₆L2] (Ts)₆ (δ_P = − 1.404 ppm) and (c) TBAH₂PO₄ + 2 equivalents of [H₆L1] (Ts)₆ (δ_P = − 1.6357 ppm)

The phosphate complex of L1 was obtained by reacting the free macrocycle with phosphoric acid. Crystals suitable for X-ray analysis were obtained by recrystallization of the salt in methanol/water. Crystallographic data are presented in Table 2. The structural analysis of the phosphate complex reveals that the salt crystallizes in the triclinic space group (P1) and the asymmetric unit consists of two hexaprotonated macrocycles, three monohydrogen phosphate (HPO₄²⁻), six di-hydrogen phosphate (H₂PO₄⁻) and eight water molecules to yield a molecular formula of 2(C₂₀H₄₀N₆S₂)⁶⁺·3(HPO₄)²⁻·6(H₂PO₄)⁻·8(H₂O). One hydrogen phosphate (P9) is disordered about 90:10 into two orientations. One water molecule (O8W) is also found to be disordered about 80:20. Each macrocycle is fully protonated and the total positive charges (12+) from the two hexaprotonated receptors are balanced by the negative charges provided by three doubly-charged monohydrogen phosphates and six singly-charged dihydrogen phosphate anions.

As shown in Figure 3, two identical macrocycles in the asymmetric unit possess a non-crystallographic inversion center, forming a sandwich type complex with four bound phosphate groups and two water molecules. Two water molecules serve as linkers to macrocycles as well as to bound dihydrogen phosphates locating between the macrocycles (Figure 3a). Each macrocycle adopts the shape of a flat ellipsoid and binds two phosphate groups; one is monohydrogen phosphate and the other is dihydrogen phosphate, located above and below the elliptical plane. The complex is stabilized through electrostatic interactions and multiple H-bonds. A list of H-bond distances with bond angles are shown in Table 3. Figure 3c shows one macrocycle of the asymmetric unit, with two bound phosphates held by hydrogen bonding interactions with the macrocyclic cation. The monohydrogen phosphate (P76) is held by two NH···O (2.684 and 2.747 Å) and two CH···O (3.266 and 3.388 Å) bonds, while the dihydrogen phosphate interacts through two strong NH···O bonds (N15H···O70 = 2.932 and N15H···O73 = 2.677 Å) and one weak NH···O bonds (N39H···O80 = 3.266 Å). As a result, the monohydrogen phosphate is closer to the cavity than the dihydrogen phosphate. The involvement of CH···O interactions is well documented in the literature [21]. Extensive hydrogen bonds between the macrocycle and water molecules are also present.

Figure 3.

Top: crystal structure of the phosphate complex showing [(H₆L1)₂·(HPO₄)₂·(H₂PO₄)₂·(H₂O)₂]⁶⁺ moiety in capped-stick view (a), and space filling view (b). Bottom: crystal structure of the phosphate complex showing [(H₆L1)·(HPO₄)·(H₂PO₄)]³⁺ moiety in capped-stick view (c), and space filling view (d).

In conclusion, we report the phosphate binding properties of two simple thiophene-based monocyclic receptors L1 and L2 by ¹H NMR and ³¹P NMR studies in two different polar solvents (D₂O and DMSO-d₆). Both ligands show lessened interactions with the anion in competitive D₂O due to their weak hydrogen bonding ability in the highly polar solvent. However, after switching the solvent from D₂O to DMSO-d₆, the binding affinity is significantly increased, exhibiting the formation of 1:2 complexes. The ³¹P NMR has been successfully used as a probe to identify the chemical environment of bound and free phosphates in D₂O. Structural characterization of the phosphate complex with L1 suggests that both NH···O and CH···O interactions play roles in stabilizing the host-guest complex.