Reversed Cation Selectivity of G₈-Octamer and G₁₆-Hexadecamer towards Monovalent and Divalent Cations

Abstract

A reverse-binding-selectivity between monovalent and divalent cations was observed for two different self-assembly G₁₆-hexadecamer and G₈-octamer systems.The dissociation constant between G₄-quadruplex and monomer was calculated via VT-¹H NMR experiments. Quantitative energy profiles revealed entropy as the key factor for the weaker binding toward Ba²⁺ compared with K⁺ in the G₈-octamer system despite stronger ion-dipole interactions. This study is the first direct comparison of the G₄-quartet binding affinity between mono and divalent cations and will benefit future applications of G-quadruplex-related research. Further competition experiments between the G₈-octamer and 18-crown-6 with K⁺ demonstrated the potential of this G₈ system as a new potassium receptor.

Graphical Abstract

G8-octamer and G16-hexadecamer showed reversed cation selectivity! Excellent K+ selectivity was achieved with a G8-octamer system. Energetic binding profiles were determined to account for this phenomenon. This exceptional binding ability toward K+ was further demonstrated through competition experiments with crown ether 18C6.

One strength of supramolecular chemistry is the ability to produce large and highly organized structures from simple building blocks through the direct assembly.^[1–5] Non-covalent interactions (H-bonding, van der Waals force, ion-dipole interaction, and π-π stacking) play a crucial role in supramolecular self-assembly, providing thermodynamic and kinetic driving force to form supermolecules.^[6–8] Hence, detailed analysis of the weak interactions is key to understanding the nature of self-assembly systems. Guanosine quadruplex is a unique supramolecular scaffold that originates from biological mimics in the DNA strand.^[9–15] As shown in Scheme 1A, this non-covalent structure contains two hierarchical orders of self-assembly: A) H-bond interaction between four guanine unit to form G₄-quartet; B) vertical stacking of G₄-quartets to give G-quadruplexes. With four carbonyl oxygens positioned in the center of a G₄-quartet, it is believed that the hard alkali or alkali earth metal cations can serve as templates to facilitate the stacking of quartet layers through ion-dipole interactions.^[16–19]

Scheme 1.

Dynamic equilibrium of self-assembly and H-bond distances.

According to the literature, the binding affinity of G₄-quartet toward different cations followed the general trend as K⁺>Rb⁺>Na⁺>Cs⁺/Li⁺ and Sr²⁺>Ba²⁺>Ca²⁺>Mg²⁺.^[20] This observation was explained based on the matching of cation size (radii) with the G₈-octamer (binding pocket). For cations of similar size, divalent cations were hypothesized to have higher binding affinity over monovalent cations because of higher charge density. However, considering the difference of counter anions (one X⁻ vs two X⁻ or one X²⁻), the overall supramolecular assembly properties may change dramatically with different cations. Thus, the assumption that divalent cations will generally bind G₄-quartet better than monovalent cations could be misleading and oversimplifying. Moreover, while binding affinity studies have been done among either monovalent cations or divalent cations,^[21–24] very few studies have been shown to compare energy and binding selectivity between these two types of charged cations, likely due to the complexity of the overall assembly.^[25,26] Thus, understanding how cations interact with G₄-quartets is one of the fundamental tasks for construction of well-defined G-quadruplexes for potential applications.^[27–32] Herein, we report the detailed investigations on cation selectivity between two reported and well-characterized G-quadruplexes supramolecular systems, the anion bridged G₁₆^[33] and anion-binding-free G₈ assembly (based on G-monomer conformation control).³⁴ The interesting reversed binding ability between mono-valent K⁺ and divalent Ba²⁺ was revealed. To the best of our knowledge, this reverse cation selectivity was the first example of direct binding evaluation of G₄ toward cations with similar size but different charges (M⁺ vs M²⁺).

Our group recently reported the influence of guanosine monomer conformation on controlling the G₄-quartet stacking molecularity for the construction of well-defined G-quadruplex.^[34] These efforts led to the discovery of a new G_8-octamer system of which the conformations have been fully characterized in solution (NMR), gas-phase (ESI-MS) and solid-state (the first G₈ X-ray structure). With this important structural information, we compared all the H-bond distances of previously reported G₄-quartets.^[33–36] As shown in Scheme 1B, no significant difference for H-bond distances of d(N1…O6) and d (N2…N7) was observed.

This result is interesting because H-bonded quartets of similar size were formed with both small cations like Na⁺ (r= 1.18 Å)^[37] and large cations like Rb⁺ (r=1.61 Å)^[37]. Thus, it is important to realize that G₄-quartet is a preorganized and independent assembly of which the size is likely only influenced by H-bonds. This makes us wonder if cation binding affinity of any discrete G-quadruplex is determined solely by size-matching and charge density. To explore cation binding selectivity and affinity, we focused on two reported and well-defined G-quadruplex systems: A) anion bridged G₁₆-hexadecamer developed by Davis’s group^[33,38,39] and B) G₈-octamer recently reported by our group (Scheme 2).^[34] Notably, both structures have been fully characterized with confirmed G₁₆ and G₈ assemblies maintained intact in solution NMR, solid state X-ray and gas phase MS.

Scheme 2.

Well-defined G-quadruplexes formation using guanosine 1a and 1b.

As shown in Figure 1A, ¹H NMR spectra revealed the excellent divalent cation selectivity of G₁₆ complexes [(1a)₁₆M_n]⁴⁺(Pic¯)₄ over monovalent cations, which was previously reported by Davis and coworkers.^[33] Using a mixture of K⁺ and Ba²⁺ with 1:1 molar ratio, only [(1a)₁₆Ba₂]⁴⁺(Pic⁻)_4·was observed in ¹H NMR spectra with no formation of [(1a)₁₆K₄]⁴⁺(Pic⁻)_4·

Figure 1.

(A) ¹H NMR experiments of 1a with Ba²⁺ and K⁺ mixture at 25°C in CDCl₃. (a) crystalline [(1a)₁₆K₄]⁴⁺(Pic⁻)₄ , Ba²⁺:K⁺ molar ratio of : (b) 1:100, (c) 1:10, (d) 1:1, (e) crystalline [(1a)₁₆Ba₂]⁴⁺(Pic⁻)₄. The concentrations of Ba²⁺ in aqueous solution are the same in all cases. (B) ¹H NMR experiments of 1b with Ba²⁺ and K⁺ mixture at 25°C in CDCl_3. (a’) crystalline [(1b)₈Ba]²⁺(Pic⁻)₂ , K⁺: Ba²⁺ molar ratio of (b’) 1:100, (c’) 1:10, (d’) 1:1, (e’) crystalline [(1b)₈K]⁺(Pic⁻). The concentrations of K⁺ in aqueous solution are the same in all cases. The portion of the spectra shows the region of the N (1)H peaks.

While K⁺ and Ba²⁺ has similar cation radii, it is believed that the good Ba²⁺ selectivity is associated with the higher charge density. In this sense, similar Ba²⁺ selectivity was expected for G₈-octamer system. However, to our great surprise, treating 1b with 1:1 K⁺/Ba²⁺ mixture gave the formation of [(1b)₈K]⁺(Pic⁻) as only complex with almost no [(1b)₈Ba]²⁺(Pic⁻)₂ observed based on ¹H NMR spectra. To confirm this reversed selectivity, assembly of guanosine derivatives 1a and 1b with cation mixtures (K⁺ and Ba²⁺) under various molar ratios (from 1:1 to 100:1) were performed. As revealed by the ¹H NMR spectra in Figure 1B, 1b demonstrated overwhelming selectivity towards K⁺ over Ba²⁺ when cations of the same concentration and molarity were added. Impressively, even with excess Ba²⁺ cation applied (10 times), only a trace amount of [(1b)₈Ba]²⁺(Pic⁻)₂ was observed with K⁺ complex dominated in solution (>93%). Further increasing the ratio of Ba²⁺:K⁺ to 100:1 led to slightly reduce of [(1b)₈K]⁺(Pic⁻) to 67%. (See ESI for detailed calculation) Switching cation from Ba²⁺ to Sr²⁺ gave similar results with dominate selectivity towards K⁺ over Sr²⁺ (See ESI).

To further confirm this reversal selectivity between two supramolecular systems, cation titration experiments were performed. As shown in Figure 2A, adding Ba²⁺(Pic⁻)₂ into a solution of 1a and 1b monomer mixtures (1:1) gave preferred formation of [(1a)₁₆Ba₂]⁴⁺(Pic⁻)₄ over [(1b)₈Ba]²⁺(Pic⁻)₂ complexes. In contrast, using K⁺Pic⁻ for the same titration experiments (Figure 2B), preferred formation of [(1b)₈K]⁺(Pic⁻) over [(1a)₁₆K₄]⁴⁺(Pic⁻)₄ was observed, which confirmed the reversal cation selectivity of these two systems (G₁₆ vs G₈) from 1a and 1b monomers.

Figure 2.

¹H NMR spectra of titration experiments. (A) ¹H NMR spectra from titration experiment of compound 1a and 1b with Ba²⁺(Pic⁻)₂ of increasing molar ratio: (a) [(1b)₈Ba]²⁺(Pic⁻)₂ (b)[(1a)₁₆Ba₂]⁴⁺ 1H NMR spectra from titration experiment of compound 1a and 1b with Ba²+(Pic⁻)2 of increasing molar ratio: (a) [(1b)₈Ba]²+(Pic⁻)₂ (b)[(1a)₁₆Ba₂]⁴⁺(Pic⁻)₄ (c) 1/2 eq. Ba²⁺(Pic⁻)₂ (d) 1/4 eq. Ba²⁺(Pic⁻)₂ (e) 1/64 eq. Ba²⁺(Pic⁻)₂ ; (B) ¹H NMR spectra of titration experiment between compound 1a and 1b with K⁺Pic⁻of increasing molar ratio: (a’) [(1b)₈K]⁺(Pic⁻) (b’) [(1a)₁₆ K₄]⁴⁺(Pic⁻)₄ (c’) 1/2 eq. K⁺Pic⁻(d’) 1/4 eq.K⁺Pic⁻(e’) 1/64 eq. K⁺Pic⁻. The portion of the spectra shows the region of the N(1)H peaks.

The above competition-titration studies revealed the different cation selectivity under the thermodynamic conditions for G₁₆ and G₈ systems. Clearly, it is important to understand the energetic driving force that accounts for this reversed selectivity. To quantitatively evaluate the thermodynamic stability^[40] of these two G-quadruplex systems (G₁₆ and G₈), we studied the dissociation process from complex to monomer using mixed solvent systems. As shown in Figure 3, using pure DMSO-d₆ solvent caused the complete dissociation of both G₁₆ and G₈ complexes, giving monomer 1a and 1b in solution based on ¹H NMR. Applying CDCl₃/DMSO-d₆ mixed solvents, a mixture of complex and monomer were obtained with no obvious assembled intermediates (such as G₄, G₁₂ and oligomers) observed.

Figure 3.

Complex-monomer dissociation in the solvent mixture (a) [(1a)₁₆Ba₂]⁴⁺(Pic⁻)₄ in DMSO-d₆(b–f) VT-¹H NMR spectra of [(1a)₁₆Ba₂]⁴⁺(Pic⁻)₄ in CDCl₃:DMSO-d₆=4:1 solvent mixture from 30°C to 50°C. H1 proton and TBS region were shown from 10–12 ppm and –0.5–0 ppm respectively. (g) [(1a)₁₆Ba₂]⁴⁺(Pic⁻)₄ in CDCl₃ (a’) [(1b)₈K]⁺(Pic⁻) in DMSO-d₆ (b’–f’) VT-¹H NMR spectra of [(1b)₈K]⁺(Pic⁻) in CDCl₃:DMSO-d₆=4:1 solvent mixture from 30°C to 50°C. H1 region and phenyl protons were shown from 10–13 ppm and 7.5–8.5 ppm respectively. (g’) [(1b)₈K]⁺(Pic⁻) in CDCl_3.

With this prerequisite, evaluations of complex dissociation processes of these two systems at different temperatures (VT) can be performed. Based on the VT-¹H NMR experiment, an equilibrium between G-quadruplexes and G-monomers can be established in CDCl₃/DMSO-d₆. The dissociation constant (K_dis) for the Van’t Hoff plot at each temperature can be readily obtained (see detailed calculation in SI). A series of G₈ and G₁₆ complexes were prepared, including [(1a)₁₆K₄]⁴⁺(Pic⁻)₄,[(1a)₁₆Ba₂]⁴⁺(Pic⁻)₄, [(1a)₁₆Sr₂]⁴⁺(Pic⁻)₄, [(1b)₈K]⁺(Pic⁻),[(1b)₈Ba]²⁺(Pic⁻)₂, and [(1b)₈Sr]²⁺(Pic⁻)₂. These complexes were dissolved in CDCl₃/DMSO-d₆=4:1 solvent mixture and the ¹H NMR spectra were recorded at various temperatures from 30°C to 50°C (see detailed spectra in ESI).

As revealed by VT-¹H NMR experiments, the complex-monomer equilibriums were shown unambiguously at each tested temperature with no significant chemical shift changing. Complex and monomer concentrations at each temperature were calculated based on NMR integrations. Complex dissociation constants K_dis could then be concluded in each case.Plotting these numbers into Van’t Hoff plot (lnK_dis over 1/T) gave good linear correlation for all cases (R²>0.99) as shown in Figure 4. From the slope and intercept of each curve, both enthalpy (ΔH) and entropy (ΔS) were calculated. All these results are summarized in Table 1 (see detailed calculation in ESI).

Figure 4.

Van’t Hoff plot (ln Kdis vs 1/T) of (A) 1a and (B) 1b complexes.

Table 1.

ΔH, ΔS and ΔG of G-quadruplex dissociation to monomer. ΔG is calculated at 298.15 K.

G-quadruplex	ΔH [kJ*mol 1]	ΔS [J*mol−1*K−1]	ΔG [kJ*mol−1]
[(1 a)16K4]4+ (Pic−)4	663.7	1008.2	363.1
[(1 a)16Ba2]4+(Pic−)4	1770.4	4628.0	390.6
[(1 a)16Sr2]4+(Pic−)4	997.6	2236.8	386.6
[(1 b)8K] +(Pic−)	223.5	282.6	139.3
[(1 b)8Ba]2+(Pic−)2	340.5	751.4	116.4
[(1 b)8Sr]2+(Pic−)2	314.8	650.7	120.9

As shown in Table 1, all G₁₆-complexes from 1a gave higher ΔH comparing with the G₈-complexes from 1b, likely due to the formation of greater amounts of H-bonds in G₁₆-hexadecamers. For 1a complex, ΔG values of both [(1a)₁₆Ba₂]⁴⁺(Pic⁻)₄ and [(1a)₁₆Sr₂]⁴⁺(Pic⁻)₄ at 25°C are higher than that of [(1a)₁₆K₄]⁴⁺(Pic⁻)₄, suggesting the formation of more stable complexes with Ba²⁺ over K⁺ as observed experimentally. In contrast, G₈-complexes from 1b gave similar ΔH values, indicating that the cation has little influence on the H-bond in the G-quartet (consistent with the structural information from X-ray^[33–36]). Interestingly, [(1b)₈K]⁺(Pic⁻) complex showed smaller ΔS increase upon dissociation (282.6 J_*mol⁻¹_*K⁻¹) comparing with G₈-octamers from divalent Ba²⁺ or Sr²⁺ cations (751.4 and 650.7 J_* mol⁻¹ K⁻¹ respectively). This difference in entropy contributions could be rationalized with the free anions associated with G₈ systems. As a result, ΔG of [(1b)₈K]⁺(Pic⁻) at room temperature is greater than that of [(1b)₈Ba]²⁺(Pic⁻)₂ (139.3 vs 116.4 kJ_* mol ¹), which explains the observed K⁺ selectivity in G₈ system from 1b monomer.

These thermodynamic dissociation experiments provided clear evidences for the observed reverse selectivity regarding G₁₆ and G₈ quadruplexes systems. Notably, based on the above observation, doubly charged anion X⁻ might help divalent cation binding in G₈ system by reducing unfavored entropy cost. Unfortunately, among all the divalent anions we tested so far, no well-defined G-quadruplex were obtained, mainly due to the poor solubility of these anions in CDCl₃ (see ESI). This result, again, showcased the great challenges associated with complex supramolecular assemblies. Nevertheless, the success in obtaining quantitative energy profiles for both G₁₆ and G₈ systems provided direct evaluations of G₄-quartet and cation interactions. To the best of knowledge, this is the first energetic evaluation of G₄-cation binding between monovalent and divalent cations.

Considering the excellent binding selectivity of 1b toward K⁺, we wondered if this G₈-quadruplex could be applied as a new K⁺ receptor with strong binding affinity. It is known that crown ethers are good hosts for alkali and alkali-earth metal cation recognition.^[41] In particular, 18-crown-6 (18C6) demonstrated strong binding affinity toward K⁺.^[41,42] As a result, 18C6 has been used as a phase-transfer catalyst to deliver insoluble K⁺ salts from aqueous layer to organic layer.^[43–45] To evaluate G₈-octamer as potential host for K⁺ binding, direct competition between 1b and 18C6 was conducted.

As shown in Figure 5, both [(1b)₈K]⁺(Pic⁻) and [(18C6)K]⁺(Pic⁻) had good solubility in CDCl_3. Treating crown ether complex [(18C6)K]⁺(Pic⁻) with 1b resulted in the formation of [(1b)₈K]⁺(Pic⁻) and the free crown ether 18C6 instantly (Figure 5d). In contrast, reaction of [(1b)₈K]⁺(Pic⁻) with 18C6 (1.0 equiv.), exhibited no change in ¹H NMR spectra even after long period of time (48 hours, Figure 5e). Both experiments provided clear evidences that self-assembled G₈-octamer can form stronger K⁺ coordination even over the well-known K⁺ receptor, 18C6. Thus, 1b could certainly be applied as an alternative new host for potassium cation recognition with much enriched functional group modification potentials. Applications of this K⁺ binding ability into phase transfer related catalysis is currently under investigation in our lab.

Figure 5.

Competition experiments between 1b and 18C6. (a) [(1b)₈K]⁺(Pic⁻) in CDCl₃ (b) 18C6 monomer in CDCl₃ (c) [(18C6)K]⁺(Pic⁻) in CDCl₃ (d) ¹H NMR spectra of 1:1 mixtures of [(18C6)K]⁺(Pic⁻) and 1b in CDCl₃ at rt (e) ¹H NMR spectra of 1:8 mixtures of [(1b)₈K]⁺(Pic⁻) and 18C6 monomer in CDCl₃ at rt.

In summary, evaluations of cation binding selectivity of two well-defined guanosine supramolecular assemblies have been performed. An interesting reversed binding ability between monovalent and divalent cations was observed with G₁₆ and G₈ systems. Van’t Hoff plot revealed entropy differences as the crucial factors that attribute to the observed reverse selectivity. These studies provided the first quantitative evaluation of different cation association toward G₄-quartet. An excellent K⁺ binding ability of the G₈-octamer system was further identified by competition experiments with 18-crown-6, indicating the potential of using guanosine derivative 1b as a new potassium receptor. It is our strong belief that these studies will not only provide important mechanistic understanding of G₄-quartet chemistry but also contribute to applications of G-quadruplexes in related biological and materials research.