Artem A Babaryk

Mohamed Haouas

Olena A Khaynakova

Erik Elkäim

Patricia Horcajada

Bis-3,5-Diamino-1,2,4-Triazolyl-1,2,4,5-Tetrazine: From Insensitive High Energy Density Materials to Small Molecule Organic Semiconductors

Keywords: Small Molecule Organic Semiconductor, Crystal Structure, Powder X-ray Diffraction, Solid

 C, and 15 N solid state nuclear magnetic resonance (NMR) spectroscopy. We demonstrate that IEF-12 has features of small molecule organic semiconductor, namely, lamellar π-stacked crystal packing in solid state, low energy optical bandgap = 2.27 eV and relatively high value of dielectric constant εr = 4.86. Overall, we exemplified on EIF-12 that insensitive high energy density materials (HEDM) may open an avenue for non-classically structured organic semiconductors, paving the way for optoelectronic and photovoltaic applications.

INTRODUCTION

Electron deficiency of 1,2,4,5-tetrazine or, more commonly, s-tetrazine results in unusual chemical and physical properties of generic compounds. In particular, 3,6-substituted s-tetrazines with a high-nitrogen content have attracted significant attention from researchers, due to their potential use as high energy density materials (HEDM) in civil and military pyrotechnic domains (e.g. automotive airbags, fireplaces, safety matches, oxygen candles, fireworks, igniters, ammunitions, gas-pressure blasting in mining, quarrying, and demolition). [START_REF] Gao | Azole-Based Energetic Salts[END_REF] This circumstance somewhat dwarfs other uses of s-tetrazine molecular materials for different industrially and societally relevant application domains. For instance, s-tetrazines can reversibly be reduced to stable anion-radicals, making them promising materials for electrochemical energy storage. [START_REF] Clavier | s-Tetrazines as Building Blocks for New Functional Molecules and Molecular Materials[END_REF] From the other hand, they exhibit a well-defined π-π* transition located at the UV region, which energy depends on the nature of 3,6-substituents. [START_REF] Gong | Synthesis and Physical Chemistry of s-Tetrazines: Which Ones are Fluorescent and Why?[END_REF] Coupling of both properties is successfully utilized for the fabrication of electroactive polymeric materials [START_REF] Audebert | First Example of an Electroactive Polymer Issued from an Oligothiophene Substituted Tetrazine[END_REF] and applied for photocatalysis of organic reactions. [START_REF] Samanta | Photocatalysis by 3,6-Disubstituted-s-Tetrazine: Visible-Light Driven Metal-Free Green Synthesis of 2-Substituted Benzimidazole and Benzothiazole[END_REF][START_REF] Samanta | 3,6-Di(pyridin-2-yl)-1,2,4,5-Ttetrazine (pytz) Mediated Metal-free Mild Oxidation of Thiols to Disulfides in Aqueous Medium[END_REF] Despite their inherent instable character, the conjugation of azole rings with the tetrazine core at para-position leads to remarkably high stable derivatives. For instance, Chavez & Hiskey reported the synthesis and propellant properties of the bis(tetrazolylaminotetrazine), with a low impact sensitivity, non-explosive, non-pyrotechnic and nonflammable character. 7 Kläpotke et al. [START_REF] Kläpotke | 3,6-Disubstituted 1,2,4,5-Tetrazines[END_REF] explored the nucleophilic substitution in s-tetrazine using high electrophilic properties of 3,6dichloro-1,2,4,5-tetrazine to obtain a series of five new HEDMs. Amongst them, bis-3,5diamino-1,2,4-triazolyl-1,2,4,5-tetrazine (BDTT) was distinguished by its outstanding thermal stability (> 370 °C). Further, BDTT has one order of magnitude greater heat of formation than the well-known space rocket propellant hexanitrostilbene, owing to the presence of highly endothermic N-N and C-N bonds, as it was assumed. However, it demonstrates 8-fold less sensitivity towards impact stimulus, probably due to an absence of any exothermic bonds as well.

From 80s, undoped organic semiconductors have been attracting the researchers' interest due to their application in photovoltaic cells, incorporating an organic hetero-junction of p-and nconducting materials, organic light-emitting devices (OLEDs) or logic circuits with organic field-effect transistors (OFETs) n-conducting materials. It has been shown that small molecules with acceptor-donor-acceptor structure, containing electron-accepting tetrazine moiety and electron-donating oligothiophenes, could be used as components of organic photovoltaic solar cells with efficacies as high as 3.24% at Jsc of 7.87 mAcm -2 . 9 n-Semiconducting halophenyltriazines were computationally and experimentally studied, yielding to relatively moderate charge mobilities (0.006-0.026 cm 2 V -1 s -1 ), [START_REF] Moral | Electronic Structure and Charge Transport Properties of a Series of 3,6-(Diphenyl)-s-tetrazine Derivatives: Are They Suitable Candidates for Molecular Electronics?[END_REF] that could be improved by switching to bis(aryleneethynylene)-s-tetrazines (up to 0.3-1.3 cm 2 V -1 s -1 ). [START_REF] Moral | Bis(arylene-ethynylene)-s-tetrazines: A Promising Family of n-Type Organic Semiconductors?[END_REF] Note here that the latter values are very promising on light organic photovoltaics.

BDTT crystal structure (named IEF-12, IEF for IMDEA Energy Framework) was obtained as a bulk dark-red precipitate, exhibiting optical absorption in the range of 1.5-2.5 eV. These values are typical for π-π* transitions in organic semiconducting materials (OSM) like, for instance in pentacene, a classical long-standing and well-studied representative OSM. Considering the fact that van der Waals forces have major importance for molecular packing in crystalline organic solids, [START_REF] Wang | Semiconducting π-Conjugated Systems in Field-Effect Transistors: A Material Odyssey of Organic Electronics[END_REF] the delocalization of electronic wavefunctions over adjacent molecules has important direct implications on charge carrier transport. Precisely, the transfer integral value is responsible for degenerating of highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) into zones, similarly to conventional inorganic semiconductors. When a charge carrier passes through a molecule, it losses some portion of energy (reorganization energy) which, in due course, depends on the conjugation length, degree, and packing of the organic molecules in the crystal. The very low solubility of EIF-12 suggests the presence of strong intermolecular interactions, contributing to the general cohesion of the crystal and, hence, knowledge of the spatial organization is a precondition for the rational selection of OSMs for optoelectronic applications. The typical fashions of π-bonded systems arrangement are schematically given in Figure 1. It is believed that the most efficient arrangement, in terms of the higher charge transfer integral and the lesser reorganization energy, is the gliding π-stacked lamellar motif (Fig. 1d). In this paper, the synthesis of IEF-12 structure was successfully replicated to elucidate the spatial organization and bulk electronic properties, proving to be interesting candidates not only for traditional HEDM application fields (pyrotechnics), but also for emerging OSMs (e.g. OLEDs, OFETs, solar cells). We report the accurate ab initio determination of the BDTT crystal structure (IEF-12) by high resolution powder X ray diffraction (PXRD) and 1 H, [START_REF] Madsen | Collection and Analysis of Powder Diffraction Data with Near-Constant Counting Statistics[END_REF] C, and [START_REF] Lee | Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron-Density[END_REF] N solid state nuclear magnetic resonance (NMR). IEF-12 structure is intrinsically lamellar due to the net of strongly H-bonded molecules, facilitating additional π-bonding between lateral donor amino groups of tetrazolyl substituent and tetrazine core acceptor. This is the first structural determination of s-tetrazine based HEMD, free of crystallization solvent. We put into agreement the optical bandgap measured by UV-vis spectroscopy with electronic band structure calculations. In addition, the static dielectric constant of IEF-12 was determined by the density functional perturbation theory calculations calibrated on the Fourier transformed infrared (FTIR) and Raman spectroscopies. Further, we rationalize here the low solubility of the compound from the post-refinement crystal structure analysis and establish a tentative charge transfer pathway from the interplay of solid and solute state (wherever it is possible) using a combination of UVvis and 1 H NMR spectroscopies in solution and time-dependent density functional theory (TD-DFT) molecular simulations.

MATERIALS AND METHODS

Synthesis. IEF-12 was synthesized using a slightly modified protocol previously reported by Kläpotke et al. [START_REF] Kläpotke | 3,6-Disubstituted 1,2,4,5-Tetrazines[END_REF] Briefly, 3,5-diamino-1,2,4-triazole (DATR; 1 g, 10.08 mmol) was dissolved in 25 mL of N,N'-dimethylformamide (DMF) at a room temperature (RT) using extensive alternate sonication and stirring until bulky crystals of DATR completely disappeared, yielding paleyellow solution. Then, a solution of dichlorotetrazine (0.377 g, 2.50 mmol) in 5 mL of acetonitrile was added dropwisely for 0.5 h. The product started to precipitate immediately. The suspension was stirred for 2 h at RT and then, refluxed at 120 °C overnight. After filtration, the product was successively washed with large amounts of DMF (about 200 mL, until the washing residues turned completely colorless), 150 mL of distilled water and 150 mL of acetonitrile to obtain 0.9 g (5.07 mmol, yield ∼ 86 wt%) of IEF-12, a deep-red fine powder as it was confirmed by scanning electron microscopy (SEM; see Figure S1 in the Supporting Information-SI). Final product was dried at open air and then, dried at 60 °C in an air-ventilated oven.

Powder X-ray diffraction (PXRD). About 50 mg of IEF-12 were loaded in a 1.0×20 mm borosilicate glass capillary (Hilgenberg GmbH). The data were collected in the parallel beam geometry using a Bruker D8 Advance system equipped with Cu-anode tube, Göbbel mirror, 0.2 mm collimating slit and LynxEye PSD detector, at ambient conditions. The variable counting time strategy [START_REF] Madsen | Collection and Analysis of Powder Diffraction Data with Near-Constant Counting Statistics[END_REF] was chosen to improve the quality of the experimental data for the structure determination process [START_REF] Shankland | Routine Ab Initio Structure Determination of Chlorothiazideby X-ray Powder Diffraction Using Optimised Data Collection and Analysis Strategies[END_REF] and statistically meaningful dataset of sinΘ•λ -1 = 0.77 Å -1 resolution was collected.

The high-resolution synchrotron data were collected at the CRISTAL beamline (SOLEIL, Saint-Aubin, France) with Si(111)-monochromated beam and the wavelength of λ = 0.7289 Å.

The capillary specimen was mounted on a 2-circle diffractometer and linear PSD Mythen (PSI, Switzerland) detector at RT. UV-vis Spectroscopy. UV-vis data were collected using a Perkin-Elmer spectrometer equipped with a tungsten-krypton light source. The spectra of dimethylsulfoxide (DMSO) solutions (1×10 -7 M) were recorded at a RT (10 mm path length). Solid state measurements were performed relatively to BaSO4 using a Praying Mantis attachment with an integrating sphere to measure diffuse reflectance. The measurements on the powder sample consisted of a small amount of powder pressed into a micro sample holder, which was exposed to light of 250-800 nm wavelengths segment. The Kubelka-Munk function F(R) was calculated from the diffuse reflectance measurements R, using the equation F(R) = (1-R) 2 (2R) -1 . Tauc plots were then plotted using (hνF(R)) n = f(hν) where n = 1/2 and 2 for direct and indirect transitions, respectively.

Solid State Nuclear Magnetic Resonance. Solid-state NMR spectra were recorded on Bruker

Avance II spectrometer equipped with a 11.7 T wide-bore superconducting magnet. Samples were packed into 2.5 mm zirconia rotors and rotated at the magic angle spinning (MAS) at 30 kHz for 1 H or at 10 KHz for [START_REF] Madsen | Collection and Analysis of Powder Diffraction Data with Near-Constant Counting Statistics[END_REF] C and [START_REF] Lee | Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron-Density[END_REF] N. [START_REF] Gao | Azole-Based Energetic Salts[END_REF] H NMR spectra were recorded using one-pulse sequence with signal averaging for 8 transients with a repeat interval of 55 s. [START_REF] Madsen | Collection and Analysis of Powder Diffraction Data with Near-Constant Counting Statistics[END_REF] C and [START_REF] Lee | Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron-Density[END_REF] N NMR spectra were recorded using cross-polarization (CP) from 1 H with a spin lock pulse (ramped for 1 H) of 10 ms. SPINAL-64 decoupling of 1 H (ν1 = 50 kHz) was applied during acquisition. Signal averaging was carried out for 1024 transients for [START_REF] Madsen | Collection and Analysis of Powder Diffraction Data with Near-Constant Counting Statistics[END_REF] C and 8704 transients for [START_REF] Lee | Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron-Density[END_REF] N with a repeat interval of 7 s.

Chemical shifts are reported relative to tetramethylsilane (TMS; 1 H and 13 C) and nitromethane CH3NO2; 15 N), using secondary solid references of adamantane (CH2 = 1.7 ppm, and CH2 = 38.5 ppm) and histidine (Nε2 = -212.0 ppm).

Vibrational Spectroscopy. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectrum was recorded using a Nicolet 6700 instrument. Three consequent scans were collected with 128 accumulations per scan in a range of 500-4000 cm -1 and averaged to minimize experimental signal errors.

Raman spectra were recorded with a JASCO NRS-5100 confocal Raman microscope using glass plate. A Nd:YAG laser excitation was operated at 0.2 mW power and Stokes Raman scattering was monitored with a Peltier cooled CCD detector in the range of 50-3650 cm -1 .

Computational details.

Gas Phase Calculations. Unconstrained structures of geometric isomers were optimized using density functional theory (DFT) method B3LYP hybrid functional [START_REF] Lee | Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron-Density[END_REF] together with 6-311++G(d,p) Pople basis set. [START_REF] Krishnan | Self-Consistent Molecular Orbital Methods. XX. A Basis Set for Correlated Wave Functions[END_REF] Frequency calculations were performed following geometry optimizations to ensure minimum-energy structures. Orbital and spectral predictions included the solute molecule density model (SMD) for dimethylsulfoxide (DMSO) solvation (ε = 47.2). All the excitation energies were converged at the cc-pVQZ basis set. [START_REF] Woon | Gaussian Basis Sets for Use in Correlated Molecular Calculations. III. The Atoms Aluminum through Argon[END_REF] 1 H, [START_REF] Madsen | Collection and Analysis of Powder Diffraction Data with Near-Constant Counting Statistics[END_REF] C, and [START_REF] Lee | Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron-Density[END_REF] N NMR shifts were calculated at def2-TZVPP basis set. [START_REF] Weigend | Balanced Basis Sets of Split Valence, Triple Zeta Valence and Quadruple Zeta Valence Quality for H to Rn: Design and assessment of accuracy[END_REF] All the calculations were carried out using the Orca 4.0 program. [START_REF] Neese | The ORCA Program System[END_REF] The Avogadro program was used to edit xyz-files. [START_REF] Hanwell | An Advanced Semantic Chemical Editor, Visualization, and Analysis Platform[END_REF] Periodic Boundary Phase Calculations. Periodic boundary density functional theory (PB-DFT) calculations were carried out using the plane-wave pseudopotential method, as implemented in the CASTEP code. [START_REF] Clark | First Principles Methods Using CASTEP[END_REF] Initial coordinates of atoms in the unit cell were taken from PXRD refined crystal structure and used for further optimizations. Generalized gradient approximation of DFT in Perdew-Becke-Ernzerhof parametrization [START_REF] Perdew | Generalized Gradient Approximation Made Simple[END_REF] was applied to account for electron exchange and correlational. Van der Waals interactions were accounted with aid of Tkatchenko-Scheffler method. [START_REF] Tkatchenko | Accurate Molecular van der Waals Interactions from Ground-State Electron Density and Free-Atom Reference Data[END_REF] The plane-wave cutoff energy was defined as1200 eV upon tests of convergence of self-consistent field energy calculations using on-the-fly generated normconserving pseudopotentials. [START_REF] Hamman | Optimized Norm-Conserving Vanderbilt Pseudopotentials[END_REF][START_REF] Hamman | Erratum: Optimized Norm-Conserving Vanderbilt Pseudopotentials[END_REF] Brillouin zone sampling of electronic states was performed on 6×4×4 Monkhorst-Pack grid. The structure at equilibrium, an essential prerequisite for lattice dynamics calculations, was established by Broyden-Fletcher-Goldfarb-Shanno algorithm geometry optimization, after which the residual forces were converged to < 10 -2 eV Å -1 . Phonon frequencies were obtained by diagonalization of dynamical matrices computed using density functional perturbation theory (DFPT). [START_REF] Gonze | Perturbation Expansion of Variational Principles at Arbitrary Order[END_REF][START_REF] Gonze | Adiabatic Density-Functional Perturbation Theory[END_REF] The determination of solid-state NMR parameters was performed with aid of the GIPAW method. [START_REF] Pickard | All-electron Magnetic Response with Pseudopotentials: NMR Chemical Shifts[END_REF] The same as given for above control parameters were used for computations of absolute chemical shielding. The predicted chemical shifts were calculated using the relationship δi so = σref-σi so, where σref corresponds to magnetic shielding for the reference compound and σi so= (σ11 + σ22 + σ33)/3. The magnetic shielding for 1 H, [START_REF] Madsen | Collection and Analysis of Powder Diffraction Data with Near-Constant Counting Statistics[END_REF] C, and [START_REF] Lee | Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron-Density[END_REF] N signal reference compounds were set at 30.97, 171 (TMS) and -154.3 ppm (CH3NO2). [START_REF] Gervais | Combined First-Principles Computational and Experimental Multinuclear Solid-State NMR[END_REF]

RESULTS AND DISCUSSION

X-ray Structure Analysis. Remarkably, to the best of our knowledge, the IEF-12 crystal structure is the first example of a s-tetrazine based polynitrogen HEDM determined in its pristine state. Other derivatives were isolated solely at the solvated state, typically with the inclusion of DMSO molecules. [START_REF] Chavez | 3,3 -Azobis(6-amino-1,2,4,5-tetrazine): A Novel High-Nitrogen Energetic Material[END_REF][START_REF] Shlomovich | Energetic Isomers of 1,2,4,5-Tetrazine-Bis-1,2,4-Triazoles With Low Toxicity[END_REF][START_REF] Parvari | Proposed Proton-Transfer Mechanism for the Initial Decomposition Steps of BTATz[END_REF] The presently reported material does not exhibit (S=O) vibrational modes in an agreement with referenced infrared (IR) observations of Kläpotke et al., [START_REF] Kläpotke | 3,6-Disubstituted 1,2,4,5-Tetrazines[END_REF] characteristic of DMSO, ruling out the solvated crystal hypothesis (as we will show further). From basic considerations, BDTT molecule could appear as one of two possible geometrical isomers, in trans or cis configuration (Figure 2 and Table S1-S2). To account for both alternative structures, we constructed initial molecular models and optimized their geometries in vacuo. space group. Implying the presence of an inversion center in both geometries, a reasonable choice of the space group is P-1, albeit it is difficult to select properly from P1 or P-1 basing solely on powder diffraction data. From the [START_REF] Madsen | Collection and Analysis of Powder Diffraction Data with Near-Constant Counting Statistics[END_REF] C and [START_REF] Lee | Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron-Density[END_REF] N solid state NMR experiments (Figure 4; see below), two independent signals of carbon and seven signals of nitrogen are detected, which indicates that half of the complete molecule resides in the asymmetric unit cell (Z' = 0.5; Scheme S1). The structure solution was performed using direct space methods. [START_REF] Altomare | The Hybrid Big Bang-Big Crunch Method for Solving Crystal Structure from Powder Diffraction Data[END_REF] Only (N1-C1-N3-C2) or (N1-C1-N3-N5) torsion angles (see Figure 4d or Scheme 1 for the numeration), corresponding to conjugated tetrazine core and triazole cycles rotation across C-C axis, were used as internal molecular degrees of freedom (translations and rotations of a rigid body taken as an entire during simulated annealing cycles). The best solution was obtained at wR = 10.5%, based on the Rietveld's definition for the cost functions of solution quality. It is worth to note that employing both initial geometries led to a unique solution, in favor of the trans-isomer. Initial crystal atomic coordinates were refined using Rietveld method (Rwp = 4.12%, Rp = 3.19%, GoF = 4.03, see SI, Figure S2 and Table S3 for further details).

Principally, molecular conformation of BDTT ought to adopt planar configuration due to the conjugation of electron donating properties of guanazole cycle [START_REF] Rabie | Charge-Transfer Molecular Complexes of Guanazole and Some 1,2,4-Triazoles with σ-and π-Acceptors Involving a New Acceptor: Resulting In Situ; DDQH2[END_REF] with electron-deficient π-system of tetrazine. Expectedly, the final interplanar angle value between the tetrazine and triazole ring is equal to 4.6(9)°. The donor-acceptor-donor (D-A-D) type of molecule strongly affects the intra-and inter-molecular hydrogen bond system. The hydrogen bond net can be efficiently rationalized using a graph set description (SI). The closer amino group in third position of triazole ring forms two symmetric S(6) rings (here and after, we will use notations as given in ref. 36 of intramolecular H-bonds with participation of N-acceptor atoms of tetrazine core; see detailed description in SI).

It is known that a crystal packing of a π-conjugated system defines the performance of organic semiconductors, affecting the transfer integral and reorganization energy of molecules. In the present case, the dense formed hydrogen bond net favors desirable lamellar motif of packed molecules: i.e., free hydrogen atom of the same terminal amino-group is engaged into R2 2 (8) cyclic H-bonding with a molecule of the next layer (Figure 3a). Essentially, the same type of cycle occurred with participation of the amino-group at C2 of triazolyl ring through the in-plane head-to-tail arrangement, as can be seen from Figure 3b. The particular D-A-D molecular organization has an impact on crystal structuring enabling non-covalent interactions of electron enriched terminal amino-groups as σ-donors with electron poor tetrazine π-acceptor rings at a distance of 2.944 Å. A 2D π-stacked lamellar motif of the crystal building oughts to be the most efficient for the charge transport, [START_REF] Wang | Semiconducting π-Conjugated Systems in Field-Effect Transistors: A Material Odyssey of Organic Electronics[END_REF] and hence, the IEF-12 crystal structure is a good candidate for further charge transport studies. Following this, herewith we focus our attention on the strongest interacting parts of the crystalline lattice, which could be representative molecular aggregates for charge transfer.

The empirical "UNI" pairwise interatomic potential parameters [START_REF] Gavezzotti | Are Crystal Structures Predictable?[END_REF] allow to identify the strongest interactions between molecular aggregates in the crystal for C, H, N, O species by quantifying the inter-molecular potentials in a crystal. Two molecules ensemble supported with nitrogen-π contacts are stacked along the b-axis (Figure S3) at a distance of 6.79 Å with the packing energy (PE) of 59.95 kJ mol -1 , while corresponding value for in-plain H-bonded aggregate is only 47.34 kJ mol -1 . Therefore, interaction strength at distances equal to separations between tetrazine core centroids strongly depends on the direction of intermolecular interactions. Finally, PE per unit formula (calculated from a shell of 200 molecules) yields to 233.7 kJ mol -1 , indicating a very strong cohesion of crystallized molecules. The packing energy often tightly correlates with ΔHsub and thus, with ρexp = 1.75 g cm -3 and low solubility (10 -5 M in DMSO at RT). Thus, one can suggest here a high-vacuum route for the shaping of this material as thin films (vs. wet deposition methods), as commonly used for insoluble conjugated organic semiconductors. [START_REF] Ruiz | Pentacene Thin Film Growth[END_REF] Solid state NMR. To validate the structural model obtained from the XRD study, solid-state NMR spectroscopy was employed. Figure 4 presents the obtained 1 H, [START_REF] Madsen | Collection and Analysis of Powder Diffraction Data with Near-Constant Counting Statistics[END_REF] C and 15 N NMR spectra.

Assignment of all signals is based on comparison with DFT calculations, which are summarized in Table 1.

I have a problem with the Table 1 content. Please revise: i) "calculated symmetric components of cs" where are they, δ11, δ22, δ33 or σ11, σ22, σ33 ??? ii) efg tensors , where are they? Instead I see quadrupolar coupling, no sense !!!! Do you have Vxx, Vyy, Vzz ? In my opinion, put in Table 1: σiso (ppm), σ11 (ppm), σ22 (ppm), σ33 (ppm), η (σ11-σ22/σ33), δcalc (ppm), δexp (ppm), δcalcδexp (ppm). (EFG not necessary) The 1 H MAS NMR exhibits a composite spectrum with at least three overlapped peaks with apparent maxima at 3.8 and 7.1 ppm and a shoulder at ca. 9.8 ppm. The calculated isotropic shifts for "inactive" N-H bond is 4.2 ppm, falling into ±0.5 ppm accuracy of proton shift, determined with GIPAW. The signal from H-atoms takes part in the intramolecular hydrogen bonds, being observed at 7.3 and 8.3 ppm. However, that difference is probably experimentally smaller, resulting in their merging and leading to observed peak integral accounting for two protons. The hint on the shoulder at 9.8 ppm should be associated with a poorly resolved low field peak of strong intermolecular H-bonded protons having the shortest H-N distances (< 2 Å). As N6H2 is involved into intramolecular hydrogen bonding, the corresponding [START_REF] Lee | Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron-Density[END_REF] N signal is shifted to lower field respecting to N7 amino group. The latter is, thus, able to participate in through-the-space charge transfer to s-tetrazine rings, in good agreement with our crystallographic analysis.

Vibrational properties. High dielectric constant organic semiconductors are of great importance for organic photovoltaic material research. [START_REF] Camaioni | Pushing the Envelope of the Intrinsic Limitation of Organic Solar Cells[END_REF] Commonly, the light photon absorption generates electron-hole excitonic pair, in which electron and hole are bounded by Coulomb attraction. The potential of the latter is inversely proportional to the dielectric constant (εr) of the material. Thus, the smaller potential is better for the charge separation, leading to the photocurrent increase. Otherwise, an electron-hole pair recombines into photon (radiatively or non-radiatively). The majority of organic semiconducting materials have εr = 2-4, that is far less than inorganic photovoltaic materials have. Therefore, the strong D-A bipolar nature of the IEF-12 could offer unusual dielectric properties.

Under the framework of DFPT, it is possible to get the response of a solid on an imposed homogeneous electric field, which in due course, permits accessing the dielectric constant numerically. [START_REF] Gonze | First-Principles Responses of Solids to Atomic Displacements and Homogeneous Electric Fields: Implementation of a Conjugate-Gradient Algorithm[END_REF][START_REF] Refson | Variational Density-Functional Perturbation Theory for Dielectrics and Lattice Dynamics[END_REF] This procedure includes calculation of all Γ-phonons (q = 0), which could be directly compared with our experimental vibrational spectroscopic data. For the present case, we used simple and rather accurate ATR-FTIR and confocal Raman spectroscopies to detect and compare the response of the material to a perturbation induced by probing electric field (Figure 16 S4). The correlation plot of observed and calculated frequencies (Figure 5) obeys a linear dependency with a small intercept value, suggesting a good quality of the fit. All six components of dielectric tensor are independent for -1Laue class (Table 2). The averaged value for polycrystalline IFE-12 is 4.86, which is a very promising result for a small molecule organic semiconductor, [START_REF] Camaioni | Pushing the Envelope of the Intrinsic Limitation of Organic Solar Cells[END_REF] even considering a maximal error of about 16%, using the Perdew, Burke, and Ernzerhof (PBE) exchange-correlation functional benchmarked on inorganic semiconductors. [START_REF] Petousis | Benchmarking Density Functional Perturbation Theory To Enable High-Throughput Screening of Materials for Dielectric Constant and Refractive Index[END_REF] Solid state optical measurement and electronic band structure. At UV-vis spectrum (3.3x10 -6 M in DMSO), three broad peaks are detected in the range of 255-600 nm (Figure S5). The low-energy band is centered at 520 nm (ε = 8455 l mol -1 cm -1 ). The absorption bands at 332 and 285 nm are relatively intense by factor ~25 and ~31, respectively.

The interpretation of optical transition can be greatly facilitated by TD-DFT, which is widely adopted for organic molecules of 20-200 atoms at reasonable accuracy and moderate calculation cost. [START_REF] Laurent | DFT Benchmarks: A Review[END_REF] The transition electron densities cannot be analyzed straightforwardly or by Kohn-Sham frontier orbitals (HOMO, LUMO at Figure 7), but their differences have more chemical sense.

Thus, lower energy transitions (#1, #6) are of π* π type, likely due to the depletion of electron density on 3,5-diamino-triazolyl rings and its accumulation on s-tetrazine cycle. The non-bonding states of nitrogen at triazole cycle donate electron density to the higher anti-bonding states of tetrazine also, resulting in an intense peak at 4.85 eV. Overall, these results agree with the solid state properties of IEF-12: i) the most interacting aggregate is the π-π stacked system, and ii) population analysis of electronic bands reveals identical results, showing dominate occupation of VB with 3,5-diamino-triazole and CB with 1,2,4,5-tetrazine states.

CONCLUSIONS

This work points to the field of insensitive high energy as a potential source of small molecule organic semiconductors for optoelectronics (photoresistors, photodiodes, etc.). As exemplified on bis-3,5-diamino-1,2,4-triazolyl-1,2,4,5-tetrazine, such compounds can adopt the lamellar πstaking of trans-isomers, owing the combination of electron donor (3,5-diamino-triazole) and electron-withdrawing rings (s-tetrazole), a relatively low bandgap and a higher dielectric constant, which is highly favorable for organic semiconducting materials.

For their practical use, small molecule or polymeric organic semiconductors are usually processed to thin films via wet methods (e.g. dip-, spin-coating, inkjet printing). However, in the present case, the strength of the H-bonds network (on par with other materials such as covalentorganic frameworks-COFs) strongly limits the solubility. Thus, based on our results, to facilitate the manufacturing and device fabrication of these organic semiconductors, chemical exfoliation [START_REF] Wang | Recent Progress in Covalent Organic Framework Thin Films: Fabrications, Applications and Perspectives[END_REF] or high vacuum evaporation route may be appropriate method for future thin film preparation.

Figure 1 .

 1 Figure 1. Usual schematic crystal packing types of OSMs: (a) herringbone packing (face-toedge) without π-π overlap (face-to-face) between adjacent molecules; (b) herringbone packing with π-π overlap between adjacent molecules; (c) lamellar motif, parallel π-stacking; (d) lamellar motif with gliding π-stacking.

Figure 2 .

 2 Figure 2. Probable (left) cis-and (right) trans-isomers of BDTT.

Figure 3 .

 3 Figure 3. View of the (a) lateral head-to-tail H-bonded ensembles, and (b) vertical inter-sheet interaction of BDTT molecules. Symmetry codes: (i) -x, 1y, 1z; (ii): x, y, 1 + z; (iii): -x, 1 -y, 2 -z; (iv): 1 -x, 1 -y, -z.

Figure 4 .

 4 Figure 4. (a) 1 H MAS, (b) 13 C{ 1 H} CPMAS, and (c) 15 N{ 1 H} CPMAS spectra of IEF-12 solid. (d) Molecular structure of BDTT in the crystal packing, showing the inequivalent atom labels used for spectral assignment according to the DFT-GIPAW calculations. "ssb" denotes spinning side bands.

Figure 5 .

 5 Figure 5. Correlation plot of observed (IR, Raman) and calculated vibration modes of IEF-12.

Figure 6 ,

 6 depicting a usual Tauc plot, is addressed to determine optical bandgap of IEF-12. As it can be seen, the indirect transition takes place at 2.27 eV, corresponding to the deep-red shade of bulk sample (see picture inset). According to band structure analysis, conditional T(0,½,½) Γ(0,0,0) pathway from valence band (VB) to conduction band (CB) defines a bandgap of 1.1 eV. Observed underestimation is known to be a limitation of the generalized gradient approximation (GGA), so we applied rigid shift of CB for comparison purposes. The visualization of orbitals (square of the absolute value of the wavefunction for a given electronic band, summed over all k-points) helps to recognize filled π-electron densities of DATR at the top of VB and empty π-states of stetrazine.

Figure 6 .

 6 Figure 6. (a) Tauc plot of processed solid state optical absorption data (green and red dashed lines are given for the guide only), a view of prepared bulk sample is given in inset. (b) A frame of calculated band structure of IEF-12 superimposed with Wigner-Seitz cell (reciprocal vectors and Brillouin zone vectors are shown with blue and maroon lines).

Figure 7 .

 7 Figure 7. Electron densities difference of ground and excited state orbitals for most intense UVvis optical transitions.

Table 1 .

 1 Calculated symmetric components of chemical shielding, electric field gradient tensors, and chemical shifts, compared to experimental values from 1 H,[START_REF] Madsen | Collection and Analysis of Powder Diffraction Data with Near-Constant Counting Statistics[END_REF] C, and 15 N MAS data.

	Atom	σi so(ppm) σ aniso(ppm)	κ	CQ(MHz)	η	δcal c (ppm)	δexp (ppm)	δcal c-δ exp (ppm)
	H1	22.63	17.97	0.29	0.197	0.17	8.34	7.1	1.2
	H2	26.80	12.73	0.23	0.243	0.15	4.17	3.8	0.4
	H3	23.71	13.33	0.50	0.222	0.15	7.26	7.1	0.2
	H4	21.66	18.02	0.37	0.188	0.20	9.31	9.8	-0.5
	C1	11.79	-165.74	0.68	-1.197	0.94	159.21	157.5	1.7
	C2	9.05	108.28	0.65	1.836	0.48	161.95	157.5	4.4
	C3	16.77	98.07	0.89	-2.032	0.47	154.23	163.3	-9.1

Table 2 .

 2 Calculated

	Dielectric tensor component	ε11	ε12	ε13	ε22	ε23	ε33	εpol y
	Calculated value	3.120 -0.752	0.009	6.189	-1.341	5.262	4.86

independent components of ( symmetry) static dielectric tensor and averaged value in polycrystalline state assumption.

Table 3 .

 3 Table 3 summarizes the main results of such calculations. Predicted vertical transition energies (singlets only), oscillator strengths in comparison to observed UV-vis absorption maxima.

	Excited state Transition	eV	TD-DFT nm	f	Experimental nm
	#1	HOMO-3 LUMO HOMO LUMO	2.14	579	0.0097	520
	#3	HOMO-2 LUMO	2.46	545	0.0059
	#4	HOMO-3 LUMO HOMO LUMO+1	3.60	344	0.0038
	#6	HOMO-3 LUMO HOMO LUMO+1	3.80	327	0.7954	332
		HOMO-16 LUMO+1
	#11	HOMO-7 LUMO	4.85	256	0.0254	284
		HOMO-3 LUMO+1
	#13	HOMO-1 LUMO+3 HOMO LUMO+2	5.10	243	0.0012
	#14	HOMO-7 LUMO HOMO-3 LUMO+1	5.16	240	0.4287

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. SEM image of IEF-12, Cartesian coordinates of DFT optimized geometry of cis-and transisomers in a gas phase, details on Rietveld refinements of synchrotron PXRD and final crystallographic parameters summary, experimental ATR-FTIR and Raman spectra, PBC-DFT calculated vibrational frequencies, UV-vis absorption spectrum in DMSO and miscellaneous information.

AUTHOR INFORMATION

Corresponding Author *patricia.horcajada@imdea.org

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources

The authors acknowledge RaPhuel (ENE2016-79608-C2-1-R). The authors would like to thank the European Synchrotron Radiation Facility (Grenoble, France) and the French National Synchrotron Radiation Source "Synchrotron Soleil" (Saint-Aubin, France) for beamtime at the CRISTAL beamline.


Dr Alessio Bonucci

Olivier Ouari

Bruno Guigliarelli

Valérie Belle

Dr Elisabetta Mileo
email: emileo@imm.cnrs.fr

Pr B Guigliarelli

Pr V Belle

In-cell EPR: progress towards structural studies inside cells

Keywords: EPR, in-cell spectroscopy, spin labels, SDSL, structural biology

Exploring biomolecules' structure and dynamics in the context of their intracellular environment has become the ultimate challenge for structural biology. As the cellular environment is barely reproducible in vitro, investigation of biomolecules directly inside cells has attracted a growing interest. Among magnetic resonance approaches, Site-Directed Spin Labeling (SDSL) coupled to Electron Paramagnetic Resonance (EPR) spectroscopy provides competitive and advantageous features to capture protein structure and dynamics inside cells. To date, several in-cell EPR approaches have been successfully applied to both bacterial and eukaryotic cells. In this review, the major advances of in-cell EPR are summarized, as well as the challenges this approach still poses.

Introduction

As Lisa Charlton and Gary Pielak stated some years ago, looking at protein structural dynamics at molecular level in their physiological environment represents the "nirvana" for scientists. [1] The intracellular environment is indeed extremely heterogeneous, complex, and crowded. Exploring biomolecule structure and dynamics, as well as following structural changes and interactions in the intracellular environment, have become the ultimate challenge for structural biology. The viscosity of the cytoplasm, the macromolecular crowding, specific and nonspecific weak interactions, as well as a myriad of potential interactors constitute parameters that can have a huge impact on protein structure, dynamics and protein-partner interaction. [2] As the cellular environment is difficult to reproduce in vitro, investigation of biomolecules directly inside cells has attracted a growing interest in the last few decades. Mass Spectrometry has been successfully employed to probe protein structure and protein-protein interactions in crude lysates, [3] and the rapid improvements of the technique will soon allow in cell studies. [4] Fluorescence-based microscopy has permitted impressive studies of subcellular dynamics, protein-biomolecule interactions and protein aggregation in a physiological setting. [5] Since 2006, efforts in Magnetic Resonance Spectroscopies have enabled important improvements in the study of structural dynamics directly in the cellular context. Liquid state and, more recently, DNP solid state NMR have both been already applied to probe the structure and dynamic features of globular, membrane and disordered proteins, [2,6] as well as nucleic acids in several cell types. [7] Among magnetic resonances approaches, Site-Directed Spin Labeling coupled to Electron Paramagnetic Resonance spectroscopy (SDSL-EPR) has demonstrated to be an accurate and powerful approach to study structural properties of biomolecules, such as soluble and membrane proteins, as well as nucleic acids. [START_REF] Ag | Emerging Topics in Life Sciences[END_REF] SDSL-EPR has been broadly applied in vitro and, since 2010, the increasing number of in-cell studies has proved its potential in investigating biological macromolecules inside cells. In this review, we provide a description of the in-cell EPR approach. In particular, we report on recent milestones, as well as on the challenges the approach still poses.

In vitro and in-cell EPR

In vitro SDSL-EPR

The SDSL approach is based on the introduction of a paramagnetic label (nitroxide radicals, lanthanide-or trityl-based tags) at a specific position of a protein or a nucleic acid followed by its study by EPR spectroscopy.

By far, the largest family of spin labels in SDSL-EPR is nitroxide radicals, with Gadolinium(III) complexes and Trityl radicals being interesting alternatives. One of the main advantages of nitroxide spin labels is their high sensitivity to the local environment which, in the SDSL-EPR approach, is used to obtain structural and dynamic information on the biomolecule on which the nitroxide is attached. [8c, 8g, 9] In the case of nitroxide labels, SDSL-EPR, combined with protein mutagenesis, is based on the selective grafting on cysteine. [10] Even if Cys residues represent the main grafting site, few examples of labeling on tyrosine residues were also reported. [11] The consecutive study of the EPR spectra and their parameters provides detailed information on the local mobility of the grafted label. Spin label dynamics is related to local structural properties of the protein under investigation and can thus be used to follow protein's structural changes [12] and to detect interaction sites in complexes [13] or monitor folding events. [8g] This kind of EPR studies is conducted in a continuous wave mode (cw-based EPR) and is carried out in solution. This represents a very interesting feature in the case of cellular studies because it allows the investigation of a biological system in conditions as close as possible to the physiological ones. In the following sections, we will define this approach "solution state EPR". On the other hand, Pulsed Dipolar EPR techniques combined with doubly labeled systems, allow accurate distance measurements between two paramagnetic centers, thus enabling the characterization of the global structure of a biomolecule. [14] Distance measurements by Pulsed Dipolar EPR are based on the dipole-dipole coupling measurements between the unpaired electrons. Commonly, distances between spin centers are measured by applying the Double Electron-Electron Resonance (DEER) sequence, a two-frequencies experiment. The distances that can be investigated lie typically between 1.5 and 8.0 nm and, by employing perdeuterated proteins, it even is possible to reach 16 nm. [15] As an alternative to DEER, we can mention pulse sequences working with a single microwave frequency such as RIDME [16] (Relaxation-Induced Dipolar Modulation Enhancement), DCQ [17] (Double Quantum Coherence) and SIFTER [18] (Single-Frequency TEchnique for Refocusing Dipolar Couplings). Pulsed Dipolar experiments are usually carried out at cryogenic temperature (below 80K) due to fast electron transverse relaxation of paramagnetic centers at room temperature. In contrast to cw-based EPR, the samples are in a frozen glassy state.

Methods to label nucleic acids are also well established and can be successfully used to probe DNA and RNA conformational flexibility and dynamics. [8b, 19] In the case of nucleic acids, the nitroxide label can be incorporated during the chemical synthesis of the nucleic acid or can be introduced after synthesis of the Author copy oligonucleotide, on a modified nucleotide containing a uniquely reactive functional group. After proper labeling, the paramagnetic nucleic acid can be followed by both "solution-state" and Pulsed Dipolar EPR approaches. A recently reported labeling strategy for nucleic acids is based on noncovalent interactions (hydrogen bonding, ionic-and Van der Waals interactions) between the spin label and the nucleic acids. The 'non-covalent' labeling is very interesting because is free from several drawbacks that affect the 'covalent' labeling approach, such as the reduction of the nitroxide during its exposure to the reagents in the course of the nucleotide synthesis or the non-specific conjugation of the nitroxide with other functional groups (as in the case of isocyanates, for example). [20] More recently, lanthanide tags based on the complexation of Gd 3+ ions have shown to be promising probes for distance measurements on proteins. [14a, 21] Transition metals, such as Cu 2+ , Mn 2+ , Mo 5+ and Fe 3+ have also been shown to be interesting paramagnetic centers in protein studies. [22] The availability of different spin labels, mainly characterized by different spectral features, allows orthogonal spin labeling, in which the use of two non-identical spin labels represents an attractive strategy for studying biomolecule complexes. [23]

In-cell EPR

EPR studies in cellular contexts date back to more than 60 years. The light-induced paramagnetism of chloroplast observed by Commoner et al. [24] or the investigation of nitroxide behavior in cells, tissues and whole animals for EPR and magnetic resonance imaging (EPRI and MRI, respectively) are some examples. [25] However, in the past decade, the flowering of structural studies of biological macromolecules in their native environment was associated with the introduction of the concept of "in-cell spectroscopies", such as in-cell FRET, in-cell NMR and in-cell EPR. Thus, in this paper we adopted the "in-cell" definition for structural studies of proteins or nucleic acids, inside cells.

Since the first in-cell EPR work, reported in 2010, [26] several studies have highlighted the advantageous features and the potential of SDSL-EPR in studying protein and nucleic acid structural dynamics inside cells. Besides the fact that EPR spectroscopy is a non-destructive technique that can provide structural details and conformational exchange details over a wide range of temperature and timescales (ns to ms), SDSL-EPR is characterized by a high sensitivity (few µL at ~ 30-50 µM concentration), negligible background interference and no molecular size limit of the biomolecule or complex under study.

In order to study biological macromolecules inside cells by EPR spectroscopy, experiments are generally conducted by labeling the system of interest prior to its delivery inside cells.

In 2010, Tochio and Shirakawa [19] measured inter-spin distances in two variants of the human ubiquitin protein, labeled with a commercially available nitroxide (1, Figure 1), and then microinjected inside Xenopus laevis oocytes. In order to perform DEER-EPR experiments, the injected oocytes were previously frozen in liquid nitrogen.

In this study the MTSL nitroxide (2), routinely employed for SDSL-EPR studies, was not chosen as spin label because of the cleavage of the S-S bond between the protein and the label inside cells. In contrast to MTSL, maleimido-proxyl 1 was employed because the maleimide group reacts with cysteine residues by forming a C-S bond, expected to be more stable to reductive cleavage inside cells than the S-S one.

The reported work represents the proof-of-principle of in-cell EPR, since it demonstrated that EPR spectroscopy can potentially be used to follow protein conformational changes in the cellular environment and, at the same time, highlighted its forthcoming limits and challenges. 4), Gd 3+ -DOTA-maleimide ( 5), BrPSPy-DO3MA-Gd3 + 6 and BrPSPy-DO3A-Gd 3+ 7.

One limitation is related to the short lifetime of nitroxide labels in the cellular context. Their decay occurs because of the presence of antioxidants and enzymes in the cytoplasm that reduce the nitroxide group to the corresponding EPR silent hydroxylamine. Among the cellular reducing species, we can mention semi-quinone radicals, glutathione, species generated from the mitochondrial electron transport chain, NAD(P)Hdependent enzymes, thioredoxin and other enzymes containing sulfhydryl groups. The nitroxide reduction thus limits the time window to acquire spectra of in-cell samples and, in the case of distance measurements, this leads to the loss of dipolar-coupled spin pairs. [27] Another point concerns the bond between the spin label and the Cys residue on the choose system, as it has to be not reductively cleavable inside cells.

In addition, the restricted number of delivering methods of the labeled protein inside cells, that are currently available, represent another limitation of in-cell EPR.

In the following sections we will describe how the EPR community actively search for new methodologies and tools to tackle these limitations and make in-cell EPR spectroscopy a powerful and complementary tool for investigating biomolecule structure and dynamics while preserving its physiological environment.

Spin labels for in-cell EPR studies

Currently, among all the spin labels families, nitroxide radicals are the sole molecules able to combine the investigation of local protein structural dynamics in solution at physiological temperature ('solution-state' EPR) with the study of the Author copy biomolecule structure by inter-spin distance measurements (DEER).

Unfortunately, as already mentioned, a major hurdle is the short lifetime of nitroxide spin labels in the cellular context (see Figure 2). As reported in most of the in-cell EPR articles, nitroxide reduction can be limited by shock-freezing the sample in liquid nitrogen as soon as possible and to carry out EPR measurements on frozen solution. Even if quite successful, this approach limits in-cell studies to distance measurements with limited incubation period. To mitigate nitroxide reduction in cell, Saxena and coworkers mixed the nitroxide-labeled protein with a solution of an oxidizing agent such as K3Fe(CN)6. [28] The use of cell strains deficient of thioredoxin reductase and glutathione reductase (for example E. coli Origami®) was also suggested as a possible means to delay nitroxide reduction in cell. [START_REF] Dunkel | Physical chemistry chemical physics[END_REF] Besides these possibilities, the development of spin labels stable in the cellular environment has appeared as an important step to push in-cell EPR researches forward. In the last years, several studies dealing with the design and synthesis of spin labels stable in the cellular context have been carried out. Shielded-nitroxides, Gd 3+ -and trityl-based spin labels have shown to be promising options.

In the following sections we provide a description of the principal families of spin labels currently employed for in-cell EPR studies. As already mentioned above, for SDSL-EPR studies in cell the spin label's reactive group responsible of the ligation of the target molecules needs to be stable to cleavage. In this manuscript, when possible, we will point up the reactive group properties of the depicted spin labels but we will not discuss them in depth. For an exhaustive discussion we suggest an interesting and detailed review recently appeared. [30]

Nitroxide-based spin labels

Interest in nitroxide lifetime in biological context started more than 40 years ago. Several research groups investigated nitroxides EPR signal loss in different conditions in order to characterize the relationship between nitroxide structure and their reduction to hydroxylamine by ascorbate and other reductants. Generally, the reduction decay kinetics of nitroxides is obtained by following the low-field line of the nitroxide spectrum as function of time in the presence of a reducing agent. The work of Keana et al. showed that the ring size of nitroxides has an impact on their reduction by ascorbate, i.e. pyrrolidine nitroxides (nitroxide 'a', 'b' and 'c' in Figure 2) being more resistant to reduction than piperidine (see nitroxide 'd', Figure 2) or doxylbased nitroxides [25a] . Later, Rassat demonstrated that steric shielding of the nitroxide moiety changing the gem-dimethyl by gem-diethyl groups on isoindoline nitroxide efficiently slows down the reduction process. [31] More recently, significant efforts have been made in the design and synthesis of nitroxides and nitroxidebased spin labels stable in the cellular context. Rajca and collaborators synthetized a series of sterically shielded pyrrolidine nitroxides and focused on their reduction properties in the presence of ascorbate and glutathione. [32] Large excess of ascorbate is usually employed as a common reducing agent for such studies. The comparison of reduction rates of the nitroxides under investigation by Rajca and collaborators, suggested that spirocyclohexyl nitroxides [33] (nitroxide 'b' in Figure 2) are reduced slower than the corresponding gem-methyl nitroxides (nitroxide 'c' in Figure 2), while gem-ethyl nitroxides (nitroxide 'a' in Figure 2) are reduced at significantly slower rates, by a factor 20-70 compared to spirocyclohexyl nitroxides. Spirocyclohexyl groups thus confer some stability towards reduction but are less efficient in steric shielding compared to the ethyl groups. [32a] The reduction properties of nitroxides bearing gem-tetracarboxylate groups, expected to be sterically bulkier than ethyl groups on the basis of DFT calculations, were also investigated. [34] However, these nitroxides undergo very fast reduction in an ascorbate/glutathione solution mixture. The authors argue that, beside steric factors, other contributions governing nitroxide reduction, such as the polarity and the withdrawing/donating properties of substituents, have to be taken in account. (20), 5322-5325). Copyright (2012) American Chemical Society. [32a] In parallel, Prisner and Sigurdsson and collaborators have studied and compared the effects of substituents on the free radical stability in four nitroxide families (specifically piperidine-, imidazolidine-, pyrrolidine-, and isoindoline-based nitroxides) in cytoplasmic extracts and in intact X. laevis oocytes. [35] Besides confirming that bulky ethyl groups next to the nitroxide moiety represent the more effective stabilisation against reduction, this study highlighted that the reduction profiles obtained in cell extracts are slower than those obtained in cell and do not follow an exponential decay. [35] The authors explain this result by admitting a competition between reducing (ascorbate, glutathione, NADH and NADPH) and oxidizing (metal enzymes, metal ion release, ROS and O2) processes present in the cell extract. Indeed, oxidizing agents, enzymes and Reactive Oxygen Species can re-oxidize the hydroxylamine back to nitroxide slowing down the reduction process. A similar trend has also been reported in other studies. [28a, 36] Moreover, because of the drastic process of cell extract formation, the redox properties of the extracts are difficult to control and poorly reproducible. Cell extracts experiments are not equivalent to intact cell studies, but provide information that are difficult to obtain through in vitro experiment using one or a mixture of antioxidants.

Despite many studies have shed light on the design of nitroxides resistant to cellular reduction and the investigation of their redox properties in a physiological environment, only a few works concern the design and synthesis of new nitroxide spin labels (able to be grafted on a biomolecule). In the following sections, we describe three shielded nitroxide spin labels recently reported.

Spin label 3, an isoindoline-derived aromatic isothiocyanate, is able to react with amino groups. In the study reported by Saha et al. [37] , it is employed for the covalent grafting of nucleic acids via its specific reactivity towards 2' amino groups that can be inserted at the 2' position on the ribose moiety of uridine in RNA. Solution-state EPR studies of labeled oligonucleotides were necessary to investigate the ability of nitroxide 3 in reporting on structural features. Interestingly, cw-EPR spectra of RNA duplexes were found to be broader than those obtained for the single labeled oligonucleotides, demonstrating that this nitroxide can be employed to follow conformational flexibility of nucleic acids. Concerning the resistance to reduction, although the stability of spin label 3 was demonstrated only in ascorbate solution and more investigations need to be conducted, the preliminary results presented clearly suggest that 3 is a promising spin label for incell EPR studies. [37] Spin label 4 (M-TETPO), exhibiting gem-diethyl groups and a maleimide moiety for protein conjugation via cysteine labeling, showed high resistance of the nitroxide moiety to reduction and of the linker to cleavage in ascorbate solutions, cell extracts and in oocytes. The property of M-TETPO in reporting on protein structural dynamics was investigated by labeling several regions of NarJ (25 kDa), a chaperone protein, and by comparing the solution state EPR spectra with those obtained by using the commercially available nitroxide 1. These studies clearly demonstrated that M-TETPO nitroxide is a sensitive spin label for structural EPR studies of protein in solution and at physiological temperature. Following, the global structural changes occurring during the binding process between NarJ and its biological partner (NarGH), M-TETPO was proven to be a suitable spin label in distances measurements performed by DEER. [36a] After having established that the new nitroxide spin label is resistant to reduction and able to report on conformational changes on a model protein, M-TETPO has been used for the investigation of NarJ structural dynamics by performing X-band DEER experiments in intact X. laevis oocytes at endogenous concentration using 20µM of labeled protein ("bulk concentration").

During the review process for this review, an interesting article on nitroxide labels for in-cell EPR studies by Bleicken et al. appeared. In particular, they reported a gem-diethyl nitroxide spin label (IAG) able to target Cys residues by a iodo-acetamide group and, thus, leading to the generation of a C-S bond between the protein and the paramagnetic label.

Taken together, these results open up the perspective of achieving high resolution EPR studies of biomolecules in their native environment in solution as well as at cryogenic temperature.

Metal cations-based spin labels

Spin labels based on paramagnetic metal centers represent an interesting alternative to nitroxide labels for in-cell EPR studies principally because they are insensitive to reduction. In general, a metal-based spin label is composed by a ligand, able to coordinate a paramagnetic metal, and by a linking group, able to specifically react with a residue into the protein, more often Cys residues. Several paramagnetic metals (Cu 2+ , Mn 2+ , Mo 5+ , Fe 3+ , Gd 3+ for example) have already been successfully employed for distance measurements by DEER spectroscopy in vitro. [14a, 21-22, 22d, 38] Concerning in-cell EPR applications, Gd 3+ based spin labels are currently the most employed tags. Contrary to nitroxides, spin labels based on Gd 3+ (S=7/2) complexes are characterized by an enhanced sensitivity at high frequency and are free from orientation selection effects. Further details on inter-spin orientation selection studies can be found in several published works. [START_REF] Ab | The journal of physical chemistry[END_REF] In general, Gd 3+ complexes are characterized by a broad frozen solution EPR spectrum except for the central transition line (-½ +½). As the width of the central transition line narrows by increasing the frequency, experiments involving Gd 3+ tags are carried out at high frequency, with 95 GHz being the most employed one. Several studies demonstrated that the EPR signal of Gd 3+ -tags is unaffected in the cellular environment. [6c, 40] On the other hand, for in-cell applications, Gd 3+ -tags must have a very high affinity for the coordinated metal to avoid the presence of free Gd 3+ ions in the cellular suspension that leads to an increase of the background signal and a decrease of the modulation depth in DEER experiments.

Concerning in-cell EPR experiments, an elegant study showed DEER measurements successfully carried out on the ubiquitin protein (8.5 kDa) doubly labeled with Gd 3+ -DOTAmaleimide tag (5, Figure 1) after its delivery in eukaryotic cells (HeLa) by hypotonic swelling. These experiments were conducted at 10K, at 95 GHz (W-band) and at a bulk concentration of ~5 µM. [40b] Since the distance distributions obtained in cell were substantially the same as those obtained in vitro, the authors hypothesized that the overall structure of ubiquitin does not change inside cells. This could also be explained considering that ubiquitin is a globular, well-structured protein, for which the impact of the cellular environment could be negligible. Nevertheless, the background decay of the DEER trace observed in cell was stronger than that observed in vitro. This is a direct consequence of the crowding in the cells which leads to higher local concentrations of the delivered protein, namely inhomogeneous distribution within the cell.

Currently, several research groups are highly active in the design of new Gd 3+ -based spin labels with optimized properties, such as i) a linker stable in the cellular environment; ii) a very high affinity for the coordinated metal to avoid the presence of Gd 3+ ions free in the cellular suspension; iii) a short and rigid linker to decrease the width of distance distribution; [41] iv) a small ZFS (Zero Field Splitting) parameter in order to have the narrower central transition and thus the best signal amplitude [42] and v) a smaller tag size. [41,43] For example, a newly designed Gd 3+ -based tag (4-vinyl-PyMTA) was demonstrated to be a valuable spin label to selectively target Cys residues of a proline rich peptide thanks to a vinyl group. In particular, the grafting reaction is a Michael addition of the thiol group of Cys to the vinyl group of the spin label, which results in a S-C bond between the label and the protein, a bond expected to be more resistant than S-S towards cleavage in cellular environment. The stability of 4-vinyl-PyMTA in cellular conditions was evaluated in cell extracts and after micro-injection in X. laevis oocytes. [40a] More recently, Goldfarb and collaborators reported on two spin labels, BrPSPy-DO3MA-Gd3 + 6 and BrPSPy-DO3A-Gd 3+ 7. [44] Concerning these two spin labels, the large Gd 3+ binding constant of DOTA-like group gives them high stability, the coordination of the Gd 3+ by a pyridine nitrogen gives higher rigidity than the DOTA-like one and the 3-bromo-phenylsulfonylpyridine moiety allows selective grafting of Cys residues of ubiquitin variants in mild conditions (pH 8) in a rather short time (6 hours of reaction) with 80% of labeling yield. [44a] Note that, in the case of spin label 7, also immunoglobulin G-binding protein (GB1) was successfully labeled. [44b] After the delivery of the labeled protein by hypotonic swelling and electroporation into HeLa cells, spin labels 6 and 7 were successfully employed to perform in-cell DEER measurements at W-band under cryogenic temperature. [44a] In particular, in the case of protein labeled with 7, the sensitivity of in-cell DEER was higher than in the case of labeling with 6. This being related to a higher phase memory time and a smaller ZFS of spin label 7, which differs from 6 in the absence of a methyl group from the acetate arms.

Trityl-based labels

Triarylmethyl radicals (trityl or TAM) are carbon-centered radicals combining a high stability in the cellular environment and a narrow EPR line with relaxation properties allowing distance measurements at physiological temperatures. For these reasons they appeared as promising candidates for in-cell EPR experiments.

Trityl chemical structure is characterized by six thioacetonide moieties, important for the stability of the radical, and by para-position substituents important to modulate their solubility. Examples of in vitro SDSL-EPR involving trityl spin labels were already reported for proteins, [17,45] polymers [46] and oligonucleotides [47] . However, reduction processes of trityl exist in vivo. While trityl radicals' stability has already been well characterized in human blood, [48] few studies report on the investigations of their stability inside cells. [35,49] In a recent study, a trityl radical linked to a poly-arginine chain, that could cross the membrane of human cells (MDA-MB-231) was prepared and its EPR signal was found to be stable at least for two hours. [49] As the reduction properties depend on the chemical structure, further improvement of trityl structure could tune their bio-reduction properties.

A recent work reported in details on the synthesis of new trityl-based tags (8, Figure 3) and their investigation as spin labels for in vitro and in-cell EPR studies. [50] Spin label 8 (R1, R2 and R3) showed to be soluble and stable in aqueous solutions, at room temperature and in the presence of molecular oxygen during at least 3 hours. Its ability to react with cysteine residues was investigated by labeling a cytochrome P450 protein, called CYP101. The stability towards cleavage in a cellular context of the thioether bond and of the triazole linkage makes SL7-R2 and SL7-R3 suitable for in-cell EPR studies, respectively. Indeed, distance measurements involving an intrinsic paramagnetic cofactor (an iron (III)), present in the CYP101 protein, and the trityl spin label were performed in cell. To this purpose, about twenty X. laevis oocytes were microinjected with a solution of the labeled CYP101 protein. After injection, the oocytes were inserted in a capillary and shockfrozen. The final in-cell concentration (bulk) was ~250 µM. The time traces obtained by measuring trityl-Fe III distances by RIDME at Q-band and at 25 K in cell show a SNR 10 times less than obtained for in vitro distance measurements. The authors explain this result by considering that they have a difficulty in controlling the iron content of the protein, that seems to be lower than the expected one. Moreover, the presence of intrinsic cellular Mn 2+ could enhance the relaxation of the Fe 3+ . However, even with this low SNR, the distance distribution obtained in cell was found to be very similar to the one obtained in vitro.

Despite the size of trityl-based spin labels, which could perturb the structure of the protein under investigation, the results achieved in this work are an interesting demonstration of the feasibility of in-cell distances measurements.

As mentioned before, the possibility to perform distance measurements at physiological condition (in solution and at room temperature) is very attractive, especially when studying biological systems. However, to this aim further improvements in the design of trityl-based spin labels are necessary, for example in modulating spin label properties which are key factor for the increase of Tm. [51] Author copy

Cellular systems and protein delivery

In order to perform in-cell EPR experiments, biological macromolecules are generally labeled with the appropriate spin label prior to its delivery inside cells.

X. laevis oocytes (Figure 4) have been widely used cells [40a, 52] owing to the fact that they can be easily micro-injected, that they are large cells (~1μL in volume) and that the amount of introduced protein can be controlled. Oocytes can be studied at X or Q band. At X-band between 50 and 100 oocytes are necessary, while at Q-band few oocytes (5) are enough. [53] Despite the fact that they are useful model cells, at least two drawbacks can be related to the use of oocytes as host cells for in-cell experiments. First, they require highly concentrated labeled protein solutions before injection, to avoid excessive dilution in the oocyte Secondly, by taking into account that prokaryotic and eukaryotic cells show important dissimilarities in terms of compartmentation and viscosity and that these differences could have a repercussion on the structural properties of the studied system, oocytes could not be the appropriate cellular environment to study functional interactions between bacterial proteins, for example. Effectively, while eukaryotic cells contain organelles (as the endoplasmic reticulum, Golgi body and mitochondria for example), have a defined nucleus and an organized cytoskeleton, bacterial cells do not contain a well-defined nucleus, thus DNA molecules are in an open region in the cytosol (the nucleoid), they do not contain membrane-bound organelle and they present elements of a cytoskeletal structure. Moreover, bacterial cells are smaller (0.5-3µM) than eukaryotic ones (2-100µM). A research line in in-cell EPR and NMR studies is related to the improvement of protein delivery methods inside cells with the aim to reach as much as possible the physiological concentration.

Permeabilisation of the membrane by hypotonic swelling [40b] and electroporation [44a, 54] are two approaches that have been successfully used in the field of in-cell EPR to deliver exogenous proteins in eukaryotic cells. An osmotic gradient between the intracellular and the extracellular compartment (hypotonic swelling) or a high-voltage electric pulse applied to a cell suspension (electroporation), allow the temporary destabilization of the cellular membrane. The result is that the membrane permeability increased and, thanks to a concentration gradient, the labeled protein can then cross the membrane and entry the cell. Not internalized proteins are then removed by a washing procedure. To confirm the delivery occurred, in general, the internalization is also monitored by microscopy using a fluorescently labeled protein. Hypotonic swelling and electroporation are particularly suited to treat a large number of cells at the same time and in a relatively short time. Moreover, no chemical conjugation is required. However, these delivery approaches can affect in an irreversible way the cellular membrane and consequently affect cell viability, cell morphology and proliferation rate. [55] In the case of electroporation, the percentage of viable cells is in general lower than for cells treated by hypotonic swelling, while the amount of apoptotic cells and the production of ROS was found to be higher.

A milder approach in term of cell viability is that based on the direct biosynthesis of the spin-labeled protein in vivo. This elegant method, based on the genetic encoding of nitroxide amino acids in response to the amber stop codon (TAG) by a tRNAPyl/pyrrolysyl-tRNA-synthetase pairs in E. coli, enables the incorporation of the nitroxide-modified amino acid at defined sites of the protein with a minimal perturbation of the natural environment of the protein. [56] This approach imposes the design and the synthesis of amino acids carrying the nitroxide moiety.

Recently, a highly efficient incorporation of lysine carrying a nitroxide side-chain (SLK-1, Figure 5) into E. coli thioredoxin has been reported. [56a] The authors demonstrated an high degree of incorporation of spin labeled Lys (99%) into several variants of the protein (mono and double SLK-1 variants). Moreover, they were able to demonstrate the detection of an EPR signal in E. coli cells at room temperature owing to the labeled protein. The EPR signal of the free spin labeled amino acid present in the cytoplasm was minor and showed a different spectral shape than that of the in vivo spin-labeled thioredoxin. This result also confirms that the spin labeled protein reaches sufficient expression levels to be detected by EPR. Despite these encouraging results, inter-spin distance measurements were not possible because of the spinspin interaction loss, related to nitroxide signal decay. This can be principally due to intracellular reduction of the nitroxide and to a possible degradation of the protein by enzymes (proteases). [57] An additional point needing improvement concerns the spin label flexibility. The high distance of the spin labeled moiety from the Author copy protein backbone and its flexibility results in a very sharp room temperature cw spectrum and provides limited dynamic information associated to broad distance distributions in DEER experiments. Schmidt et al. J. Am. Chem. Soc. 2014, 136, 4, 1238-1241). [56a] Copyright American Chemical Society.

Another work, based on in vivo bio-synthesis of the labeled protein and self-assembled metal spin labels (SAMSL) was recently showed. [58] A genetically encodable metal ion-binding tag was introduced at each of the terminus of a model protein, a 3helix-bundle. In this case the binding tag, a peptide sequence (YIDTNNDGWIEGDEL) capable of specifically bind lanthanides (Gd 3+ ), was genetically encoded and incorporated in vivo. The 3helix-bundle was overexpressed in E. coli cells, grown on a medium supplemented with Gd 3+ (500 µM). At least 4h of growth on Gd 3+ medium were necessary to reach a good level of Gd 3+ uptake. Despite the low binding constant of Gd 3+ and the excess of free Gd 3+ limiting the signal-to-noise ratio, the authors were able to detect dipolar modulation signal for the "Gd 3+ : 3-helix-bundle Gd 3+ " and the distance distribution obtained in cell was very similar to that obtained in vitro. 1NQE). On the right: a-c, background corrected Q-band DEER traces for the double mutant in E. coli cells (red) and in isolated outer membrane (black) in the absence and in the presence of ligands; d-f, distance distributions and their simulations. Adapted with permission (B. Joseph et al. J. Am. Chem. Soc. 2016, 138, 6, 1844-1847). [48b] Copyright (2016) American Chemical Society.

Without a doubt, the approaches presented in this paragraph are very interesting and appear as powerful tools for in-cell EPR studies.

Membrane proteins in whole cells

Among in-cell EPR studies, a big challenge is the investigation of membrane protein structure in the native lipid environment.

An excellent example concerns the investigation of the structural dynamics of the outer membrane cobalamin transporter BtuB, a β-barrel protein. [59] Notably, it was possible to detect structural changes induced by ligand binding in intact E. coli cells. All the BtuB variants used in this work carry cysteine residues in extracellular loops of the membrane protein. The cysteine variants were overexpressed in E. coli and the whole cell suspension was then labeled with MTSL (2, Figure 1), an already established procedure. [60] Thanks to several control experiments and assuming that outer membrane proteins in E. coli are in general free from Cys residues, the authors were able to demonstrate that the EPR signal observed after labeling of the whole cell suspension is specific of the BtuB protein. In fact, the free MTSL label which could reach the periplasm or, eventually, the cytoplasm and thus react with Cys of other proteins is rapidly reduced to an EPR silent specie, in a similar way for what already observed for nitroxide labels. [35-36, 52c] In this case the reduction of nitroxide is desired to eliminate the unwanted signals.

A double BtuB mutant, bearing 2 Cys residues (188 and 399, Figure 6) in two extra cellular loops (the second and the seventh loop) was overexpressed in the cells and successfully labeled with MTSL. The spin concentration reached with the labeling of BtuB in the cellular suspension was of 30 µM, and this concentration allowed good quality DEER measurements in intact E. coli cells. The core of this work, however, is the detection of changes in distance distribution after the addition of BtuB ligands (Ca 2+ ions and cyanocobalamin) to the cell suspension. These changes were explained taking into account conformational changes of Author copy one of the investigated extracellular loop (second loop) and are also supported by X-ray crystallography data. This is the first evidence of structural changes captured in the native cellular environment.

Another example of membrane protein studied by SDSL-EPR in native conditions concerns E. coli colicin A (ColA), a poreforming toxin produced by E. coli. Steinhoff and collaborators characterized ColA oligomerisation upon insertion in E. coli membrane by both room temperature cw-EPR and DEER-EPR. [START_REF] Dunkel | Physical chemistry chemical physics[END_REF] To this purpose, ColA was previously spin labeled in vitro and then incubated with liposomes prepared from E. coli polar lipids (artificial membrane) and with Origami ® E.coli cells.

The solution-state EPR spectra of labeled ColA in artificial or native membranes showed very similar lineshapes, suggesting that the nitroxide mobility is very similar in the two conditions. Thus, the authors conclude admitting that ColA should bind to artificial and to native membranes in a very similar way. Concerning DEER experiments, even if the signal-to-noise ratio was poor, a dipolar interaction was detected in intact cells, and most important, at the native concentration of the membrane protein.

Conclusion

All the studies presented in this review show that SDSL-EPR can be successfully applied to investigate biological macromolecules in cellular conditions. However, a lot of work still need to be done and both methodological and technical developments are required. For example, while distance measurements in-cell are quite established, the structural dynamics study of a protein or a nucleic acid in-cell in physiological conditions by solution-state EPR is still very challenging. In the near future, efforts will be partly directed toward the design of spin labels and paramagnetic non-natural amino acids with improved chemical properties (for example concerning their stability in the cellular environment) and characterized by enhanced spectroscopic features that will have a high impact on the biomacromolecule study directly inside cells.

Particular effort should be directed in order to make possible the investigation of protein-protein or protein-ligand interaction. In particular, this will be possible by improving tools to engineer proteins in living cells based on genetic code expansion and unnatural amino acid mutagenesis and on a better control of the expression of two or more proteins with the use of inducible promoters.

Obviously, questions concerning the cellular viability, protein compartmentation and localization should be addressed in-depth in the future.

Understanding how the natural cellular environment affects protein structure, dynamics and consequently protein function, and unravelling protein-protein interactions in physiologically relevant conditions represent an extraordinary step forward in the field of cellular structural biology and will provide fundamental elements to understand how cells work.

MINIREVIEW

Among magnetic resonances approaches, Site-Directed Spin Labeling (SDSL) coupled to Electron Paramagnetic Resonance (EPR) spectroscopy exhibits competitive and advantageous features to capture protein structure and dynamics inside cells. To date, several in-cell EPR approaches have been successfully applied to both bacterial and eukaryotic cells. In this review, the major advances of in-cell EPR are summarized, as well as the challenges this approach still holds. In-cell EPR: progress towards structural studies inside cells.

Figure 1 .

 1 Figure 1. Spin Labels cited in this review: 3-maleimido-PROXYL (1), MTSL (2), 1,1,3,3-tetramethylisoindolin-5-isothiocyanate-2oxyl (3), M-TETPO (4), Gd 3+ -DOTA-maleimide (5), BrPSPy-DO3MA-Gd3 + 6 and BrPSPy-DO3A-Gd 3+ 7.

Figure 2 .

 2 Figure 2. Example of a) gem-ethyl, b) spirocyclohexyl, c) gem-methyl and d) piperidine-based nitroxides and their reduction profiles (0.2 mM) in 4mM ascorbate solution. The figure has been adapted with permission (J. T. Palettaet al. Org. Lett. 2012, 14 (20), 5322-5325). Copyright (2012) American Chemical Society.[32a]

Figure 3 .

 3 Figure 3. Trityl-based spin labels.

Figure 4 .

 4 Figure 4. A) and B) Xenopus laevis oocytes at stage VI. C) An X-band EPR tube filled with ~40 X. laevis oocytes.

Figure 5 .

 5 Figure 5. A) Nitroxide-based unnatural amino acid SLK-1. B) Expression kinetics of SLK-1-labeled protein in E. coli strains Top10 (TT) and JX33 analyzed by SDS-PAGE. C) EPR spectra of SLK-1-labeled protein samples from panel B (Top10 and JX33 shown in red and black, respectively). D) X-band EPR spectrum of SLK-1 in buffer. E) X-band EPR spectrum of purified thioredoxin labeled with SLK-1 in buffer. Experimental and simulated spectra are shown in red and black, respectively. Adapted with permission (M. J.Schmidt et al. J. Am. Chem. Soc. 2014, 136, 4, 1238-1241).[56a] Copyright American Chemical Society.

Figure 6 .

 6 Figure 6.On the left, apo-BtuB structure (PDB 1NQE). On the right: a-c, background corrected Q-band DEER traces for the double mutant in E. coli cells (red) and in isolated outer membrane (black) in the absence and in the presence of ligands; d-f, distance distributions and their simulations. Adapted with permission (B.Joseph et al. J. Am. Chem. Soc. 2016, 138, 6, 1844-1847).[48b] Copyright (2016) American Chemical Society.

Acknowledgements

We thank A. Magalon and D. Normanno for helpful discussion. We are grateful to E. Etienne and G. Gerbaud from the national TGE RPE facilities (IR 3443). We acknowledge financial support from the Agence Nationale de la Recherche (contract ANR15-CE29-0022-02 and ANR-18-CE11-0007-01) and from the Conseil Régional of Région Sud (APEX-2015).

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