Enantiospecific formation of a metal-mediated base pair inside a DNA duplex

Accepted Manuscript Enantiospecific formation of a metal-mediated base pair inside a DNA duplex Biswarup Jash, Johannes Neugebauer, Jens Müller PII: DOI: Reference:

S0020-1693(16)30028-7 http://dx.doi.org/10.1016/j.ica.2016.02.012 ICA 16882

To appear in:

Inorganica Chimica Acta

Received Date: Revised Date: Accepted Date:

30 November 2015 19 January 2016 2 February 2016

Please cite this article as: B. Jash, J. Neugebauer, J. Müller, Enantiospecific formation of a metal-mediated base pair inside a DNA duplex, Inorganica Chimica Acta (2016), doi: http://dx.doi.org/10.1016/j.ica.2016.02.012

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Enantiospecific formation of a metal-mediated base pair inside a DNA duplex Biswarup Jash,a Johannes Neugebauer,b Jens Müller*,a a) Westfälische Wilhelms-Universität Münster, Institut für Anorganische und Analytische Chemie and NRW Graduate School of Chemistry, Corrensstraße 28/30, 48149 Münster, Germany, fax: +49 251 8336007, e-mail: [email protected] b) Westfälische Wilhelms-Universität Münster, Organisch-Chemisches Institut and Center for Multiscale Theory and Computation, Corrensstraße 40, 48149 Münster, Germany

ABSTRACT The metal-mediated base pair P–Ag(I)–P involving the bidentate ligand 1H-imidazo[4,5-f][1,10]phenanthroline (P) was investigated within a B-DNA duplex. The underlying tetrahedral complex exists as a pair of enantiomers. Interestingly, one of the isomers forms preferentially within the B-DNA context, as was shown in a combined computational and experimental approach by means of circular dichroism spectroscopy. Towards this end, the homoleptic Ag(I) complexes of 1-methyl-1H-imidazo[4,5-f][1,10]phenanthroline and both enantiomers of 3(1H-imidazo[4,5-f][1,10]phenanthrolin-1-yl)propane-1,2-diol were investigated outside the DNA context as well as the P–Ag(I)–P base pair within the DNA context. In this paper, we also propose a simplified nomenclature for the enantiomers of undistorted tetrahedral complexes bearing asymmetric bidentate ligands.

Keywords: bioinorganic chemistry, nucleic acids, chirality, silver

1

1

Introduction

Nucleic acids provide a plethora of binding sites for metal ions [1]. To be nevertheless able to exploit the superb self-assembly properties of nucleic acids for a precise arrangement of specific metal ions, so-called metal-mediated base pairs have been developed [2-6]. These base pairs rely on the use of mostly artificial nucleobases with a high affinity towards transition metal ions, so that duplexes with pre-designed metalbinding sites can be devised. Hence, in metal-mediated base pairs the hydrogen bonds found in canonical base pairs are formally replaced by coordinate bonds. The resulting metal-modified DNA is of superior thermal [7] and mechanical [8, 9] stability. In some cases, even two metal ions can be introduced per base pair [10-13]. Several X-ray structures and NMR structures confirm the preferred localization of the associated metal ions inside the duplex, as exemplified for DNA [14-18], RNA [19], and GNA [20] (GNA = glycol nucleic acid). Nucleic acids with metal-mediated base pairs have found application as metal-ion sensors [21], in expanding the genetic alphabet [18], in the recognition of nucleic acid sequences [22], and in increasing the charge transfer capabilities of DNA [23, 24]. We recently introduced the bidentate ligand 1H-imidazo[4,5-f][1,10]phenanthroline (imphen, P, Scheme 1a) as an artificial GNA-based nucleoside into metal-mediated base pairing [25], using a monodentate imidazole nucleoside [26] as the complementary nucleoside.

+

Scheme 1. Structural representation of a) imphen and b) the two enantiomeric [Ag(imphen)2] complexes (only the σ bonds are shown, hydrogen atoms omitted for clarity).

This publication reports the introduction of the imphen-containing metal-mediated homo base pair P–Ag(I)–P into a nucleic acid. It is a general presumption that metal-mediated base pairs should ideally be planar to fit into the base pair stack [27], even though this is apparently not strictly required in duplexes comprising metalmediated base pairs only [10, 28]. The P–Ag(I)–P pair needs to adopt a tetrahedrally distorted coordination environment around the Ag(I) ion to avoid a steric clash of the H2 and H9 hydrogen atoms of the participating

2

1,10-phenanthroline moieties (Scheme 1b). The (distorted) tetrahedral geometry of the metal-mediated base pair raises a fundamental question that is as yet unexplored: As homoleptic tetrahedral metal complexes of asymmetrically substituted bidentate ligands exist as pairs of enantiomers, is there an enantiospecific formation of the corresponding metal-mediated base pair, induced by the inherent chirality of a DNA duplex? To

answer

this

question,

we

investigated

the

Ag(I)

complexes

of

1-methyl-1H-imidazo[4,5-

f][1,10]phenanthroline (1) and the chiral GNA building blocks 3-(1H-imidazo[4,5-f][1,10]phenanthrolin-1yl)propane-1,2-diol ((S)-2 and (R)-2)) (Scheme 2) as well as the metal-mediated base pairs P–Ag(I)–P inside DNA duplexes with either (S,S)- or (R,R)-configured GNA nucleosides. Compound 1 thereby acts as a model nucleobase, in which the glycosidic bond is formally replaced by the bond to a methyl substituent. The use of model nucleobases has proven highly valuable in evaluating possible structures of metal-mediated base pairs [29-31].

Scheme 2. Bidentate 1H-imidazo[4,5-f][1,10]phenanthroline-derived ligands investigated in this study.

2

Experimental Section

2.1

Measurements and Computations

DNA syntheses were performed in the DMT-off mode on a K&A Laborgeräte H8 DNA/RNA synthesizer following standard protocols (except for a threefold coupling time for the artificial phosphoramidite). Workup of the oligonucleotides was performed as recently reported [25]. The desalted oligonucleotides were identified by MALDI-ToF mass spectrometry (Table S1, Figures S6–S9, Supporting Information). MALDI-ToF mass spectra were recorded on a Bruker Reflex IV instrument using a 3-hydroxypicolinic acid/ammonium citrate matrix. The specific rotation was measured on a Perkin Elmer 341 polarimeter. For quantification of the oligonucleotides, a 2

–1

1

13

31

molar extinction coefficient ε260 = 10.0 cm mmol was used for P. H, C and P NMR spectra were recorded in CD2Cl2 on Bruker Avance (I) 400 and Avance (III) 400 spectrometers and are referenced relative to TMS and H3PO4, respectively (δ / ppm = 0). UV/Vis spectra were recorded on a CARY BIO 100 spectrophotometer.

3

Temperature-dependent UV spectra were recorded between 10 °C and 70 °C with a heating / cooling rate of 1 –1

°C min and a data interval of 1 °C. Absorbance was normalized according to Anorm = (A – Amin)/(Amax – Amin) at 260 nm. Melting temperatures have been determined as the maximum of the derivative of the annealing curves. CD spectra were recorded at 10 °C on a Jasco J-815 spectrometer. The spectra were smoothed, and a manual baseline correction was applied. Oligonucleotide solutions for the spectroscopic characterization contained 1 µM oligonucleotide duplex, 150 mM NaClO4, and 5 mM MOPS buffer (pH 6.8). For quantum chemical calculations, we employed the program package Turbomole [32, 33]. Structure optimizations were carried out using the TPSS functional [34], applying the D3-dispersion correction with Becke-Johnson damping [35, 36], dubbed TPSS-D3(BJ), with Ahlrichs’ def2-TZVP basis set [37] and a Stuttgart effective-core potential for Ag [38]. We confirmed that the optimized structures are minima on the potentialenergy surface by means of a frequency analysis with the program SNF [39] of the MoViPac suite [40]. Excitation energies and rotatory strengths have been calculated using the Amsterdam Density Functional (ADF) package [41, 42], with the range-separated CAMY-B3LYP functional [43] and a TZP basis from the ADF basis set library. Test calculations with the larger TZ2P basis set did not lead to significant changes in the results. In the ADF calculations, relativistic effects have been accounted for in terms of the zeroth-order regular approximation (ZORA) [44]. For comparison, we also calculated CD spectra with Becke’s three-parameter hybrid functional B3LYP [45-48] and the def2-TZVP basis using Turbomole. It has been observed that global hybrids like B3LYP may outperform range-separated hybrids in CD spectra calculations for transition metal complexes with bidentate nitrogen-containing ligands [49], although less accurate results may be obtained in case of simple DNA duplexes [50]. For the final spectra simulations, a Gaussian broadening with a half-width of 0.3 eV was applied to obtain the difference in extinction as a function of photon energy, following the procedure in reference [51]. The photon energy axis has been converted to a wavelength scale for consistency with experiment.

2.2

Syntheses

1H-Imidazo[4,5-f][1,10]phenanthroline, the DMT-protected (S)- and (R)-glycidol, and compounds (S)-3 and (S)-4 were synthesized according to published procedures [52-54]. Synthesis of (R)-1-(4,4’-dimethoxytrityl)-3-(1H-imidazo[4,5-f][1,10]phenanthrolin-1-yl)propan-2-ol (R)-3. 1HImidazo[4,5-f][1,10]phenanthroline (757 mg, 3.44 mmol) and NaH (60% in mineral oil, 34 mg, 0.85 mmol) were

4

suspended in dry DMF (10 mL). After stirring for 1 h at 0 °C, (R)-glycidyl 4,4’-dimethoxytrityl ether (1.941 g, 5.156 mmol) in dry DMF (5 mL) was added. The resulting mixture was stirred for 18 h at 110 °C. After removing the solvent in vacuo, the oily mass was treated with ethyl acetate, and the precipitate was filtered off. After removal of the solvent, (R)-3 was purified by column chromatography (SiO2, CH2Cl2/MeOH/Et3N (10/1/0.01)) 1

and was obtained as an off-white foam (1.82 g, 3.04 3mmol, 89%). H NMR (400 MHz, CD2Cl2): δ = 8.57–8.51 (m, 2H, H2, H9), 8.48–8.35 (m, 2H, H4, H7), 7.80 (s, 1H, H2imi), 7.66–7.61 (m, 2H, DMT), 7.53–7.46 (m, 4H, DMT), 7.42 (dd, J = 8.4 Hz, 6.9 Hz, 2H, DMT), 7.36–7.29 (m, 2H, H3, DMT), 7.23 (dd, J = 8.3 Hz, 4.3 Hz, 1H, H8), 6.99– 6.93 (m, 4H, DMT), 5.08–4.83 (m, 2H, H3GNA), 4.30–4.11 (m, 1H, H2GNA), 3.82 (s, 6H, 2 × CH3), 3.78–3.69 (m, 1H, H1GNA), 3.54–3.37 (m, 1H, H1GNA) ppm.

13

C NMR (101 MHz, CD2Cl2): δ = 159.2 (DMT), 148.2 (C2), 147.1 (C9),

145.5 (C1a), 144.7 (DMT), 143.2 (C10a), 142.2 (C2imi), 142.0 (C5), 137.0 (DMT), 136.2 (DMT), 130.4 (DMT), 128.8 (DMT), 128.4 (C4), 127.4 (C7), 123.7 (DMT), 123.3 (DMT), 122.4 (C4a), 119.7 (C3), 119.4 (C6a), 113.7 (C6), 113.5 +

(C8), 113.4 (DMT), 87.2 (DMT), 67.8 (C2GNA), 66.2 (C1GNA), 55.7 (2 × CH3), 54.2 (C3GNA) ppm. HRMS: m/z [M+H] calcd. for C37H33N4O4: 597.2502, found: 597.2504. Synthesis

of

(R)-1-(4,4’-dimethoxytrityl)-3-(1H-imidazo[4,5-f][1,10]phenanthrolin-1-yl)propan-2-yl

(2-

cyanoethyl) diisopropylphosphoramidite (R)-4. To a solution of (R)-3 (200 mg, 0.335 mmol) and diisopropyethylamine (292 µL, 1.68 mmol) in dry CH2Cl2 (10 mL), N,N-diisopropylchlorophosphoramidite (149 µL, 0.670 mmol) was added. After 1 h, the reaction mixture was poured into saturated aqueous NaHCO3 solution and extracted with DCM (2 × 25 mL). The organic layer was dried (MgSO4) and evaporated. The residue was purified by chromatography (SiO2, CH2Cl2/MeOH/Et3N (12/1/0.1)), affording (R)-4 as a mixture of diastereomers as a colorless oil (140 mg, 0.176 mmol, 52%). 1H NMR (400 MHz, CD2Cl2): δ = 9.12–9.03 (m, 2H, H2, H9), 8.98–8.88 (m, 2H, H4, H7), 7.98 (s, 0.5H, H2imi), 7.94 (s, 0.5H, H2imi), 7.77–7.62 (m, 2H, H3, H8), 7.57– 7.50 (m, 2H, DMT), 7.48–7.27 (m, 7H, DMT), 6.92–6.82 (m, 4H, DMT), 5.21–5.01 (m, 1H, H3GNA), 4.69–4.36 (m, i

2H, H3GNA, H2GNA), 4.60–4.45 (m, 2H, OCH2), 3.79 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.58–3.27 (m, 4H, 2 × Pr-CH, i

31

H1GNA), 2.29–2.14 (m, 2H, CH2CN), 1.01–0.77 (m, 12H, 4 × Pr-CH3) ppm. P NMR (81 MHz, CD2Cl2): δ = 150.2, +

149.8 ppm. HRMS: m/z [M+H] calcd. for C46H50N6O5P: 797.3580, found: 797.3555.

Synthesis of (S)-3-(1H-imidazo[4,5-f][1,10]phenanthrolin-1-yl)propan-2-ol (S)-2. To a solution of (S)-3 (110 mg, 0.184 mmol) in CH2Cl2 at room temperature, 6 mL of TCA solution (3% trichloroacetic acid in CH2Cl2) were added in three intervals and stirred for 30 min. After reducing the solvent in vacuo, the product was extracted

5

with water and subsequently neutralized by NaOH. The solution was evaporated to dryness and the product (S)-2 obtained as off-white solid (43 mg, 0.145 mmol, 79%). (R)-2 was obtained analogously from (R)-3. 1

Characterization of (R)-2. H NMR (400 MHz, D2O, pD 6.7): δ = 8.26–8.06 (dd, 2H), 7.39 (d, 2H), 7.15 (s, 1H), +

7.11–6.92 (m, 2H), 3.83 (m, 2H) 3.16 (q, 1H), 1.24 (t, 2H) ppm. HRMS: m/z [M+H] calcd. for C16H15N4O2: 1

295.1195, found: 295.1206. Characterization of (S)-2. H NMR (400 MHz, D2O, pD 6.9): δ = 8.20–8.09 (dd, 2H), 7.37 (d, 2H), 7.15 (s, 1H), 7.00 (dd, 2H), 3.84 (m, 2H), 3.22 (q, 1H), 1.30 (t, 2H). HRMS: m/z [M+H]+ calcd. for C16H15N4O2: 295.1195, found: 295.1207.

3

Results and Discussion

3.1

Characterization of the Chiral Tetrahedral Complexes +

The molecular structures of the enantiomeric tetrahedral metal complexes [Ag(1)2] have been computed using TPSS-D3(BJ)/def2-TZVP (Figure 1). An asymmetric substitution pattern of the ligand was obtained by using the N-methylated derivative 1 in the computations. To date, no simple nomenclature exists to distinguish the enantiomers of undistorted tetrahedral complexes bearing asymmetric bidentate ligands. The R/S nomenclature based on the Cahn-Ingold-Prelog priority rules is not very meaningful here, because in the case of [Ag(1)2]+ the priority is determined by the identity of an atom at distance six from the stereocenter. Hence, +

we suggest a simplified nomenclature for [Ag(1)2] and related complexes, using the affixes ∆ and Λ well-know from chiral octahedral metal complexes. As no helical chirality exists in undistorted tetrahedral complexes bearing asymmetric bidentate ligands, a definition of ∆ and Λ different from the general skew-lines convention [55] needs to be applied. We suggest that one of the (planar) ligands be oriented vertically with the substituent giving rise to the asymmetry of the ligand being located in the back pointing upwards (if more than one substituent is present, the one with the highest priority points upwards). In an undistorted tetrahedral complex, the other ligand is now oriented horizontally. In order to superimpose the substituents on the different ligands, the ligand in the back needs to be rotated by 90° either to the right or to the left (Figure 1). The former complex is the ∆-isomer, the latter the Λ-isomer. For the complex under investigation here, ∆+

+

[Ag(1)2] corresponds to (S)-[Ag(1)2] .

6

Figure 1. Geometry-optimized molecular structures of ∆-[Ag(1)2]+ and Λ-[Ag(1)2]+.

To investigate the CD spectroscopic features of the ∆- and Λ-enantiomers, we prepared the ligand 3-(1Himidazo[4,5-f][1,10]phenanthrolin-1-yl)propane-1,2-diol 2. Both (R)-2 and (S)-2 were obtained in a regioselective and stereospecific fashion by a ring-opening reaction starting from (S)- and (R)-glycidol, respectively. These ligands represent the GNA-based nucleosides of the imphen ligand. Because the ligands contain a stereocenter at their alkyl substituent, the ∆- and Λ-isomers of their metal complexes are no longer enantiomers but diastereomers. As a result, a diastereoselective formation of one of the isomers can be anticipated. In fact, the preferred formation of one diastereomer has previously been observed for octahedral complexes bearing an asymmetrically substituted chiral 2,2’-bipyridine ligand [56, 57]. Figure 2 shows the CD +

+

spectra of [Ag{(R)-2}2] and [Ag{(S)-2}2] . As can be seen, the spectra are mirror images of each other, clearly indicating the preferred formation of opposite diastereomers, depending on the chirality of the ligand. As the propane-1,2-diol moiety does not show any absorbance at the investigated wavelengths, the CD signals must be due to electronic transitions of the (achiral) imphen moieties, hence must result from the metal-centered chirality of the complex. Three distinct Cotton effect are observed at 248, 274 and 292 nm.

7

Figure 2. CD spectra of [Ag{(R)-2}2]+ (dotted line) and [Ag{(S)-2}2]+ (solid line).

To assign the experimental CD spectra to the respective isomers of the metal complexes, CD spectra were +

+

calculated for complexes ∆-[Ag(1)2] and Λ-[Ag(1)2] . The calculations were carried out independently as a cross-check of our methodology. Since the resulting spectra are, within a small numerical error, mirror images of each other, we only discuss the results of the ∆-isomer. To assess the accuracy of the vertical excitation energies underlying the spectra, we first compared the main peaks in the corresponding UV absorption spectra. In the experimental spectrum, two peak maxima occur at 277 and 244 nm (Figure S1, Supporting Information). This is well reproduced by B3LYP/def2-TZVP calculations (highest oscillator strengths for transitions at 287 and 248 nm), whereas the CAMY-B3LYP/TZP calculations yield slightly blue-shifted results (269 and 236 nm). Keeping this blue-shift in mind, we can assign the negative band with minimum at 237 nm in the simulated +

CAMY-B3LYP/TZP circular dichroism spectrum of ∆-[Ag(1)2] (Figure 3) to the negative band at 248 nm in the +

experimental spectrum of [Ag{(S)-2}2] , and the positive band at 274 nm to the experimental band at 292 nm. The corresponding peaks in the simulated B3LYP/def2-TZVP spectra for ∆-[Ag(1)2]+ occur at 245 nm (negative) and 297 nm (positive), which corroborates our assignment. The region between these two peaks is hard to interpret in the simulated spectra, since several positive and negative bands are superimposed. However, we note that both the CAMY-B3LYP/TZP and the B3LYP/def2-TZVP spectra show another maximum, followed by a minimum on the long-wavelength side, in this intermediate regime. Again, this is qualitatively in line with the +

experimental spectrum for [Ag{(S)-2}2] . The agreement between experimental and computed CD spectra leads +

to the conclusion that the diastereomeric mixture of [Ag{(R)-2}2] contains an excess of the Λ-isomer, whereas

8

+

the diastereomeric mixture of [Ag{(S)-2}2] contains excess ∆-isomer. Hence, the configuration of the stereocenter of the GNA-based nucleoside analogue has a direct influence on the preferred chirality of the metal complex.

Figure 3. Simulated CD spectra of Λ-[Ag(1)2]+ (dotted line) and ∆-[Ag(1)2]+ (solid line) from CAMY-B3LYP/TZP (left) and B3LYP/def2-TZVP (right) calculations.

3.2

Investigation of the Ag(I)-Mediated Base Pairs

To incorporate an imphen-derived artificial nucleoside into a DNA oligonucleotide, the (R)- and (S)-configured GNA building blocks were synthesized as shown in Scheme 3 [25]. Addition of (R)-glycidyl 4,4’-dimethoxytrityl ether to a solution of in situ deprotonated imphen gave the ring-opened product (R)-3 in a stereospecific manner. The next step comprised the conversion to the phosphoramidite (R)-4 by means of 2-cyanoethyl-N,Ndiisopropylchlorophosphoramidite. Compound (S)-4 was obtained from (S)-glycidyl 4,4’-dimethoxytrityl ether in an analogous fashion. Chiral HPLC was used to confirm the enantiomeric purity of the compounds (Figure S2, Supporting Information).

9

Scheme 3. Synthesis of the phosphoramidite building block 4 required for automated solid-phase DNA synthesis. Shown is the route from DMT-protected (R)-glycidol leading to the (R)-phosphoramidite. a) NaH, DMF; b) CEDIP-Cl, DIPEA, CH2Cl2 (CEDIP = cyanoethyl-N,N-diisopropyl phosphoramidite, DIPEA = N,Ndiisopropylethylamine, DMT = 4,4’-dimethoxytrityl).

The sequence of the DNA duplex containing one central P:P mispair (designed to form a P–Ag(I)–P metalmediated base pair) is shown in Scheme 4. It comprises canonical 2’-deoxyribonucleotides and the GNA-based imphen nucleoside P. The sequence was chosen because it had been shown to form comparatively stable metal-mediated base pairs compared with related sequences [25]. As (S)- and (R)-configured GNA nucleoside analogues are both capable of forming base pairs [54, 58], both an (S,S)-DNA duplex and an (R,R)-DNA duplex were synthesized. In this notation, the prefixes indicate the configuration of the stereocenters of the imphencontaining GNA building blocks.

Scheme 4. Sequence of the DNA duplexes under investigation (P = GNA-based imphen nucleoside).

To characterize the Ag(I)-binding behavior of the DNA duplexes, their melting temperatures Tm were determined in the presence of increasing amounts of Ag(I). Figure 4 shows the melting curves of the (R,R)-DNA duplex, those of the (S,S)-duplex are given in Figure S3 (Supporting Information). As can be seen, the addition of one equivalent of Ag(I) leads to a significant thermal stabilization of the duplex. Excess Ag(I) does not influence the melting temperature any further, confirming a 1:1 binding stoichiometry between duplex and metal ion and thereby clearly indicating the formation of a metal-mediated base pair [2]. It is interesting to note that substoichiometric amounts of Ag(I) lead to a biphasic melting behavior of the duplex. This

10

observation is not unprecedented [59, 60] and suggests that the Ag(I)-mediated base pair is kinetically inert, meaning that once formed it does not dissociate on the time scale of the temperature-dependent UV measurements.

Figure 4. Melting curves of (R,R)-DNA duplex in the presence of increasing amounts of Ag(I) (0; 0.25; 0.5; 0.75; 1; 1.5; 2 equiv.). The inset shows how Tm depends on the equivalents of Ag(I).

Table 1 summarizes the melting temperatures Tm and the increase in thermal stability ∆Tm. The melting temperature in the absence of Ag(I) amounts to 36 °C for both duplexes, indicating that the (R,R)-configured P:P mispair has the same net effect on the duplex stability as the (S,S)-configured P:P mispair. A comparison with closely related natural duplexes comprising a central A:T or G:C base pair shows that the P:P mispair significantly destabilizes the duplex (Tm = 47 °C for the A:T-containing and 53 °C for the G:C-containing duplex). Upon formation of the Ag(I)-mediated base pair, Tm increases much more significantly for the (R,R)-DNA, suggesting that the P–Ag(I)–P complex formed from the two (R)-configured artificial nucleosides fits better into the DNA duplex than the corresponding complex formed from the two (S)-configured nucleoside analogues. The duplexes containing a canonical base pair in the center rather than an P:P mispair do not show a significant change of Tm upon the addition of Ag(I) (Figure S4, Supporting Information), thereby confirming that the large increase in Tm observed for the P:P-containing duplexes is indeed due to the formation of P–Ag(I)–P base pairs.

11

Table 1. Melting temperature Tm / °C and increase in thermal stability ∆Tm / °C for both duplexes.

0 equiv. Ag(I)

1 equiv. Ag(I)

2 equiv. Ag(I)

∆Tm (0→1 equiv. Ag(I))

(R,R)-DNA

36

59

59

23

(S,S)-DNA

36

52

53

16

CD spectroscopy was applied to characterize the Ag(I)-binding behavior of the DNA duplexes further. The CD spectra of the (S,S)-DNA duplex and the (R,R)-DNA duplex in the presence of increasing amounts of Ag(I) are displayed in Figure 5. Several interesting observations can be made. 1) In the absence of any Ag(I), the CD spectra of both duplexes look very much alike, with a positive Cotton effect around 275 nm and a negative one around 247 nm. The spectra resemble that of regular B-DNA [61], indicating that the incorporation of the GNAbased P:P mispair does not induce a major conformational change to the duplex. 2) Upon formation of the P– Ag(I)–P base pair, a new negative Cotton effect appears at ∼294 nm for both duplexes. 3) In the case of the (S,S)-DNA duplex, an additional positive Cotton effect evolves at 258 nm. For the (R,R)-DNA duplex, this effect is less obvious but still discernible.

Figure 5. CD spectra of a) (S,S)-DNA duplex and b) (R,R)-DNA duplex in the presence of increasing amounts of Ag(I) (0; 0.5; 1; 2 equiv.). Major changes in the molar ellipticity as discussed in the text are indicated by arrows.

A comparison with the CD spectra of related canonical duplexes without a central P:P mispair (Figure S5, Supporting Information) provides proof that the new Cotton effects are the result of the formation of a P– Ag(I)–P base pair rather than of unspecific binding. Taken together, these data can be explained as follows: On first sight, the change of the sign of the molar ellipticity at around 290 nm upon the addition of Ag(I) could be

12

attributed to a B- to Z-DNA conformational transition. This would mean that the duplex becomes left-handed after the formation of the central P–Ag(I)–P base pair, as had for example been reported for another metalmediated base pair [62]. However, a transition to a Z-DNA duplex can be ruled out upon further inspection, particularly because an intense negative Cotton effect remains at ∼250 nm. For Z-DNA, a positive molar ellipticity would be expected from about 220 – 260 nm, because a change from a right-handed to a left-handed double helix inverts its chirality and hence alters the sign of all Cotton effects [61]. Alternatively, it could be assumed that the formation of the non-planar P–Ag(I)–P perturbs the helical structure of the adjacent canonical base pairs. For example, a study on the formation of the P–Ag(I)–Im base pair (with Im = imidazole 2’deoxyribonucleoside) shows that the metal-mediated base pair formation can indeed influence the geometry of the neighboring canonical base pairs [25]. However, that study also showed that a stabilizing metalmediated base pair hardly distorts the B-DNA duplex, whereas a significant distortion is accompanied by a lack of thermal stabilization. Hence, as the P–Ag(I)–P base pair strongly stabilizes the DNA duplex, a significant perturbation of the helical structure is rather unlikely. Finally, a third explanation is based on a comparison +

with the CD spectra of [Ag(2)2] (Figure 2). Obviously, the newly appearing negative Cotton effect at ∼294 nm in +

the CD spectra of the DNA perfectly coincides with the one observed for [Ag{(R)-2}2] at 292 nm. Moreover, the evolution of a positive Cotton effect at 258 nm for the DNA is in good agreement with the positive Cotton effect at 248 nm in the CD spectrum of [Ag{(R)-2}2]+. Hence, the spectral changes indicate that the P–Ag(I)–P base pair adopts a Λ-configuration in both DNA duplexes, i.e. irrespective of the configuration of the GNA stereocenter. It appears that the helical structure of the duplex accommodates the Λ-isomer better than the ∆isomer. A similar phenomenon has previously been established for the intercalation of Λ- and ∆-configured octahedral complexes into duplex DNA, where the ∆-enantiomer was found to preferentially bind to righthanded B-DNA [63-65]. Moreover, short oligonucleotides comprising artificial ligand-based nucleosides only were shown to incorporate Fe(III) to form octahedral complexes in an enantiospecific manner, giving rise to the formation of triple-stranded helicates [66]. Remarkably, the larger thermal stabilization of the (R,R)-DNA duplex upon formation of the P–Ag(I)–P base pair (Table 1) coincides with the preferred formation of the Λisomer by the (R)-configured GNA nucleoside 2 (Figure 2). Conversely, it can be concluded that because the preference of the (S)-configured GNA nucleoside to form the ∆-isomer is overruled by the geometric requirements of the DNA duplex, the resulting (S,S)-DNA duplex with an P–Ag(I)–P base pair is thermally less stable than its (R,R)-DNA counterpart. In principle, the different stability of the (S,S)- and (R,R)-DNA duplexes

13

could also be simply due to the differing geometry of the GNA building blocks. This hypothesis could be evaluated based on the stability of DNA duplexes with a single GNA-based Watson-Crick base pair. Unfortunately, no such study exits. However, a detailed study involving a closely related nucleoside analogue (amino propyl nucleic acid (APNA), formal replacement of the primary OH group of the GNA nucleoside by an NH2 group) within a DNA duplex shows a rather small influence of the chirality [67]. If one nucleoside of an A:T base pair is replaced by an APNA nucleoside, the duplex stability in the presence of the (S)-APNA vs. (R)-APNA nucleoside differs by only 0.0 to 1.2 °C, depending on the oligonucleotide sequence. If both nucleosides of the A:T base pair are replaced by APNA nucleosides, the melting temperature of the DNA duplex comprising the (S,S)-configured base pair is more stable by 4.5 – 5.4 °C than that of the double helix with an (R,R)-configured base pair. It can be expected that a similar trend exists for DNA duplexes comprising a single GNA base pair. Notably, the differential stabilization observed for the APNA-containing duplexes is smaller than the one observed for the P–Ag(I)–P base pair (∆Tm between (S,S) and (R,R) = 7 °C, Table 1). Moreover, the latter shows the exact opposite trend, favoring the (R,R)-configured base pair. Hence, taken together with the fact that the P:P-containing duplexes (i.e. in the absence of Ag(I)) show that same melting temperature irrespective of the geometry of the GNA nucleoside, this indicates that the different stabilization of the duplexes with the (S,S)and (R,R)-configured P–Ag(I)–P base pair originates from the chirality of the metal complex rather than from the geometry of the GNA stereocenter.

4

Conclusions

Several metal-mediated base pairs have been reported in which two bidentate ligands bind to a central metal ion in a tetrahedrally distorted fashion, both by other groups [68-72] and by us [59, 60]. As asymmetrically substituted bidentate ligands form enantiomeric tetrahedral complexes, it appeared obvious to study whether the chirality of the DNA duplex induces the enantiospecific formation of such a metal-mediated base pair. We were able to investigate this enantiospecificity in a combined computational and experimental approach by using the imphen-containing P–Ag(I)–P base pair. By applying CD spectroscopy we could clearly demonstrate that the Λ-configured P–Ag(I)–P base pair is formed preferentially within a B-DNA context. We predict that all other non-planar metal-mediated base pairs comprising two bidentate ligands exhibit a related preference for one enantiomer of the chiral metal complex.

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5

Acknowledgement

Financial support by the DFG (SFB 858) and the NRW Graduate School of Chemistry is gratefully acknowledged.

6

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A chiral Ag(I)-mediated base pair involving achiral phenanthroline-derived nucleobases is formed enantiospecifically within a B-DNA duplex.

•

A novel Ag(I)-mediated base pair with an artificial phenanthroline-derived nucleobase is reported.

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The B-DNA duplex induces an enantiospecific formation of the chiral Ag(I)-mediated base pair.

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A simplified nomenclature is suggested for chiral tetrahedral complexes with achiral bidentate ligands.

20