Time-Resolved Spectroscopic Characterization of a Novel Photodecarboxylation Reaction Mediated by Homolysis of a Carbon α‑Bond in Flurbiprofen

Flurbiprofen (Fp), a nonsteroidal anti-inflammatory drug (NSAID) currently in use for arthritis pain relief and in clinical trials for metastatic prostate cancer, can induce photosensitization and phototoxicity upon exposure to sunlight. The mechanisms responsible for Fp phototoxicity are poorly understood and deserve investigation. In this study, the photodecarboxylation reaction of Fp, which has been assumed to underpin its photoinduced side effects, was explored by femtosecond transient absorption (fs-TA), nanosecond transient absorption (ns-TA), and nanosecond time-resolved resonance Raman (ns-TR³) spectroscopic techniques in pure acetonitrile (MeCN) solvent. Density functional theory (DFT) calculations were also performed to facilitate the assignments of transient species. The resonance Raman and DFT calculation results reveal that the neutral form of Fp was the predominant species present in MeCN. Analysis of the ultraviolet/visible absorption spectrum and results from TD-DFT calculations indicate that the second excited singlet (S₂) can be excited by 266 nm light. Due to its intrinsic instability, S₂ rapidly underwent internal conversion (IC) to decay to the lowest lying excited singlet (S₁), which was observed in the fs-TA spectra at very early delay times. Intriguingly, three distinct pathways for S₁ decay seem to coexist. Specifically, other than fluorescence emission back to the ground state and transformation to the lowest triplet state T₁ through intersystem crossing (ISC), the homolysis of the carbon α-bond decarboxylation reaction proceeded simultaneously to give rise to two radical species, one being carboxyl and another being the residual, denoted as FpR. The coexistence of the triplet Fp (T₁) and FpR species was verified by means of TR³ spectra along with ns-TA spectra. As a consequence of its apparent high reactivity, the FpR intermediate was observed to undergo oxidation under oxygen-saturated conditions to yield another radical species, denoted as FOR, which subsequently underwent intramolecular hydrogen transfer (IHT) and dehydroxylation (DHO) to form a final product, which could react with the carboxyl from the decarboxylation reaction to generate a minor final product. TD-DFT and transient state (TS) calculations for predicting the absorption bands and activation energies of the transient species produced in the photodecarboxylation reaction have provided valuable mechanistic insights for the assignment of the intermediate species observed in the time-resolved spectroscopy experiments reported here. The results of the time-resolved spectroscopy experiments and DFT calculations were used to elucidate the reaction mechanisms and intermediates involved in the photochemistry of Fp

Femtosecond Transient Absorption Spectroscopy Study of the Early Events of Norfloxacin in Aqueous Solutions with Varying pH Values

The photophysics and photochemistry of norfloxacin (NF) have been investigated in aqueous solutions of different pH using femtosecond transient absorption spectroscopy (fs-TA). Resonance Raman spectroscopic experiments on NF have also been conducted in aqueous solutions of different pH to characterize the vibrational and structural information on the initial forms of NF. The experimental results in combination with density functional theory calculations of the key intermediates help us to elucidate the early events for NF after photoexcitation in aqueous solutions with varying pH values. The fs-TA results indicate that NF mainly underwent photophysical processes on the early delay time scale (before 3 ns), and no photochemical reactions occurred on this time scale. Specifically, after the irradiation of NF, the molecule reaches a higher excited singlet S_<i>n</i> and then decays to the lowest-lying excited singlet state S₁ followed by intersystem crossing to transform into the lowest-lying triplet state T₁ with a high efficiency, with an exception that there is a lower efficiency observed in basic aqueous solution due to the generation of an intramolecular electron transfer as an additional pathway to waste energy

<i>meta</i> versus <i>para</i> Substitution: How Does C–H Activation in a Methyl Group Occur in 3‑Methylbenzophenone but Does Not Take Place in 4‑Methylbenzophenone?

The photophysical and photochemical reactions of 3-methylbenzophenone (3-MeBP) and 4-methylbenzophenone (4-MeBP) were investigated using femtosecond transient absorption (fs-TA) and nanosecond time-resolved resonance Raman (ns-TR³) spectroscopy and density functional theory (DFT) calculations. 3-MeBP and 4-MeBP were observed to behave similarly to their parent compound benzophenone (BP) in acetonitrile and isopropyl alcohol solvents. However, in acidic aqueous solutions, an unusual acid-catalyzed proton exchange reaction (denoted the <i>m</i>-methyl activation) of 3-MeBP (with a maximum efficiency at pH 0) is detected to compete with a photohydration reaction. In contrast, only the photohydration reaction was observed for 4-MeBP under the acidic pH conditions investigated. How the <i>m</i>-methyl activation takes place after photolysis of 3-MeBP in acid aqueous solutions is briefly discussed and compared to related photochemistry of other <i>meta</i>-substituted aromatic carbonyl compounds

Time-Resolved Spectroscopic Study of the Photochemistry of Tiaprofenic Acid in a Neutral Phosphate Buffered Aqueous Solution from Femtoseconds to Final Products

The photo-decarboxylation and overall reaction mechanism of tiaprofenic acid (TPA) was investigated by femtosecond transient absorption (fs-TA), nanosecond transient absorption (ns-TA), and nanosecond time-resolved resonance Raman (ns-TR³) spectroscopic experiments in a neutral phosphate buffered solution (PBS). In addition, density functional theory (DFT) calculations were presented to help interpret the experimental results. Resonance Raman and DFT calculation results revealed that the deprotonated tiaprofenic acid (TPA^–) form was the primary species that is photoexcited in a near neutral PBS aqueous solution. The fs-TA experimental data indicated that the lowest lying excited singlet state S₁ underwent an efficient intersystem crossing process (ISC) to quickly transform into the lowest lying excited triplet state T₁ that then undergoes decarboxylation to generate a triplet biradical species (TB³). ns-TA and ns-TR³ results observed a protonation process for TB³ to produce a neutral species (TBP³) that then decayed via ISC to produce a singlet TBP species that further reacted to make the final product (DTPA). A comparison of the present results for TPA^– with similar results for the deprotonated form of ketoprofen (KP^–) in the literature was done to investigate how the thiophene moiety in TPA^– that replaces one phenyl ring in KP^– affects the reaction mechanism and photochemistry of these nonsteroidal anti-inflammatory drugs (NSAIDs)

Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in <i>Catharanthus roseus</i>

<div><p>In <i>Catharanthus roseus</i>, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive <i>in vivo</i> technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for <i>DXS1</i> were repressed by both inhibitors, whereas transcripts for <i>DXS2A</i>&<i>B</i>, <i>DXR</i> and <i>HDS</i> increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation <i>in planta</i> and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in <i>C. roseus</i> in response to MEP-pathway perturbation.</p></div

Peroxiredoxin Q (PrxQ) is required for cellular resistance to diverse stresses in <i>Corynebacterium glutamicum</i>.

<p><i>C</i>. <i>glutamicum</i> wild-type WT(Vector), Δ<i>prxQ</i>(Vector), and Δ<i>prxQ</i>(<i>prxQ</i>) strains grown to stationary phase were exposed to the adverse stresses for 30 min. (<b>a</b>) The mutant lacking PrxQ was highly sensitive to adverse stresses. The viability of the cells was determined. Mean values with standard deviations (error bars) from at least three repeats are shown. **: <i>P</i>≤0.01; *: <i>P</i>≤0.05. (<b>b</b>) Deletion of <i>prxQ</i> led to accumulation of intracellular ROS. The intracellular levels of ROS were determined with the DCFH-DA probe after exposure of stationary phase strains to adverse stresses. **: <i>P</i>≤0.01; *: <i>P</i>≤0.05. (<b>c</b>) The mutant lacking PrxQ had enhanced protein carbonyl levels under adverse stresses. 20 μg of each DNPH-derivatized protein were loaded and electrophoresis was conducted on a 15% SDS-PAGE gel. The protein carbonyl levels were measured with anti-dinitrophenyl antibody (Upper panel). A parallel run was stained with Coomassie Brilliant Blue (Bottom panel). Similar results were obtained in three independent experiments, and the data shown are from one representative experiment.</p

Time course of <i>in vivo</i> clomazone treatment on the expression of MEP pathway genes, and subsequent degradation of DXS and DXR proteins in isolated chloroplasts.

<p>For each plant, two pairs of mature leaves were injected with a 50 µM clomazone solution (or water for control) until the entire leaf blades were fully soaked, using a 1 ml needleless syringe applied to the lower epidermis. For each time point, young leaves were pooled from three independent plants and processed for MEP pathway protein and transcript analysis, respectively. <b>(A)</b> DXS, DXR and HDS proteins were detected by immunoblot. Equal sample loading was confirmed by Coomassie staining. The arrow marks the position of mature CrDXS2A protein. <b>(B)</b> transcript amounts for DXS isoforms (1, 2A & 2B), DXR and HDS were determined by qPCR relative to the geometric mean of multiple reference genes according to Vandesompele et al.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062467#pone.0062467-Vandesompele1" target="_blank">[60]</a>. The experiment was performed 3 times, values from a representative experiment are presented ± SD. <b>(C)</b> chloroplasts were isolated from young leaves of 6-week-old soil-grown <i>C. rosues</i> control and 50 µM clomazone-treated plants (see <b>(A)</b> this Figure) 78 hours after treatment. Chloroplasts were incubated for 1 h in the light (100 µmol m⁻² s⁻¹) at 25°C in the presence of 5 mM ATP. Aliquots were taken at 0, 15, 30 and 60 minutes and used for protein extraction. DXS and DXR proteins were detected by immunoblot. Note that to obtain similar signal intensity at time point 0, the loading amount of protein from control samples (chloroplast isolated from water infiltrated plants) was twice that of clomazone samples.</p

Oxidizing substrate of PrxQ.

<p>(<b>a</b>) The Michaelis-Menten plots of PrxQ activity versus different substrates by NADPH-coupled spectrophotometric method.The reaction mixtures containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM EDTA, 250 μM NADPH, 1 μM PrxQ, 15 μM TrxR, and 40 μM Trx1 and 0–2 mM peroxides (upper left), or 40 μM Trx2 and 0–2 mM peroxides (upper right). The data were analyzed by nonlinear regression using the program GraphPad Prism 5 and were presented as means of the values obtained from three independent assays. (<b>b</b>) Kinetics of peroxynitrite reduction by PrxQ. Peroxynitrite (1 μM) in 10 mM NaOH was rapidly mixed with HRP (5 μM) in the absence or presence of increasing concentrations of PrxQ in 100 mM sodium phosphate buffer (pH 7.4) at 25°C. The inset shows the experimental traces corresponding to HRP compound I formation without PrxQ (control) and with different concentration of PrxQ (0.0, 5.0, 7.5 μM) (lower left). Experimental data were fitted to single exponentials from which observed rate constants of HRP compound I formation were determined. The latter were plotted against PrxQ concentrations (lower right). The data were presented as means of the values obtained from three independent assays.</p

Determination of the molecular weights of native and oxidized peroxiredoxin Q (PrxQ).

<p>(<b>a</b>) The purified recombinant PrxQ was mixed with loading buffer containing 250 mM Tris-HCl (pH 6.8), 0.5% (m V^-1) bromophenol blue, and 50% (V V^-1) glycerol, resolved on 15% polyacrylamide gel electrophoresis (PAGE) (pH 8.8), and then stained with Coomassie Brilliant Blue. M, molecular weight markers. (<b>b</b>) Molecular weight standard curve. (<b>c</b>) Gel filtration of the native PrxQ. Molecular weight of the purified PrxQ was estimated using the above molecular weight standard curve. (<b>d</b>) Redox response of PrxQ. 50 mM dithiothreitol (DTT)-treated proteins (10 μM) were incubated with (+) or without (−) 1 mM H₂O₂, and then the resulting samples were resolved on 15% non-reducing SDS-PAGE.</p

Model of resistance to diverse stresses in <i>Corynebacterium glutamicum</i> based on peroxiredoxin Q/thioredoxin (PrxQ/Trx).

<p>The first step consists of H₂O₂ reduction with the concomitant formation of a stable sulfenic acid intermediate on catalytic Cys49 of PrxQ. Nucleophilic attack by Cys54 on the sulfenic acid intermediate leads to the release of one molecule of H₂O and the formation of a transient disulfide bond between Cys49 and Cys54. This disulfide bond is reduced by the Trx/thioreductase recycling system to reform the reduced peroxidatic Cys. Then, the regenerated PrxQ is ready for another catalytic cycle.</p