Synthesis and stereochemical assignment of DNA spore photoproduct analogues

Investigation of the DNA repair process performed by the spore photoproduct (SP) lyase repair enzyme is strongly hampered by the lack of defined substrates needed for detailed enzymatic studies. The problem is particularly severe because the repair enzyme belongs to the class of strongly oxygen-sensitive radical (S)-adenosylmethionine (SAM) enzymes, which are notoriously difficult to handle. We report the synthesis of the spore photoproduct analogues 1 a and 1 b, which have open backbones and are diastereoisomers. In order to solve the problem of stereochemical assignment, two further derivatives 2 a and 2 b with closed backbones were prepared. The key step of the synthesis of 2 a/b is a metathesis-based macrocyclization that strongly increases the conformational rigidity of the synthetic spore photoproduct derivatives. NOESY experiments of the cyclic isomers furnished a clear cross-peak pattern that allowed the unequivocal assignment of the stereochemistry. The results were transferred to the data for isomers 1 a and 1 b, which were subsequently used for enzymatic-repair studies. These studies were performed with the novel spore photoproduct lyase repair enzyme from Geobacillus stearothermophilus. The studies showed an accordance with a recent investigation performed by us with the spore photoproduct lyase from Bacillus subtilis, in that only the S isomer 1 a is recognized and repaired. The ability to prepare a defined functioning substrate now paves the way for detailed enzymatic studies of the SP-lyase lesion recognition and repair process.     

Crystal structures and repair studies reveal the identity and the base-pairing properties of the UV-induced spore photoproduct DNA lesion

UV light is one of the major causes of DNA damage. In spore DNA, due to an unusual packing of the genetic material, a special spore photoproduct lesion (SP lesion) is formed, which is repaired by the enzyme spore photoproduct lyase (Spl), a radical S-adenosylmethionine (SAM) enzyme. We report here the synthesis and DNA incorporation of a DNA SP lesion analogue lacking the phosphodiester backbone. The oligonucleotides were used for repair studies and they were cocrystallized with a polymerase enzyme as a template to clarify the configuration of the SP lesion and to provide information about the base-pairing properties of the lesion. The structural analysis together with repair studies allowed us to clarify the identity of the preferentially repaired lesion diastereoisomer.     

Photosensitized Thymine Dimerization via Delocalized Triplet Excited States

A new mechanism of photosensitized formation of thymine (Thy) dimers is proposed, which involves generation of a delocalized triplet excited state as the key step. This is supported by chemical evidence obtained by combining one benzophenone and two Thy units with different degrees of freedom, whereby the photoreactivity is switched from a clean Paternò–Büchi reaction to a fully chemo‐, regio‐, and stereoselective [2+2] cycloaddition.     

Mechanistic Study on the Photogeneration of Hydrogen by Decamethylruthenocene

Detailed studies on hydrogen evolution by decamethylruthenocene ([Cp*₂Ru^II]) highlighted that metallocenes are capable of photoreducing hydrogen without the need for an additional sensitizer. Electrochemical, gas chromatographic, and spectroscopic (UV/Vis, ¹H and ¹³C NMR) measurements corroborated by DFT calculations indicated that the production of hydrogen occurs by a two-step process. First, decamethylruthenocene hydride [Cp*₂Ru^IV(H)]⁺ is formed in the presence of an organic acid. Subsequently, [Cp*₂Ru^IV(H)]⁺ is reversibly reduced in a heterolytic reaction with one-photon excitation leading to a first release of hydrogen. Thereafter, the resultant decamethylruthenocenium ion [Cp*₂Ru^III]⁺ is further reduced with a second release of hydrogen by deprotonation of a methyl group of [Cp*₂Ru^III]⁺. Experimental and computational data show spontaneous conversion of [Cp*₂Ru^II] to [Cp*₂Ru^IV(H)]⁺ in the presence of protons. Calculations highlight that the first reduction is endergonic (ΔG⁰=108 kJ mol⁻¹) and needs an input of energy by light for the reaction to occur. The hydricity of the methyl protons of [Cp*₂Ru^II] was also considered.     

2′‐Methoxyacetophenone: An Efficient Photosensitizer for Cyclobutane Pyrimidine Dimer Formation

Stationary and time‐resolved experiments show that 2′‐methoxyacetophenone (2‐M) is an interesting compound for the investigation of triplet states in thymine samples. Time‐resolved emission experiments show that the fluorescence lifetime of 2‐M is 660 ps. A similar time constant of 680 ps is found in transient IR experiments. The data indicate efficient intersystem crossing (≈97 %) from the fluorescent singlet state to the triplet state. The lifetime of the triplet state of 2‐M dissolved in D₂O at room temperature and ambient oxygen concentration is 400 ns. 2‐M has a strong absorption in the UV‐A range and can photosensitize the triplet state of a thymidine dinucleotide with light at a wavelength of 320 nm. The experiments show that 2‐M is well‐suited for time‐resolved experiments on the triplet‐sensitizing process.     

Photoreaction of [Ru(hat)2phen]2+ with guanosine-5'-monophosphate and DNA: formation of new types of photoadducts

[Ru(hat)2phen]2+ (HAT=1,4,5,8,9,12-hexaazatriphenylene, phen=1,10-phenanthroline) interacts with a good affinity with polynucleotides and DNA by intercalation, despite the presence of a second voluminous ancillary HAT ligand. It photoreacts with guanosine-5'-monophosphate (GMP). From HPLC, ESMS and NMR analyses, it can be concluded that this complex forms photoadducts with GMP. In contrast to the photoadducts isolated with Ru-TAP complexes (TAP=1,4,5,8-tetraazaphenanthrene), the photoadducts with [Ru(hat)2phen]2+ contain a covalent link between the oxygen atom of the guanine unit and a HAT ligand. Formation of oxidised photoadducts and compounds resulting from the addition of two GMP entities to the complex are also detected as side products. In the presence of oligo- and polynucleotides, [Ru(hat)2phen]2+ yields photoadducts when guanine bases are present.     

Reactions of ·OH with thymine studied using density functional theory

We studied three possible reactions of a free ·OH radical with thymine by using density functional theory calculations. The results indicate that there is no energy barrier in the reactions of a free ·OH radical adding to the C₆ position of thymine, while the reactions of a free ·OH radical adding to the C₅ position of thymine and the reaction of an ·OH abstracting a hydrogen from the C₅ methyl group should overcome the energy barriers of ～0.70 kcal/mol and ～1.88 kcal/mol, respectively. The C₆‐hydroxylated radical formed is energetically more favorable than the C₅‐hydroxylated radical. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

Facile synthesis of a cis-syn thymine dimer building block and its incorporation into oligodeoxynucleotides

The synthesis of a building block containing the photobiologically relevant cis-syn thymine cyclobutane photoproduct and its incorporation into oligonucleotides by the phosphoramidite-based solid-phase synthesis is reported. Compared to previous syntheses, this route is extremely short and allows such modified oligonucleotides to be easily available for biological studies.     

Gas‐Phase Photodissociation of CH3CHBrCOCl at 248 nm: Detection of Molecular Fragments by Time‐Resolved FT‐IR Spectroscopy

By employing time‐resolved Fourier transform infrared emission spectroscopy, the fragments HCl (v=1–3), HBr (v=1), and CO (v=1‐3) are detected in one‐photon dissociation of 2‐bromopropionyl chloride (CH₃CHBrCOCl) at 248 nm. Ar gas is added to induce internal conversion and to enhance the fragment yields. The time‐resolved high‐resolution spectra of HCl and CO were analyzed to determine the rovibrational energy deposition of 10.0±0.2 and 7.4±0.6 kcal mol^?1, respectively, while the rotational energy in HBr is evaluated to be 0.9±0.1 kcal mol^?1. The branching ratio of HCl(v>0)/HBr(v>0) is estimated to be 1:0.53. The bond selectivity of halide formation in the photolysis follows the same trend as the halogen atom elimination. The probability of HCl contribution from a hot Cl reaction with the precursor is negligible according to the measurements of HCl amount by adding an active reagent, Br₂, in the system. The HCl elimination channel under Ar addition is verified to be slower by two orders of magnitude than the Cl elimination channel. With the aid of ab initio calculations, the observed fragments are dissociated from the hot ground state CH₃CHBrCOCl. A two‐body dissociation channel is favored leading to either HCl+CH₃CBrCO or HBr+CH₂CHCOCl, in which the CH₃CBrCO moiety may further undergo secondary dissociation to release CO.     

Visible‐Light‐Enabled Stereodivergent Synthesis of E‐ and Z‐Configured 1,4‐Dienes by Photoredox/Nickel Dual Catalysis

A stereodivergent reductive coupling reaction between allylic carbonates and vinyl triflates to furnish both E‐ and Z‐configured 1,4‐dienes has been achieved by visible‐light‐induced photoredox/nickel dual catalysis. The mild reaction conditions allow good compatibility of both vinyl triflates and allylic carbonates. Notably, the stereoselectivity of this synergistic cross‐electrophile coupling can be tuned by an appropriate photocatalyst with a suitable triplet‐state energy, providing a practical and stereodivergent means to alkene synthesis. Preliminary mechanistic studies shed some light on the coupling step as well as the control of the stereoselectivity step.     

Metal‐Free and Redox‐Neutral Conversion of Organotrifluoroborates into Radicals Enabled by Visible Light

Converting organoboron compounds into the corresponding radicals has broad synthetic applications in organic chemistry. To achieve these transformations, various strong oxidants such as Mn(OAc)₃, AgNO₃/K₂S₂O₈, and Cu(OAc)₂, in stoichiometric amounts are required, proceeding by a single‐electron transfer mechanism. Established herein is a distinct strategy for generating both aryl and alkyl radicals from organotrifluoroborates through an S_H2 process. This strategy is enabled by using water as the solvent, visible light as the energy input, and diacetyl as the promoter in the absence of any metal catalyst or redox reagent, thereby eliminating metal waste. To demonstrate its synthetic utility, an efficient acetylation to prepare valuable aryl (alkyl) methyl ketones is described and applications to construct C?C, C?I, C?Br, and C?S bonds are also feasible. Experimental evidence suggests that triplet diacetyl serves as the key intermediate in this process.     

Variable Time Normalization Analysis: General Graphical Elucidation of Reaction Orders from Concentration Profiles

The recent technological evolution of reaction monitoring techniques has not been paralleled by the development of modern kinetic analyses. The analyses currently used disregard part of the data acquired, thus requiring an increased number of experiments to obtain sufficient kinetic information for a given chemical reaction. Herein, we present a simple graphical analysis method that takes advantage of the data‐rich results provided by modern reaction monitoring tools. This analysis uses a variable normalization of the time scale to enable the visual comparison of entire concentration reaction profiles. As a result, the order in each component of the reaction, as well as k_obs , is determined with just a few experiments using a simple and quick mathematical data treatment. This analysis facilitates the rapid extraction of relevant kinetic information and will be a valuable tool for the study of reaction mechanisms.     

A Visible‐Light‐Promoted O‐Glycosylation with a Thioglycoside Donor

Visible‐light irradiation of 4‐p‐methoxyphenyl‐3‐butenylthioglucoside donors in the presence of Umemoto's reagent and alcohol acceptors serves as a mild approach to O‐glycosylation. Visible‐light photocatalysts are not required for activation, and alkyl‐ and arylthioglycosides not bearing the p‐methoxystyrene are inert to these conditions. Experimental and computational evidence for an intervening electron donor–acceptor complex, which is necessary for reactivity, is provided. Yields with primary, secondary, and tertiary alcohol acceptors range from moderate to high. Complete β‐selectivity can be attained through neighboring‐group participation.     

A QM/MM Investigation of Thymine Dimer Radical Anion Splitting Catalyzed by DNA Photolyase

On the mend : The repair reaction of the thymine dimer by DNA photolyase (see picture) is studied by hybrid quantum mechanical/molecular mechanical dynamics simulations based on the X‐ray structure of the enzyme–DNA complex. The dynamics of splitting of the thymine dimer radical anion within the DNA photolyase active site is characterized. The model includes the protein environment.