Tailored Transition‐Metal Coordination Environments in Imidazole‐Modified DNA G‐Quadruplexes

Abstract Two types of imidazole ligands were introduced both at the end of tetramolecular and into the loop region of unimolecular DNA G‐quadruplexes. The modified oligonucleotides were shown to complex a range of different transition‐metal cations including NiII, CuII, ZnII and CoII, as indicated by UV/Vis absorption spectroscopy and ion mobility mass spectrometry. Molecular dynamics simulations were performed to obtain structural insight into the investigated systems. Variation of ligand number and position in the loop region of unimolecular sequences derived from the human telomer region (htel) allows for a controlled design of distinct coordination environments with fine‐tuned metal affinities. It is shown that CuII, which is typically square‐planar coordinated, has a higher affinity for systems offering four ligands, whereas NiII prefers G‐quadruplexes with six ligands. Likewise, the positioning of ligands in a square‐planar versus tetrahedral fashion affects binding affinities of CuII and ZnII cations, respectively. Gaining control over ligand arrangement patterns will spur the rational development of transition‐metal‐modified DNAzymes. Furthermore, this method is suited to combine different types of ligands, for example, those typically found in metalloenzymes, inside a single DNA architecture.


Synthesis
All chemicals were obtained from commercial sources and used without further purification. Gel permeation chromatography (GPC) purification of ligands was performed on a LC-9210 II NEXT system with CHCl3 (HPLC grade) as eluent. NMR measurements were conducted at 298 K on Avance-600 and Avance-700 instruments from Bruker and on 500 MHz Bruker Avance neo NMR. Chemical shifts for 1 H and 13 C are reported in ppm on the δ scale; 1 H and 13 C signals were referenced to the residual solvent peak. The following abbreviations are used to describe signal multiplicity for 1 H-NMR spectra: s: singlet, d: doublet, t: triplet, dd: doublet of doublets; dt: doublet of triplets; m: multiplet, br: broad. High resolution electrospray ionization mass spectrometry (ESI HRMS) was performed on Bruker Apex IV FTICR, Bruker compact and Bruker timsTOF ESI mass spectrometers.
DMT-protected glycidol 2 DMT-protected glycidol 2 was synthesized enantiomerically pure according to a modified literature procedure. 1 To a solution of (R or S)-glycidol (1.0 mL, 15.1 mmol, 1 equiv.) and Et3N (5.4 mL, 40.7 mmol, 2.7 equiv.) in CH2Cl2 (34 mL) DMT-Cl (4,4ʹ-Dimethoxytrityl chloride; 6.45 g, 19 mmol, 1.26 equiv.) was added and the mixture stirred at 20 °C for 16 h. The reaction mixture was washed with half saturated aq. NaHCO3 (50 mL) and extracted with CH2Cl2 (2 × 30 mL). The combined organic layers were dried over MgSO4 and the solvent was removed under reduced pressure to afford a dark red oil. The product was purified by column chromatography (n-pentane/EtOAc, 98/2 → n-pentane/EtOAc, 90/10) to afford the DMT-protected glycidol 2 as a viscous colourless oil. 2 Solid phase DNA synthesis Solid phase DNA synthesis was carried out on a K&A Laborgeräte GbR H-8 synthesizer using the standard phosphoramidite method on a 1 µmol scale. Cartridges (Biosearch Technologies or Link Technologies) for solid phase synthesis were self-packed with CPG (1000 Å, 33 µmol/g) purchased from Sigma Aldrich. First, the cartridge was treated three times with 3 % DCA (dichloroacetic acid in CH2Cl2). Second, the coupling step was performed by mixing activator (5-(benzylthio)-1H-tetrazole, BTT) and phosphoramidite in a 1:1 ratio. The incubation time for the standard canonical nucleotides was 0.5 min whereas for artificial nucleotides the coupling time was extended to 3.5 min. Next, unreacted 5'-OH groups were acetylated with a 1:1 mixture of capping solutions Cap A and Cap B followed by a final oxidation step (see table 1). After each step, the cartridge was washed with acetonitrile followed by a drying step using argon gas. The described cycle was repeated for every incorporated base.

G5L
LGG GGG 4 °C at 0.5 °C min -1 . Tetramolecular samples were then frozen at -20 °C for one hour to ensure full G-quadruplex formation. [31] Thermal difference spectra (TDS) were recorded from 220 to 350 nm by subtracting the low temperature spectrum (4 °C) from the high temperature spectrum (85 °C). The absorption at 350 nm was then set to 0.
Thermal denaturation experiments were carried out to determine the thermal stability expressed in the melting temperature Tm. Therefore, the change in absorbance, as the temperature was increased with 0.5 °C min -1 , was followed at 295 nm indicative for G-quadruplex denaturation and at 350 nm as control. Note that for unimolecular G-quadruplexes at every temperature an equilibrium between folded and unfolded species is given. This is not true for tetramolecular G-quadruplexes which is the reason why the melting temperature for tetramolecular G-quadruplexes depends on the heating rate. The spectrometer bandwidth was set to 2 nm. To control the temperature, a cuvette containing water was equipped with a temperature probe connected to the spectrometer. Water evaporation from the samples was avoided by addition of small amounts of silicon oil. To ensure equal heating rates between different experiments, always five samples were measured at a time. The measured absorbance was plotted against the temperature after the absorbance at 350 nm was subtracted to remove background absorption. Spectra were normalized to fraction folded values between 1 and 0 corresponding to fully folded or unfolded G-quadruplexes, respectively.

Circular dichroism (CD) measurements
CD measurements were carried out on a Chirascan qCD spectrometer in black quartz cuvettes. Samples were prepared as described under UV/VIS spectroscopy. Temperature was controlled using a Quantum Northwest temperature control attached to a sample probe. Spectra were recorded from 205 to 350 nm (120 nm min -1 ) with a 1 nm interval and 0.5 nm bandwidth three times and averaged using the built-in software. The averaged spectra were smoothed (adjacent averaging) with a factor of 5 and the smoothed background (sample containing only buffer, smooth factor 10) was subtracted. The background was recorded once from 205 to 350 nm with the same settings as for the samples in the same cuvette.                          a stepwise reduction of the electric field strength leads to a release of ion packages separated by their mobility. After a subsequent focussing, the separated ions are transferred to the TOF-analyser. [28][29][30] The ion mobility K was directly calculated from the trapping electric field strength E and the velocity of the carrier gas stream to obtain the reduced mobility K0, where P is the pressure and T is the temperature. By using the Mason-Schamp equation, the collisional cross-section Ω can be calculated: where ze is the ion charge, kB is the Boltzmann constant, µ is the reduced mass of analyte and carrier gas and N0 is the number density of the neutral gas. [28][29][30] For calibration of both the TIMS and TOF analysers, commercially available Agilent ESI tuning mix was used. The instrument was calibrated before each measurement, including each change in the ion mobility resolution mode ("imeX" settings: survey, detect or ultra).

MD simulations
Simulations were performed using GROMACS 2016.1 and GROMACS 2019.2. [3][4][5][6][7][8][9] The AMBER force field ff99bsc1 was modified with new force field parameters regarding the imidazole ligand according to a previously published protocol. [10][11][12][13][14] Missing forcefield parameters for the imidazole ligand were based on analogy to existing force field parameters. For both enantiomers, the same set of parameters was used. Parameters regarding the Cu(II) and Zn(II) complex were derived using VFFDT (Visual Force Field Derivation Toolkit) with a Gaussian '09 geometry optimized structure (B3LYP/6-311+G(d,p)). [10,[15][16][17][18] To obtain a square-planar and tetrahedral coordination environment for Cu(II) and Zn(II), respectively, adjacent ligands were named differently, to avoid ambiguity in the parameterization. Improper dihedrals were used to maintain coplanarity between the imidazole plane and the imidazole -metal plane. The respective parameters for Cu(II) and Zn(II) were estimated based on literature values (Table 5). [17,18] As starting structure, the crystal structure of a tetramolecular parallel stranded (pdb entry: 2O4F) and the NMR solution structure of an unimolecular antiparallel (143D, 1C35) G-quadruplex were used and modified. [19,20] Obtained starting structures where solvated in a rhombic bounding box with TIP3P water. Negative charges on the phosphates were neutralized with Na + (tetramolecular) or K + (unimolecular) cations and the concentration of Na/KCl was set to 100 mM. Prior to the MD simulation, the starting structure was subjected to three rounds of energy minimization and equilibrated at 298 K and 1 bar.
The final MD was simulated for 100 ns in an NPT ensemble. For details see a previous publication. [10] Partial charges (RESP) Partial point charges were derived by RESP charge fitting. Therefore the imidazole ligands and the imidazole Cu(II) complex were fragmented according to the original capping scheme, structure optimized (B3LYP/6-311+G(d,p)) and submitted to the REDServer-Development (Table 4). Inter-molecular and intra-molecular charge constraints were used to maintain the correct total charge of the ligand. In all cases, the constrained charges were similar to the unconstrained values. [21][22][23][24] For the Zn(II) complex a different procedure was applied. The complex was structure optimized (HF/6-31+G*) and used as input for antechamber to calculate the RESP charges. 25