Redox Transformations of the OX063 Radical in Biological Media: Oxidative Decay of Initial Trityl with Further Formation of Structurally-Modified TAM.

Being a low-toxic and hydrophilic representative of TAM, OX063 has shown its suitability for in-vivo and in-cell EPR experiments and design of spin labels. Using 13C labeling, we investigated the course of oxidative degradation of OX063 into quinone-methide (QM) under the influence of superoxide as well as further thiol-promoted reduction of QM into TAM radical, which formally corresponds to substitution of a carboxyl function by a hydroxyl group. We found these transformations being quantitative in model reactions mimicking specific features of biological media and confirmed the presence of these reactions in the blood and liver homogenate of mice in vitro. The emergence of the trityl with the hydroxyl group can be masked by an initial TAM in EPR spectra and may introduce distortions into EPR-derived oximetry data if they have been obtained for objects under hypoxia. 13C labeling allows one to detect its presence, considering its different hyperfine splitting constant on 13C1 (2.04 mT) as compared to OX063 (2.30 mT). The potential involvement of these reactions should be considered when using TAM in spin-labeling of biopolymers intended for subsequent EPR experiments, as well as in the successful application of TAM in experiments in vivo and in cell.

In Situ Generation and High Bioresistance of Trityl-based Semiquinone Methide Radicals Under Anaerobic Conditions in Cellular Systems.

Bioreduction of spin labels and polarizing agents (generally stable radicals) has been an obstacle limiting the in-cell applications of pulsed electron paramagnetic resonance (EPR) spectroscopy and dynamic nuclear polarization (DNP). In this work, we have demonstrated that two semiquinone methide radicals (OXQM⋅ and CTQM⋅) can be easily produced from the trityl-based quinone methides (OXQM and CTQM) via reduction by various reducing agents including biothiols and ascorbate under anaerobic conditions. Both radicals have relatively low pKa's and exhibit EPR single line signals at physiological pH. Moreover, the bioreduction of OXQM in three cell lysates enables quantitative generation of OXQM⋅ which was most likely mediated by flavoenzymes. Importantly, the resulting OXQM⋅ exhibited extremely high stability in the E.coli lysate under anaerobic conditions with 76- and 14.3-fold slower decay kinetics as compared to the trityl OX063 and a gem-diethyl pyrrolidine nitroxide, respectively. Intracellular delivery of OXQM into HeLa cells was also achieved by covalent conjugation with a cell-permeable peptide as evidenced by the stable intracellular EPR signal from the OXQM⋅ moiety. Owing to extremely high resistance of OXQM⋅ towards bioreduction, OXQM and its derivatives show great application potential in in-cell EPR and in-cell DNP studies for various cells which can endure short-term anoxic treatments.

Solution and gaseous phase sensing of formaldehyde with economical triphenylmethane based sensors: a tool to estimate formaldehyde content in stored fish samples.

The use of formalin to preserve raw food items such as fish, meat, vegetables etc. is very commonly practiced in the present day. Also, formaldehyde (FA), which is the main constituent of formalin solution, is known to cause serious health issues on exposure. Considering the ill effects of formaldehyde, herein we report synthesis of highly sensitive triphenylmethane based formaldehyde (FA) sensors from a single step reaction of inexpensive reagents namely 4-hydroxy benzaldehyde and 2,6-dimethyl phenol. The synthetic method also provides highly pure product in bulk quantity. The analytical activity of the triphenylmethane sensor 1 with a limit of detection (LOD) value of 2.31 × 10-6 M for FA was significantly enhanced through induced deprotonation and thereafter a LOD value of 1.82 × 10-8 M could be achieved. To the best of our knowledge, the LOD value of the deprotonated form (sensor 2) for FA was superior to those of all the FA optical sensors reported so far. The mechanism of sensing was demonstrated by 1H-NMR titration and recording mass spectra before and after addition of FA to a solution of sensor 2. Both sensor 1 and sensor 2 exhibit quenching in emission upon addition of FA. A fluorescence study also demonstrates enhancement in analytical activity of the sensor upon induced deprotonation. Then the sensor was effectively immobilized into a hydrophilic and biocompatible starch-PVA polymer matrix which enabled detection of FA in a 100% aqueous system reversibly. Again, quick and effective sensing of FA in real food samples (stored fish) with the help of a computational application was demonstrated. The sensors have significant practical applicability as they effectively detect FA in real food samples qualitatively and quantitatively.

Effect of molecular structure on membrane diffusion: Triphenylmethanes across Escherichia coli studied by second harmonic light scattering.

Understanding how the structure of molecules affects their permeability across cell membranes is crucial for many topics in biomedical research, including the development of drugs. In this work, we examine the transport rates of structurally similar triphenylmethane dyes, malachite green (MG) and brilliant green (BG), across the membranes of living Escherichia coli (E. coli) cells and biomimetic liposomes. Using the time-resolved second harmonic light scattering technique, we found that BG passively diffuses across the E. coli cytoplasmic membrane (CM) 3.8 times faster than MG. In addition, BG exhibits a diffusion rate 3.1 times higher than MG across the membranes of liposomes made from E. coli polar lipid extracts. Measurements on these two molecules, alongside previously studied crystal violet (CV), another triphenylmethane molecule, are compared against the set of propensity rules developed by Lipinski and co-workers for assessing the permeability of hydrophobic ion-like drug molecules through biomembranes. It indicates that BG's increased diffusion rate is due to its higher lipophilicity, with a distribution coefficient 25 times greater than MG. In contrast, CV, despite having similar lipophilicity to MG, shows negligible permeation through the E. coli CM on the observation scale, attributed to its more hydrogen bonding sites and larger polar surface area. Importantly, cell viability tests revealed that BG's antimicrobial efficacy is ∼2.4 times greater than that of MG, which aligns well with its enhanced diffusion into the E. coli cytosol. These findings offer valuable insights for drug design and development, especially for improving the permeability of poorly permeable drug molecules.

Characterization and Degradation of Triphenylmethane Dyes and Their Leuco-Derivatives by Heterologously Expressed Laccase From Coprinus cinerea.

Laccase is a copper-containing polyphenol oxidase that can oxidize phenolic and non-phenolic organic substrates. In the past decades, laccases had received considerable attention because of the ability to degrade various organic substances. Based on the codon preference of the Pichia pastoris expression system, this study optimized the gene structure of the laccase gene Lcc1 from Coprius cinerea through synthetic biology methods. A new gene Lcc1I was synthesized and heterologously expressed in P. pastoris. After 3 days of cultivation in a shake flask at 30°C, the transformants produced at a yield of 890 mg L-1protein. The highest production level of the recombinant laccase was 2760 U L-1. The molecular mass of the recombinant laccase was estimated at 60 kDa. The enzyme showed highest activity at pH 3.4 and 45°C. It possessed better stability at higher pH and lower temperature condition. Using 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as the substrate, the Km and Vmax values were 0.136 mM and 9778 µM min-1 mg-1, respectively. The recombinant laccase could directly oxidize some triphenylmethane dyes like leuco-crystal violet (LCV) and leuco-malachite green (LMG). With the help of ABTS mediator, it could oxidize and degrade 77.7% crystal violet (CV) and 79.2% malachite green (MG) within 1 h. Our results indicate that optimization of the laccase gene achieves good expression results in the host system. The dye degradation model constructed in this study may also be applied to the degradation of other organic pollutants and toxic substances, providing new solutions for environmental remediation against the increasingly severe environmental pollution.

Efficient degradation and detoxification of structurally different dyes and mixed dyes by LAC-4 laccase purified from white-rot fungi Ganoderma lucidum.

The purpose of this study is to evaluate the decolorization ability and detoxification effect of LAC-4 laccase on various types of single and mixed dyes, and lay a good foundation for better application of laccase in the efficient treatment of dye pollutants. The reaction system of the LAC-4 decolorizing single dyes (azo, anthraquinone, triphenylmethane, and indigo dyes, 17 dyes in total) were established. To explore the decolorization effect of the dye mixture by LAC-4, two dyes of the same type or different types were mixed at the same concentration (100 mg/L) in the reaction system containing 0.5 U laccase, and time-course decolorization were performed on the dye mixture. The combined dye mixtures consisted of azo + azo, azo + anthraquinone, azo + indigo, azo + triphenylmethane, indigo + triphenylmethane, and triphenylmethane + triphenylmethane. The results obtained in this study were as follows. Under optimal conditions of 30 °C and pH 5.0, LAC-4 (0.5 U) can efficiently decolorize four different types of dyes. The 24-hour decolorization efficiencies of LAC-4 for 800 mg/L Orange G and Acid Orange 7 (azo), Remazol Brilliant Blue R (anthraquinone), Bromophenol Blue and Methyl Green (triphenylmethane), and Indigo Carmine (indigo) were 75.94%, 93.30%, 96.56%, 99.94%, 96.37%, and 37.23%, respectively. LAC-4 could also efficiently decolorize mixed dyes with different structures. LAC-4 can achieve a decolorization efficiency of over 80% for various dye mixtures such as Orange G + Indigo Carmine (100 mg/L+100 mg/L), Reactive Orange 16 + Methyl Green (100 mg/L+100 mg/L), and Remazol Brilliant Blue R + Methyl Green (100 mg/L+100 mg/L). During the decolorization process of the mixed dyes by laccase, four different interaction relationships were observed between the dyes. Decolorization efficiencies and rates of the dyes that were difficult to be degraded by laccase could be greatly improved when mixed with other dyes. Degradable dyes could greatly enhance the ability of LAC-4 to decolorize extremely difficult-to-degrade dyes. It was also found that the decolorization efficiencies of the two dyes significantly increased after mixing. The possible mechanisms underlying the different interaction relationships were further discussed. Free, but not immobilized, LAC-4 showed a strong continuous batch decolorization ability for single dyes, two-dye mixtures, and four-dye mixtures with different structures. LAC-4 exhibited high stability, sustainable degradability, and good reusability in the continuous batch decolorization. The LAC-4-catalyzed decolorization markedly reduced or fully abolished the toxic effects of single dyes (azo, anthraquinone, and indigo dye) and mix dyes (nine dye mixtures containing four structural types of dyes) on plants. Our findings indicated that LAC-4 laccase had significant potential for use in bioremediation due to its efficient degradation and detoxification of single and mixed dyes with different structural types.

Biocompatible Monophosphonated Trityl Spin Probe, HOPE71, for In Vivo Measurement of pO2, pH, and [Pi] by Electron Paramagnetic Resonance Spectroscopy.

Hypoxia, acidosis, and elevated inorganic phosphate concentration are characteristics of the tumor microenvironment in solid tumors. There are a number of methods for measuring each parameter individually in vivo, but the only method to date for noninvasive measurement of all three variables simultaneously in vivo is electron paramagnetic spectroscopy paired with a monophosphonated trityl radical, pTAM/HOPE. While HOPE has been successfully used for in vivo studies upon intratissue injection, it cannot be delivered intravenously due to systemic toxicity and albumin binding, which causes significant signal loss. Therefore, we present HOPE71, a monophosphonated trityl radical derived from the very biocompatible trityl probe, Ox071. Here, we describe a straightforward synthesis of HOPE71 starting with Ox071 and report its EPR sensitivities to pO2, pH, and [Pi] with X-band and L-band EPR spectroscopy. We also confirm that HOPE71 lacks albumin binding, shows low cytotoxicity, and has systemic tolerance. Finally, we demonstrate its ability to profile the tumor microenvironment in vivo in a mouse model of breast cancer.

Perfluoro-tert-butanol for selective on-resin detritylation: a mild alternative to traditionally used methods.

Solid-phase synthesis of cyclic, branched or side-chain-modified peptides typically involves introduction of a residue carrying a temporary side-chain protecting group that undergoes selective on-resin removal. In particular, Nα-Fmoc-Nε-(4-methyltriphenylmethyl) (Mtt)-protected lysine and its shorter analogues are commercially available and extensively used in this context. Nevertheless, rapid reliable methods for on-resin removal of Mtt groups in the presence of tert-butyloxycarbonyl (Boc) groups are needed. Current commonly used conditions involve low concentrations (1-3%) of trifluoroacetic acid (TFA) in dichloromethane, albeit adjustment to each specific application is required to avoid premature removal of Boc groups or cleavage from the linker. Hence, a head-to-head comparison of several deprotection conditions was performed. The selected acids represent a wide range of acidity from TFA to trifluoroethanol. Also, on-resin removal of the N-(4-methoxytriphenylmethyl) (Mmt) and O-trityl groups (on serine) was investigated under similar conditions. The mildest conditions identified for Mtt deprotection involve successive treatments with 30% hexafluoroisopropanol (HFIP) or 30% perfluoro-tert-butanol [(CF3)3COH] in dichloromethane (3 × 5 or 3 × 15 min, respectively), while 30% HFIP, 30% (CF3)3COH, or 10% AcOH-20% trifluoroethanol (TFE) in CH2Cl2 (3 × 5 min) as well as 5% trichloroacetic acid in CH2Cl2 (3 × 2 min) enabled Mmt removal. Treatment with 1% TFA with/without 2% triisopropylsilane added (3 × 5 min), but also prolonged treatment with 30% (CF3)3COH (5 × 15 min), led to selective deprotection of an O-Trt group on a serine residue. In all cases, the sequences also contained N-Boc or O-tBu protecting groups, which were not affected by 30% HFIP or 30% (CF3)3COH even after a prolonged reaction time of 4 h. Finally, the optimized conditions involving HFIP or (CF3)3COH proved applicable also for selective deprotection of a longer resin-bound peptide [i.e., Ac-Gly-Leu-Leu-Lys(Mtt)-Arg(Pbf)-Ile-Lys(Boc)-Ser(tBu)-Leu-Leu-RAM-PS] as well as allowed for an almost complete deprotection of a Dab(Mtt) residue.

Biological Applications of Electron Paramagnetic Resonance Viscometry Using a 13C-Labeled Trityl Spin Probe.

Alterations in viscosity of biological fluids and tissues play an important role in health and diseases. It has been demonstrated that the electron paramagnetic resonance (EPR) spectrum of a 13C-labeled trityl spin probe (13C-dFT) is highly sensitive to the local viscosity of its microenvironment. In the present study, we demonstrate that X-band (9.5 GHz) EPR viscometry using 13C-dFT provides a simple tool to accurately measure the microviscosity of human blood in microliter volumes obtained from healthy volunteers. An application of low-field L-band (1.2 GHz) EPR with a penetration depth of 1-2 cm allowed for microviscosity measurements using 13C-dFT in the living tissues from isolated organs and in vivo in anesthetized mice. In summary, this study demonstrates that EPR viscometry using a 13C-dFT probe can be used to noninvasively and rapidly measure the microviscosity of blood and interstitial fluids in living tissues and potentially to evaluate this biophysical marker of microenvironment under various physiological and pathological conditions in preclinical and clinical settings.

Rapid, High-Yielding Solid-Phase Synthesis of Cathepsin-B Cleavable Linkers for Targeted Cancer Therapeutics.

Antibody-drug conjugates (ADCs) constitute an emerging class of anticancer agents that deliver potent payloads selectively to tumors while avoiding systemic toxicity associated with conventional chemotherapeutics. Critical to ADC development is a serum-stable linker designed to decompose inside targeted cells thereby releasing the toxic payload. A protease-cleavable linker comprising a valine-citrulline (Val-Cit) motif has been successfully incorporated into three FDA-approved ADCs and is found in numerous preclinical candidates. Herein, we present a high-yielding and facile synthetic strategy for a Val-Cit linker that avoids extensive protecting group manipulation and laborious chromatography associated with previous syntheses and provides yields that are up to 10-fold higher than by standard methods. This method is easily scalable and takes advantage of cost-effective coupling reagents and high loading 2-chlorotrityl chloride (2-CTC) resin. Modularity allows for introduction of various conjugation handles in final stages of the synthesis. Facile access to such analogues serves to expand the repertoire of available enzymatically cleavable linkers for ADC generation. This methodology empowers a robust and facile library generation and future exploration into linker analogues containing unnatural amino acids as a selectivity tuning tool.

Band-Gap Energies of Choline Chloride and Triphenylmethylphosphoniumbromide-Based Systems.

UV-VIS spectroscopy analysis of six mixtures containing choline chloride or triphenylmethylphosphonium bromide as the hydrogen bond acceptor (HBA) and different hydrogen bond donors (HBDs, nickel sulphate, imidazole, d-glucose, ethylene glycol, and glycerol) allowed to determine the indirect and direct band-gap energies through the Tauc plot method. Band-gap energies were compared to those relative to known choline chloride-containing deep band-gap systems. The measurements reported here confirmed the tendency of alcohols or Lewis acids to increment band-gap energy when employed as HBDs. Indirect band-gap energy of 3.74 eV was obtained in the case of the triphenylmethylphosphonium bromide/ethylene glycol system, which represents the smallest transition energy ever reported to date for such kind of systems.

Optimization of Nazarov Cyclization of 2,4-Dimethyl-1,5-diphenylpenta-1,4-dien-3-one in Deep Eutectic Solvents by a Design of Experiments Approach.

The unprecedented Nazarov cyclization of a model divinyl ketone using phosphonium-based Deep Eutectic Solvents as sustainable non-innocent reaction media is described. A two-level full factorial Design of Experiments was conducted for elucidating the effect of the components of the eutectic mixture and optimizing the reaction conditions in terms of temperature, time, and substrate concentration. In the presence of the Deep Eutectic Solvent (DES) triphenylmethylphosphonium bromide/ethylene glycol, it was possible to convert more than 80% of the 2,4-dimethyl-1,5-diphenylpenta-1,4-dien-3-one, with a specific conversion, into the cyclopentenone Nazarov derivative of 62% (16 h, 60 °C). For the reactions conducted in the DES triphenylmethylphosphonium bromide/acetic acid, quantitative conversions were obtained with percentages of the Nazarov product above 95% even at 25 °C. Surface Responding Analysis of the optimized data furnished a useful tool to determine the best operating conditions leading to quantitative conversion of the starting material, with complete suppression of undesired side-reactions, high yields and selectivity. After optimization, it was possible to convert more than 90% of the model substrate into the desired cyclopentenone with cis percentages up to 77%. Experimental validation of the implemented model confirmed the robustness and the suitability of the procedure, leading to possible further extension to this specific combination of experimental designs to other substrates or even to other synthetic processes of industrial interest.

Identification of Aroma Composition and Key Odorants Contributing to Aroma Characteristics of White Teas.

The identification of aroma composition and key odorants contributing to aroma characteristics of white tea is urgently needed, owing to white tea's charming flavors and significant health benefits. In this study, a total of 238 volatile components were identified in the three subtypes of white teas using headspace solid-phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOFMS). The multivariate statistical analysis demonstrated that the contents of 103 volatile compounds showed extremely significant differences, of which 44 compounds presented higher contents in Baihaoyinzhen and Baimudan, while the other 59 compounds exhibited higher contents in Shoumei. The sensory evaluation experiment carried out by gas chromatography-olfactometry/mass spectrometry (GC-O/MS) revealed 44 aroma-active compounds, of which 25 compounds were identified, including 9 alcohols, 6 aldehydes, 5 ketones, and 5 other compounds. These odorants mostly presented green, fresh, floral, fruity, or sweet odors. Multivariate analyses of chemical characterization and sensory evaluation results showed that high proportions of alcohols and aldehydes form the basis of green and fresh aroma characteristic of white teas, and phenylethyl alcohol, γ-Nonalactone, trans-ß-ionone, trans-linalool oxide (furanoid), α-ionone, and cis-3-hexenyl butyrate were considered as the key odorants accounting for the different aroma characteristics of the three subtypes of white tea. The results will contribute to in-depth understand chemical and sensory markers associated with different subtypes of white tea, and provide a solid foundation for tea aroma quality control and improvement.

SLIM: A Short-Linked, Highly Redox-Stable Trityl Label for High-Sensitivity In-Cell EPR Distance Measurements.

The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules, preferably acquired under native conditions. In this regard, pulsed dipolar EPR spectroscopy (PDS) in conjunction with site-directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However, the currently available spin labels have diverse deficiencies for in-cell applications, for example, low radical stability or long bioconjugation linkers. In this work, a synthesis strategy is introduced for the derivatization of trityl radicals with a maleimide-functionalized methylene group. The resulting trityl spin label, called SLIM, yields narrow distance distributions, enables highly sensitive distance measurements down to concentrations of 90 nm, and shows high stability against reduction. Using this label, the guanine-nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O (YopO) is shown to change its conformation within eukaryotic cells.

Exploring the Nature of Cationic Blocker Recognition by the Anthrax Toxin Channel.

Obstructing conductive pathways of the channel-forming toxins with targeted blockers is a promising drug design approach. Nearly all tested positively charged ligands have been shown to reversibly block the cation-selective channel-forming protective antigen (PA63) component of the binary anthrax toxin. The cationic ligands with more hydrophobic surfaces, particularly those carrying aromatic moieties, inhibited PA63 more effectively. To understand the physical basis of PA63 selectivity for a particular ligand, detailed information is required on how the blocker structural elements (e.g., positively charged and aromatic groups) influence the molecular kinetics of the blocker/channel binding reactions. In this study, we address this problem using the high-resolution single-channel planar lipid bilayer technique. Several structurally distinct cationic blockers, namely per-6-S-(3-amino) propyl-ß-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-α-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-ß-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-γ-cyclodextrin, methyltriphenylphosphonium ion, and G0 polyamidoamine dendrimer are tested for their ability to inhibit the heptameric and octameric PA63 variants and PA63F427A mutant. The F427 residues form a hydrophobic constriction region inside the channel, known as the "ϕ-clamp." We show that the cationic blockers interact with PA63 through a combination of forces. Analysis of the binding reaction kinetics suggests the involvement of cation-π, Coulomb, and salt-concentration-independent π-π or hydrophobic interactions in the cationic cyclodextrin binding. It is possible that these blockers bind to the ϕ-clamp and are also stabilized by the Coulomb interactions of their terminal amino groups with the water-exposed negatively charged channel residues. In PA63F427A, only the suggested Coulomb component of the cyclodextrin interaction remains. Methyltriphenylphosphonium ion and G0 polyamidoamine dendrimer, despite being positively charged, interact primarily with the ϕ-clamp. We also show that seven- and eightfold symmetric cyclodextrins effectively block the heptameric and octameric forms of PA63 interchangeably, adding flexibility to the earlier formulated blocker/target symmetry match requirement.

Detecting Functional and Accessible Folate Receptor Expression in Cancer and Polycystic Kidneys.

Folate-based small molecule drug conjugates (SMDCs) are currently under development and have shown promising preclinical and clinical results against various cancers and polycystic kidney disease. Two requisites for response to a folate-based SMDC are (i) folate receptor alpha (FRα) protein is expressed in the diseased tissues, and (ii) FRα in those tissues is accessible and functionally competent to bind systemically administered SMDCs. Here we report on the development of a small molecule reporter conjugate (SMRC), called EC2220, which is composed of a folate ligand for FRα binding, a multilysine containing linker that can cross-link to FRα in the presence of formaldehyde fixation, and a small hapten (fluorescein) used for immunohistochemical detection. Data show that EC2220 produces a far greater IHC signal in FRα-positive tissues over that produced with EC17, a folate-fluorescein SMRC that is released from the formaldehyde-denatured FRα protein. Furthermore, the extent of the EC2220 IHC signal was proportional to the level of FRα expression. This EC2220-based assay was qualified both in vitro and in vivo using normal tissue, cancer tissue, and polycystic kidneys. Overall, EC2220 is a sensitive and effective reagent for evaluating functional and accessible receptor expression in vitro and in vivo.

The versatile use of solubilizing trityl tags for difficult peptide/protein synthesis.

Solubilizing trityl tags (Trt-oligoLys/Arg) proved applicable to metal-free radical-triggered desulfurization and an Ag-mediated thioester method. Additionally, using the solubilizing trityl tag strategy, synthesis of the influenza BM2 proton channel, which previously required organic solvent-aided native chemical ligation (NCL) and desulfurization due to its low solubility, was achieved without using organic solvents.

Direct dynamic nuclear polarization of 15N and 13C spins at 14.1 T using a trityl radical and magic angle spinning.

We investigate solid-state dynamic nuclear polarization of 13C and 15N nuclei using monoradical trityl OX063 as a polarizing agent in a magnetic field of 14.1 T with magic angle spinning at ∼100 K. We monitored the field dependence of direct 13C and 15N polarization for frozen [13C, 15N] urea and achieved maximum absolute enhancement factors of 240 and 470, respectively. The field profiles are consistent with polarization of 15N spins via either the solid effect or the cross effect, and polarization of 13C spins via a combination of cross effect and solid effect. For microcrystalline, 15N-enriched tryptophan synthase sample containing trityl radical, a 1500-fold increase in 15N signal was observed under microwave irradiation. These results show the promise of trityl radicals and their derivatives for direct polarization of low gamma, spin-½ nuclei at high magnetic fields and suggest a novel approach for selectively polarizing specific moieties or for polarizing systems which have low levels of protonation.

Site Selective and Efficient Spin Labeling of Proteins with a Maleimide-Functionalized Trityl Radical for Pulsed Dipolar EPR Spectroscopy.

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) in combination with site-directed spin labeling (SDSL) of proteins and oligonucleotides is a powerful tool in structural biology. Instead of using the commonly employed gem-dimethyl-nitroxide labels, triarylmethyl (trityl) spin labels enable such studies at room temperature, within the cells and with single-frequency electron paramagnetic resonance (EPR) experiments. However, it has been repeatedly reported that labeling of proteins with trityl radicals led to low labeling efficiencies, unspecific labeling and label aggregation. Therefore, this work introduces the synthesis and characterization of a maleimide-functionalized trityl spin label and its corresponding labeling protocol for cysteine residues in proteins. The label is highly cysteine-selective, provides high labeling efficiencies and outperforms the previously employed methanethiosulfonate-functionalized trityl label. Finally, the new label is successfully tested in PDS measurements on a set of doubly labeled Yersinia outer protein O (YopO) mutants.

Suppressive Interaction Approach for Masking Stale Note of Instant Ripened Pu-Erh Tea Products.

The unpleasant stale note is a negative factor hindering the consumption of instant ripened Pu-erh tea products. This study focused on investigating volatile chemicals in instant ripened Pu-erh tea that could mask the stale note via sensory evaluation, gas chromatography-mass spectrometry (GC-MS), and gas chromatography-olfactometry (GC-O) analyses. GC-MS and GC-O analyses showed that linalool, linalool oxides, trans-ß-ionone, benzeneacetaldehyde, and methoxybenzenes were the major aroma contributors to the simultaneous distillation and extraction (SDE) extract of instant ripened Pu-erh tea. Sensory evaluation showed that the SDE extract had a strong stale note, which was due to methoxybenzenes. By investigating suppressive interaction among flavour components, the stale note from methoxybenzenes was shown to have reciprocal masking interactions with sweet, floral, and green notes. Moreover, the validation experiment showed that the addition of 40 µg/mL of trans-ß-ionone in the instant ripened Pu-erh tea completely masked the stale note and improved the overall aromatic acceptance. These results elucidate the volatile chemicals that could mask the stale note of instant ripened Pu-erh tea products, which might help to develop high quality products made from instant ripened Pu-erh tea.