[Developments of Profluorescent Nitroxide Probes for Highly Sensitive and Selective Detection of Biological Redox Molecules].

Disruption of the redox balance in vivo is closely involved in the development of various diseases associated with oxidative stress. Therefore, methods for the in vivo analysis of antioxidants and free radicals are essential to elucidate the pathogenic mechanisms of such diseases. Although profluorescent nitroxide probes can be used to evaluate redox molecules with high sensitivity, these probes have low selectivity. Recently, we developed two profluorescent nitroxide probes, 15-((9-(ethylimino)-10-methyl-9Hbenzo[a]phenoxazin-5-yl)amino)-3,11-dioxa-7-azadispiro-hexadecan-7-yloxyl (Nile-DiPy) and 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen), which had high sensitivity and selectivity toward ascorbic acid and lipid-derived radicals, respectively. These probes can react sensitively and selectively to each target molecule and can be used in animal experiments. In this paper, we review the design strategies and application of these profluorescent nitroxide probes.

Amphiphilic Nitroxide-Bearing Siloxane-Based Block Copolymer Coatings for Enhanced Marine Fouling Release.

The buildup of organic matter and organisms on surfaces exposed to marine environments, known as biofouling, is a disruptive and costly process affecting maritime operations. Previous research has identified some of the surface characteristics particularly suited to the creation of antifouling and fouling-release surfaces, but there remains room for improvement against both macrofouling and microfouling organisms. Characterization of their adhesives has shown that many rely on oxidative chemistries. In this work, we explore the incorporation of the stable radical 2,2,6,6-tetramethylpipiderin-1-oxyl (TEMPO) as a component in an amphiphilic block copolymer system to act as an inhibitor for marine cements, disrupting adhesion of macrofouling organisms. Using polystyrene-b-poly(dimethylsiloxane-r-vinylmethysiloxane) block copolymers, pendent vinyl groups were functionalized with TEMPO and poly(ethylene glycol) to construct an amphiphilic material with redox active character. The antifouling and fouling-release performance of these materials was investigated through settlement and removal assays of three model fouling organisms and correlated to surface structure and chemistry. Surfaces showed significant antifouling character and fouling-release performance was increased substantially toward barnacles by the incorporation of stable radicals, indicating their potential for marine antifouling applications.

Synthesis, Neuroprotection, and Antioxidant Activity of 1,1'-Biphenylnitrones as α-Phenyl-N-tert-butylnitrone Analogues in In Vitro Ischemia Models.

Herein, we report the neuroprotective and antioxidant activity of 1,1'-biphenyl nitrones (BPNs) 1-5 as α-phenyl-N-tert-butylnitrone analogues prepared from commercially available [1,1'-biphenyl]-4-carbaldehyde and [1,1'-biphenyl]-4,4'-dicarbaldehyde. The neuroprotection of BPNs1-5 has been measured against oligomycin A/rotenone and in an oxygen-glucose deprivation in vitro ischemia model in human neuroblastoma SH-SY5Y cells. Our results indicate that BPNs 1-5 have better neuroprotective and antioxidant properties than α-phenyl-N-tert-butylnitrone (PBN), and they are quite similar to N-acetyl-L-cysteine (NAC), which is a well-known antioxidant agent. Among the nitrones studied, homo-bis-nitrone BPHBN5, bearing two N-tert-Bu radicals at the nitrone motif, has the best neuroprotective capacity (EC50 = 13.16 ± 1.65 and 25.5 ± 3.93 µM, against the reduction in metabolic activity induced by respiratory chain blockers and oxygen-glucose deprivation in an in vitro ischemia model, respectively) as well as anti-necrotic, anti-apoptotic, and antioxidant activities (EC50 = 11.2 ± 3.94 µM), which were measured by its capacity to reduce superoxide production in human neuroblastoma SH-SY5Y cell cultures, followed by mononitrone BPMN3, with one N-Bn radical, and BPMN2, with only one N-tert-Bu substituent. The antioxidant activity of BPNs1-5 has also been analyzed for their capacity to scavenge hydroxyl free radicals (82% at 100 µM), lipoxygenase inhibition, and the inhibition of lipid peroxidation (68% at 100 µM). Results showed that although the number of nitrone groups improves the neuroprotection profile of these BPNs, the final effect is also dependent on the substitutent that is being incorporated. Thus, BPNs bearing N-tert-Bu and N-Bn groups show better neuroprotective and antioxidant properties than those substituted with Me. All these results led us to propose homo-bis-nitrone BPHBN5 as the most balanced and interesting nitrone based on its neuroprotective capacity in different neuronal models of oxidative stress and in vitro ischemia as well as its antioxidant activity.

Aerobic Cytotoxicity of Aromatic N-Oxides: The Role of NAD(P)H:Quinone Oxidoreductase (NQO1).

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities, which are typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the role of NAD(P)H:quinone oxidoreductase (NQO1) in ArN→O aerobic cytotoxicity. We synthesized 9 representatives of ArN→O with uncharacterized redox properties and examined their single-electron reduction by rat NADPH:cytochrome P-450 reductase (P-450R) and Plasmodium falciparum ferredoxin:NADP+ oxidoreductase (PfFNR), and by rat NQO1. NQO1 catalyzed both redox cycling and the formation of stable reduction products of ArN→O. The reactivity of ArN→O in NQO1-catalyzed reactions did not correlate with the geometric average of their activity towards P-450R- and PfFNR, which was taken for the parameter of their redox cycling efficacy. The cytotoxicity of compounds in murine hepatoma MH22a cells was decreased by antioxidants and the inhibitor of NQO1, dicoumarol. The multiparameter regression analysis of the data of this and a previous study (DOI: 10.3390/ijms20184602) shows that the cytotoxicity of ArN→O (n = 18) in MH22a and human colon carcinoma HCT-116 cells increases with the geometric average of their reactivity towards P-450R and PfFNR, and with their reactivity towards NQO1. These data demonstrate that NQO1 is a potentially important target of action of heteroaromatic N-oxides.

Heterocyclic N-Oxides as Small-Molecule Fluorogenic Scaffolds: Rational Design and Applications of Their "On-Off" Fluorescence.

Small-molecule fluorescent probes are powerful tools in chemical analysis and biological imaging. However, as the foundation of probe design, the meager existing set of core fluorophores have largely limited the diversity of current probes. Consequently, there is a high demand to discover fluorophores with new scaffolds and optimize the existing fluorophores. Here, we put forward a facile strategy of heterocyclic N-oxidation to address these challenges. The introduced N-O bond reconstructs the electron "push-pull" system of heterocyclic scaffolds and dramatically improves their photophysical properties by red-shifting the spectra and increasing the Stokes shift. Meanwhile, the heterocyclic N-O bond also enables a function of the fluorescence switch. It can turn on the fluorescence of pyridine and increase the fluorescence of quinoline and, conversely, decrease the fluorescence of acridines and resorufin. As a further practical application, we successfully utilized the quinoline N-oxide scaffold to design fluorogenic probes for H2S (8) and formaldehyde (FA, 9). Given their ultraviolet-visible spectra, both probes with high selectivity and sensitivity could be conveniently used in the naked eye detection of target analytes under illumination with a portable UV lamp. More interestingly, the probes could be effectively used in the imaging of nuclear and cytoplasmic H2S or nuclear and perinuclear FA. This potentially overcomes the weaknesses of existing H2S or FA probes that can only work in the cytoplasm. These interesting findings demonstrate the ability to rapidly expand and optimize the existing fluorophore library through heterocyclic N-oxidation.

Facile Fabrication of Hierarchical MOF-Metal Nanoparticle Tandem Catalysts for the Synthesis of Bioactive Molecules.

Multifunctional metal-organic frameworks (MOFs) that possess permanent porosity are promising catalysts in organic transformation. Herein, we report the construction of a hierarchical MOF functionalized with basic aliphatic amine groups and polyvinylpyrrolidone-capped platinum nanoparticles (Pt NPs). The postsynthetic covalent modification of organic ligands increases basic site density in the MOF and simultaneously introduces mesopores to create a hierarchically porous structure. The multifunctional MOF is capable of catalyzing a sequential Knoevenagel condensation-hydrogenation-intramolecular cyclization reaction. The unique selective reduction of the nitro group to intermediate hydroxylamine by Pt NPs supported on MOF followed by intramolecular cyclization with a cyano group affords an excellent yield (up to 92%) to the uncommon quinoline N-oxides over quinolines. The hierarchical MOF and polyvinylpyrrolidone capping agent on Pt NPs synergistically facilitate the enrichment of substrates and thus lead to high activity in the reduction-intramolecular cyclization reaction. The bioactivity assay indicates that the synthesized quinoline N-oxides evidently inhibit the proliferation of lung cancer cells. Our findings demonstrate the feasibility of MOF-catalyzed direct synthesis of bioactive molecules from readily available compounds under mild conditions.

Revision of the Regioselectivity of the Beirut Reaction of Monosubstituted Benzofuroxans with Benzoylacetonitrile. 6-Substituted quinoxaline-2-carbonitrile 1,4- dioxides: Structural Characterization and Estimation of Anticancer Activity and Hypoxia Selectivity.

BACKGROUND: Quinoxaline 1,4-dioxides have a broad range of biological activity that causes a growing interest in their derivatives for drug discovery. Recent studies demonstrated that quinoxaline 1,4- dioxides have a promising anticancer activity and good hypoxia-selectivity. OBJECTIVE: The preparation, isolation, structure characterization, and screening for anticancer activity of the first representatives of 6-substituted quinoxaline-2-carbonitrile 1,4-dioxides have been described. MATERIALS AND METHODS: A series of 7- and 6-halogeno-3-phenylquinoxaline-2-carbonitrile 1,4-dioxides was synthesized by the Beirut reaction. The cytotoxicity was assessed by MTT test (72 h incubation) in normoxia (21% O2) and hypoxia (1% O2) conditions. RESULTS: We found that during the Beirut reaction between a benzofuroxan bearing an electron withdrawing group and benzoylacetonitrile in the presence of triethylamine, in addition to well-known 7-substituted quinoxaline-2-carbonitrile 1,4-dioxides 7-11a, the 6-isomers 7-11b are formed. Moreover, the yield of the 6- isomers increased with the increase in the electron-withdrawing character of the substituent. For benzofuroxans with CO2Me and CF3 groups, 6-substituted quinoxaline-2-carbonitrile 1,4-dioxides 10-11b were the major products. Despite similarities in physicochemical and spectroscopic properties, the obtained isomers exhibit considerable differences in their anticancer activity and hypoxia selectivity. CONCLUSION: Substituents and their electronic effects play a key role in the formation of 7- and 6-substituted quinoxaline-2-carbonitrile 1,4-dioxides in the Beirut reaction and in the cytotoxicity properties of the obtained isomers.

In Situ Imaging of Cysteine in the Brains of Mice with Epilepsy by a Near-Infrared Emissive Fluorescent Probe.

Epilepsy is characterized by oxidative stress in the brain. As the crucial reductive biothiol, cysteine (Cys) directly regulates the occurrence of oxidative stress in the brain. Observations suggest that the decreased cysteine in plasma could potentially serve as a redox biomarker in temporal lobe epilepsy. However, due to the complexity of the brain and lack of appropriate in situ detecting tools, the concentration change and regulation of Cys in epileptic brains remain unclear. Here, we report a near-infrared imaging probe (named Mito-CP) for tracking endogenous Cys in brains of pentylenetetrazole (PTZ)-induced epileptic seizures with high sensitivity and selectivity. Using this probe, we achieved an in situ visualization of the increased Cys in PC12 cells under dithiothreitol stimulation. In addition, Mito-CP was able to real-time monitor Cys fluctuation in lipopolysaccharide-mediated oxidative stress in zebrafish. Ultimately, we directly visualized the precipitous reduction of Cys in epileptic brains for the first time. Mito-CP also revealed the fluctuation of Cys in epileptic brains during the treatment by an antiepileptic drug, curcumin. This work provides an effective tool for Cys imaging in brains and will help to expand the understanding of the pathogenesis of epilepsy.

Isoquinolinequinone N-oxides as anticancer agents effective against drug resistant cell lines.

The isoquinolinequinone (IQQ) pharmacophore is a privileged framework in known cytotoxic natural product families, caulibugulones and mansouramycins. Exploiting both families as a chemical starting point, we report on the structured development of an IQQ N-oxide anticancer framework which exhibits growth inhibition in the nM range across melanoma, ovarian and leukaemia cancer cell lines. A new lead compound (16, R6 = benzyl, R7 = H) exhibits nM GI50 values against 31/57 human tumour cell lines screened as part of the NCI60 panel and shows activity against doxorubicin resistant tumour cell lines. An electrochemical study highlights a correlation between electropositivity of the IQQ N-oxide framework and cytotoxicity. Adduct binding to sulfur based biological nucleophiles glutathione and cysteine was observed in vitro. This new framework possesses significant anticancer potential.

Synthesis and evaluation of 13C-labeled 5-5-dimethyl-1-pyrroline-N-oxide aimed at in vivo detection of reactive oxygen species using hyperpolarized 13C-MRI.

Effective means to identify the role of reactive oxygen species (ROS) mediating several diseases including cancer, ischemic heart disease, stroke, Alzheimer's and other inflammatory conditions in in vivo models would be useful. The cyclic nitrone 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) is a spin trap frequently used to detect free radicals in vitro using Electron Paramagnetic Resonance (EPR) spectroscopy. In this study, we synthesized 13C-labeled DMPO for hyperpolarization by dynamic nuclear polarization, in which 13C NMR signal increases more than 10,000-fold. This allows in vivo 13C MRI to investigate the feasibility of in vivo ROS detection by the 13C-MRI. DMPO was 13C-labeled at C5 position, and deuterated to prolong the T1 relaxation time. The overall yield achieved for 5-13C-DMPO-d9 was 15%. Hyperpolarized 5-13C-DMPO-d9 provided a single peak at 76 ppm in the 13C-spectrum, and the T1 was 60 s in phosphate buffer making it optimal for in vivo 13C MRI. The buffered solution of hyperpolarized 5-13C-DMPO-d9 was injected into a mouse placed in a 3 T scanner, and 13C-spectra were acquired every 1 s. In vivo studies showed the signal of 5-13C-DMPO-d9 was detected in the mouse, and the T1 decay of 13C signal of hyperpolarized 5-13C-DMPO-d9 was 29 s. 13C-chemical shift imaging revealed that 5-13C-DMPO-d9 was distributed throughout the body in a minute after the intravenous injection. A strong signal of 5-13C-DMPO-d9 was detected in heart/lung and kidney, whereas the signal in liver was small compared to other organs. The results indicate hyperpolarized 5-13C-DMPO-d9 provided sufficient 13C signal to be detected in the mouse in several organs, and can be used to detect ROS in vivo.

Investigation of efficiency of a novel, zinc oxide loaded TEMPO-oxidized cellulose nanofiber based hemostat for topical bleeding.

Lethal bleeding due to street accidents, natural calamities, orthopedic/dental surgeries, organ transplantation and household injuries, is the leading cause of morbidity and mortality. In the current study, zinc oxide (ZnO) was incorporated in TEMPO-oxidized cellulose nanofiber (TOCN) and polyethylene glycol (PEG) polymer system for hemorrhage control by freeze drying method. SEM and XRD data showed the presence of ZnO in the porous structure. FT-IR analysis showed that, successful conjugation occurs among the TOCN and PEG. The results revealed that, the incorporation of ZnO and higher concentrations of PEG increased the degradability but decreased swelling of the scaffolds. The increase in PEG content and ZnO incorporation significantly decreased the bleeding time in rabbit ear arterial bleeding model. Further, the incorporation of ZnO enhanced the antibacterial property of TOCN-PEG. The results suggested that excellent hemostatic and mechanical properties of the TOCN-5% PEG-ZnO might contribute in controlling bleeding and reducing post traumatic dermal bacterial infection.

Synthesis and identification of 2,4-bisanilinopyrimidines bearing 2,2,6,6-tetramethylpiperidine-N-oxyl as potential Aurora A inhibitors.

The Aurora kinases are a family of serine/threonine kinases that interact with components of the mitotic apparatus and serve as potential therapeutic targets in oncology. Herein, we reported a series of 2,4-bisanilinopyrimidines bearing 2,2,6,6-tetramethylpiperidine-N-oxyl with selective inhibition of Aurora A in either enzymatic assays or cellular phenotypic assays, and displaying more potent anti-proliferation compared with that of VX-680. The most potent compound 10a forms better interaction with Aurora A than Aurora B in molecular docking. Mechanistic studies revealed that 10a disrupt the spindle formation, block the cell cycle progression in the G2/M phase and induce apoptosis in HeLa cell. These results suggested that the produced series of compounds are potential anticancer agents for further development as selective Aurora A inhibitors.

A near-infrared heptamethine aminocyanine dye with a long-lived excited triplet state for photodynamic therapy.

A water-soluble near-infrared aminocyanine dye has been developed with a long triplet-state lifetime (τ = 9.16 µs in deaerated ethanol). Thereby, extremely high singlet oxygen quantum yield (ΦΔ = 0.20) and low dark cytotoxicity (IC50 = 715.4 µM) were achieved. The potential of the dye as a PDT photosensitizer was demonstrated.

Cholesterol-nitrone conjugates as protective agents against lipid oxidation: A model membrane study.

Free radical scavengers such as α-phenyl-N-tert-butylnitrone (PBN) have been widely used as protective agents in several biological models. We recently designed two PBN derivatives by adding a cholesterol moiety to the parent nitrone to increase its lipophilicity. In addition to the cholesterol, a sugar group was also grafted to enhance the hydrophilic properties at the same time. In the present work we report on the synthesis of a third derivative bearing only a cholesterol moiety and the physical chemical and antioxidant characterization of these three derivatives. We demonstrated they were able to form stable monolayers at the air/water interface and with the two derivatives bearing a sugar group, repulsive interactions with 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) were observed. We next investigated the interaction with DLPC on a liposome model. Fluorescence spectroscopy experiments showed the addition of a cholesterol moiety causes an ordering effect whereas the presence of the sugar group led to a disordering effect. The protective effect against lipid oxidation was then investigated using dynamic light scattering and the formation of conjugated dienes was quantified spectrophotometrically. Two oxidizing systems were tested, i.e. the AAPH-thermolysis which generates peroxyl radicals and the Fenton reagent which is responsible of the formation of hydroxyl radicals. Due to their membrane localization, the three cholesteryl-PBN derivatives are able to prevent lipid oxidation with the two types of radical inducers but with a different mode of action.

Noncovalent Immobilization of Molecular Electrocatalysts for Chemical Synthesis: Efficient Electrochemical Alcohol Oxidation with a Pyrene-TEMPO Conjugate.

Electrocatalytic methods for organic synthesis could offer sustainable alternatives to traditional redox reactions, but strategies are needed to enhance the performance of molecular catalysts designed for this purpose. The synthesis of a pyrene-tethered TEMPO derivative (TEMPO=2,2,6,6-tetramethylpiperidinyl-N-oxyl) is described, which undergoes facile in situ noncovalent immobilization onto a carbon cloth electrode. Cyclic voltammetry and controlled potential electrolysis studies demonstrate that the immobilized catalyst exhibits much higher activity relative to 4-acetamido-TEMPO, an electronically similar homogeneous catalyst. In preparative electrolysis experiments with a series of alcohol substrates and the immobilized catalyst, turnover numbers and frequencies approach 2 000 and 4 000 h-1 , respectively. The synthetic utility of the method is further demonstrated in the oxidation of a sterically hindered hydroxymethylpyrimidine precursor to the blockbuster drug, rosuvastatin.

Indole-TEMPO conjugates alleviate ischemia-reperfusion injury via attenuation of oxidative stress and preservation of mitochondrial function.

Mitochondrial oxidative damage contributes to a wide range of pathologies including ischemia/reperfusion injury. Accordingly, protecting mitochondria from oxidative damage should possess therapeutic relevance. In the present study, we have designed and synthesized a series of novel indole-TEMPO conjugates that manifested good anti-inflammatory properties in a murine model of xylene-induced ear edema. We have demonstrated that these compounds can protect cells from simulated ischemia/reperfusion (s-I/R)-induced reactive oxygen species (ROS) overproduction and mitochondrial dysfunction. Furthermore, we have demonstrated that indole-TEMPO conjugates can attenuate organ damage induced in rodents via intestinal I/R injury. We therefore propose that the pharmacological profile and mechanism of action of these indole-TEMPO conjugates involve convergent roles, including the ability to decrease free radical production via lipid peroxidation which couples to an associated decrease in ROS-mediated activation of the inflammatory process. We further hypothesize that the protective effects of indole-TEMPO conjugates partially reside in maintaining optimal mitochondrial function.

Ester of Quinoxaline-7-carboxylate 1,4-di-N-oxide as Apoptosis Inductors in K-562 Cell Line: An in vitro, QSAR and DFT Study.

BACKGROUND: Quinoxalines have shown a wide variety of biological activities including as antitumor agents. The aims of this study were to evaluate the activity of quinoxaline 1,4-di-N-oxide derivatives on K562 cells, the establishment of the mechanism of induced cell death, and the construction of predictive QSAR models. MATERIAL AND METHODS: Sixteen esters of quinoxaline-7-carboxylate 1,4-di-N-oxide were evaluated for antitumor activity on K562 chronic myelogenous leukemia cells and their IC50 values were determined. The mechanism of induced cell death by the most active molecule was assessed by flow cytometry and an in silico study was conducted to optimize and calculate theoretical descriptors of all quinoxaline 1,4-di-N-oxide derivatives. QSAR and QPAR models were created using genetic algorithms. RESULTS & CONCLUSIONS: Our results show that compounds C5, C7, C10, C12 and C15 had the lowest IC50 of the series. C15 was the most active compound (IC50= 3.02 µg/mL), inducing caspase-dependent apoptotic cell death via the intrinsic pathway. QSAR and QPAR studies are discussed.

Inhibition of fatty acid amide hydrolase by BIA 10-2474 in rat brain.

In a recent clinical trial, the drug BIA 10-2474, a putative fatty acid amide hydrolase(FAAH) inhibitor, was responsible for severe adverse events (SAEs), including one death. To date, there has been little reliable information divulged about the potency of BIA 10-2474 at FAAH in the central nervous system. We synthesised BIA 10-2474 and determined its ability to inhibit FAAH ex vivo in rat brain using a FAAH selective radiotracer. BIA 10-2474 proved to be a potent FAAH inhibitor with IC50s of 50-70 µg/kg (i.p.) in various brain regions. This information may be useful for determining the cause of the SAEs.

Novel Synthetic PEGylated Conjugate of α-Lipoic Acid and Tempol Reduces Cell Death in a Neuronal PC12 Clonal Line Subjected to Ischemia.

α-Lipoic acid (α-LA), a natural thiol antioxidant, and Tempol, a synthetic free radical scavenger, are known to confer neuroprotection following ischemic insults in both in vivo and in vitro models. The aim of this study was to synthesize and characterize a conjugate of α-LA and Tempol linked by polyethylene glycol (PEG) in order to generate a more efficacious neuroprotectant molecule. AD3 (α-Tempol ester-ω-lipo ester PEG) was synthesized, purified, and characterized by flash chromatography and reverse phase high pressure liquid chromatography and by 1H nuclear magnetic resonance, infrared spectroscopy, and mass spectrometry. AD3 conferred neuroprotection in a PC12 pheochromocytoma cell line of dopaminergic origin, exposed to oxygen and glucose deprivation (OGD) insult measured by LDH release. AD3 exhibited EC50 at 10 µM and showed a 2-3-fold higher efficacy compared to the precursor moieties, indicating an intrinsic potent neuroprotective activity. AD3 attenuated by 25% the intracellular redox potential, by 54% lipid peroxidation and prevented phosphorylation of ERK, JNK, and p38 by 57%, 22%, and 21%, respectively. Cumulatively, these findings indicate that AD3 is a novel conjugate that confers neuroprotection by attenuation of MAPK phosphorylation and by modulation of the redox potential of the cells.

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.

Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with ~17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.