Structural Factors Controlling the Spin–Spin Exchange Coupling: EPR Spectroscopic Studies of Highly Asymmetric Trityl–Nitroxide Biradicals

Highly asymmetric exchange-coupled biradicals, e.g., the trityl–nitroxides (TNs), possess particular magnetic properties that have opened new possibilities for their application in biophysical, physicochemical, and biological studies. In the present work, we investigated the effect of the linker length on the spin–spin coupling interaction (<i>J</i>) in TN biradicals using the newly synthesized biradicals CT02-GT, CT02-AT, CT02-VT, and CT02-PPT as well as the previously reported biradicals TNN14 and TN1. The results show that the magnitude of <i>J</i> can be easily tuned from ∼4 G (conformer 1 in CT02-PPT) to >1200 G (in TNN14) by varying the linker separating the two radical moieties and changing the temperature. Computer simulations of EPR spectra were carried out to estimate <i>J</i> values of the TN biradicals directly. In addition to the spin–spin coupling interaction of TN biradicals, their <i>g</i>, hyperfine-splitting, and zero-field-splitting interactions were explored at low temperature (220 K). Our present study clearly shows that varying the spin–spin interaction as a function of linker distance and temperature provides an effective strategy for the development of new TN biradicals that can find wide applications in relevant fields

Esterified Dendritic TAM Radicals with Very High Stability and Enhanced Oxygen Sensitivity

In this work, we have developed a new class of dendritic TAM radicals (TG, TdG, and dTdG) through a convergent method based on the TAM core CT-03 or its deuterated analogue dCT-03 and trifurcated Newkome-type monomer. Among these radicals, dTdG exhibits the best EPR properties with sharpest EPR singlet and highest O₂ sensitivity due to deuteration of both the ester linker groups and the TAM core CT-03. Like the previous dendritic TAM radicals, these new compounds also show extremely high stability toward various reactive species owing to the dendritic encapsulation. The highly charged nature of these molecules resulting from nine carboxylate groups prevents concentration-dependent EPR line broadening at physiological pH. Furthermore, we demonstrate that these TAM radicals can be easily derivatized (e.g., PEGylation) at the nine carboxylate groups and the resulting PEGylated analogue dTdG–PEG completely inhibits the albumin binding, thereby enhancing suitability for in vivo applications. These new dendritic TAM radicals show great potential for in vivo EPR oximetric applications and provide insights on approaches to develop improved and targeted EPR oximetric probes for biomedical applications

Methylation status of the promoter region of four salinity-responsive TFs in untreated (S0) and salinity-stressed (S1–S24) seedlings (S1: 1h, S3: 3h, S6: 6h, S12: 12h, S24: 24h).

<p>(a) The black and white boxes indicate, respectively, exon and untranslated regions. The short bars annotated with “I, II, III”, “a” or b'' indicate, respectively the sequences subjected to ChIP analysis, genomic bisulfite sequencing and those used as probes for Southern blotting. The long vertical bars marked “c” display the distribution of CG dinucleotides (marked with red vertical lines), and CNG (blue vertical lines) and CNN (black vertical lines) trinucleotides. The red vertical lines marked with a rectangular indicate CCGG sites analyzed by Southern blotting. The thick black vertical lines represent the proportion of methylated cytosine. Ten positive clones from each gene's amplicon were sequenced. The data reflect the outcome of three independent experiments, and error bars represent standard error (SD). (b) The efficiency of the bisulfite treatment to transform unmethylated cytosine to thymine. A fragment of <i>Glyma20g32730</i> with numerous cytosines was cloned into Dm- <i>E. coli</i> cells and the plasmid was treated with bisulfite in parallel with the soybean genomic DNA. All clones processed showed a transformation rate >99.7%. (c) Methylation-sensitive DNA gel blot analysis of non-stressed (S0) and salinity-stressed seedlings (S1–S24). Genomic DNA was digested to generate large fragments, then with one or other of the schizomers <i>Hpa</i>II or <i>Msp</i>I. Hybridization probes indicated. A DNA fragment amplified from the probe sequence was used as a positive control (+), and ddH₂O was used as a negative control (−).</p

Expression, DNA methylation and histone modification status of <i>Glyma11g02400, Glyma16g27950, Glyma08g41450</i> and <i>Glyma20g30840</i> in none treated (S0) and salinity-stressed (S1–S24) seedlings.

<p>(a) Relative H3K9 demethylation, acetylation and H3K4 trimethylation content (ChIP assay). A 1∶1,000 dilution of input DNA (Input) served as a control for PCR amplifications and the ChIP reactions carried out in the absence of antibody (N0 AB). Relative H3K9 acetylation, H3K9 dimethylation and H3K4 trimethylation were determined by qRT-PCR and normalized to an internal control <i>TUBULIN</i> gene (Genbank accession AY907703). Data represent the mean of three biological replicates. Asterisks indicate means differing significantly from the S0 situation. Error bars represent standard errors. <i>*P</i><0.05, <i>**P</i><0.01. (b) Gene expression (qRT-PCR) profiles. (c) Cytosine methylation level (bisulfite sequencing).</p

The expression of the 49 TFs in mock-stressed and salinity-stressed seedlings.

<p>(a) <i>GmAP2-DREBs</i>, (b) <i>GmMYBs</i>, (c) <i>GmNACs</i> and (d) <i>Gmb-ZIPs</i>. M0-M24 refer to seedlings exposed to just ddH₂O for, respectively, 0h, 1h, 3h, 6h, 12h and 24h; S0–S24 refer seedlings exposed to 150 mM NaCl for 0h, 1h, 3h, 6h, 12h and 24h, respectively. Each gene-specific region was amplified by RT–PCR using the gene-specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041274#pone-0041274-g005" target="_blank">Table S2</a>). The <i>TUBULIN</i> gene (Genbank accession AY907703) was used as an internal control. The experiment was repeated three times with similar result.</p

Promoter methylation status in four salinity-responsive TFs in non-treated (A0) and 5-ADC treated seedlings (A12–A72).

<p>For Figure legend please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041274#pone-0041274-g003" target="_blank">Figure 3</a> legend.</p

Expression of 45 salinity inducible TFs in seedlings exposed to 5-ADC treatment.

<p>(a) <i>GmAP2-DREBs</i>, (b) <i>GmMYBs</i>, (c). <i>GmNACs</i>. and (d) <i>Gmb-ZIPs</i>. M0-M72 refers to seedlings treated with water only for, respectively 0h, 12h, 24h, 48h and 72h, while A0-A72 refer to seedlings exposed to 50 µM 5-ADC for 0h, 12h, 24h, 48h and 72h, respectively. Each gene-specific region was amplified by RT–PCR using the gene-specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041274#pone-0041274-g005" target="_blank">Table S2</a>). The <i>TUBULIN</i> gene (Genbank accession AY907703) was used as an internal control. The experiment was repeated three times with similar result.</p

Thiol-Dependent Reduction of the Triester and Triamide Derivatives of Finland Trityl Radical Triggers O₂‑Dependent Superoxide Production

Tetrathiatriaylmethyl (trityl) radicals have found wide biomedical applications as magnetic resonance probes. Trityl radicals and their derivatives are generally stable toward biological reducing agents such as glutathione (GSH) and ascorbate. We demonstrate that the triester (ET-03) and triamide (AT-03) derivatives of the Finland trityl radical exhibit unique reduction by thiols such as GSH and cysteine (Cys) to generate the corresponding trityl carbanions as evidenced by the loss of EPR signal and appearance of characteristic UV–vis absorbance at 644 nm under anaerobic conditions. The trityl carbanions can be quickly converted back to the original trityl radicals by oxygen (O₂) in air, thus rendering the reaction between the trityl derivative and biothiol undetectable under aerobic conditions. The reduction product of O₂ by the trityl carbanions was shown to be superoxide radical (O₂^•–) by EPR spin-trapping. Kinetic studies showed that the reaction rate constants (<i>k</i>) depend on the types of both trityl radicals and thiols with the order of <i>k</i>_ET‑03/Cys (0.336 M^–1 s^–1) > <i>k</i>_ET‑03/GSH (0.070 M^–1 s^–1) > <i>k</i>_AT‑03/Cys (0.032 M^–1 s^–1) > <i>k</i>_AT‑03/GSH (0.027 M^–1 s^–1). The reactivity of trityl radicals with thiols is closely related to the para-substituents of trityl radicals as well as the p<i>K</i>_a of the thiols and is further reflected by the rate of O₂^•– production and consumptions of O₂ and thiols. This novel reaction represents a new metabolic process of trityl derivatives and should be considered in the design and application of new trityl radical probes

Biocompatible Folic-Acid-Strengthened Ag–Ir Quantum Dot Nanozyme for Cell and Plant Root Imaging of Cysteine/Stress and Multichannel Monitoring of Hg²⁺ and Dopamine

To boost the enzyme-like activity, biological compatibility, and antiaggregation effect of noble-metal-based nanozymes, folic-acid-strengthened Ag–Ir quantum dots (FA@Ag–Ir QDs) were developed. Not only did FA@Ag–Ir QDs exhibit excellent synergistic-enhancement peroxidase-like activity, high stability, and low toxicity, but they could also promote the lateral root propagation of Arabidopsis thaliana. Especially, ultratrace cysteine or Hg2+ could exclusively strengthen or deteriorate the inherent fluorescence property with an obvious “turn-on” or “turn-off” effect, and dopamine could alter the peroxidase-like activity with a clear hypochromic effect from blue to colorless. Under optimized conditions, FA@Ag–Ir QDs were successfully applied for the turn-on fluorescence imaging of cysteine or the stress response in cells and plant roots, the turn-off fluorescence monitoring of toxic Hg2+, or the visual detection of dopamine in aqueous, beverage, serum, or medical samples with low detection limits and satisfactory recoveries. The selective recognition mechanisms for FA@Ag–Ir QDs toward cysteine, Hg2+, and dopamine were illustrated. This work will offer insights into constructing some efficient nanozyme sensors for multichannel environmental analyses, especially for the prediagnosis of cysteine-related diseases or stress responses in organisms

Synthesis and Characterization of PEGylated Trityl Radicals: Effect of PEGylation on Physicochemical Properties

Tetrathiatriarylmethyl (TAM, trityl) radicals have attracted considerable attention as spin probes for biological electron paramagnetic resonance (EPR) spectroscopy and imaging owing to their sharp EPR singlet signals and high biostability. However, their <i>in vivo</i> applications were limited by the short blood circulation lifetimes and strong binding with albumins. Our previous results showed that PEGylation is a feasible method to overcome the issues facing <i>in vivo</i> applications of TAM radicals. In the present study, we synthesized a series of new PEGylated TAM radicals (TTP1, TPP2, TNP1, TNP2, d-TNP1, and d-TNP3) containing various lengths and numbers of mPEG chains. Our results found that the pattern of PEGylation exerts an important effect on physicochemical properties of the resulting TAM radicals. Dendritic PEGylated TAM radicals, TNP1 and TNP2, have higher water solubility and lower susceptibility for self-aggregation than their linear analogues TPP1 and TPP2. Furthermore, dendritic PEGylated TAM radicals exhibit extremely high stability toward various biological oxidoreductants as well as in rat whole blood, liver homogenate, and following <i>in vivo</i> intravenous administration in mice. Importantly, the deuterated derivatives, especially d-TNP3, exhibit excellent properties including the sharp and O₂-sensitive EPR singlet signal, good biocompatibility, and prolonged kinetics with half-life time of ≥10 h in mice. These PEGylated TAM radicals should be suitable for a wide range of applications in <i>in vivo</i> EPR spectroscopy and imaging