Solvent-dependent photophysics of a red-shifted, biocompatible coumarin photocage.

Controlling the activity of biomolecules with light-triggered photocages is an important research tool in the life sciences. We describe here a coumarin photocage that unusually combines the biocompatible optical properties of strong absorption at a long wavelength close to 500 nm and high photolysis quantum yields. The favourable properties are achieved by synthetically installing on the photocage scaffold a diethyl amino styryl moiety and a thionoester group rather than the lactone typical for coumarins. The photocage's photophysics are analysed with microsecond transient absorption spectroscopy to reveal the nature of the excited state in the photolysis pathway. The excited state is found to be strongly dependent on solvent polarity with a triplet state formed in DMSO and a charge-separated state in water that is likely due to aggregation. A long triplet lifetime is also correlated with a high photolysis quantum yield. Our study on the biocompatible photocage reveals fundamental insight for designing advanced photocages such as longer wavelengths in different solvent conditions tailored for applications in basic and applied research.

Discrete and Stereospecific Oligomers Prepared by Sequential and Alternating Single Unit Monomer Insertion.

Natural biopolymers, such as DNA and proteins, have uniform microstructures with defined molecular weight, precise monomer sequence, and stereoregularity along the polymer main chain that affords them unique biological functions. To reproduce such structurally perfect polymers and understand the mechanism of specific functions through chemical approaches, researchers have proposed using synthetic polymers as an alternative due to their broad chemical diversity and relatively simple manipulation. Herein, we report a new methodology to prepare sequence-controlled and stereospecific oligomers using alternating radical chain growth and sequential photoinduced RAFT single unit monomer insertion (photo-RAFT SUMI). Two families of cyclic monomers, the indenes and the N-substituted maleimides, can be alternatively inserted into RAFT agents, one unit at a time, allowing the monomer sequence to be controlled through sequential and alternating monomer addition. Importantly, the stereochemistry of cyclic monomer insertion into the RAFT agents is found to be trans-selective along the main chains due to steric hindrance from the repeating monomer units. All investigated cyclic monomers provide such trans-selectivity, but analogous acyclic monomers give a mixed cis- and trans-insertion.

Raphanusanin-mediated resistance to pathogens is light dependent in radish and Arabidopsis thaliana.

Raphanusanin (Ra) is a light-induced inhibitor of hypocotyl growth that responds to unilateral blue light illumination in radish seedlings. We have previously shown that Ra regulates genes that are involved in common defense mechanisms. Many genes that are induced by Ra are also positively regulated by early blue light. To extend the understanding of the role of Ra in pathogen defense, we evaluated the effects of Ra on radish and Arabidopsis thaliana (A. thaliana) infected with the necrotrophic pathogen Botrytis cinerea (B. cinerea) and biotrophic pathogen Pseudomonas syringae (P. syringae). Radish and A. thaliana were found to be resistant to both pathogens when treated with Ra, depending on the concentration used. Interestingly, Ra-mediated resistance to P. syringae is dependent on light because Ra-treated seedlings exhibited enhanced susceptibility to P. syringae infection when grown in the dark. In addition to regulating the biotic defense response, Ra inhibited seed germination and root elongation and enhanced the growth of root hairs in the presence of light in radish and A. thaliana. Our data suggest that Ra regulates the expression of a set of genes involved in defense signaling pathways and plays a role in pathogen defense and plant development. Our results show that light may be generally required not only for the accumulation of Ra but also for its activation during the pathogen defense response.

High-resolution mapping of 1D and 2D dose distributions using X-band electron paramagnetic resonance imaging.

Electron paramagnetic resonance imaging (EPRI) was performed to visualise 2D dose distributions of homogenously irradiated potassium dithionate tablets and to demonstrate determination of 1D dose profiles along the height of the tablets. Mathematical correction was applied for each relative dose profile in order to take into account the inhomogeneous response of the resonator using X-band EPRI. The dose profiles are presented with the spatial resolution of 0.6 mm from the acquired 2D images; this value is limited by pixel size, and 1D dose profiles from 1D imaging with spatial resolution of 0.3 mm limited by the intrinsic line-width of potassium dithionate. In this paper, dose profiles from 2D reconstructed electron paramagnetic resonance (EPR) images using the Xepr software package by Bruker are focussed. The conclusion is that using potassium dithionate, the resolution 0.3 mm is sufficient for mapping steep dose gradients if the dosemeters are covering only ±2 mm around the centre of the resonator.

EPR imaging of dose distributions aiming at applications in radiation therapy.

A one-dimensional electron paramagnetic resonance (EPR) imaging method for visualisation of dose distributions in photon fields has been developed. Pressed pellets of potassium dithionate were homogeneously irradiated in a (60)Co radiation field to 600 Gy. The EPR analysis was performed with an X-Band (9.6 GHz) Bruker E540 EPR and EPR imaging spectrometer equipped with an E540 GC2X two-axis X-band gradient coil set with gradients along the y axis (along the sample tube) and z axis (along B0) and an ER 4108TMHS resonator. Image reconstruction, including deconvolution, baseline corrections and corrections for the resonator sensitivity, was performed using an in-house-developed Matlab code for the purpose to have a transparent and complete algorithm for image reconstruction. With this method, it is possible to visualise a dose distribution with an accuracy of ∼5 % within ±5 mm from the centre of the resonator.

Raphanusanin-induced genes and the characterization of RsCSN3, a raphanusanin-induced gene in etiolated radish hypocotyls.

Raphanusanin is a light-induced growth inhibitor involved in inhibition of hypocotyl growth in response to unilateral blue light illumination in radish seedlings. To understand better the role of raphanusanin in growth inhibition, we randomly analyzed raphanusanin-induced genes using a modified DD-RT-PCR (differential display RT-PCR) approach. The differential expression RT-PCR approach resulted in identification of four known candidate genes, of which three encoded functional proteins known to be related to responsiveness to diverse environmental stimuli. One of these genes appeared to be an essential element in the inhibition of hypocotyl growth, and was named RsCSN3 (a homologue of subunit 3 of the COP9 signalosome). During the growth inhibition that was observed within minutes of irradiation, the expression of the RsCSN3 gene was increased by phototropic stimulation, as well as by raphanusanin treatment, suggesting that this gene is involved in light-induced growth inhibition. In addition, down-regulation of the RsCSN3 transcript, that is specifically expressed at 60 min after the onset of stimulation under blue light, green light, and raphanusanin treatment, shows a functional correlation with the phototropic response.

Visualizing the dose distribution and linear energy transfer by 1D and 2D ESR imaging: a potassium dithionate dosimeter irradiated with C6+ and N7+ ions.

We report the application of one- and two-dimensional (1D and 2D) spectral-spatial electron spin resonance imaging (ESRI) for visualizing the dose distribution and linear energy transfer (LET) in a potassium dithionate, K2S2O6 (PDT), dosimeter irradiated with the heavy ions C(6+) and N(7+). The ESR spectrum in the irradiated PDT consists of a superposition of two isotropic signals assigned to two *SO3(-) radicals, R1 and R2, with no hyperfine splittings and slightly different g values. The 1D ESRI profiles clearly indicate the spatial penetration of the beams and the location of the sharp maximum dose, the "Bragg peak", detected for each beam. The depth penetrations are different: approximately 2.3 mm for C(6+) and approximately 1.8 mm for N(7+) beams, +/-0.1 mm; beyond these limits, no radicals were detected. 2D spectral-spatial ESRI images reflect both the dose distribution and the spatial dependence of the relative intensities of radicals R1 and R2, an effect that is assigned to the depth variation of the LET. This study has demonstrated that ESRI is a promising new method for dose and LET determination. Of particular interest are applications in the field of radiotherapy with heavy ions, because in this case the Bragg peak is pronounced and the dose can be focused at specific depths while the surrounding areas are protected.

Formates and dithionates: sensitive EPR-dosimeter materials for radiation therapy.

Polycrystalline formates and dithionates are promising materials for EPR dosimetry, as large yields of radiation induced stable radicals are formed with a linear dose response. Rapid spin relaxation rates were detected in many of the substances, indicating that a high microwave power can be applied during EPR acquisition in order to improve sensitivity. Different techniques used to further improve the sensitivity, such as the replacement of 7Li with 6Li or exchange of protons with deuterons in the corresponding crystalline matrices and metal ion doping are discussed. It is concluded that formates and dithionates may be up to 10 times as sensitive as L-alpha-alanine.

Photobiological properties of hydroxy-substituted flavothiones.

Flavothione (FT) and a series of 18 hydroxy- and methoxy-substituted flavothiones were screened for photobiological activity. The 5-hydroxy-substituted compounds (group 3) and the methoxy-substituted flavothiones were inactive. FT and the remaining hydroxy-substituted compounds, all displayed photobiological activity. Among these, the 3-hydroxy-substituted compounds (group 2) were the most efficient photosensitizers overall in spite of their concurrent fast photodegradation. FT and all other hydroxyflavothiones, not substituted in the 3- or 5-positions (group 1), were inefficient compared with group 2. Detailed photobiological tests were carried out for four flavothiones of groups 1 and 2. The biological tests included fungi, several strains of Escherichia coli, Salmonella typhimurium and mammalian cells. In addition, the ability of these flavothiones to perform lipid peroxidation was evaluated. FT and 6-hydroxyflavothione (group 1) induce DNA damage via H-atom abstraction from the lowest n, pi* triplet state of the thione (oxygen independent). For 3-hydroxy and 3,6-dihydroxyflavothione (group 2), both DNA and the membrane are targets. The mechanism likely involves both energy transfer and electron transfer from the lowest pi, pi* triplet state to oxygen, to form singlet oxygen and the superoxide anion. Some of these compounds could be considered as models for environmentally safe photopesticides.

Photolysis of N-hydroxpyridinethiones: a new source of hydroxyl radicals for the direct damage of cell-free and cellular DNA.

N-Hydroxypyridine-2-thione (2-HPT), known to release hydroxyl radicals on irradiation with visible light, and two related compounds, viz. N-hydroxypyridine-4-thione (4-HPT) and N-hydroxyacridine-9-thione (HAT), were tested for their potency to induce DNA damage in L1210 mouse leukemia cells and in isolated DNA from bacteriophage PM2. DNA single-strand breaks and modifications sensitive to various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) were quantified. Illumination of cell-free DNA in the presence of 2-HPT and 4-HPT gave rise to damage profiles characteristic for hydroxyl radicals, i.e. single-strand breaks and the various endonuclease-sensitive modifications were formed in the same ratios as after exposure to established hydroxyl radical sources. In contrast, HAT plus light gave rise to a completely different DNA damage profile, namely that characteristic for singlet oxygen. Experiments with various scavengers (t-butanol, catalase, superoxide dismutase) and in D2O as solvent confirmed that hydroxyl radicals are directly responsible for the DNA damage caused by photoexcited 2-HPT and 4-HPT, while the damage by HAT plus light is mediated by singlet oxygen and type I reactions. The type of DNA damage characteristic of hydroxyl radicals was also observed in L1210 mouse leukemia cells when treated with 2-HPT plus light or with H2O2 at 0 degrees C. t-Butanol (2%) inhibited the cellular DNA damage by approximately 50%. A dose of 2-HPT plus light that generated single-strand breaks at a frequency of 5 x 10(-7)/bp was associated with 50% cell survival. No DNA damage and cytotoxicity was observed after treatment with 2-HPT in the dark. We propose that 2-HTP and 4-HTP may serve as new agents to study the consequences of DNA damage induced by hydroxyl radicals in cells. In addition, the data provide direct evidence that hydroxyl radicals are ultimately responsible for the genotoxic effects caused by H2O2 in the dark.