Or Szekely Gregory Olsen
email: gregory.olsen@univie.ac.at

Borja Mateos

Pavel Kadeřávek

Fabien Ferrage

Robert Konrat

Roberta Pierattelli

Isabella Felli

Geoffrey Bodenhausen

Dennis Kurzbach
email: dennis.kurzbach@univie.ac.at12

Sensitivity-enhanced three-dimensional and carbon-detected two-dimensional NMR of proteins using hyperpolarized water

Keywords: Hyperpolarization, Dissolution-dynamic nuclear polarization (D-DNP), Intrinsically disordered proteins (IDPs), Direct 13 C detection, 3D NMR, Non-uniform sampling, BEST-HNCO, HyperW, HYPEX

teaching and research institutions in France or abroad, or from public or private research centers.

Sensitivity-Enhanced Three-Dimensional and Carbon-Detected Two-Dimensional NMR of Proteins using Hyperpolarized Water

Gregory L. Olsen 1,2,* , Or Szekely [START_REF] Reichheld | Direct observation of structure and dynamics during phase separation of an elastomeric protein[END_REF] , Borja Mateos [START_REF] Gil | NMR Spectroscopic Studies of Intrinsically Disordered Proteins at Near-Physiological Conditions[END_REF] , Pavel Kadeřávek, [START_REF] Yuwen | CP-HISQC: a better version of HSQC experiment for intrinsically disordered proteins under physiological conditions[END_REF][START_REF] Lipso | Large dose hyperpolarized water with dissolution-DNP at high magnetic field[END_REF] Fabien Ferrage, [START_REF] Lipso | Large dose hyperpolarized water with dissolution-DNP at high magnetic field[END_REF] Robert Konrat [START_REF] Gil | NMR Spectroscopic Studies of Intrinsically Disordered Proteins at Near-Physiological Conditions[END_REF] , Roberta Pierattelli, [START_REF] Kaderavek | High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents[END_REF] Isabella C. Felli [START_REF] Kaderavek | High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents[END_REF] , Geoffrey Bodenhausen [START_REF] Lipso | Large dose hyperpolarized water with dissolution-DNP at high magnetic field[END_REF] , Dennis Kurzbach 1,5,* , Lucio Frydman 2

Introduction

Nuclear magnetic resonance (NMR) spectroscopy is one of the principal tools for the investigation of the structural dynamics and structure-function relationships of proteins in solution. 1- 4 A significant limitation of the method, however, is its low sensitivity, which can necessitate extensive signal-averaging. The acquisition of protein spectra thus often extends to hours or days, which can be prohibitive for the study of systems which are evolving rapidly or have limited stability in solution. We have recently explored the use of hyperpolarized water [START_REF] Lipso | Large dose hyperpolarized water with dissolution-DNP at high magnetic field[END_REF] to boost signals in protein spectra. Hyperpolarized HDO produced using dissolution dynamic nuclear polarization (D-DNP) can serve as a polarization reservoir, enhancing the 1 H signals of sites undergoing chemical-or polarization exchange with the hyperpolarized solvent. This approach, which has been referred to as or HYPEX [START_REF] Kaderavek | High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents[END_REF] or HyperW [START_REF] Szekely | High-resolution 2D NMR of disordered proteins enhanced by hyperpolarized water[END_REF] , is applicable to a broad range of aqueous systems without requiring any special properties or chemical modification of the molecules under study, and makes it possible to achieve over 100-fold signal enhancements in spectra of folded or intrinsically disordered proteins. [START_REF] Kaderavek | High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents[END_REF][START_REF] Szekely | High-resolution 2D NMR of disordered proteins enhanced by hyperpolarized water[END_REF][START_REF] Kurzbach | Investigation of Intrinsically Disordered Proteins through Exchange with Hyperpolarized Water[END_REF][START_REF] Olsen | Optimizing water hyperpolarization and dissolution for sensitivity-enhanced 2D biomolecular NMR[END_REF][START_REF] Kim | Modeling of Polarization Transfer Kinetics in Protein Hydration Using Hyperpolarized Water[END_REF][START_REF] Ragavan | Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy[END_REF][START_REF] Wang | Determination of Ligand Binding Epitope Structures Using Polarization Transfer from Hyperpolarized Ligands[END_REF][START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF][START_REF] Viennet | Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization[END_REF] In the present study, we expand this approach to include three-dimensional (3D) and [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] Cdetected 2D experiments [START_REF] Gil | NMR Spectroscopic Studies of Intrinsically Disordered Proteins at Near-Physiological Conditions[END_REF] , which we demonstrate using two representative protein systems: the compactly folded model protein Ubiquitin [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF][START_REF] Hamiltona | Identification of the ubiquitin interfacial residues in a ubiquitin-E2covalent complex[END_REF] , and the intrinsically disordered protein (IDP) Osteopontin (OPN), and its complex with its ligand heparin. [START_REF] Kurzbach | Cooperative Unfolding of Compact Conformations of the Intrinsically Disordered Protein Osteopontin[END_REF][START_REF] Rodrigues | The role of osteopontin in tumor progression and metastasis in breast cancer[END_REF] Using the proposed experiments, we obtain hyperpolarized 'fingerprints' of the two proteins, and are also able to detect a minor OPN population that would likely have remained undetected by conventional thermal equilibrium NMR spectroscopy.

Results and discussion

The hyperpolarized HDO-based buffers that are at the heart of this experimental strategy were produced using D-DNP systems operating at ~1.2 K and magnetic fields of either 3.35 T (Oxford HyperSense™) or 6.7 T (Bruker BioSpin). The general procedure is as follows (for details see the Experimental section): i) Hyperpolarization of a frozen H 2 O solution containing a paramagnetic polarization agent and a cryo-protectant is achieved by means of offresonance microwave irradiation. ii) After building up the polarization for 2-3 hours, the hyperpolarized H 2 O is dissolved with a burst of superheated neat or buffered D 2 O to produce a mixture containing 1-4 % HDO (depending on the system and experimental parameters), which is then injected in 1-3 s into an NMR tube waiting in a high-field detection spectrometer, where it mixes in-situ with the target protein solution. iii) Chemical and 'magnetic' exchange processes (transient Overhauser effects) between hyperpolarized HDO and the protein transfer 1 H-hyperpolarization to the protein, thereby selectively enhancing NMR signals of residues with favorable solvent interactions. [START_REF] Kaderavek | High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents[END_REF][START_REF] Szekely | High-resolution 2D NMR of disordered proteins enhanced by hyperpolarized water[END_REF][START_REF] Kurzbach | Investigation of Intrinsically Disordered Proteins through Exchange with Hyperpolarized Water[END_REF][START_REF] Olsen | Optimizing water hyperpolarization and dissolution for sensitivity-enhanced 2D biomolecular NMR[END_REF][START_REF] Kim | Modeling of Polarization Transfer Kinetics in Protein Hydration Using Hyperpolarized Water[END_REF] Signal intensities for all experiments are tabulated in the supplementary information. In most cases, even after extensive signal averaging, the signals in thermal equilibrium spectra for the D-DNP samples were too weak for a reliable determination of the enhancement factor '' (often defined as the ratio between per-scan signal amplitudes in the hyperpolarized spectrum and the corresponding spectrum acquired after return of the system to thermal equilibrium). Therefore, to quantify the signal intensities obtained with hyperpolarized HDO, we instead report signal-to-noise ratios (SNR) for the hyperpolarized datasets. (As an approximate lower bound for , one can thus assume that  ≥ SNR for instances where the corresponding reference signal remains below the limit of detection).

Hyperpolarized high-resolution 13 C-detected 2D NMR

In previous work, we demonstrated how solvent hyperpolarization can be combined with rapid 2D 1 H- [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N HMQC spectroscopy [START_REF] Schanda | SOFAST-HMQC experiments for recording twodimensional heteronuclear correlation spectra of proteins within a few seconds[END_REF] of folded proteins and small IDPs. [START_REF] Kaderavek | High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents[END_REF][START_REF] Szekely | High-resolution 2D NMR of disordered proteins enhanced by hyperpolarized water[END_REF][START_REF] Kurzbach | Investigation of Intrinsically Disordered Proteins through Exchange with Hyperpolarized Water[END_REF][START_REF] Olsen | Optimizing water hyperpolarization and dissolution for sensitivity-enhanced 2D biomolecular NMR[END_REF][START_REF] Kim | Modeling of Polarization Transfer Kinetics in Protein Hydration Using Hyperpolarized Water[END_REF] However, analysis of proton-detected hyperpolarized 2D spectra of larger proteins or IDPs is sometimes challenging, as signal overlap and broadening due to exchange, radiation damping, and potentially paramagnetic relaxation may hinder spectral analysis. To address these limitations, we expand our approach here to include [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C-detected [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C- [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N correlation experiments (H N -CON, see pulse sequence in Figure S1). [START_REF] Gil | NMR Spectroscopic Studies of Intrinsically Disordered Proteins at Near-Physiological Conditions[END_REF][START_REF] Bertini | 13C direct-detection biomolecular NMR spectroscopy in living cells[END_REF] In these experiments, chemical and magnetic exchange transfers proton hyperpolarization from HDO to protein backbone amide (and sidechain) sites. The amide hyperpolarization is then transferred by selective INEPT to the neighboring [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N nuclei, and, after an [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N evolution period, transferred onward to the adjacent 13 CO spins for detection. [START_REF] Gil | NMR Spectroscopic Studies of Intrinsically Disordered Proteins at Near-Physiological Conditions[END_REF] The amide proton polarization is thus continuously replenished in successive scans by the hyperpolarized HDO pool, while 1 H-detection is avoided. Direct [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C detection offers the added advantage that the chemical shift dispersion in the [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C dimension is greatly improved compared to [START_REF] Theillet | Structural disorder of monomeric alpha-synuclein persists in mammalian cells[END_REF] H, while at the same time the penalties associated with 1 H detection are reduced.

Application to a folded protein: Ubiquitin

A hyperpolarized H N -CON spectrum obtained for ubiquitin via this strategy is shown in Fig. 1a (red), overlaid with a conventional H N -CON spectrum of ubiquitin obtained at thermal equilibrium in a 90:10% H 2 O:D 2 O phosphate buffered solution (blue). The hyperpolarized spectrum was acquired in 69 s at 37 °C. Both spectra were obtained in a magnetic field of 14.1T (600 MHz). superimposed on the corresponding thermal equilibrium spectrum collected in 10% deuterated buffer (blue peaks). The assignments denote residue {i}, for each {i, i-1} 15 N-13 C cross peak. b) Ubiquitin crystal structure (PDB code 1ubq), with the subset of residues detected in experiments with hyperpolarized HDO highlighted in red. These consist primarily of residues undergoing rapid solvent exchange, but also include several cases where the enhanced polarization has migrated to adjacent core residues. (Signal intensities are tabulated in supplementary table S1.)

Only if chemical and magnetic exchange from HDO is sufficiently fast, will enhanced signals be observed 6-7, 10, 21-23 . In particular, the signal improvement for an amide site should be increased by faster exchange rates on the one hand, as replenishment of hyperpolarized amide protons between successive scans is improved, while on the other hand the improvement will be attenuated by losses due to proton exchange and relaxation during evolution delays and detection. Thus, as expected, the hyperpolarized spectrum contains a reduced subset of the signals seen in the corresponding conventional spectrum. Figure 1b maps the observed signals onto the crystal structure of ubiquitin, revealing that some residues in the hydrophobic core are also detected, indicating that hyperpolarization may in some cases be transferred from the solvent-exposed surface to the protein's inner core.

The set of peaks observed depends on many variables, in particular on the overall SNR characteristic of the experiment in question, the delay between successive detections, and as noted above, the hyperpolarization transfer efficiency through proton exchange, nuclear Overhauser effects (NOE), or exchange-relayed NOE. While the latter three polarization transfer pathways do not critically depend on the pulse sequence or nucleus used for detection, the SNR of course does. However, if the underlying HYPEX/HyperW exchange and NOE polarization transfer mechanisms are operating as in our earlier proton-detected work [START_REF] Kaderavek | High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents[END_REF] , one should anticipate a qualitative agreement between the set of residues observed in the hyperpolarized proton-detected 1 H/ [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N HMQC spectrum reported there and in the [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C-detected spectrum we obtain here. And indeed, the H N -CON results confirm these expectations, as all peaks observed with H N -CON detection were also observed in the hyperpolarized HMQC. Note that in the H N -CON, we do not see all of the peaks seen in the HMQC spectrum; we see instead the set of peaks which showed the highest SNR in the proton-detected experiment. The reduction in the number of detected residues is due to several factors, such as the additional coherence transfer steps in the H N -CON experiments and the intrinsically lower sensitivity of [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C detection. No new peaks appear in the H N -CON; we thus attribute the difference primarily to the sensitivity penalty accompanying [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C detection.

Moreover, as seen in Fig. 1a, the linewidths in the directly-detected dimension of the hyperpolarized H N -CON (10-15 Hz) are comparable to or narrower than those in its conventional counterpart, and approximately half of those obtained previously using 1 H-detection (25-30 Hz in hyperpolarized 1 H/ 15 N HMQC) [START_REF] Kaderavek | High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents[END_REF] , indicating that 13 C-detection reduces radiation damping and/or decoherence due to rapid proton exchange and paramagnetic relaxationfactors that limited resolution in earlier proton-detected hyperpolarized studies of protein systems. [START_REF] Kurzbach | Investigation of Intrinsically Disordered Proteins through Exchange with Hyperpolarized Water[END_REF] Application to an IDP: Osteopontin and its complex with heparin Figure 2a displays a hyperpolarized 13 C- [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N spectrum of a 220-residue truncation mutant of OPN. Fig. 2b shows a thermal equilibrium reference spectrum collected in a 10% deuterated buffer, and Fig. 2c shows a comparison of the hyperpolarized and reference spectra. The hyperpolarized spectrum was obtained in 55 s at 37 °C and pH 7.4 at 14.1 T (600 MHz for 1 H). As for Ubiquitin, the hyperpolarized and conventional H N -CON spectra show nearly identical linewidths. In contrast to the folded ubiquitin, however, a markedly larger fraction of the peaks observed in the conventional spectrum of OPN are also present in the hyperpolarized spectrum. This is not surprising, given the greater solvent exposure expected for an IDP, which would be consistent with particularly efficient chemical exchange and polarization transfer. A strikingly different spectral response is observed when OPN is monitored after mixing with its ligand heparin. [START_REF] Clemente | Osteopontin Bridging Innate and Adaptive Immunity in Autoimmune Diseases[END_REF] The heparin interaction site spans OPN residues 100 to 180. Upon binding of the large 17 kDa ligand, much of the IDP is in direct contact with heparin, and thus likely becomes somewhat shielded from the solvent. Indeed, when probed by hyperpolarized H N -CON after addition of heparin, the number of detected OPN peaks drops significantly, and the subset of observed residues changes markedly as well (Figure 2d). Due to signal overlap, numerous peaks observed in both the conventional and hyperpolarized spectra of OPN are not assignable with 2D HMQC or H N -CON data alone, making it difficult to determine the true spectral 'binding footprint' for this system. However, the fact that the pronounced drop in observed signals is not restricted to the binding site suggests that screening by the ligand alone is not the only determinant of changes in polarization transfer efficiency in the complex. Transition of OPN from a disordered to a fully-or partially-folded state upon formation of the complex would also be expected to affect solvent exposure and polarization transfer of newly-sequestered residues substantially, perhaps resulting in a sparse enhancement pattern not unlike that seen for ubiquitin.

Hyperpolarized Three-dimensional NMR

While the improvements in resolution provided by the hyperpolarized H N -CON relative to hyperpolarized 1 H- [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N HMQC experiments can be substantial, particularly for samples such as OPN, [START_REF] Mateos | NMR Characterization of Long-Range Contacts in Intrinsically Disordered Proteins from Paramagnetic Relaxation Enhancement in C-13 Direct-Detection Experiments[END_REF] in many instances 2D NMR cannot reduce signal overlap sufficiently to permit residue-specific analysis. To further reduce spectral crowding and to widen the scope of our approach, we therefore implemented hyperpolarized 3D 1 H-13 C- [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N correlation experiments -in particular, the BEST-HNCO experiment shown in Fig. S2. [START_REF] Lescop | A set of BEST triple-resonance experiments for time-optimized protein resonance assignment[END_REF]

Methodological Considerations

Given a final solvent deuteration level of 96-98% after dissolution and mixing, typical proton hyperpolarization lifetimes in these experiments are less than 2 minutes (T 1 ( 1 H)  20 s.) To permit detection of two indirectly encoded dimensions under this time constraint, the use of non-uniform sampling (NUS) [START_REF] Kim | Modeling of Polarization Transfer Kinetics in Protein Hydration Using Hyperpolarized Water[END_REF][START_REF] Ragavan | Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy[END_REF] is mandatory. In the present case, after empirical optimization, 9% NUS was chosen to provide a compromise between spectral resolution along  1 ( 13 C) and  2 ( 15 N), while allowing us to complete the 3D acquisitions within the time restriction of the HDO polarization lifetime. Poisson gap sampling [START_REF] Wang | Determination of Ligand Binding Epitope Structures Using Polarization Transfer from Hyperpolarized Ligands[END_REF] was used to generate NUS schedules, and a total of 96 FIDs were recorded in 65-75 s for a nominal digital resolution of 44 x 89 x 352 Hz in  3 ,   and  1 respectively. The hyperpolarized HNCO spectra presented here were acquired in 65 s (OPN+Heparin) or 75 s (ubiquitin, OPN) at 18.8 T, in 96% deuterated buffer, at pH 7.4 and 37°C.

The full 3D spectra were reconstructed using hmsIST [START_REF] Hyberts | Application of iterative soft thresholding for fast reconstruction of NMR data non-uniformly sampled with multidimensional Poisson Gap scheduling[END_REF] in NMRPipe [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] , and all spectra were cross-checked against conventional thermal equilibrium spectra to identify potential spectral reconstruction artifacts. While many alternative NUS strategies are available [START_REF] Kazimierczuk | Narrow peaks and high dimensionalities: Exploiting the advantages of random sampling[END_REF][START_REF] Kazimierczuk | Non-uniform frequency domain for optimal exploitation of non-uniform sampling[END_REF] , the HMS implementations of sampling and reconstruction were chosen for the proof-of-concept experiments described here, as they are readily available to the NMR community and robust in our hands. 30 Ubiquitin As for the selectively hyperpolarized H N -CON experiments described above, we first tested the hyperpolarized HNCO using ubiquitin as a representative folded protein. Figure 3a displays the projection of the 1 H- [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N plane from a hyperpolarized 3D HNCO spectrum collected in just over one minute overlaid onto a conventional 1 H-15 N 2D HSQC of ubiquitin detected in 2 h (in 90:10 H 2 O:D 2 O, at pH 7.4 and 37 °C). Representative planes of the hyperpolarized HNCO spectrum are shown in Fig. 3b. While the conventional 2D reference spectrum has better resolution than its hyperpolarized 3D counterpart, as expected, the subset of peaks obtained in the hyperpolarized HNCO spectrum are in good agreement with their counterparts in the conventional 2D trace, and the linewidths in the NUS 3D are near the nominal resolution expected for an equivalent 'fully-sampled' 75 second acquisition ( 15 N nominal resolution: 89 Hz (~1.1 ppm); [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N observed resolution: 1-2 ppm). Figure 3c maps the detected residues (in red) onto the ubiquitin crystal structure. This set of detected signals corresponds to the one obtained by the hyperpolarized H N -CON (Fig. 1b), as all peaks observed in the H N -CON are also observed in the HNCO. Notably, as was seen previously in the hyperpolarized HMQC for ubiquitin [START_REF] Kaderavek | High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents[END_REF] , significant enhancements are observed not only for solvent-exposed surface residues, but also for several residues in the protein core.

Osteopontin and its ligand heparin

Figures 4a and4b show representative planes from the hyperpolarized HNCO spectra of the heparin-free and -bound states of OPN (magenta and orange peaks, respectively), overlaid on the corresponding conventional thermal equilibrium HSQC spectra (green and blue, respectively). Figure 4c illustrates the residue-selectivity of the hyperpolarization in the presence and absence of heparin. The signal overlap in conventional 2D spectra of this system would make a transfer of assignments and spectral analysis particularly challenging (see also the Supporting Information). Here, extension of hyperpolarized experiments to encompass a third spectral dimension provides a significant improvement in resolution, and is also able to highlight several features of the OPN-heparin interaction. [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N HSQC spectrum of free OPN in 10% deuterated buffer (green) with three planes from a hyperpolarized 3D NUS HNCO in 96% deuterated buffer (magenta). The dashed box indicates signals of arginine side chains that are folded in both the [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C-and the [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N dimension. b) Overlay of the conventional 2D 1 H-15 N HSQC spectrum of OPN in the presence of 2 equivalents of heparin (blue) and three hyperpolarized 3D NUS HNCO planes (orange: with heparin; magenta: without heparin). c) Superpositions of three zoomed planes from the hyperpolarized HNCO spectrum in the presence of heparin (orange), and conventional 1 H- [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N HSQCs of OPN in the presence (blue) and absence (green) of heparin. Both ligand-free and ligand-bound states are detectable in the HNCO spectrum for residue T176, which is located in the binding site and experiences a chemical shift upon heparin binding. The signal of the heparin-bound form of OPN can be seen in the δ( 13 C) = 178.1 ppm HNCO plane (left), where the bound-form peak (in yellow) coincides with that of the heparin-bound reference (blue), while the heparin-free form is observed in the δ( 13 C) = 177.3 ppm HNCO plane (center), where the signal now coincides with the heparin-free reference (green). For residue K158 (also located in the binding site; right), only the signal of the free form is observed in the presence of heparin, whereas in the bound form this residue is broadened beyond detection in both conventional and hyperpolarized spectra. (Signal intensities are tabulated in supplementary tables S5 and S6.) d) Superposition of the zoomed 1 H-15 N planes of the HNCO shown at left and center in panel c (at δ( 13 C) = 177.3 ppm and δ( 13 C) = 178.1 ppm), at different cut-off levels showing the two distinct peaks which can be discerned for residue T176 in the 3D experiment.

Notably, in the presence of 2 equivalents of the ligand, traces of a second small population were also seen which we attribute to the unbound IDP. This could not be observed in conventionally-detected spectra of the complex. This is illustrated in Figures 4c and4d, where spectral signatures of both the free and bound forms of OPN are detected for residue T176, while in the corresponding conventional spectrum, only the bound form is seen. If we assume that free OPN is not screened by the ligand, and thus more readily enhanced, solvent-based hyperpolarization would be expected to be particularly effective in highlighting the small population of free OPN even in the presence of a much larger population of the bound state. For residue K158 this effect is indeed observed, as only the free form is seen in the presence of heparin. While the pH and temperature used here (7.4, 37 °C) differ from those used in previous studies of this system, which may affect the visibility of certain residues, in any case the ability to detect such small populations should prove valuable for the characterization of sparsely-populated states. Due to substantial overlap, the resolution in hyperpolarized 1D or 2D spectroscopy would not be sufficient to distinguish these two signals (see Fig. S9 in the Supporting Information for representative 1D projections). In the 3D experiments, however, these peaks are well isolated from other resonances and thus could be unambiguously assigned. As can be seen in Fig. 4c, the [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N chemical shifts of the two T176 signals are in good but not perfect agreement with those observed for the two peaks in this region of the static reference spectra. While we cannot exclude the possibility that alternative OPN conformations giving a similar but slightly shifted spectral signature could be present in the sample, the presence of distinct T176 signals in the 3D spectrum is consistent with the presence of both a bound and an unbound OPN species following heparin addition. Thus, by combining the improved sensitivity provided by hyperpolarized water with the improved resolution afforded by 3D detection, signatures of even small populations of minor states can be discerned.

Conclusions

The present study demonstrates how, by combining the use of hyperpolarized water with rapid 3D-and 13 C-detected 2D experiments, we are able to take advantage of the enhanced sensitivity available in earlier hyperpolarized 1D and 2D 1 H- [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N HMQC experiments while providing the improved resolution needed to extend the method to larger proteins and IDPs. Hyperpolarized HDO-based experiments such as those introduced here typically produce spectra showing non-uniform 'hyperpolarization-selective' signal enhancements. Many factors need to be considered when trying to predict the likelihood that the intensity of a given residue will be boosted by means of hyperpolarized solvents, most importantly the proton exchange rate, but also the contributions of direct-or exchange-relayed NOE [START_REF] Kaderavek | High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents[END_REF] . As noted above, the improvement in signal intensity for an amide site is increased by faster exchange rates on the one hand, as replenishment of hyperpolarized amide protons between successive scans is improved, while on the other hand the improvement is attenuated by losses due to proton exchange and relaxation during transfer, evolution, and detection. Moreover, the efficiency of the hyperpolarization also depends on the details of local solvation, the ionic strength, as well as the the polarization and relaxation rate of the HDO, such that reliable predictions are challenging. However, it is already clear that the use of hyperpolarized water to boost protein signals not only provides a more sensitive detection, but also achieves a 'hyperpolarization-selective labeling', carrying valuable insight about the rates of chemical and magnetic (NOE) polarization exchange between solvent and protein. It should also be noted that the experiments presented here suffer a substantial SNR penalty as a result of the dilution of the hyperpolarized water during the dissolution process: in contrast to the 90% H 2 O buffers commonly used in standard NMR measurements, the final H 2 O fractions available to populate the protein sites are typically reduced to 2-4% after mixing of the hyperpolarized HDO with the waiting sample. Most of the protein is thus not visible due to deuteration of the amide protons (and consequently, in most cases we could usually not detect any protein signals for the samples measured here after return to thermal equilibrium.) The required initial protein concentrations are thus comparable to those used in standard experiments. Until dilution during dissolution can be reduced, this will remain a primary limitation of the method. For exchanging sites however, the loss in signal intensity due to limited proton availability in the solvent fraction is compensated due to the typical enhancements that can be over 500-fold carried by the incoming hyperpolarized HDO, making them selectively visible, and doing so very rapidly when exchange is favorable. A key benefit of the reduced acquisition times demonstrated here should thus be in monitoring applications for rapid processes involving growth or evolution of small populations of transient species which can be made visible and resolvable by dissolution DNP with hyperpolarized water. While it has been shown here that both folded and intrinsically disordered proteins are amenable to the method, IDPs in particular suffer from well-known resolution limitations when studied by 1 H-15 N 2D correlation methods. Hyperpolarized 13 C-detected experiments can reduce these penalties, by providing improved spectral dispersion and reducing the broadening effects of rapid solvent exchange. Thus, while the H N -CON may not be particularly informative for samples like Ubiquitin, which was used here to provide proof of concept, for systems like OPN where extensive broadening renders attempts to resolve hyperpolarized 1 H/ [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N correlations difficult or even impossible, the experiment should prove valuable, as it can provide useful site-specific information when alternative D-DNP-enhanced experiments would otherwise be unavailable. Furthermore, the extensive solvent exposure of IDPs facilitates good water-based enhancements, while preserving access to site-specific information about exchange. These combined resolution and sensitivity benefits are made available in hyperpolarized 2D H N -CON NMR experiments.

Hyperpolarized 3D experiments were also shown here to be feasible via the use of NUS, which made such acquisitions compatible with the limited life time of hyperpolarized HDO. The power of the resulting method was demonstrated by its ability to discern residues involved in the binding undergone by a relatively large IDP like osteopontin. It also permitted the detection of small protein populations whose characterization would have been challenging or impossible by other NMR methods. We expect the proposed experiments to be particularly useful for the study of phenomena such as post-translational modifications [START_REF] Bah | Modulation of Intrinsically Disordered Protein Function by Post-translational Modifications[END_REF] , as such protein modification sites are frequently solvent-exposed and hence particularly well suited for investigation by water-derived hyperpolarized experiments.

Material and Methods

Dissolution and polarization conditions

For the hyperpolarized H N -CON experiments, 150 L of a 10 mM solution of 4-amino-TEMPO in an 85:15 v/v mixture of water and glycerol was vitrified in liquid helium. The glassy sample was positively hyperpolarized for ~3 h at ~1.16 K in a magnetic field of 3.35 T by partial saturation of the EPR spectrum at 94.195 GHz (Oxford HyperSense™, Weizmann Institute). Dissolution was achieved with a burst of 2.8 mL deuterated 20 mM phosphate buffered saline (PBS) at 180 °C (453 K) under 0.9 MPa. The sample was subsequently propelled to the nearby detection NMR spectrometer operating at 14.1 T (600 MHz 1 H NMR) and 37 °C (310 K) within 1.5 s with He gas [START_REF] Olsen | Optimizing water hyperpolarization and dissolution for sensitivity-enhanced 2D biomolecular NMR[END_REF] , where it was mixed with 150 L of target protein solution. Transfer and injection of hyperpolarized HDO was performed using an automated high-pressure injection system optimized to deliver reproducible volumes of solvent to the waiting protein solution while suppressing bubble and foam formation during mixing. [START_REF] Szekely | High-resolution 2D NMR of disordered proteins enhanced by hyperpolarized water[END_REF][START_REF] Kurzbach | Investigation of Intrinsically Disordered Proteins through Exchange with Hyperpolarized Water[END_REF][START_REF] Katsikis | Improved Stability and Spectral Quality in Ex Situ Dissolution DNP Using an Improved Transfer Device[END_REF] NMR experiments were then triggered after a 3 s delay for mixing and settling.

For the hyperpolarized HNCO experiments, 180 L of a 15 mM TEMPOL solution in a 0:85:0.15 v/v mixture of water and glycerol was vitrified in liquid helium. The glassy sample was positively hyperpolarized at 1.2 K in a magnetic field of 6.7 T by partial saturation of the EPR spectrum at 187.7 GHz 9 (Bruker Biospin, ENS Paris). Under these conditions, the hyperpolarization build-up rates were found to be R build-up = (4.8 ± 0.1) 10 -5 s -1 , hence hyperpolarization times of 3h were used. Dissolution was achieved with a burst of 5 mL D 2 O at 180 °C (453 K) under 1.05 MPa. The sample was subsequently propelled to a detection NMR spectrometer operating at 18.8 T (800 MHz for 1 H) and 37 °C (310 K) within 1.3 s with He gas under 0.7 MPa through a "magnetic tunnel" maintaining a constant magnetic field of 0.9 T over a distance of approximately 4 m. The delay for mixing with the target protein solution (150 L) and settling of turbulence prior to initiation of the NMR experiments was again 3 s.

NMR Spectroscopy 1 H and [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C chemical shifts were referenced to DSS, [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N chemical shifts were referenced according to the coefficients reported in ref. 33.

H N -CON experiments

The H N -CON experiments were implemented as described by Felli and co-workers [START_REF] Gil | NMR Spectroscopic Studies of Intrinsically Disordered Proteins at Near-Physiological Conditions[END_REF] . All H N -CON spectra were recorded on a 14.1T (600 MHz) Bruker Avance III ® NMR spectrometer equipped with a 5mm Prodigy ® cryoprobe. Bandwidths and offsets of shaped pulses were optimized empirically to maximize excitation of amide protons while minimizing unwanted depletion of the hyperpolarized HDO pool, as well as radiation damping effects that would result if the hyperpolarized water resonance were excited. As the HDO resonance in hyperpolarized water experiments is typically quite broad, the bandwidths of the proton excitation and refocusing pulses are more tightly constrained than in experiments at thermal equilibrium. In our hands, a good compromise for avoiding excitation of the water resonance while still exciting the target H N amide protons of interest was obtained by applying the proton excitation and refocusing pulses at 9.5-10.5 ppm while increasing their bandwidths to ~6 ppm. Selective 1 H excitation and refocusing were achieved using PC9 34 (bandwidth 6.25 ppm, duration 2001 µs), and REBURP [START_REF] Geen | Band-selective radiofrequency pulses[END_REF] (bandwidths 6.0-6.05 ppm, durations 1600-1614 µs) pulses, respectively. Selective proton pulses were centered at 9.75 ppm (OPN) or 10.5 ppm (ubiquitin).

Selective [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C excitation and refocusing were achieved using Q5 (bandwidth 136 ppm, duration 300 µs), and Q3 (bandwidth 115 ppm, duration 199 µs) pulses. [START_REF] Emsley | Gaussian pulse cascades: New analytical functions for rectangular selective inversion and in-phase excitation in NMR[END_REF] Selective 13 C pulses were centered at 173 and 54 ppm for CO and Cα, respectively. IPAP acquisition was used to suppress CO-Cα couplings during acquisition. Non-selective [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N 90 ⸰ pulse durations were 31.5 µs (ubiquitin) and 32.9 µs (OPN).

The spectral widths used were 19.9 and 26.3 ppm for [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C and [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N for ubiquitin, and 30.0 and 26.3 ppm for OPN.

For ubiquitin, the acquisition time in the direct dimension was 85 ms, the recycle delay was 350 ms, and the total time per transient was 537 ms. A total of 64 FIDs (in-phase plus antiphase) were collected, which were combined to give 16 complex increments. Two scans were collected per increment, to give a total experimental duration of 69 s.

For OPN, the acquisition time in the direct dimension was 56 ms, the recycle delay was 50 ms, and the total time per transient 210 ms. A total of 128 FIDs (in-phase plus anti-phase) were collected, which were combined to give 32 complex increments. Two scans were again collected per increment, to give a total experimental duration of 55 s.

Reference H N -CON spectra for ubiquitin and unbound OPN were collected at thermal equilibrium using experimental parameters matched to the corresponding hyperpolarized experiments, but with longer experimental durations, and using 2 mM samples in 90% protonated PBS buffer. For the ubiquitin reference, the number of scans was again 2, the recycle delay was increased to 1 s, and the number of t 1 increments collected was increased to 80 (20 complex t 1 increments), to give a total experimental duration of 3 minutes 6 s. For OPN, the number of scans was increased to 16, the recycle delay was increased to 300 ms, and the number of t 1 increments collected was doubled to 256 (64 complex t 1 increments), to give a total experimental duration of 32 minutes.

HNCO experiments 1 H, [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C, [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N BEST HNCO spectra were recorded on a wide-bore 18.8 T Bruker Avance III HD NMR spectrometer equipped with a TXI probe, using the Bruker library pulse sequence 'b_hncogp3d' (modified to generate shaped 1 H pulses directly from input values of offsets, flip-angles, and bandwidths). [START_REF] Lescop | A set of BEST triple-resonance experiments for time-optimized protein resonance assignment[END_REF] As for the H N -CON experiments, shaped 1 H pulse bandwidth/offset combinations were again optimized empirically.

Selective 1 H pulses were PC9 (bandwidth 4.17 ppm, duration 2251 µs), REBURP (bandwidth 4.85 ppm, duration 1498 µs), EBURP2 and EBURP2tr (bandwidths 4.3 ppm, durations 1439 µs), and BIP720, 50, 20.1 (150 µs). [START_REF] Smith | Improved Broadband Inversion Performance for NMR in Liquids[END_REF] Except for the BIP pulse (set on resonance at 4.7 ppm), all 1 H selective pulses were centered at 10.5 ppm (ubiquitin and OPN) or 9.9 ppm (OPN-heparin).

Selective [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C pulses were G4 and G4tr (bandwidth 125 ppm, duration 308 µs), and Q3 (bandwidth 82 ppm, duration 210 µs). [START_REF] Emsley | Gaussian pulse cascades: New analytical functions for rectangular selective inversion and in-phase excitation in NMR[END_REF] Selective 13 C pulses were centered at 173 ppm for CO, or 53.2 ppm for Cα. Non-selective [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N 90 ⸰ pulse durations were 39.75 µs.

Spectral widths acquired were 13.95 ppm for 1 H, 35.0 ppm for [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N, and 14.0 ppm for [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C. Carrier frequencies were 4.7 ppm ( 1 H), 173.5 ppm ( 13 C) and 117 ppm ( 15 N). Poisson-gap NUS at 9.4% sampling was used, with sampling schedules generated using the HMSist 'nusPGSv3' AU macro. [START_REF] Hyberts | Application of iterative soft thresholding for fast reconstruction of NMR data non-uniformly sampled with multidimensional Poisson Gap scheduling[END_REF] Twenty-four {t 1 , t 2 } increment pairs were collected, to represent the 32x8 complex pairs (TD = 512x64x16) of the corresponding fully-sampled HNCO experiment (or 512x32x32, and 24 increment pairs, for the OPN+heparin sample). The acquisition time in the direct dimension was 23 ms, the recycle delay 600 ms (500 ms for OPN+heparin), and the total time per transient 664 ms, to give a total experimental time of 75 s (65 s for OPN+heparin). The full 3D spectra were reconstructed using the hmsIST [START_REF] Hyberts | Application of iterative soft thresholding for fast reconstruction of NMR data non-uniformly sampled with multidimensional Poisson Gap scheduling[END_REF] add-on in NMRPipe [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] and analyzed with the Sparky 38 program package. Reference HSQC spectra were collected at thermal equilibrium using the standard Bruker sequence 'fhsqcf3gpph'. [START_REF] Mori | Improved Sensitivity of HSQC Spectra of Exchanging Protons at Short Interscan Delays Using a New Fast HSQC (FHSQC) Detection Scheme That Avoids Water Saturation[END_REF] Signals were averaged over 16 scans, and 256 real and 256 imaginary t 1 points (t 1 = 280 s) were collected, with a recovery delay of 1 s. The 1 H and [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N 90 ⸰ pulse durations were 9.7 and 39.7 µs, respectively. To avoid extended signal averaging which would be required for samples in 96-98% deuterated buffers, the HSQC reference spectra were acquired using fresh samples of the same proteins prepared in a 90% protonated PBS buffer, at pH 7.4 and 37 °C.

Protein Sample Preparation.

Uniformly 15 N and [START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C enriched ubiquitin and OPN samples were prepared as described. [START_REF] Kaderavek | High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents[END_REF][START_REF] Platzer | The Metastasis-Associated Extracellular Matrix Protein Osteopontin Forms Transient Structure in Ligand Interaction Sites[END_REF] Before dilution by hyperpolarized HDO, protein concentrations were 2 mM in all cases.

Figure 1 .

 1 Figure 1. a) Hyperpolarized H N -CON correlation spectrum of ubiquitin in 98% deuterated buffer (red signals), superimposed on the corresponding thermal equilibrium spectrum collected in 10% deuterated buffer (blue peaks). The assignments denote residue {i}, for each {i, i-1} 15 N-13 C cross peak. b) Ubiquitin crystal structure (PDB code 1ubq), with the subset of residues detected in experiments with hyperpolarized HDO highlighted in red. These consist primarily of residues undergoing rapid solvent exchange, but also include several cases where

Figure 2 .

 2 Figure 2. a) Hyperpolarized H N -CON correlation spectrum of Osteopontin detected in 55 s using hyperpolarized HDO, in ca. 98% deuterated buffer, with assignments of a few well-resolved residues. b) conventional reference spectrum acquired in 10% deuterated buffer. c) Overlay of the hyperpolarized spectrum (red) and the reference spectrum (black). d) Hyperpolarized H N -CON correlation spectrum of OPN in the presence of 2 equivalents of heparin, with assignments of some well-resolved residues. (Signal intensities are tabulated in supplementary tables S2 and S3.)

Figure 3 .

 3 Figure 3. a) Overlay of a conventional H-15 N HSQC spectrum of ubiquitin obtained in 2 hours (blue) and the projection onto the 1 H-15 N plane of a selectively hyperpolarized 3D HNCO spectrum of ubiquitin obtained in 75 s using NUS (red). Assignments for ubiquitin at pH 7.4 were based upon pH titration experiments (see Fig. S10.) Asterisks denote possible reconstruction artifacts. b) Planes representative of the selectively hyperpolarized HNCO. c) Selectively hyperpolarized residues (red) mapped onto the crystal structure (PDB code 1ubq) of ubiquitin. (Signal intensities are tabulated in supplementary table S4.)

Figure 4 .

 4 Figure 4. Detection of heparin binding to OPN: a) Overlay of a conventional thermal equilibrium 2D 1 H-[START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N HSQC spectrum of free OPN in 10% deuterated buffer (green) with three planes from a hyperpolarized 3D NUS HNCO in 96% deuterated buffer (magenta). The dashed box indicates signals of arginine side chains that are folded in both the[START_REF] Doll | Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures[END_REF] C-and the[START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N dimension. b) Overlay of the conventional 2D 1 H-15 N HSQC spectrum of OPN in the presence of 2 equivalents of heparin (blue) and three hyperpolarized 3D NUS HNCO planes (orange: with heparin; magenta: without heparin). c) Superpositions of three zoomed planes from the hyperpolarized HNCO spectrum in the presence of heparin (orange), and conventional 1 H-[START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF] N HSQCs of OPN in the presence (blue) and absence (green) of heparin. Both ligand-free and ligand-bound states are detectable in the HNCO spectrum for residue T176, which is located in the binding site and experiences a chemical shift upon heparin binding. The signal of the heparin-bound form of OPN can be seen in the δ( 13 C) = 178.1 ppm HNCO plane (left), where the bound-form peak (in yellow) coincides with that of the heparin-bound reference (blue), while the heparin-free form is observed in the δ( 13 C) = 177.3 ppm HNCO plane (center), where the signal now coincides with the heparin-free reference (green). For residue K158 (also located in the binding site; right), only the signal of the free form is observed in the presence of heparin, whereas in the bound form this residue is broadened beyond detection in both conventional and hyperpolarized spectra. (Signal intensities are tabulated in supplementary tables S5 and S6.) d) Superposition of the zoomed 1 H-15 N planes of the HNCO shown at left and center in panel c (at δ( 13 C) = 177.3 ppm and δ( 13 C) = 178.1 ppm), at different cut-off levels showing the two distinct peaks which can be discerned for residue T176 in the 3D experiment.

Acknowledgments

Bruker BioSpin provided a prototype of a D-DNP polarizer to ENS in Paris. This work was supported by the French CNRS, the ERC (contracts 'dilute para water', grant agreement 339754, 'HYPROTIN', grant agreement 801936 and '2F4BIODYN', grant agreement 279519), by the CNRS infrastructure network TGIR-RMN-THC (FR 3050), Equipex grant 'Paris-en-Résonance' (contract ANR-10-EQPX-09), Israel Science Foundation Grant 965/18, the Kimmel Institute of Magnetic Resonance (Weizmann Institute), and the generosity of the Perlman Family Foundation. We are grateful to Kobi Zibzener for his help throughout the project.


Jérôme Médard

Philippe Decorse

Claire Mangeney

Jean Pinson

Maurizio Fagnoni

Stefano Protti

Simultaneous Photografting of Two Organic Groupson a Gold Surface by using Arylazo Sulfones As the Single Precursors

Arylazo sulfones have been exploited as photoactivable substrates for the simultaneous photografting of both aryl and methanesulfonyl groups on a gold surface. The obtained samples have been characterized by different spectroscopic techniques including Infrared reflection absorption (IRRAS), Surface-enhanced Raman (SERS), XPS spectroscopy and AFM.

INTRODUCTION.

The surface of many objects for industrial use or daily life is modified either for aesthetic purposes (plating of metals by chromium or gold), for preventing corrosion (painting of mild steel objects), or for adding new properties to a material (self-cleaning glasses). As concerning organic coatings, the simplest one is painting that protects a steel gate from rusting, but there is an interest in modifying surfaces with more strongly bonded organic films.

A significant example of the potentialities of surface modification in medicine is provided by stents insertion into arteries, where the release of an antimitotic agent (Paclitaxel for example) prevents restenosis. [START_REF] Haroun | Drug eluting with a biodegradable release layer attached with an electro-grafted primer coating[END_REF][START_REF] Vergnol | Electrografting of a biodegradable layer as a primer adhesion coating onto a metallic stent: in vitro and in vivo evaluation[END_REF] Binding of the drug on the metal surface must be strong enough to resist friction during implantation; this has been achieved by electrochemical reduction of a diazonium salt in the presence of butyl methacrylate, using the stainless steel stent as a cathode. [START_REF] Haroun | Drug eluting with a biodegradable release layer attached with an electro-grafted primer coating[END_REF][START_REF] Vergnol | Electrografting of a biodegradable layer as a primer adhesion coating onto a metallic stent: in vitro and in vivo evaluation[END_REF] Several methods to obtain such strongly (covalently) bonded films are available. Selfassembled monolayers (SAMs) of silanes, [START_REF] Pujari | Covalent surface modification of oxide surfaces[END_REF][START_REF] Zharov | Surface-Modified Silica Colloidal Crystals: Nanoporous Films and Membranes with Controlled Ionic and Molecular Transport[END_REF][START_REF] Chandekar | Thermal stability of thiol and silane monolayers: A comparative study[END_REF] and phosphonates, [START_REF] Sahoo | Alkyl Pho pho /Pho ph Co g o M g N op r c : A Co p r o w h F Ac d[END_REF] have been prepared on oxide surfaces, whereas alkenes and alkynes have been covalently bonded to hydrogenated silicon [START_REF] Collins | Palladium-Catalyzed Coupling Reactions for the Functionalization of Si Surfaces: Superior Stability of Alkenyl Monolayers[END_REF]8 or carbon. [START_REF] Ssenyange | Covalent Bonding of Alkene and Alkyne Reagents to Graphitic Carbon Surfaces[END_REF] Analogously, long alkyl chain thiols SAMs have been attached to different metals, including Au, Ag and Cu. [START_REF] Love | Self-Assembled Monolayers of Thiolates on Metals as a Form of Nanotechnology[END_REF] Electro-grafting strategies were also adopted in the presence of diazonium salts as grafting agents, able to provide aryl films covalently bonded to a variety of surfaces (carbon, metals, oxides, polymers). [START_REF] Berisha | Electrode surface modification using diazonium salts[END_REF][START_REF] Langer | Electrografting: a powerful method for surface modification[END_REF] Photoinduced functionalization of surfaces by means of visible-or UV-light irradiation of benzoin derivatives, [START_REF] Frick | Toward a quantitative description of radical photoinitiator structure-reactivity correlations[END_REF] azobisisobutyronitrile [START_REF] Qi | Fluorine modification on titanium dioxide particles: Improving the anti-icing performance through a very hydrophobic surface[END_REF] and benzophenone [START_REF] You F | UV-light assisted single step route to functional PEEK surfaces[END_REF] was likewise exploited in radical-triggered polymerization. [START_REF] Prucker | Synthesis of poly(styrene) monolayers attached to high surface area silica gels through self-assembled monolayers of azo initiators[END_REF] Benzophenone has been commonly used as a photoinitiator, since the biradical character of its triplet excited state, acts as a hydrogen atom abstractor to generate the radical responsible of the polymerization event. For example, the photografting of porous three-dimensional materials has been achieved via the benzophenone photoinitiated surface polymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid within the pores of a macroporous polymer monolith contained in a fused silica capillary. [START_REF] Rohr | Photografting and the Control of Surface Chemistry in Three-Dimensional Porous Polymer Monoliths[END_REF] In another a hydrogen donor (the N,N-dimethyl aminophenyl group, DMA) was first electrografted on the surface, and benzophenone in solution was used as the photosensitizer to strip hydrogen from the grafted DMA. This system permitted efficient surface initiation of photopolymerization of 2-hydroxyethyl methacrylate. [START_REF] Gam-Derouich | Diazonium salt-derived 4-(dimethylamino)phenyl groups as hydrogen donors in surface-confined radical photopolymerization for bioactive poly(2-hydroxyethyl methacrylate) grafts[END_REF] Short lived intermediates such as singlets or triplets carbenes and nitrenes presenting free doublets [START_REF] Rch | Adv c d Org c Ch r[END_REF] can be involved in insertion processes occurring into alkyl and aryl C-H and X-H (X = O, N, S) bonds for the surface modification of polymers. Among these species, diarylcarbenes, obtained by thermolysis or photolysis of the corresponding diazo compounds, have been investigated [START_REF] Wentrup | Carbenes and Nitrenes: Recent Developments in Fundamental Chemistry Angew[END_REF] for the preparation of highly colored polystyrene beads. 21 Diazonium salts (Ar-N 2 + ) or iodonium salts (Ar-I + -Ar') under UV 22 or visible light exposure (in the presence of a photoredox catalyst) [START_REF] Bouriga | Sensitized Photografting of Diazonium Salts by Visible Light[END_REF]24,[START_REF] Garcia | Polymer Grafting by Inkjet Printing: A Direct Chemical Writing Toolset[END_REF][START_REF] Schroll | Photocatalytic surface patterning of cellulose using diazonium salts and visible light[END_REF] cleave to aryl radicals that react with metals or polymers. In this way it was possible to pattern gold surfaces, 24 polyethyleneterephthalate (PET) [START_REF] Garcia | Polymer Grafting by Inkjet Printing: A Direct Chemical Writing Toolset[END_REF] or paper. [START_REF] Schroll | Photocatalytic surface patterning of cellulose using diazonium salts and visible light[END_REF] However, all the above reactions allow for the functionalization of the surface by a single chemical group obtained from the starting precursor. On the other hand, the preparation of mixed layers in order to enrich the surface chemistry has received less attention. As an example, mixed monomers providing charge transfer complexes such as styrene and vinyl acetate or maleic anhydride have been photografted to improve the surface properties of PET. [START_REF] Zhou | Ultraviolet Grafting of Styrene and Maleic Anhydride on Polyethylene-Terephthalate Film[END_REF][START_REF] Jianping | Surface Photografting Polymerization of Vinyl Acetate, Maleic Anhydride, and Their Charge-Transfer Complex.VIII. Charge-Transfer Complex[END_REF] Two incompatible enzymes (trypsin and transglutaminase) were separately encapsulated into different poly(ethylene glycol) (PEG) network layer grafted on a polyethylene film via visible light induced living photografting polymerization. [START_REF] Zhu | Separated Immobilization of Incompatible Enzymes on Polymer Substrate via Visible Light Induced Living Photografting Polymerization[END_REF] Electrografting also allows the preparation of mixed layers. Thus, a binary mixture of aryl diazonium salts bearing oppositely charged para substituents (-SO 3 -and -N(CH 3 ) 2 + ) has been electrografted and the surface ratio of the two phenyl groups is consistently 1:1 regardless of the ratio of the two aryl diazonium salts in the modification solutions. [START_REF] Gui | Using Supramolecular Binding Motifs to Provide Precise Control over the Ratio and Distribution of Species in Multiple Component Films Grafted on Surfaces: Demonstration Using Electrochemical Assembly from Aryl Diazonium Salts[END_REF] The grafting of the 1-(bithien-2-yl)-4-benzenediazonium (BTB) salt complexed by β-cyclodextrin (CD) provides a layer of grafted BTB oligomers complexed by CD. When CD is removed from the surface, pinholes are created and the 4-nitrobenzenediazonium salt is reduced to graft a second component. [START_REF] Santos | Formation of Mixed Organic Layers by Stepwise Electrochemical Reduction of Diazonium Compounds[END_REF] Following the same strategy, after electrografting of the sterically hindered 4-((triisopropylsilyl)ethynyl)phenyl groups, the cleavage of the isopropyl groups and further grafting of nitrophenyl groups also permits to obtain mixed aryl layers. [START_REF] Lee | Mixed Monolayer Organic Films via Sequential Electrografting from Aryldiazonium Ion and Arylhydrazine Solutions[END_REF] Mixed layers of aminophenyl and perfluoroalkyl groups electrografted on carbon fibers (obtained from aryldiazonium salt and perfluoroalkyliodide) permit to obtained hydrophobic carbon-epoxy composites with improved mechanical properties. [START_REF] Arnold | Simultaneously increasing the hydrophobicity and interfacial adhesion of carbon fibres: a simple pathway to install passive functionality into composites[END_REF] Mixed layers have also been prepared by combined electro and photografting: a multilayer film of nitrophenyl groups was electrografted to a carbon surface and 4-aminophenylazide was further photografted to this surface from a spin-coated solution. [START_REF] Gross | Two-Component Mixed and Patterned Films on Carbon Surfaces through the Photografting of Arylazides[END_REF] We present herein a protocol for the simultaneous gold surface modification by two different functional groups, starting from arylazo sulfones 1a-c as photoactivable precursors.

The presence of a dyedauxiliary group -N 2 SO 2 CH 3 in such bench stable derivatives is responsible for both color and photoreactivity of the molecules that, when irradiated, undergo homolytic cleavage of the N-S bond, and the formation of the corresponding aryl radical (Ar • ). [START_REF] Crespi | Wavelength Selective Generation of Aryl Radicals and Aryl Cations for Metal-free Photoarylations[END_REF] Such substrates have found recent application in organic synthesis, for the visible-light induced formation, under metal-and

(photo)catalyst free conditions, of both C-C [START_REF] Malacarne | A visible light driven, metal-free route to aromatic amides via radical arylation of isonitriles[END_REF][START_REF] Dossena | Visible light promoted metal-and photocatalyst-free synthesis of allylarenes[END_REF]38,[START_REF] Sauer | Photocatalyst-free, visible light driven, gold promoted Suzuki synthesis of (hetero) biaryls[END_REF] and C-heteroatom bonds. [START_REF] Blank | Visible Light-Promoted Formation of C-B and C-S Bonds under Metal-and Photocatalyst-Free Conditions[END_REF][START_REF] Liu | Catalyst-free visible-light-initiated oxidative coupling of aryldiazo sulfones with thiols leading to unsymmetrical sulfoxides in air[END_REF] In contrast, the use of arylazo sulfones in material chemistry received only few attentions in the past as the use of arylazo tosylsulfones as thermal initiators in the radical polymerization of acrylate monomers. [START_REF] Crespi | Wavelength Selective Generation of Aryl Radicals and Aryl Cations for Metal-free Photoarylations[END_REF] In this paper we present the photografting of azosulfones and demonstrate that both substituents of the N=N bond are effectively bonded to the surface.

EXPERIMENTAL.

Chemicals. Arylazo sulfones were prepared from the corresponding anilines by following a known method. [START_REF] Malacarne | A visible light driven, metal-free route to aromatic amides via radical arylation of isonitriles[END_REF] Spectroscopical data of the compounds are in accordance with the literature. Dry and purified acetonitrile was obtained from a Pure Solv system from Innovative Technology.

Photografting was achieved by placing a gold plate in a test tube with a septum screw cap; the tube was filled with argon and a 0.1 mM solution of the chosen azosulfone in ACN was deposited with a needle, through the septum, on the gold surface. Irradiation was performed using a Kessil Lamp (40 W, 456 nm) as the light source for 8 h (Scheme 1).

Scheme 1. Experimental setup.

Substrates. Gold coated (100 nm) silicon wafers obtained from Sigma-Aldrich, were cut into 1 × 1 cm 2 pieces and successively rinsed in a bath of sulfuric acid (95%) for 2 s, and with distilled water and under sonication in distilled water, acetone, and absolute ethanol (380 s each) and finally dried by an argon stream.

Infrared reflection absorption spectroscopy (IRRAS). Spectra of modified plates were recorded using a purged (low CO 2 , dry air) Jasco FT/IR-6100 Fourier Transform InfraRed Spectrometer equipped with an MCT (mercury-cadmium-telluride) detector. For each spectrum, 1000 scans were accumulated with a spectral resolution of 4 cm -1 . The background recorded before each spectrum was that of a cleaned substrate.

Surface-enhanced Raman spectroscopy (SERS).

The SERS spectra were recorded on gold SERS substrates modified by arylazo sulfone using a Horiba XploRA PLUS Raman microscope with an excitation wavelength of 633 nm and a power of 0.6 mW. The spectral resolution was less than 3 cm -1 and the accumulation time was set to 20 s with two repetitions.

XPS Spectra. XPS measurements were performed using a K-Alpha system (Thermo Fisher Scientific, East-Grinstead, UK) fitted with a microfocused and monochromatic Al Kα X-ray source (1486.6 eV, spot size: 400 μm). The pass energy was set to 150 and 40 eV for the survey and the high-resolution spectra, respectively. The spectra were calibrated against the C-C/C-H C 1s component set at 285 eV.

The chemical composition was determined with version 5.9902 Avantage software, by using the manufacturer sensitivity factors. The spectra were calibrated against C 1s set at 285 eV.

Contact Angle Measurements. The water interaction properties were characterized with a drop shape r ss DSA30S and a Drop Shape Analysis 1.92.1.1 version software.

AFM Images. AFM images were recorded with a NT-MDT Solver pro equipment. AFM topography was performed in the intermittent contact mode with standard silicon cantilevers. Image analysis was achieved with the free software WSxM. IRRAS. After irradiation of the azosulfone solution deposited on a gold plate (in deoxygenated ACN), the plate was ultrasonicated for 180 s in EtOH and dried under argon, an IRRAS spectrum was recorded. The modified gold plates are referred as Au-NO 2 , Au-CN, Au-COCH 3 . The IR and Raman spectra indicate the presence of the aromatic groups and also of the substituents (NO 2 , CN, C(=O)CH 3 ), in the absence of irradiation these IR signals are not observed (Table 1, Figure 1). We also searched for the presence of azo bonds either as Au-N=N-or as -N=N-inside the film. The Ar-N=N-SO 2 Me stretching bands are weak in the starting azosulfones (1414, 1404 and 1407 cm -1 for, respectively, 1a, 1b, 1c) and are not observed in Au-NO 2 , Au-CN, Au-COCH 3 . [START_REF] Socrates | Infrared and Raman Characteristic Group Frequencies[END_REF] SO 2 stretching ( sym(SO2) at ca. 1110-1170 cm -1 ) and bending ( (SO2) at ca. 325-365 cm -1 ) vibrations [START_REF] Bazylewski | In situ Raman spectroscopy distinguishes between reversible and irreversible thiol modifications in L-cysteine[END_REF][START_REF] Wang | Enhanced gas sorption and breathing properties of the new sulfone functionalized COMOC-2 metal organic framework[END_REF][START_REF] Diaz-Fleming | Surface-enhanced Raman scattering and density functional theory studies of bis(4-aminophenyl)sulfone[END_REF] cannot be safely assigned by IR while they clearly appear in the surface-enhanced Raman spectrum at 1174 cm -1 and 1363 cm -1 (Table 1, Figure 2). XPS. The modified Au plates were also examined by XPS. The three samples present a contribution of Au4f, this indicates that the organic is thinner than 5-10 nm. The observation of the signals assigned to the substituents confirms the grafting of the substituted aryl rings and of the sulfone group on the gold surface (Table 2, Figure 3). It is difficult to differentiate a grafted substituted phenyl ring from a substituted azophenyl group; however, the Au-N=N-Ar structure has been observed during the grafting of diazonium salts on Au through an N1s signal at 397.5 eV. [START_REF] Deniau | Spontaneous Grafting of Diazonium Salts: Chemical Mechanism on Metallic Surfaces[END_REF] In our case, the deconvolution of the N1s peaks, does not indicate the presence of this bond except after grafting the NO 2 substituted azosulfone where a very small contribution is observed at this energy. In addition, the XPS spectra indicate the presence of oxidized sulfur that corresponds to sulfone groups, but also of unoxidized sulfur similar to that of thiol SAMS, the origin of reduced sulfur results, most probably, from a reduction by the X-ray beam.

RESULTS AND DISCUSSION

The spectrum of Au-NO 2 present a N1s contribution at 406.0 eV characteristic of the nitro groups, the N1s contribution at 399.1 and 400.1 eV (corresponding to a nitrogen and its protonated form), result respectively from i) some contamination, ii) the reduction of the nitro group under the X-ray beam, but could also be assigned to iii) azo bonds inside the film as with diazonium salts. The C1s peak presents three contributions at 284.8, 286.0 and 288.4 eV that can be assigned to aromatic carbons but also to the carbons bonded to sulfur (284.7, 285.3 and 286.3 for polyethersulfone [START_REF]NIST X-ray Photoelectron Spectroscopy Database[END_REF] ). The sulfur is mostly present in its oxidized form at 166.9 eV assigned to the sulfone group (Figure 4).

Au-CN presents two N1s contributions at 399.3 and 401.7 eV that can be assigned to the cyano group but also to some contamination. Nearly equal amounts of oxidized and reduced sulfur are observed.

The C1s peak also presents three contributions at 284.8, 286.3, 287.9 eV). Au-COCH 3 , the C1S peak presents three contribution at 284.8, 286.2, and 287.8 eV, the last two correspond to oxidized carbon C(=O) and C-S. The above experiments indicate that the film is a thin multilayer with very high needles, they also show that both the aryl group and the sulfone groups are part of this film. The formation of multilayers is a common feature of all radical grafting reactions but the photografting of two groups deriving from the starting compound is unexpected; therefore, the problem arises of determining the structure of the film.

The previously described mechanism permits to get a first insight into the formation of this film. methanesulfonyl radicals. The corresponding aryl radical is obtained from N 2 loss of the diazenyl radical (path c). [START_REF] Arnold | Simultaneously increasing the hydrophobicity and interfacial adhesion of carbon fibres: a simple pathway to install passive functionality into composites[END_REF] Trapping by the gold surface of the diazenyl radical/ methanesulfonyl radical pair allowed the formation of the mixed functionalized surface (path d, Scheme 2). The presence of the aryl groups in the film and on the surface, has been well documented during of diazonium salts and the IRRAS spectra of surfaces grafted by arylazo sulfones indicate the presence of these groups. On the other hand, the presence of the methanesulfone moiety on the film is attested by Raman and XPS; however, we also noticed that the methanesulfonyl radical can either i) attach to the surface (path d) or ii) react on grafted aryl groups (p h d') or both; to test this hypothesis we have abraded the surface with clusters of 2000 Ar + ions with a total energy of 4 keV and recorded spectra at different times (note that the total thickness of the film is analyzed). Figure 6 presents the spectra recorded after 0, 60, 120 and 180 s and the profile of the atomic % of the nitro group and of sulfur. The NO 2 % decreases linearly with abrasion time, while S2p % increases slightly (due the decrease of the NO 2 %) and decreases when reaching the surface. This indicates that the methanesulfonyl radical only reacts on the surface and not on the aryl group as in Scheme 2, path d''. In the case of 4-nitrophenylazo sulfone, early results suggested that trapping of the diazenyl radical could occur faster than N 2 extrusion, and the formation of the Au-N 2 -Ar bond being in competition with that of the Au-Ar bond (path c'). We thus examined the possibility of grafting a diazenyl radical.

This radical generated by photochemistry of arylthiodiazene has been observed by means of ESR analyses, the decomposition rate of the phenyl diazenyl radical was measured at -96 °C : k = 34 s -1 . [START_REF] Suehiro | Decay Reactions of Aryldiazenyl Radicals in Solution[END_REF] The bond dissociation energy of the Au-N bond in Au-N=N-Ar structures has been calculated by DFT methods, it is much smaller (7.91 kcal mol -1 ) 54 than that of an Au-Ar bond (38.5 kcal mol -1 in water).

Nevertheless, an Au-N bond has been characterized by an XPS signal at 397.5 eV assigned to gold nitride during the spontaneous grafting of 4-nitrobenzenediazonium in unbuffered water. [START_REF] Deniau | Spontaneous Grafting of Diazonium Salts: Chemical Mechanism on Metallic Surfaces[END_REF] In the spectra above, there is no sign of -N=N-bonds, however a very small shoulder appears at 397.6 on the C1s XPS of Au-NO (arrow in Figure 3A). Therefore Au-N=N-bonds should be present only in Au-NO 2 (path c'). In this case trapping of the diazenyl radical occurs faster than N 2 extrusion, and the formation of the Ar-N 2 -Au bond competes with that of the Ar-Au bond (path d).

Conclusion

The surface modification of gold by photolysis of arylazo sulfone permits the attachment of both the substituted aryl groups and of the S(=O) 2 CH 3 group; two groups are then grafted from a single precursor. This results from the photocleavage mechanism of azosulfones that provide two radicals, while aryl radicals react both on the surface and the first grafted groups the methanesulfonyl radical reacts only with the surface. Due to its small size it is buried in the polyphenylene oligomers film; it appears as a diluent of the aryl oligomers, a feature that is looked for when attaching large molecules to the extremities of the aryl oligomers. [START_REF] Liu | An Interface Comprising Molecular Wires and Poly(ethylene glycol) Spacer Units Self-Assembled on Carbon Electrodes for Studies of Protein Electrochemistry[END_REF]

Figure 2 .

 2 Figure 2. Surface-enhanced Raman spectrum of Au-NO 2 .

Figure 3 .

 3 Figure 3. XPS spectra (N1s contribution) of A) Au-NO 2 and B) Au-CN.

Figure 4 .Figure 5 .

 45 Figure 4. XPS spectra (S2p contribution) of Au-NO 2

  Visible light irradiation of arylazo sulfones resulted in the homolytic cleavage of the N-S bond (via the 1 π* g xc d , (Scheme 2, paths a, b) and the generation, of the diazenyl and

Figure 6 .Scheme 2 .

 62 Figure 6. Abrasion of the Au-NO 2 film and profile through the film of the NO 2 and S signals

Table 1 .

 1 IR and Raman spectra of Au-NO 2 , Au-CN, Au-COCH 3 , wavenumbers (cm -1 )

	Azosulfones		Aromatic	NO 2	a)	CN	COCH 3	CH 3 S(=O) 2
			bands
	1a, R = NO 2 IR	1598	1528			-
			1580	1350
		Raman	1591	1335			1174, 1363
	1b, R= CN	IR	1577			2229 b)		-
	1c, R =	IR	1560,				1694 c)
	COCH 3		1604,
			1576

-Figure 1. IRRAS spectra of Au-NO 2 , Au-CN, Au-COCH 3 .

Table 2 .

 2 XPS Spectra (Binding Energy eV, and % elemental composition) ) Polyacrylonitrile N1s at 399.6 eV,[START_REF] Beamson | High Resolution XPS of Organic Compounds[END_REF] benzonitrile N1s a.t 399.2, 50 polymethacrylonitrile C1s at 286.4 eV[START_REF] Beamson | High Resolution XPS of Organic Compounds[END_REF] , b) poly(3methyl-but-3-en-2-one) at 287.4 eV. 51 c) see reference.[START_REF] Deniau | Spontaneous Grafting of Diazonium Salts: Chemical Mechanism on Metallic Surfaces[END_REF] d) corresponds to an oxidized sulfur, for example Na 2 SO 4 at 168.4 eV) e) corresponds to an Au-S-R bond, for example Au-S-C 18 H 37 .

	Au-NO 2 , CN, COCH 3	NO 2	CN	COCH 3	Au-N	S
	R = NO 2	N1s 406.0 (2.1)			N1s 397.5 c) (<0.1)	S2p 168.9 d) (0.3)	S2p 161.8 e) (1.0)
			C1s
	R = CN		286.3 a) (6.2) N1s 400.0		-	S2p 168.9 d) (0.4)	S2p 162.2 e) (1.9)
			5.2
	R = COCH 3			C(1s) 287.9 b (5.5)	-	S2p 168.7 d) (0.6)	S2p 162.3 e) (1.3)

a

By comparison,48 nitrobenzene signals were observed at 1521 and 1347 cm -1 ; b) benzonitrile at 2230 cm -1 ; c) acetophenone at 1686 cm-1

Acknowledgments CNRS, Paris Diderot University, ANR (Agence Nationale de la Recherche) and CGI (Commissariat a l'Investissement d'Avenir) are gratefully acknowledged for their financial support through Labex SEAM (Science and Engineering for Advanced Materials and devices) ANR 11 LABEX 086, ANR 11 IDEX 05 02.