Automated Synthesis of 3‘-Metalated Oligonucleotides

We report the first synthesis of a metallonucleoside bound to a solid support and subsequent oligonucleotide synthesis with this precursor. Large-scale syntheses of metal-containing oligonucleotides are achieved using a solid support modified with [Ru(bpy)_2(impy‘)]^(2+) (bpy is 2,2‘-bipyridine; impy‘ is 2‘-iminomethylpyridyl-2‘-deoxyuridine). A duplex formed with the metal-containing oligonucleotide exhibits superior thermal stability when compared to the corresponding unmetalated duplex (Tm = 50 °C vs T_m = 48 °C). Electrochemical (E_(1/2) = 1.3 V vs NHE), absorption (λ_(max) = 480 nm), and emission (λ_(max) = 720 nm, τ = 44 ns, Φ = 0.11 × 10^(-3)) data for the ruthenium-modified oligonucleotides indicate that the presence of the oligonucleotide does not perturb the electronic properties of the ruthenium complex. The absence of any change in the emission properties upon duplex formation suggests that the [Ru(bpy)_2(impy)]^(2+) chromophore will be a valuable probe for DNA-mediated electron-transfer studies. Despite the relatively high Ru(III/II) reduction potential, oxidative quenching of photoexcited [Ru(bpy)_2(impy)]^(2+) does not lead to oxidative damage of guanine or other DNA bases

Synthesis and Characterization of Ruthenium and Rhenium Nucleosides

We report the synthesis and characterization of new ruthenium and rhenium nucleosides [Ru(tolyl-acac)_2(IMPy)-T] (tolyl-acac = di(p-methylbenzonatemethane), IMPy = 2‘-iminomethylpyridine, T = thymidine) (5) and [Re(CO)_3(IMPy)-T]Cl (9), respectively. Structural analysis of 9 shows that the incorporation of this metal complex causes minimal perturbation to the sugar backbone and the nucleobase. Eletrochemical (5, E_(1/2) = 0.265 V vs NHE; 9, E_(1/2) = 1.67 V vs NHE), absorption (5, λ_(max) = 600, 486 nm; 9, λ_(max) = 388 nm), and emission (9, λ_(max) = 770 nm, π = 17 ns) data indicate that 5 and 9 are suitable probes for DNA-mediated ground-state electron-transfer studies. The separation and characterization of diastereoisomers of 5 and bipyridyl-based ruthenium nucleoside [Ru(bpy)_2(IMPy)-T]^(2+) (7) are reported

Synthesis, Crystal Structure, Magnetic, and Electron Paramagnetic Resonance Properties of a Spiroconjugated Biradical. Evidence for Spiroconjugation Exchange Pathway

A spiroconjugated nitronyl nitroxide biradical, 6,6‘-(4,4,5,5-tetramethylimidazolidine-3-oxide-1-oxyl)-3,3,3‘,3‘-tetramethyl-1,1‘-spirobisindane (1), has been prepared by functionalization of a 3,3,3‘,3‘-tetramethyl-1,1‘-spirobisindane framework followed by Ullman condensation and subsequent oxidation. The biradical crystallizes in the monoclinic space group C2/c with four molecules in the unit cell of dimensions a = 24.861(10) Å, b = 12.129(3) Å, and c = 12.258(6) Å. X-ray analysis of a blue-plate single crystal has revealed dihedral angles of 28° between the nitronyl nitroxide moiety and aromatic ring with intramolecular through space radical−radical distances of 8.25 and 10.11 Å. In the solid state, the temperature dependence of the molar magnetic susceptibility reveals antiferromagnetic interactions. These interactions are best fit using a pair model, affording the value J = −4.0 cm^(-1) where J is the interaction parameter appearing in the spin Hamiltonian H = −JS_1·S_2. The field dependence of the magnetization measured at 2 K is consistent with a pair model. Frozen matrix EPR spectra of biradical 1 in CH_2Cl_2 at 100 K shows a half field transition at 1700 G. Temperature dependence of the half field transition intensity has been found to be consistent with a ground singlet state and thermally accessible triplet state. The magnetic interaction observed in the solid state is also observed in solution. Thus, room-temperature solution spectra display a nine-line pattern, with hyperfine coupling to four “equivalent” nitrogen atoms and a hyperfine coupling constant a_N = 3.8 G. Temperature dependence of the solution EPR spectra of biradical 1 displays alternating line width effects caused by conformational dynamics in solution. This behavior has been attributed to modulation of exchange and hyperfine interactions most likely caused by rotational motion about the nitronyl nitroxide−phenyl bond. Biradical 1 therefore exists as a ground-state singlet with a thermally accessible triplet at ca. 4 cm^(-1) higher in energy with a conformational dependence of intramolecular exchange in solution. This coupling may present evidence for spiroconjugation as an exchange pathway. Density functional calculations (B3/6-311G(D)) have been performed to investigate this possibility

5‘ Modification of Duplex DNA with a Ruthenium Electron Donor−Acceptor Pair Using Solid-Phase DNA Synthesis

Incorporation of metalated nucleosides into DNA through covalent modification is crucial to measurement of thermal electron-transfer rates and the dependence of these rates with structure, distance, and position. Here, we report the first synthesis of an electron donor−acceptor pair of 5‘ metallonucleosides and their subsequent incorporation into oligonucleotides using solid-phase DNA synthesis techniques. Large-scale syntheses of metal-containing oligonucleotides are achieved using 5‘ modified phosporamidites containing [Ru(acac)_2(IMPy)]^(2+) (acac is acetylacetonato; IMPy is 2‘-iminomethylpyridyl-2‘-deoxyuridine) (3) and [Ru(bpy)_2(IMPy)]^(2+) (bpy is 2,2‘-bipyridine; IMPy is 2‘-iminomethylpyridyl-2‘-deoxyuridine) (4). Duplexes formed with the metal-containing oligonucleotides exhibit thermal stability comparable to the corresponding unmetalated duplexes (T_m of modified duplex = 49 °C vs T_m of unmodified duplex = 47 °C). Electrochemical (3, E_(1/2) = −0.04 V vs NHE; 4, E_(1/2) = 1.12 V vs NHE), absorption (3, λ_(max) = 568, 369 nm; 4, λ_(max) = 480 nm), and emission (4, λ_(max) = 720 nm, τ = 55 ns, Φ = 1.2 × 10-4) data for the ruthenium-modified nucleosides and oligonucleotides indicate that incorporation into an oligonucleotide does not perturb the electronic properties of the ruthenium complex or the DNA significantly. In addition, the absence of any change in the emission properties upon metalated duplex formation suggests that the [Ru(bpy)_2(IMPy)]^(2+)[Ru(acac)_2(IMPy)]^(2+) pair will provide a valuable probe for DNA-mediated electron-transfer studies

Modulating Short Wavelength Fluorescence with Long Wavelength Light

Two molecules in which the intensity of shorter-wavelength fluorescence from a strong fluorophore is modulated by longer-wavelength irradiation of an attached merocyanine-spirooxazine reverse photochromic moiety have been synthesized and studied. This unusual fluorescence behavior is the result of quenching of fluorophore fluorescence by the thermally stable, open, zwitterionic form of the spirooxazine, whereas the photogenerated closed, spirocyclic form has no effect on the fluorophore excited state. The population ratio of the closed and open forms of the spirooxazine is controlled by the intensity of the longer-wavelength modulated light. Both square wave and sine wave modulation were investigated. Because the merocyanine-spirooxazine is an unusual reverse photochrome with a thermally stable long-wavelength absorbing form and a short-wavelength absorbing photogenerated isomer with a very short lifetime, this phenomenon does not require irradiation of the molecules with potentially damaging ultraviolet light, and rapid modulation of fluorescence is possible. Molecules demonstrating these properties may be useful in fluorescent probes, as their use can discriminate between probe fluorescence and various types of adventitious autofluorescence from other molecules in the system being studied

Antioxidant sensing by spiropyrans: Substituent effects and NMR spectroscopic studies

The development of stimuli-responsive small molecules for probing biologically active antioxidants such as glutathione (GSH) has important ramifications in the detection of oxidative stress. An ideal sensor for biological applications should exhibit sufficient sensitivity and selectivity for detection at physiological concentrations and be reversible to allow continuous and dynamic monitoring of antioxidant levels. Designing a suitable sensor thus requires a detailed understanding of activation properties and mechanism of action. In this work, we report a new set of GSH-responsive spiropyrans and demonstrate how changes in the electronic structure of spiropyrans influence GSH sensing with high specificity versus other structurally similar and biologically relevant redox-active molecules. The sensitivity, selectivity, kinetics, binding constant, and reversibility of GSH-responsive-substituted spiropyrans were investigated using UV-vis spectroscopy and laser irradiation experiments. Detailed studies of the mechanism of interaction between spiropyrans with GSH were investigated using NMR spectroscopy. Understanding how electronic effects impact the sensing ability of spiropyrans toward antioxidants and elucidating the mechanism of the spiropyran-GSH interaction will facilitate the design of more effective sensors for detection of antioxidants in vivo. © 2019 American Chemical Society

Modulating Short Wavelength Fluorescence with Long Wavelength Light

Two molecules in which the intensity of shorter-wavelength fluorescence from a strong fluorophore is modulated by longer-wavelength irradiation of an attached merocyanine–spirooxazine reverse photochromic moiety have been synthesized and studied. This unusual fluorescence behavior is the result of quenching of fluorophore fluorescence by the thermally stable, open, zwitterionic form of the spirooxazine, whereas the photogenerated closed, spirocyclic form has no effect on the fluorophore excited state. The population ratio of the closed and open forms of the spirooxazine is controlled by the intensity of the longer-wavelength modulated light. Both square wave and sine wave modulation were investigated. Because the merocyanine–spirooxazine is an unusual reverse photochrome with a thermally stable long-wavelength absorbing form and a short-wavelength absorbing photogenerated isomer with a very short lifetime, this phenomenon does not require irradiation of the molecules with potentially damaging ultraviolet light, and rapid modulation of fluorescence is possible. Molecules demonstrating these properties may be useful in fluorescent probes, as their use can discriminate between probe fluorescence and various types of adventitious “autofluorescence” from other molecules in the system being studied