"Dynamical Docking" of Cyclic Dinuclear Au(I) Bis-N-heterocyclic Complexes Facilitates Their Binding to G-Quadruplexes

Abstract

With the aim to improve the design of metal complexes as stabilizers of noncanonical DNA secondary structures, namely, G-quadruplexes (G4s), a series of cyclic dinuclear Au(I) N-heterocyclic carbene complexes based on xanthine and benzimidazole ligands has been synthesized and characterized by various methods, including X-ray diffraction. Fluorescence resonance energy transfer (FRET) and CD DNA melting assays unraveled the compounds' stabilization properties toward G4s of different topologies of physiological relevance. Initial structure-activity relationships have been identified and recognize the family of xanthine derivatives as those more selective toward G4s versus duplex DNA. The binding modes and free-energy landscape of the most active xanthine derivative (featuring a propyl linker) with the promoter sequence cKIT1 have been studied by metadynamics. The atomistic simulations evidenced that the Au(I) compound interacts noncovalently with the top G4 tetrad. The theoretical results on the Au(I) complex/DNA Gibbs free energy of binding were experimentally validated by FRET DNA melting assays. The compounds have also been tested for their antiproliferative properties in human cancer cells in vitro, showing generally moderate activity. This study provides further insights into the biological activity of Au(I) organometallics acting via noncovalent interactions and underlines their promise for tunable targeted applications by appropriate chemical modifications.

Figure 1

(A) General structure of Au(I) NHC complexes and possible modifications and (B) structure of the cationic bis-NHC Au(I) complex [Au(9-methylcaffeine-8-ylidene)₂]⁺ (AuTMX₂) as a selective G4 stabilizer.

Scheme 1. Reaction Scheme Leading to the Metallacyclic Au(I) Purine- (A) and Benzimidazole-Based (B) NHC Complexes

Figure 2

Molecular structures of the cyclic dinuclear Au(I) bis-NHC complexes AuB2, AuB3, AuC1, and AuC2. Compound AuB3 features two independent molecules (a,b) with different conformations. Ellipsoids are displayed at the 50% probability level. Hydrogen atoms as well as co-crystallized solvent molecules and counterions are omitted for clarity. Color code: Au (yellow), C (gray), N (blue), and O (red).

Figure 3

(A) ¹H NMR spectra in DMSO-d₆/D₂O (80:20) of AuB3 alone and in the presence of an equimolar amount of NAC; the NAC spectrum is also reported as reference. Newly formed peaks are labeled with *. (B) Evolution of the 4.10 (blue) and 1.83 ppm ¹H NMR signals (green) over 24 h reaction. (C) HR-ESI-MS of the reaction mixture after 24 h.

Figure 4

G-quadruplex stabilization effects induced by the Au(I) NHC complexes studied by the FRET DNA melting assay. (a) ΔT_m (°C) of selected G4s solutions in 60 mM potassium cacodylate (pH = 7.4) in the presence of Au(I) NHC complexes (5 equiv); (b) representation of the ΔT_m (°C) in a radial plot (from 0 to 30 °C). ΔT_m (°C) of hTelo (c) and cKIT1 (d) samples in 60 mM potassium cacodylate (pH = 7.4) in the presence of 5 equiv of selected Au(I) NHC complexes and 50 equiv of CT-DNA. A pattern is shown to differentiate the purine series from the benzimidazole one. Data are shown as mean ± SEM of at least three independent experiments.

Figure 5

Stabilization effects induced by 1–5 equiv of AuB3 (sky blue dashed lines) or AuC3 (dark blue dashed lines) complexes toward hTelo (squares) or cKIT1 (circles) studied by FRET DNA melting in 60 mM potassium cacodylate (pH = 7.4).

Figure 6

CD spectra of hTelo (a,c) and cKIT1 solutions (b,d) in the presence of increasing amounts (1–10 equiv) of AuB3 (a,b) and AuC3 (c,d) recorded in Tris–HCl/KCl (10/50 mM, pH = 7.4) buffer. Directions of spectral changes are shown by the arrows.

Figure 7

(A) Position of AuB3 and cKIT1 at the beginning of the metaD simulation, showing in stick the upper tetrad and upper K⁺ ion (purple sphere) and in ball-and-stick the cyclic dinuclear Au(I) bis-NHC complex. CVs (CV1 and CV2) correspond to distances of Au⁺ ions from the K⁺ ion (Å). (B) Representative FES depicted as a heat map showing two energy minima based on the two CV distances. (C) Comparison of averaged ΔG for both metaD and FRET DNA melting assays for AuTMX₂(28) and AuB3, defined as a bar graph with standard deviation added (state I and state II refer to CV1 < CV2 and CV2 < CV1 respectively).

Figure 8

Positions and interactions of the upper G-tetrad nucleobases of cKIT1 with AuB3 (top) and AuTMX₂ (bottom) as studied by metaD. (A) G4-adducts of AuB3 and AuTMX₂ with cKIT1 in their lowest energy conformations (state I in the case of AuB3). (B) Zoom into the interactions of AuB3 and AuTMX₂ with the upper G4 tetrad with π–π stacking interactions shown as an orange surface. (C) 2D interaction diagram shows increased π–π stacking with AuTMX₂ when compared to AuB3 with nucleobases G6 (DG6) and G10 (DG10).

Figure 9

Positions of AuB3 on the upper G-tetrad of cKIT1 in the “parallel” orientation with respect to A1 (A) observed in 21% of the simulation and in the “perpendicular” orientation (B) observed in 47% of the simulations.

Figure 10

Position of AuB3 with the upper G-tetrad in both the “open” (A) and “closed” (B) conformations of A1 (AuB3 and A1 shown in ball and stick representation, cKIT1 shown as a ribbon with bases as a wire. A1 is colored in green for clarity).