Radical-Induced Changes in Transition Metal Interfacial Magnetic Properties: A Blatter Derivative on Polycrystalline Cobalt.

In this work, we study the interface obtained by depositing a monolayer of a Blatter radical derivative on polycrystalline cobalt. By examining the occupied and unoccupied states at the interface, using soft X-ray techniques, combined with electronic structure calculations, we could simultaneously determine the electronic structure of both the molecular and ferromagnetic sides of the interface, thus obtaining a full understanding of the interfacial magnetic properties. We found that the molecule is strongly hybridized with the surface. Changes in the core level spectra reflect the modification of the molecule and the cobalt electronic structures inducing a decrease in the magnetic moment of the cobalt atoms bonded to the molecules which, in turn, lose their radical character. Our method allowed us to screen, beforehand, organic/ferromagnetic interfaces in view of their potential applications in spintronics.

Chiral π-Conjugated Double Helical Aminyl Diradical with the Triplet Ground State.

We report a neutral high-spin diradical of chiral C2-symmetric bis[5]diazahelicene with ΔEST ≈ 0.4 kcal mol-1, as determined by EPR spectroscopy/SQUID magnetometry. The diradical is the most persistent among all high-spin aminyl radicals reported to date by a factor of 20, with a half-life of up to 6 days in 2-MeTHF at room temperature. Its triplet ground state and excellent persistence may be associated with the unique spin density distribution within the dihydrophenazine moiety, which characterizes two effective 3-electron C-N bonds analogous to the N-O bond of a nitroxide radical. The enantiomerically enriched (ee ≥ 94%) (MM)- and (PP)-enantiomers of the precursors to the diradicals are obtained by either preparative chiral supercritical fluid chromatography or resolution via functionalization with the chiral auxiliary of the C2-symmetric racemic tetraamine. The barrier for the racemization of the solid tetraamine is ΔG‡ = 43 ± 0.01 kcal mol-1 in the 483-523 K range. The experimentally estimated lower limit of the barrier for the racemization of a diradical, ΔG‡ ≥ 26 kcal mol-1 in 2-MeTHF at 293 K, is comparable to the DFT-determined barrier of ΔG‡ = 31 kcal mol-1 in the gas phase at 298 K. While the enantiomerically pure tetraamine displays strong chiroptical properties, with anisotropy factor |g| = |Δε|/ε = 0.036 at 376 nm, |g| ≈ 0.005 at 548 nm of the high-spin diradical is comparable to that recently reported triplet ground-state diradical dication. Notably, the radical anion intermediate in the generation of diradical exhibits a large SOMO-HOMO inversion, SHI = 35 kcal mol-1.

Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 × 106 M-1, is at least 1 order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase in the rotational correlation time, τrot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τrot and electron spin coherence time, Tm, along with the narrowing of interspin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4-9, compared to the common spin label such as MTSL, which is not affected by CB-7. Interspin distances of 3 nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating an undisturbed structure and conformation of the protein.

From Stable Radicals to Thermally Robust High-Spin Diradicals and Triradicals.

Stable radicals and thermally robust high-spin di- and triradicals have emerged as important organic materials due to their promising applications in diverse fields. New fundamental properties, such as SOMO/HOMO inversion of orbital energies, are explored for the design of new stable radicals, including highly luminescent ones with good photostability. A relation with the singlet-triplet energy gap in the corresponding diradicals is proposed. Thermally robust high-spin di- and triradicals, with energy gaps that are comparable to or greater than a thermal energy at room temperature, are more challenging to synthesize but more rewarding. We summarize a number of high-spin di- and triradicals, based on nitronyl nitroxides that provide a relation between the experimental pairwise exchange coupling constant J/k in the high-spin species vs experimental hyperfine coupling constants in the corresponding monoradicals. This relation allows us to identify outliers, which may correspond to radicals where J/k is not measured with sufficient accuracy. Double helical high-spin diradicals, in which spin density is delocalized over the chiral π-system, have been barely explored, with the sole example of such high-spin diradical possessing alternant π-system with Kekulé resonance form. Finally, we discuss a high-spin diradical with electrical conductivity and derivatives of triangulene diradicals.

Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 × 106 M-1, is at least one order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase of the rotational correlation time, τrot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τrot and electron spin coherence time, Tm, along with the narrowing of inter-spin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4 - 9, compared to the common spin label such as MTSL, which is not affected by CB-7. Inter-spin distances of 3-nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating undisturbed structure and conformation of the protein.

Thermally Ultrarobust S = 1/2 Tetrazolinyl Radicals: Synthesis, Electronic Structure, Magnetism, and Nanoneedle Assemblies on Silicon Surface.

Open-shell organic molecules, including S = 1/2 radicals, may provide enhanced properties for several emerging technologies; however, relatively few synthesized to date possess robust thermal stability and processability. We report the synthesis of S = 1/2 biphenylene-fused tetrazolinyl radicals 1 and 2. Both radicals possess near-perfect planar structures based on their X-ray structures and density-functional theory (DFT) computations. Radical 1 possesses outstanding thermal stability as indicated by the onset of decomposition at 269 °C, based on thermogravimetric analysis (TGA) data. Both radicals possess very low oxidation potentials <0 V (vs. SCE) and their electrochemical energy gaps, Ecell ≈ 0.9 eV, are rather low. Magnetic properties of polycrystalline 1 are characterized by superconducting quantum interference device (SQUID) magnetometry revealing a one-dimensional S = 1/2 antiferromagnetic Heisenberg chain with exchange coupling constant J'/k ≈ -22.0 K. Radical 1 in toluene glass possesses a long electron spin coherence time, Tm ≈ 7 µs in the 40-80 K temperature range, a property advantageous for potential applications as a molecular spin qubit. Radical 1 is evaporated under ultrahigh vacuum (UHV) forming assemblies of intact radicals on a silicon substrate, as confirmed by high-resolution X-ray photoelectron spectroscopy (XPS). Scanning electron microscope (SEM) images indicate that the radical molecules form nanoneedles on the substrate. The nanoneedles are stable for at least 64 hours under air as monitored by using X-ray photoelectron spectroscopy. Electron paramagnetic resonance (EPR) studies of the thicker assemblies, prepared by UHV evaporation, indicate radical decay according to first-order kinetics with a long half-life of 50 ± 4 days at ambient conditions.

Long-Term Degradation Mechanisms in Application-Implemented Radical Thin Films.

Blatter radical derivatives are very attractive due to their potential applications, ranging from batteries to quantum technologies. In this work, we focus on the latest insights regarding the fundamental mechanisms of radical thin film (long-term) degradation, by comparing two Blatter radical derivatives. We find that the interaction with different contaminants (such as atomic H, Ar, N, and O and molecular H2, N2, O2, H2O, and NH2) affects the chemical and magnetic properties of the thin films upon air exposure. Also, the radical-specific site, where the contaminant interaction takes place, plays a role. Atomic H and NH2 are detrimental to the magnetic properties of Blatter radicals, while the presence of molecular water influences more specifically the magnetic properties of the diradical thin films, and it is believed to be the major cause of the shorter diradical thin film lifetime in air.

Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids.

Site-directed spin-labeling (SDSL)─in combination with double electron-electron resonance (DEER) spectroscopy─has emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. DEER combined with in situ SDSL in live cells is challenging since current bioorthogonal labeling approaches are too slow to allow for complete labeling with low concentrations of spin label prior to loss of signal from cellular reduction. Here, we overcome this limitation by genetically encoding a novel family of small, tetrazine-bearing noncanonical amino acids (Tet-v4.0) at multiple sites in proteins expressed in Escherichia coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans-cyclooctene (sTCO)-functionalized nitroxides─including a gem-diethyl-substituted nitroxide with enhanced stability in cells─with rate constants that can exceed 106 M-1 s-1. The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro. Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and support assignment of the conformational state of an MBP mutant within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.

Aromatic C-C Bond Cleavage in a Curved π-System of Aminocorannulene.

We report a metal- and oxidant-free aromatic C-C bond cleavage in the curved corannulene skeleton. Reaction of 1-aminocorannulene with hydrazonyl chloride generates an amidrazone intermediate that undergoes facile intramolecular proton migrations and ring annulation to give a 1,2,4-triazole derivative of planar benzo[ghi]fluoranthene, in which the release of strain associated with the curved π-surface and the formation of an aromatic triazole moiety are the driving forces. This report provides new insights into the aromatic C-C bond cleavage.

Ultra-Fast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids.

Studying protein structures and dynamics directly in the cellular environments in which they function is essential to fully understand the molecular mechanisms underlying cellular processes. Site-directed spin-labeling (SDSL)-in combination with double electron-electron resonance (DEER) spectroscopy-has emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. In-cell DEER spectroscopy on proteins in mammalian cells has thus far not been possible due to the notable challenges of spin-labeling in live cells. In-cell SDSL requires exquisite biorthogonality, high labeling reaction rates and low background signal from unreacted residual spin label. While the bioorthogonal reaction must be highly specific and proceed under physiological conditions, many spin labels display time-dependent instability in the reducing cellular environment. Additionally, high concentrations of spin label can be toxic. Thus, an exceptionally fast bioorthogonal reaction is required that can allow for complete labeling with low concentrations of spin-label prior to loss of signal. Here we utilized genetic code expansion to site-specifically encode a novel family of small, tetrazine-bearing non-canonical amino acids (Tet-v4.0) at multiple sites in green fluorescent protein (GFP) and maltose binding protein (MBP) expressed both in E. coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans -cyclooctene (sTCO)-functionalized nitroxides-including a gem -diethyl-substituted nitroxide with enhanced stability in cells-with rate constants that can exceed 10 6 M -1 s -1 . The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live HEK293T cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide added directly to the culture medium. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro . Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and successfully discerned the conformational state of MBP within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.

High-Spin S = 3/2 Ground-State Aminyl Triradicals: Toward High-Spin Oligo-Aza Nanographenes.

We report high-spin aminyl triradicals with near-planar triphenylene backbones. Near-planarity of the fused aminyl radicals and the 2,6,10-triphenylene ferromagnetic coupling unit (FCU), magnetically equivalent to three fused 3,4'-biphenyl FCUs, assures an effective 2pπ-2pπ overlap within the cross-conjugated π-system, leading to an S = 3/2 (quartet) ground state that is well separated from low-spin excited doublet states. Thermal populations of the low-spin (S = 1/2) excited states are detectable both by SQUID magnetometry and electron paramagnetic resonance (EPR) spectroscopy, providing doublet-quartet energy gaps, ΔEDQ, corresponding to >85% population of the quartet ground states at room temperature. Notably, EPR-based determination of ΔEDQ relies on direct detection of the quartet ground state and doublet excited states. The ΔEDQ values are 1.0-1.1 kcal mol-1, with the more sterically shielded triradical having the larger value. The half-life of the more sterically shielded triradical in 2-methyltetrahydrofuran (2-MeTHF) is about 6 h at room temperature. The less sterically shielded triradical in 2-MeTHF decomposes at 158 K with a half-life of about 4 h, while at 195 K, the half-life is still about 2 h. The dominant products of the decay of triradicals are the corresponding triamines, suggesting hydrogen atom abstraction from the solvent as the primary mechanism. This study expands the frontier of the open-shell PAHs/nanographenes, of which the unique electronic, nonlinear optical, and magnetic properties could be useful in the development of novel organic electronics, photonics, and spintronics.

High-Spin (S = 1) Blatter-Based Diradical with Robust Stability and Electrical Conductivity.

Triplet ground-state organic molecules are of interest with respect to several emerging technologies but usually show limited stability, especially as thin films. We report an organic diradical, consisting of two Blatter radicals, that possesses a triplet ground state with a singlet-triplet energy gap, ΔEST ≈ 0.4-0.5 kcal mol-1 (2J/k ≈ 220-275 K). The diradical possesses robust thermal stability, with an onset of decomposition above 264 °C (TGA). In toluene/chloroform, glassy matrix, and fluid solution, an equilibrium between two conformations with ΔEST ≈ 0.4 kcal mol-1 and ΔEST ≈ -0.7 kcal mol-1 is observed, favoring the triplet ground state over the singlet ground-state conformation in the 110-330 K temperature range. The diradical with the triplet ground-state conformation is found exclusively in crystals and in a polystyrene matrix. The crystalline neutral diradical is a good electrical conductor with conductivity comparable to the thoroughly optimized bis(thiazolyl)-related monoradicals. This is surprising because the triplet ground state implies that the underlying π-system is cross-conjugated and thus is not compatible with either good conductance or electron delocalization. The diradical is evaporated under ultra-high vacuum to form thin films, which are stable in air for at least 18 h, as demonstrated by X-ray photoelectron and electron paramagnetic resonance (EPR) spectroscopies.

Bis-Spiro-Oxetane and Bis-Spiro-Tetrahydrofuran Pyrroline Nitroxide Radicals: Synthesis and Electron Spin Relaxation Studies.

Synthesis of bis-spiro-oxetane and bis-spiro-tetrahydrofuran pyrroline nitroxide radicals relies on the Mitsunobu reaction-mediated double cyclizations of N-Boc protected pyrroline tetraols. Structures of the nitroxide radicals are supported by X-ray crystallography. In a trehalose/sucrose matrix at room temperature, the bis-spiro-oxetane nitroxide radical possesses electron spin coherence time, Tm ≈ 0.7 µs. The observed enhanced Tm is most likely associated with strong hydrogen bonding of oxetane moieties to the trehalose/sucrose matrix.

Thiophene-Based Double Helices: Radical Cations with SOMO-HOMO Energy Level Inversion.

We report relatively persistent, open-shell thiophene-based double helices, radical cations 1•+ -TMS12 and 2•+ -TMS8 . Closed-shell neutral double helices, 1-TMS12 and 2-TMS8 , have nearly identical first oxidation potentials, E +/0 ≈ +1.33 V, corresponding to reversible oxidation to their radical cations. The radical cations are generated, using tungsten hexachloride in dichloromethane (DCM) as an oxidant, E +/0 ≈ +1.56 V. EPR spectra consist of a relatively sharp singlet peak with an unusually low g-value of 2.001-2.002, thus suggesting exclusive delocalization of spin density over π-conjugated system consisting of carbon atoms only. DFT computations confirm these findings, as only negligible fraction of spin density is found on sulfur and silicon atoms and the spin density is delocalized over a single tetrathiophene moiety. For radical cation, 1•+ -TMS12 , energy level of the singly occupied molecular orbital (SOMO) lies below the four highest occupied molecular orbitals (HOMOs), thus indicating the SOMO-HOMO inversion (SHI) and therefore, violating the Aufbau principle. 1•+ -TMS12 has a half-life of the order of only 5 min at room temperature. EPR peak intensity of 2•+ -TMS8 , which does not show SHI, is practically unchanged over at least 2 h.

Synthesis and Thin Films of Thermally Robust Quartet (S = 3/2) Ground State Triradical.

High-spin (S = 3/2) organic triradicals may offer enhanced properties with respect to several emerging technologies, but those synthesized to date typically exhibit small doublet quartet energy gaps and/or possess limited thermal stability and processability. We report a quartet ground state triradical 3, synthesized by a Pd(0)-catalyzed radical-radical cross-coupling reaction, which possesses two doublet-quartet energy gaps, ΔEDQ ≈ 0.2-0.3 kcal mol-1 and ΔEDQ2 ≈ 1.2-1.8 kcal mol-1. The triradical has a 70+% population of the quartet ground state at room temperature and good thermal stability with onset of decomposition at >160 °C under an inert atmosphere. Magnetic properties of 3 are characterized by SQUID magnetometry in polystyrene glass and by quantitative EPR spectroscopy. Triradical 3 is evaporated under ultrahigh vacuum to form thin films of intact triradicals on silicon substrate, as confirmed by high-resolution X-ray photoelectron spectroscopy. AFM and SEM images of the ∼1 nm thick films indicate that the triradical molecules form islands on the substrate. The films are stable under ultrahigh vacuum for at least 17 h but show onset of decomposition after 4 h at ambient conditions. The drop-cast films are less prone to degradation in air and have a longer lifetime.

Supramolecular Approach to Electron Paramagnetic Resonance Distance Measurement of Spin-Labeled Proteins.

We demonstrate a host-guest molecular recognition approach to advance double electron-electron resonance (DEER) distance measurements of spin-labeled proteins. We synthesized an iodoacetamide derivative of 2,6-diazaadamantane nitroxide (DZD) spin label that could be doubly incorporated into T4 Lysozyme (T4L) by site-directed spin labeling with efficiency up to 50% per cysteine. The rigidity of the fused ring structure and absence of mobile methyl groups increase the spin echo dephasing time (Tm) at temperatures above 80 K. This enables DEER measurements of distances >4 nm in DZD-labeled T4L in glycerol/water at temperatures up to 150 K with increased sensitivity compared to that of a common spin label such as MTSL. Addition of ß-cyclodextrin reduces the rotational correlation time of the label, slightly increases Tm, and most importantly, narrows (and slightly lengthens) the interspin distance distributions. The distance distributions are in good agreement with simulated distance distributions obtained by rotamer libraries. These results provide a foundation for developing supramolecular recognition to facilitate long-distance DEER measurements at near physiological temperatures.

Pro-organic radical contrast agents ("pro-ORCAs") for real-time MRI of pro-drug activation in biological systems.

Nitroxide-based organic-radical contrast agents (ORCAs) are promising as safe, next-generation magnetic resonance imaging (MRI) tools. Nevertheless, stimuli-responsive ORCAs that enable MRI monitoring of prodrug activation have not been reported; such systems could open new avenues for prodrug validation and image-guided drug delivery. Here, we introduce a novel "pro-ORCA" concept that addresses this challenge. By covalent conjugation of nitroxides and drug molecules (doxorubicin, DOX) to the same brush-arm star polymer (BASP) through chemically identical cleavable linkers, we demonstrate that pro-ORCA and prodrug activation, i.e., ORCA and DOX release, leads to significant changes in MRI contrast that correlate with cytotoxicity. This approach is shown to be general for a range of commonly used linker cleavage mechanisms (e.g., photolysis and hydrolysis) and release rates. Pro-ORCAs could find applications as research tools or clinically viable "reporter theranostics" for in vitro and in vivo MRI-correlated prodrug activation.

High-Spin Diradical Dication of Chiral π-Conjugated Double Helical Molecule.

We report an air-stable diradical dication of chiral D2-symmetric conjoined bis[5]diazahelicene with an unprecedented high-spin (triplet) ground state, singlet triplet energy gap, ΔEST = 0.3 kcal mol-1. The diradical dication possesses closed-shell (Kekulé) resonance forms with 16 π-electron perimeters. The diradical dication is monomeric in dibutyl phthalate (DBP) matrix at low temperatures, and it has a half-life of more than 2 weeks at ambient conditions in the presence of excess oxidant. A barrier of ∼35 kcal mol-1 has been experimentally determined for inversion of configuration in the neutral conjoined bis[5]diazahelicene, while the inversion barriers in its radical cation and diradical dication were predicted by the DFT computations to be within a few kcal mol-1 of that in the neutral species. Chiral HPLC resolution provides the chiral D2-symmetric conjoined bis[5]diazahelicene, enriched in (P,P)- or (M,M)-enantiomers. The enantiomerically enriched triplet diradical dication is configurationally stable for 48 h at room temperature, thus providing the lower limit for inversion barrier of configuration of 27 kcal mol-1. The enantiomers of conjoined bis[5]diazahelicene and its diradical dication show strong chirooptical properties that are comparable to [6]helicene or carbon-sulfur [7]helicene, as determined by the anisotropy factors, |g| = |Δε|/ε = 0.007 at 348 nm (neutral) and |g| = 0.005 at 385 nm (diradical dication). DFT computations of the radical cation suggest that SOMO and HOMO energy levels are near-degenerate.

A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels.

Bax is a Bcl-2 protein crucial for apoptosis initiation and execution, whose active conformation is only partially understood. Dipolar EPR spectroscopy has proven to be a valuable tool to determine coarse-grained models of membrane-embedded Bcl-2 proteins. Here we show how the combination of spectroscopically distinguishable nitroxide and gadolinium spin labels and Double Electron-Electron Resonance can help to gain new insights into the quaternary structure of active, membrane-embedded Bax oligomers. We show that attaching labels bulkier than the conventional MTSL may affect Bax fold and activity, depending on the protein/label combination. However, we identified a suitable pair of spectroscopically distinguishable labels, which allows to study complex distance networks in the oligomers that could not be disentangled before. Additionally, we compared the stability of the different spin-labeled protein variants in E. coli and HeLa cell extracts. We found that the gem-diethyl nitroxide-labeled Bax variants were reasonably stable in HeLa cell extracts. However, when transferred into human cells, Bax was found to be mislocalized, thus preventing its characterization in a physiological environment. The successful use of spectroscopically distinguishable labels on membrane-embedded Bax-oligomers opens an exciting new path towards structure determination of membrane-embedded homo- or hetero-oligomeric Bcl-2 proteins via EPR.

gem-Diethyl Pyrroline Nitroxide Spin Labels: Synthesis, EPR Characterization, Rotamer Libraries and Biocompatibility.

The availability of bioresistant spin labels is crucial for the optimization of site-directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins' fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin-labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide- and maleimide-functionalized spin labels based on the gem-diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem-dimethyl analogs by site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the "true" biocompatibility of the gem-diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations.