O2 Binding to Heme is Strongly Facilitated by Near‐Degeneracy of Electronic States

This paper reports the computed O₂ binding to heme, which for the first time explains experimental enthalpies for this process of central importance to bioinorganic chemistry. All four spin states along the relaxed Fe? O₂‐binding curves were optimized using the full heme system with dispersion, thermodynamic, and scalar‐relativistic corrections, applying several density functionals. When including all these physical terms, the experimental enthalpy of O₂ binding (?59 kJ mol^?1) is closely reproduced by TPSSh‐D3 (?66 kJ mol^?1). Dispersion changes the potential energy surfaces and leads to the correct electronic singlet and heptet states for bound and dissociated O₂. The experimental activation enthalpy of dissociation (～82 kJ mol^?1) was also accurately computed (～75 kJ mol^?1) with an actual barrier height of ～60 kJ mol^?1 plus a vibrational component of ～10 and ～5 kJ mol^?1 due to the spin‐forbidden nature of the process, explaining the experimentally observed difference of ～20 kJ mol^?1 in enthalpies of binding and activation. Most importantly, the work shows how the nearly degenerate singlet and triplet states increase crossover probability up to ～0.5 and accelerate binding by ～100 times, explaining why the spin‐forbidden binding of O₂ to heme, so fundamental to higher life forms, is fast and reversible.     

Reversible Soft Mechanochemical Control of Biaryl Conformations through Crosslinking in a 3D Macromolecular Network

Tuning the dihedral angle (DA) of axially chiral compounds can impact biological activity, catalyst efficiency, molecular motor performance, or chiroptical properties. Herein, we report gradual, controlled, and reversible changes in molecular conformation of a covalently linked binaphthyl moiety within a 3D polymeric network by application of a macroscopic stretching force. We managed direct observation of DA changes by measuring the circular dichroism signal of an optically pure BINOL-crosslinked elastomer network. Stretching the elastomer resulted in a widening of the DA between naphthyl rings when the BINOL was doubly grafted to the elastomer network; no effect was observed when a single naphthyl ring of the BINOL was grafted to the elastomer network. We have determined that ca. 170 % extension of the elastomers led to the transfer of a mechanical force to the BINOL moiety of 2.5 kcal mol⁻¹ Å⁻¹ (ca. 175 pN) in magnitude and results in the opening of the DA of BINOL up to 130°.     

Synthesis and structural and DNA binding studies of mono‐ and dinuclear copper(II) complexes constructed with ─O and ─N donor ligands: Potential anti‐skin cancer drugs

Mononuclear and dinuclear copper(II) complexes with thiophenecarboxylic acid, [Cu(3‐TCA)₂(2,2′‐bpy)] ( 1 ), [Cu(3‐Me‐2‐TCA)₂(H₂O)(2,2′‐bpy)] ( 2 ), [Cu(5‐Me‐2‐TCA)₂(H₂O)(2,2′‐bpy)] ( 3 ) and [Cu₂(2,5‐TDCA)(DMF)₂(H₂O)₂(2,2′‐bpy)₂](ClO₄)₂ ( 4 ) (where 3‐TCA = 3‐thiophenecarboxylic acid; 3‐Me‐2‐TCA = 3‐methyl‐2‐thiophenecarboxylic acid; 5‐Me‐2‐TCA = 5‐methyl‐2‐thiophenecarboxylic acid; 2,5‐TDCA = thiophene‐2,5‐dicarboxylic acid; 2,2′‐bpy = 2,2′‐bipyridyl; DMF = N,N‐dimethylformamide), were synthesized. Compounds 1 – 4 were extensively characterized using both analytical and spectroscopic methods. Additionally, the solid‐state structures of 1 and 4 were unambiguously established from single‐crystal X‐ray diffraction studies. The hexacoordinated Cu(II) centre in 1 (CuO₄N₂) is a distorted octahedral geometry whereas the pentacoodinated 4 (CuO₃N₂) has distorted square pyramidal geometry. Compounds 1 and 4 exhibit intermolecular hydrogen bonding which leads to the formation of two‐ and three‐dimensional supramolecular architectures, respectively. Spectrophotometric and computational investigations suggest that these compounds bind with DNA in minor groove binding such that K_b = 4.9 × 10⁵ M^?1 and K_sv = 3.4 × 10⁵ M^?1, and binding score of ?5.26 kcal mol^?1. The binding affinity of these complexes to calf thymus DNA is in the order 2 > 3 > 4 > 1 . Methyl‐substituted thiophene ring increases the DNA binding affinity whereas unsubstituted thiophene ring DNA binding rate is reduced. The methyl group on the thiophene ring would sterically hinder π–π stacking of the ring with DNA base pairs, and subsequently they are involved in hydrophobic interaction with the DNA surface rather than partial intercalative interaction. Compounds 1 – 4 show pronounced activity against B16 mouse melanoma skin cancer cell lines as measured by MTT assay yielding IC₅₀ values in the micromolar concentration range. The compounds could prove to be efficient anti‐cancer agents, since at a concentration as low as 2.1 μg ml^?1 they exerted a significant cytotoxic effect in cancer cells whereas cell viability was not affected in normal cells.     

Imidazolo‐dC Metal‐Mediated Base Pairs: Purine Nucleosides Capture Two Ag+ Ions and Form a Duplex with the Stability of a Covalent DNA Cross‐Link

8‐Phenylimidazolo‐dC (^phImidC, 2 ) forms metal‐mediated DNA base pairs by entrapping two silver ions. To this end, the fluorescent “purine” 2′‐deoxyribonucleoside 2 has been synthesised and converted into the phosphoramidite 6 . Owing to the ease of nucleobase deprotonation, the new Ag⁺‐mediated base pair containing a “purine” skeleton is much stronger than that derived from the pyrrolo‐ [3,4‐d]pyrimidine system (^phPyrdC, 1 ). The silver‐mediated ^phImidC–^phImidC base pair fits well into the DNA double helix and has the stability of a covalent cross‐link. The formation of such artificial metal base pairs might not be limited to DNA but may be applicable to other nucleic acids such as RNA, PNA and GNA as well as other biopolymers.     

Experimental Study of Hydrogen Bonding Potentially Stabilizing the 5′‐Deoxyadenosyl Radical from Coenzyme B12

Coenzyme B₁₂ can assist radical enzymes that accomplish the vicinal interchange of a hydrogen atom with a functional group. It has been proposed that the Co? C bond homolysis of coenzyme B₁₂ to cob(II)alamin and the 5′‐deoxyadenosyl radical is aided by hydrogen bonding of the corrin C19? H to the 3′‐O of the ribose moiety of the incipient 5′‐deoxyadenosyl radical, which is stabilized by 30 kJ mol^?1 (B. Durbeej et al., Chem. Eur. J. 2009 , 15, 8578–8585). The diastereoisomers (R)‐ and (S)‐2,3‐dihydroxypropylcobalamin were used as models for coenzyme B₁₂. A downfield shift of the NMR signal for the C19? H proton was observed for the (R)‐isomer (δ=4.45 versus 4.01 ppm for the (S)‐isomer) and can be ascribed to an intramolecular hydrogen bond between the C19? H and the oxygen of CHOH. Crystal structures of (R)‐ and (S)‐2,3‐dihydroxypropylcobalamin showed C19? H???O distances of 3.214(7) Å (R‐isomer) and 3.281(11) Å (S‐isomer), which suggest weak hydrogen‐bond interactions (?ΔG<6 kJ mol^?1) between the CHOH of the dihydroxypropyl ligand and the C19? H. Exchange of the C19? H, which is dependent on the cobalt redox state, was investigated with cob(I)alamin, cob(II)alamin, and cob(III)alamin by using NMR spectroscopy to monitor the uptake of deuterium from deuterated water in the pH range 3–11. No exchange was found for any of the cobalt oxidation states. 3′,5′‐Dideoxyadenosylcobalamin, but not the 2′,5′‐isomer, was found to act as a coenzyme for glutamate mutase, with a 15‐fold lower k_cat/K_M than 5′‐deoxyadenosylcobalamin. This indicates that stabilization of the 5′‐deoxyadenosyl radical by a hydrogen bond that involves the C19? H and the 3′‐OH group of the cofactor is, at most, 7 kJ mol^?1 (?ΔG). Examination of the crystal structure of glutamate mutase revealed additional stabilizing factors: hydrogen bonds between both the 2′‐OH and 3′‐OH groups and glutamate 330. The actual strength of a hydrogen bond between the C19? H and the 3′‐O of the ribose moiety of the 5′‐deoxyadenosyl group is concluded not to exceed 6 kJ mol^?1 (?ΔG).     

Stabilization of a Bimolecular Triplex by 3′‐S‐Phosphorothiolate Modifications: An NMR and UV Thermal Melting Investigation

Triplexes formed from oligonucleic acids are key to a number of biological processes. They have attracted attention as molecular biology tools and as a result of their relevance in novel therapeutic strategies. The recognition properties of single‐stranded nucleic acids are also relevant in third‐strand binding. Thus, there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes, composed of Watson–Crick (W–C) base‐paired DNA duplexes and a Hoogsteen base‐paired RNA strand, are reported to be more thermodynamically stable than those in which the third strand is DNA. Consequently, synthetic efforts have been focused on developing TFOs with RNA‐like structural properties. Here, the structural and stability studies of such a TFO, composed of deoxynucleic acids, but with 3′‐S‐phosphorothiolate (3′‐SP) linkages at two sites is described. The modification results in an increase in triplex melting temperature as determined by UV absorption measurements. ¹H NMR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altered by the major groove binding of either TFO. However, the triplex involving the 3′‐SP modifications is more compact. The 3′‐SP modification was previously shown to stabilise G‐quadruplex and i‐motif structures and therefore is now proposed as a generic solution to stabilising multi‐stranded DNA structures.     

A cationic dye triplet as a unique "glue" that can connect fully matched termini of DNA duplexes

In this study, we propose that three consecutive cationic p‐methylstilbazoles tethered on D ‐threoninols ( Z residues) at 5′ termini act as a unique “glue” connecting DNA duplexes by their interstrand cluster formation. Interstrand clustering of p‐methylstilbazoles ( ZZZ triplets) induces narrowing and hypsochromic shift of bands at 350 nm, which can be assigned to the absorption of p‐methylstilbazole. However, single‐stranded DNA conjugates involving a ZZZ triplet at the 5′ terminus of 8‐mer native nucleotides is found not to induce such large spectral changes, which implies that the intrinsic self‐assembling property of ZZZ triplets is weak. Interestingly, when this conjugate is hybridized with a complementary 8‐mer native oligonucleotide, a remarkable spectral change is observed, indicating the dimerization of a duplex through the interstrand clustering of ZZZ triplets. Dimerization of the duplex is also evidenced by cold‐spray ionization mass spectrometry. This interstrand clustering is observed only when a ZZZ triplet is tethered to a 5′ rather than 3′ terminus. Furthermore, the stability of the interstrand cluster increases by increasing the number of nucleobases of the DNA portion, and when mismatched base pairs are incorporated or when a base next to the Z residue is deleted, the stability substantially drops. When we apply the ZZZ triplet to the formation of a nanowire using two complementary DNA conjugates, each of which has a ZZZ triplet at the 5′ termini as overhang, we demonstrate the successful formation of a nanowire by native PAGE analysis. Since native sticky ends that have three nucleotides do not serve as “glue”, ZZZ triplets with their unique glue‐like properties are prime candidates for constructing DNA‐based nanoarchitectures.     

Contiguous metal-mediated base pairs comprising two Ag(I) ions

The incorporation of transition‐metal ions into nucleic acids by using metal‐mediated base pairs has proved to be a promising strategy for the site‐specific functionalization of these biomolecules. We report herein the formation of Ag⁺‐mediated Hoogsteen‐type base pairs comprising 1,3‐dideaza‐2′‐deoxyadenosine and thymidine. By defunctionalizing the Watson–Crick edge of adenine, the formation of regular base pairs is prohibited. The additional substitution of the N3 nitrogen atom of adenine by a methine moiety increases the basicity of the exocyclic amino group. Hence, 1,3‐dideazaadenine and thymine are able to incorporate two Ag⁺ ions into their Hoogsteen‐type base pair (as compared with one Ag⁺ ion in base pairs with 1‐deazaadenine and thymine). We show by using a combination of experimental techniques (UV and circular dichroism (CD) spectroscopies, dynamic light scattering, and mass spectrometry) that this type of base pair is compatible with different sequence contexts and can be used contiguously in DNA double helices. The most stable duplexes were observed when using a sequence containing alternating purine and pyrimidine nucleosides. Dispersion‐corrected density functional theory calculations have been performed to provide insight into the structure, formation and stabilization of the twofold metalated base pair. They revealed that the metal ions within a base pair are separated by an Ag???Ag distance of about 2.88 Å. The Ag–Ag interaction contributes some 16 kcal mol^?1 to the overall stability of the doubly metal‐mediated base pair, with the dominant contribution to the Ag–Ag bonding resulting from a donor–acceptor interaction between silver 4d‐type and 4s orbitals. These Hoogsteen‐type base pairs enable a higher functionalization of nucleic acids with metal ions than previously reported metal‐mediated base pairs, thereby increasing the potential of DNA‐based nanotechnology.     

DNA Interstrand Cross‐Links of an Antitumor Trinuclear Platinum(II) Complex: Thermodynamic Analysis and Chemical Probing

The trinuclear platinum compound [{trans‐PtCl(NH₃)₂}₂(μ‐trans‐Pt(NH₃)₂{NH₂(CH₂)₆NH₂}₂)]⁴⁺ (BBR3464) belongs to the polynuclear class of platinum‐based anticancer agents. These agents form in DNA long‐range (Pt,Pt) interstrand cross‐links, whose role in the antitumor effects of BBR3464 predominates. Our results show for the first time that the interstrand cross‐links formed by BBR3464 between two guanine bases in opposite strands separated by two base pairs (1,4‐interstrand cross‐links) exist as two distinct conformers, which are not interconvertible, not only if these cross‐links are formed in the 5′‐5′, but also in the less‐usual 3′‐3’ direction. Analysis of the conformers by differential scanning calorimetry, chemical probes of DNA conformation, and minor groove binder Hoechst 33258 demonstrate that each of the four conformers affects DNA in a distinctly different way and adopts a different conformation. The results also support the thesis that the molecule of antitumor BBR3464 when forming DNA interstrand cross‐links may adopt different global structures, including different configurations of the linker chain of BBR3464 in the minor groove of DNA. Our findings suggest that the multiple DNA interstrand cross‐links available to BBR3464 may all contribute substantially to its cytotoxicity.     

Time‐Resolved Thermodynamic Profile upon Photoexcitation of a Nitrospiropyran in Cycloalkanes and of the Corresponding Merocyanine in Aqueous Solutions

The photoconversion of 2′,3′‐dihydro‐6‐nitro‐1′,3′,3′‐trimethylspiro[2H‐1‐benzopyran‐2,2′‐indole] ( Sp ) to its open merocyanine form ( Mc ) in a series of aerated cycloalkanes (cyclopentane, cyclohexane, and trans‐ and cis‐decalin) and of the protonated merocyanine ( McH ⁺ ) to Sp in aqueous solution were studied by laser‐induced optoacoustic spectroscopy (LIOAS). The +(11±2) ml mol⁻¹ expansion determined for the ring closure is due to deprotonation of McH ⁺ plus the reaction of the ejected proton with the monoanion of malonic acid (added to stabilize Mc ), an intrinsic expansion and a small electrostriction term. The energy difference between Sp and initial McH ⁺ is (282±110) kJ mol⁻¹. An intrinsic contraction of −(47±15) ml mol⁻¹ occurs upon ring opening, forming triplet ³Mc in the cycloalkanes, whereas no volume change was detected for the ³Mc to Mc relaxation. Electrostriction decreases the ³Mc energy, (165±18) kJ mol⁻¹, to 135 kJ mol⁻¹. The difference in the values of the ring‐opening ( Sp to Mc ) reaction enthalpy in cycloalkanes as derived from the temperature dependence of the Sp ⇌ Mc equilibrium, (29±8) kJ mol⁻¹, and from the LIOAS data, −(9±25) kJ mol⁻¹, is due to the formation of Mc‐Sp aggregates during steady‐state measurements. The Sp ‐sensitized singlet molecular oxygen, O₂(¹Δ_g), quantum yield (average Φ_Δ=0.58±0.03) derived from the near‐IR emission of O₂(¹Δ_g), was taken as a measure of Mc production in the cycloalkanes. These solvents, albeit troublesome in their handling, provide an additional series for the determination of structural volume changes in nonaqueous media, besides the alkanes already used.     

Reactivity of common functional groups with urethanes: Models for reactive compatibilization of thermoplastic polyurethane blends

Two model urethane compounds, dibutyl 4,4′‐methylenebis(phenyl carbamate) (BMB) and dioctyl 4,4′‐methylenebis(phenyl carbamate) (OMO) were prepared by capping 4,4′‐methylenebis(phenyl isocyanate) with n‐butanol and n‐octanol, respectively. The reactions of the two model urethane compounds with several small monofunctional compounds as well as two model poly(ethylene glycols) were carried out with neat mixtures at elevated temperatures. The ranking of reactivity of the functional groups with the urethanes was determined as follows—primary amine > secondary amine ? hydroxyl ～ acid ～ anhydride ? epoxide. Nuclear magnetic resonance spectroscopy (NMR) was used for the quantitative analysis. Fourier transform infrared spectroscopy was used to complement the NMR analysis. Conversions of carbamate in each reaction were monitored over time at constant temperature (200 °C). The reactions between OMO and primary amine were conducted at 170, 180, 190, and 200 °C and best described with a second‐order bimolecular reaction model. The rate constant was estimated to be 1.8 × 10^?3 L · mol^?1 · s^?1 and activation energy 115 kJ · mol^?1. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 2310–2328, 2002     

Spectroscopic studies on the interaction of alpha‐eleostearic acid with calf thymus DNA

Interaction between alpha‐eleostearic acid (α‐ESA) and calf thymus DNA in Tris‐HCl buffer (pH = 7.4) using neutral red (NR) dye as a spectral probe was investigated using UV–Vis absorption and fluorescence spectroscopy. Spectral data matrix of the complexed reaction between α‐ESA and NR with DNA was processed with an alternative least‐squares (ALS) algorithm, the obtained concentration profiles and the corresponding pure spectra for species (NR, DNA–NR, and DNA–NR–ESA) demonstrated three kinds of reactions might occur in the system. The major groove binding between α‐ESA and DNA was further validated using circular dichroism, viscosity, DNA melting, and ionic strength effect measurements. Moreover, the calculated values of thermodynamic parameters, such as enthalpy (ΔH^θ, ?22.04 kJ/mol) and entropy change (ΔS^θ, 91.52 J K^?1 mol^?1), suggested binding between α‐ESA and DNA was mainly driven by hydrophobic interactions and hydrogen bonds without electrostatic force.     

Evaluation of Weak Interactions in [2]Pseudorotaxanes

Viologens readily thread bis‐p‐phenylene crown ethers to form [2]pseudorotaxanes. However, the binding of sterically hindered 3,3′‐dimethylviologens is very weak. Density functional theory (DFT) calculations indicated that the additional energy cost of “flattening” is substantial, 55 kJ mol^?1, and prevents the formation of a stable host–guest complex. The structures of [2]pseudorotaxanes determined by X‐ray crystallography are in good agreement with the NMR characterisation and DFT results. Their association constants and thermodynamic parameters in solution were measured by using a dilution method and, for the first time, by host–guest nuclear Overhauser effect (NOE) correlations. The NOE approach was subsequently applied to study the sterically hindered analogues and it was shown that the binding in 3,3′‐dimethyl‐N,N‐dibenzyl [2]pseudorotaxane is by 8.5 kJ mol^?1 weaker than in its regular analogue. The proposed technique helps to quantify weak interactions in [2]pseudorotaxanes and can be applied to other host‐guest complexes.     

Bis-ortho-substitution by methyl groups dramatically increases the racemization barrier of Tröger bases

We have shown through racemization kinetics studies that the enantiomerization barriers of the bis‐ortho‐methyl substituted Tröger bases 2 and 3 in acidic media are raised by 30 kJ mol^?1 relative to the parent compound 1 , that is 130.4(4) and 131.6(4) kJ mol^?1, respectively (105 °C, pH 1, ethylene glycol). The enantiomerization barrier of para‐methoxy‐para‐nitro substituted Tröger base 4 was determined by dynamic capillary electrophoresis to 96.3(2) kJ mol^?1 (25 °C, pH 2.2, H₂O), which is lower by 5 kJ mol^?1 relative to 1 . The influence of deutero‐substitution on the racemization rates was also studied. The influence of steric and electronic factors on the enantiomerization barrier was investigated by quantum‐mechanical (DFT) calculations. It is shown that enantiomerization takes place in two steps: ring‐opening and further interconversion of the monocyclic intermediate. For the interconversion to occur a transition state has to be passed which is sensitive to steric effects. Ortho‐substitution by methyl groups significantly increases the energy of this state. Thus, compounds 2 and 3 are the simplest Tröger bases which are configurationally stable in acidic media.     

Initial DNA Interactions of the Binuclear Threading Intercalator Λ,Λ‐[μ‐bidppz(bipy)4Ru2]4+: An NMR Study with [d(CGCGAATTCGCG)]2

Binuclear polypyridine ruthenium compounds have been shown to slowly intercalate into DNA, following a fast initial binding on the DNA surface. For these compounds, intercalation requires threading of a bulky substituent, containing one Ru^II, through the DNA base‐pair stack, and the accompanying DNA duplex distortions are much more severe than with intercalation of mononuclear compounds. Structural understanding of the process of intercalation may greatly gain from a characterisation of the initial interactions between binuclear Ru^II compounds and DNA. We report a structural NMR study on the binuclear Ru^II intercalator Λ,Λ‐B (Λ,Λ‐[μ‐bidppz(bipy)₄Ru₂]⁴⁺; bidppz=11,11′‐bis(dipyrido[3,2‐a:2′,3′‐c]phenazinyl, bipy = 2,2′‐bipyridine) mixed with the palindromic DNA [d(CGCGAATTCGCG)]₂. Threading of Λ,Λ‐B depends on the presence and length of AT stretches in the DNA. Therefore, the latter was selected to promote initial binding, but due to the short stretch of AT base pairs, final intercalation is prevented. Structural calculations provide a model for the interaction: Λ,Λ‐B is trapped in a well‐defined surface‐bound state consisting of an eccentric minor‐groove binding. Most of the interaction enthalpy originates from electrostatic and van der Waals contacts, whereas intermolecular hydrogen bonds may help to define a unique position of Λ,Λ‐B. Molecular dynamics simulations show that this minor‐groove binding mode is stable on a nanosecond scale. To the best of our knowledge, this is the first structural study by NMR spectroscopy on a binuclear Ru compound bound to DNA. In the calculated structure, one of the positively charged Ru²⁺ moieties is near the central AATT region; this is favourable in view of potential intercalation as observed by optical methods for DNA with longer AT stretches. Circular dichroism (CD) spectroscopy suggests that a similar binding geometry is formed in mixtures of Λ,Λ‐B with natural calf thymus DNA. The present minor‐groove binding mode is proposed to represent the initial surface interactions of binuclear Ru^II compounds prior to intercalation into AT‐rich DNA.     

Ligand Dynamics in the Solid: Lithium‐fluorenide and Lithium‐benzo[b]fluorenide N,N,N′,N′‐Tetramethylethylenediamine (tmeda) Complexes

The dynamic behavior of the N,N,N′,N′‐tetramethylethylenediamine (tmeda) ligand has been studied in solid lithium‐fluorenide(tmeda) ( 3 ) and lithium‐benzo[b]fluorenide(tmeda) ( 4 ) using CP/MAS solid‐state ¹³C‐ and ¹⁵N‐NMR spectroscopy. It is shown that, in the ground state, the tmeda ligand is oriented parallel to the long molecular axis of the fluorenide and benzo[b]fluorenide systems. At low temperature (<250 K), the ¹³C‐NMR spectrum exhibits two MeN signals. A dynamic process, assigned to a 180° rotation of the five‐membered metallacycle (π‐flip), leads at elevated temperatures to coalescence of these signals. Line‐shape calculations yield ΔH^?=42.7 kJ mol^?1, ΔS^?=?5.3 J mol^?1 K^?1, and =44.3 kJ mol^?1 for 3 , and ΔH^?=36.8 kJ mol^?1, ΔS^?=?17.7 J mol^?1 K^?1, and =42.1 kJ mol^?1 for 4 , respectively. A second dynamic process, assigned to ring inversion of the tmeda ligand, was detected from the temperature dependence of T_1ρ, the ¹³C spin‐lattice relaxation time in the rotating frame, and led to ΔH^?=24.8 kJ mol^?1, ΔS^?=?49.2 J mol^?1 K^?1, and =39.5 kJ mol^?1 for 3 , and ΔH^?=18.2 kJ mol^?1, ΔS^?=?65.3 J mol^?1 K^?1, and =37.7 kJ mol^?1 for 4 , respectively. For (D₁₂)‐ 3 , the rotation of the CD₃ groups has also been studied, and a barrier E_a of 14.1 kJ mol^?1 was found.     

Fluorescence Quenching over Short Range in a Donor‐DNA‐Acceptor System

A new donor‐DNA‐acceptor system has been synthesized containing Nile red‐modified 2′‐deoxyuridine as charge donor and 6‐N,N‐dimethylaminopyrene‐modified 2′‐deoxyuridine as acceptor to investigate the charge transfer in DNA duplexes using fluorescence spectroscopy and time‐resolved femtosecond pump‐probe techniques. Fluorescence quenching experiments revealed that the quenching efficiency of Nile red depends on two components: 1) the presence of a charge acceptor and 2) the number of intervening CG and AT base pairs between donor and acceptor. Surprisingly, the quenching efficiency of two base pairs (73 % for CG and the same for AT) is higher than that for one base pair (68 % for CG and 37 % for AT), while at a separation of three base pairs less than 10 % quenching is observed. A comparison with the results of time‐resolved measurements revealed a correlation between quenching efficiency and the first ultrafast time constant suggesting that quenching proceeds via a charge transfer from the donor to the acceptor. All transients are satisfactorily described with two decays: a rapid charge transfer with 600 fs (～10¹² s^?1) that depends strongly and in a non‐linear fashion on the distance between donor and acceptor, and a slower time constant of a few picoseconds (～10¹¹ s^?1) with weak distance dependence. A third time constant on a nanosecond time scale represents the fluorescence lifetime of the donor molecule. According to these results and time‐dependent density functional theory (TDDFT) calculations a combination of single‐step superexchange and multistep hopping mechanisms can be proposed for this short‐range charge transfer. Furthermore, significantly less quenching efficiency and slower charge transfer rates at very short distances indicate that the direct interaction between donor and acceptor leads to a local structural distortion of DNA duplexes which may provide some uncertainty in identifying the charge transfer rates in short‐range systems.     

AFM‐Correlated CSM‐Coupled Raman/Fluorescence Studies on Selective Interactions of Water Soluble Porphyrins with DNA

The Raman and fluorescence spectroscopic properties of water‐soluble oxo‐titanium(IV) mesotetrakis (1‐methyl pyridium‐4‐yl) porphyrin (O=Ti(TMPyP)⁴⁺) bound with calf thymus DNA and artificial DNAs such as double stranded poly[d(A‐T)₂] and poly[d(G‐C)₂] have been investigated on the single DNA molecule basis by AFM‐correlated confocal scanning microscope (CSM)‐coupled Raman and fluorescence spectroscopic techniques as well as the ensemble‐averaged spectroscopy. The ensemble‐averaged spectroscopic studies imply that the porphyrin interacts with DNA in different groove binding patterns depending on the base pairs. AFM‐images of the different DNAs bound with O=Ti(TMPyP)⁴⁺ were measured, and their morphologies are found to depend on kind of base pairs interacting with O=Ti(TMPyP)⁴⁺. Being correlated with the AFM images, the CSM‐coupled Raman and fluorescence spectral properties of the three different single O=Ti(TMPyP)⁴⁺‐DNA complexes were observed to be highly resolved and sensitive to base pair‐dependent axial ligation of Ti‐O bond as compared to the corresponding ensemble‐averaged spectral properties, which affect the groove binding and its strength of the O=Ti(TMPyP)⁴⁺ with DNA. The axial ligation was found to be accompanied by vibration structural change of the porphyrin ring, leading to keep the shape of double stranded poly[d(A‐T)₂] rigid while poly‐[d(G‐C)₂] and calf thymus DNA flexible after binding with the oxo‐titanyl porphyrin. The base pair dependence of the fluorescence decay times of the DNA‐bound porphyrins was also observed, implying that an excited‐state charge transfer takes place in the G‐C rich major groove in calf thymus DNA. These results suggest that binding of O=Ti(TMPyP)⁴⁺ is more preferential with the G‐C rich major groove than with the A‐T rich minor groove in calf thymus DNA so that the morphology of DNA is changed.     

Site‐Directed Spin‐Labeling of DNA by the Azide–Alkyne ‘Click’ Reaction: Nanometer Distance Measurements on 7‐Deaza‐2′‐deoxyadenosine and 2′‐Deoxyuridine Nitroxide Conjugates Spatially Separated or Linked to a ‘dA‐dT’ Base Pair

Nucleobase‐directed spin‐labeling by the azide‐alkyne ‘click’ (CuAAC) reaction has been performed for the first time with oligonucleotides. 7‐Deaza‐7‐ethynyl‐2′‐deoxyadenosine ( 1 ) and 5‐ethynyl‐2′‐deoxyuridine ( 2 ) were chosen to incorporate terminal triple bonds into DNA. Oligonucleotides containing 1 or 2 were synthesized on a solid phase and spin labeling with 4‐azido‐2,2,6,6‐tetramethylpiperidine 1‐oxyl (4‐azido‐TEMPO, 3 ) was performed by post‐modification in solution. Two spin labels ( 3 ) were incorporated with high efficiency into the DNA duplex at spatially separated positions or into a ‘dA‐dT’ base pair. Modification at the 5‐position of the pyrimidine base or at the 7‐position of the 7‐deazapurine residue gave steric freedom to the spin label in the major groove of duplex DNA. By applying cw and pulse EPR spectroscopy, very accurate distances between spin labels, within the range of 1–2 nm, were measured. The spin–spin distance was 1.8±0.2 nm for DNA duplex 17 ( dA*^7,10 ) ?11 containing two spin labels that are separated by two nucleotides within one individual strand. A distance of 1.4±0.2 nm was found for the spin‐labeled ‘dA‐dT’ base pair 15 ( dA*⁷ ) ?16 ( dT*⁶ ). The ‘click’ approach has the potential to be applied to all four constituents of DNA, which indicates the universal applicability of the method. New insights into the structural changes of canonical or modified DNA are expected to provide additional information on novel DNA structures, protein interaction, DNA architecture, and synthetic biology.