Pauline Gosset

Grégory Taupier

Olivier Crégut

Johanna Brazard

Yves Mély

Kokou-Dodzi Dorkenoo

Jérémie Léonard
email: jeremie.leonard@ipcms.unistra.fr

Pascal Didier
email: pascal.didier@unistra.fr

Excited-State Proton Transfer in Oxyluciferin and its Analogues

One of the most characterized bioluminescent reactions involves the firefly luciferase that catalyzes the oxidation of the luciferin producing oxyluciferin in its first excited state. While relaxing to the ground state, oxyluciferin emits visible light with an emission maximum that can vary from green to red. Oxyluciferin exists under six different chemical forms resulting from a keto/enol tautomerization and the deprotonation of the phenol or enol moieties. The optical properties of each chemical form have been recently characterized by the investigations of a variety of oxyluciferin derivatives, indicating unresolved excited-state proton transfer (ESPT) reactions. In this work, femtosecond pump-probe spectroscopy and picosecond-resolved fluorescence are used to investigate the picosecond kinetics of the ESPT reactions and demonstrate the excited state keto to enol conversion of oxyluciferin and its derivatives in aqueous buffer as a function of pH. A comprehensive photophysical scheme is provided describing the complex luminescence pathways of oxyluciferin in protic solution.

TOC Graphic

Bioluminescence refers to the fascinating phenomenon of natural conversion of chemical energy into light 1 by living organisms. It is observed in the deep sea from sharks 2 , dinoflagellates 3,4 , jellyfishes 3 or on earth from bacteria 3,5 or worms 6 . Up to now, firefly is the most studied bioluminescent organism due to the high efficiency of its light-emissive enzymatic

reaction [START_REF] Ando | Firefly Bioluminescence Quantum Yield and Colour Change by PH-Sensitive Green Emission[END_REF] . The light is emitted through the luciferase-catalyzed oxidation of D-luciferin resulting in the formation of oxyluciferin (OxyLH 2 ) in its first singlet excited state (S 1 ), which then decays radiatively to the ground state [START_REF] White | The Chemi and Bioluminescence of Firefly Luciferin : An Efficient Chemical Production of Electronically Excited States[END_REF][START_REF] Mccapra | Chemical Mechanisms in Bioluminescence[END_REF][START_REF] Mccapra | The Chemistry of Bioluminescence[END_REF][START_REF] Branchini | Yellow-Green and Red Firefly Bioluminescence from 5,5-Dimethyloxyluciferin[END_REF] . Due to its high quantum yield and the absence of autofluorescence (no photo-excitation) that enable a high signal-to-noise ratio, the luciferin/luciferase complex is very popular and widely used as a luminescent probe [START_REF] Branchini | Yellow-Green and Red Firefly Bioluminescence from 5,5-Dimethyloxyluciferin[END_REF][START_REF] Branchini | Red-Emitting Chimeric Firefly Luciferase for in Vivo Imaging in Low ATP Cellular Environments[END_REF] for bioassays [START_REF] Roura | Bioluminescence Imaging: A Shining Future for Cardiac Regeneration[END_REF][START_REF] Minekawa | Practical Application of Bioluminescence Enzyme Immunoassay Using Enhancer for Firefly Luciferin-Luciferase Bioluminescence[END_REF][START_REF] Takakura | Aminoluciferins as Functional Bioluminogenic Substrates of Firefly Luciferase[END_REF] and for in vivo and in vitro bioimaging [START_REF] Sun | D-Luciferin Analogues: A Multicolor Toolbox for Bioluminescence Imaging[END_REF][START_REF] Greer | Imaging of Light Emission from the Expression of Luciferases in Living Cells and Organisms: A Review[END_REF][START_REF] Shinde | Luciferin Derivatives for Enhanced in Vitro and in Vivo Bioluminescence Assays[END_REF][START_REF] Tian | Visualization of in Vivo Hydrogen Sulfide Production by a Bioluminescence Probe in Cancer Cells and Nude Mice[END_REF][START_REF] Buckley | In Vivo Bioimaging with Tissue-Specific Transcription Factor Activated Luciferase Reporters[END_REF] .

Natural bioluminescence wavelengths are observed to vary from green to red [START_REF] Seliger | The Colors Of Firefly Bioluminescence: Enzyme Configuration And Species Specificity[END_REF][START_REF] Cheng | Theoretical Development of Near-Infrared Bioluminescent Systems[END_REF] . Due to its increasing number of applications, it is essential to understand the origin of the color modulation which depends on several factors such as temperature, pH or nature of the luciferase enzyme [START_REF] Seliger | The Colors Of Firefly Bioluminescence: Enzyme Configuration And Species Specificity[END_REF][START_REF] Hiyama | Theoretical Insights into the Effect of PH Values on Oxidation Processes in the Emission of Firefly Luciferin in Aqueous Solution[END_REF][START_REF] Orlova | Theoretical Study of the Amazing Firefly Bioluminescence: The Formation and Structures of the Light Emitters[END_REF][START_REF] Hosseinkhani | Molecular Enigma of Multicolor Bioluminescence of Firefly Luciferase[END_REF] . OxyLH 2 can exist under six different chemical forms resulting from keto/enol tautomerization (see Scheme 1) and the deprotonation of the phenol and enol moieties [START_REF] Snellenburg | Excited-State Dynamics of Oxyluciferin in Firefly Luciferase[END_REF][START_REF] Ran | The Spectral-Structural Relationship of a Series of Oxyluciferin Derivatives[END_REF] . The main obstacle to the understanding of the OxyLH 2 fluorescence emission is the high complexity of its environment-sensitive photochemistry, which implies several ground-state equilibria and excited-state interconversions between various protonated/deprotonated and keto/enol forms.

In addition, a major limiting experimental factor has long been related to the chemical synthesis of OxyLH 2 , its lability being due to the presence of both keto and enol forms in protic solution that permits a Mannich-type dimerization [START_REF] Maltsev | Why Is Firefly Oxyluciferin a Notoriously Labile Substance?[END_REF] . For these reasons, most spectroscopic investigations were performed on luciferin in solution in the absence of the enzyme [START_REF] Cheng | What Exactly Is the Light Emitter of a Firefly?[END_REF][START_REF] White | Analogs of Firefly Luciferin[END_REF][START_REF] Kakiuchi | Spectroscopic Properties of Amine-Substituted Analogues of Firefly Luciferin and Oxyluciferin[END_REF][START_REF] Ghose | Emission Properties of Oxyluciferin and Its Derivatives in Water: Revealing the Nature of the Emissive Species in Firefly Bioluminescence[END_REF][START_REF] Solntsev | Photoinduced Dynamics of Oxyluciferin Analogues: Unusual Enol "Super"Photoacidity and Evidence for Keto-Enol Isomerization[END_REF] or only recently on OxyLH 2 and model compounds where excited state proton transfer (ESPT) or ketoenol tautomerization are blocked. The steady-state optical properties of these derivatives have been extensively investigated both theoretically [START_REF] Ran | The Spectral-Structural Relationship of a Series of Oxyluciferin Derivatives[END_REF][START_REF] García-Iriepa | Simulation and Analysis of the Spectroscopic Properties of Oxyluciferin and Its Analogues in Water[END_REF][START_REF] Pinto Da Silva | Theoretical Study of the Nontraditional Enol-Based Photoacidity of Firefly Oxyluciferin[END_REF][START_REF] Pinto Da Silva | Quantum/Molecular Mechanics Study of Firefly Bioluminescence on Luciferase Oxidative Conformation[END_REF][START_REF] Pinto Da Silva | Theoretical Study of the Efficient Fluorescence Quenching Process of the Firefly Luciferin[END_REF][START_REF] Pinto Da Silva | Theoretical Analysis of the Color Tuning Mechanism of Oxyluciferin and 5-Hydroxyoxyluciferin[END_REF][START_REF] Silva | Computational Investigation of the Effect of PH on the Color of Firefly Bioluminescence by DFT[END_REF][START_REF] Chen | Systematic Theoretical Investigation on the Light Emitter of Firefly[END_REF][START_REF] Navizet | Are the Bio-and Chemiluminescence States of the Firefly Oxyluciferin the Same as the Fluorescence State?[END_REF][START_REF] Falklöf | Distinguishing between Keto-Enol and Acid-Base Forms of Firefly Oxyluciferin through Calculation of Excited-State Equilibrium Constants[END_REF] and experimentally [START_REF] Ghose | Emission Properties of Oxyluciferin and Its Derivatives in Water: Revealing the Nature of the Emissive Species in Firefly Bioluminescence[END_REF][START_REF] Rebarz | Deciphering the Protonation and Tautomeric Equilibria of Firefly Oxyluciferin by Molecular Engineering and Multivariate Curve Resolution[END_REF][START_REF] Hirano | Spectroscopic Studies of the Light-Color Modulation Mechanism of Firefly (Beetle) Bioluminescence[END_REF] allowing for instance to determine the relative concentrations of each chemical form as a function of pH [START_REF] Ghose | Emission Properties of Oxyluciferin and Its Derivatives in Water: Revealing the Nature of the Emissive Species in Firefly Bioluminescence[END_REF][START_REF] Rebarz | Deciphering the Protonation and Tautomeric Equilibria of Firefly Oxyluciferin by Molecular Engineering and Multivariate Curve Resolution[END_REF] .

We recently characterized the pH dependence of oxyluciferin photophysics by using the 5,5-Cpr-OxyLH (Cpr = (spiro) cyclopropyl) and the 6'-Me-5,5-Cpr-OxyL to mimic the keto species The above mentioned excited state reaction is also supported by theoretical calculations performed to model the proton transfer for OxyLH 2 and its analogues [START_REF] Pinto Da Silva | Chemiexcitation Induced Proton Transfer: Enolate Oxyluciferin as the Firefly Bioluminophore[END_REF][START_REF] Pinto Da Silva | Oxyluciferin Photoacidity: The Missing Element for Solving the Keto-Enol Mystery?[END_REF][START_REF] Pinto Da Silva | Efficient Firefly Chemi/Bioluminescence: Evidence for Chemiexcitation Resulting from the Decomposition of a Neutral Firefly Dioxetanone Molecule[END_REF][START_REF] Silva | Firefly Chemiluminescence and Bioluminescence: Efficient Generation of Excited States[END_REF] . Another key step of the OxyLH 2 photophysics relies on the keto-enol tautomerization. Indeed, according to the wellestablished enzymatic reaction model, the bioluminescence emitter is created in its keto form which can, during its excited state lifetime, undergo partial enolization and deprotonation.

Experiments performed by Naumov et al. using oxyluciferin analogues evidenced that in a nonpolar and basic environment the keto form in the excited state can tautomerize into the enol form, which subsequently undergoes excited state proton transfer to produce the enolate ion [START_REF] Ran | The Spectral-Structural Relationship of a Series of Oxyluciferin Derivatives[END_REF][START_REF] Solntsev | Photoinduced Dynamics of Oxyluciferin Analogues: Unusual Enol "Super"Photoacidity and Evidence for Keto-Enol Isomerization[END_REF] .

These authors were however unable to determine the time scale of the keto-enol conversion.

To decipher the ps kinetics associated to the conversion between the OxyLH 2 species, we report here on femtosecond transient absorption (TA) and nanosecond fluorescence decay measurements (see experimental details in the SI) performed on OxyLH 2 and its analogues (Scheme 2) in acidic (pH = 5) and basic (pH = 11) aqueous buffer solutions for which it is possible to avoid a mixture between protonated and deprotonated forms in the ground state. Hence, we first studied compounds 1a and 1b which mimic the keto species, since the Cpr cycle prevents the conversion to the enol form (see Scheme1). In 1a, the phenol form is "locked" since the hydroxyl group is replaced by a methoxy substituent (red) that precludes deprotonation in S 0 and in S 1 (ESPT is no longer possible). Conversely, 1b is used to investigate the phenol-phenolate conversion of the keto form, in both S 0 and S 1 . Figure 1 displays the TA data obtained for these compounds as a function of pH. The absorption and emission maxima wavelengths of 1a are 388 nm and 525 nm respectively, independent of the pH value (Figure 1a). At pH=5, 1b is protonated and its absorption spectrum is the same as 1a, characterizing the phenol-keto form. At pH=11, 1b deprotonated (i.e. phenolate-keto) and its absorption maximum is shifted to 482 nm. For both pH values, the emission spectrum of 1b is the same, centered at 640 nm, indicating that the same emissive state is observed.

For 1a, two contributions are clearly apparent in the TA data (Figure 1d), a positive signal (in red) at 460 nm associated to an Excited-State Absorption (ESA) and a negative signal (in blue) at 525 nm corresponding to Stimulated Emission (SE) band that matches the steady-state emission spectrum of the phenol-keto form later denoted R = . For 1b at pH=5, the early ESA and SE bands at 470 nm and 525 nm respectively, are associated to the same S 1. signature of the phenol-keto form (R = ). After a few picoseconds, these bands undergo a red-shift to 550 nm and 640 nm respectively, which correspond to the S 1 signature of the phenolate-keto form ( -R = ) form as evidenced by the TA data obtained at pH=11 in Figure 1f. We conclude that the TA data for 1b at pH=5, reveal the deprotonation of the phenol-keto form in S 1 , namely an ESPT reaction. At long time delays at pH=5 (Figure 1e), a positive signal at 480 nm remains up to 4 ns (and beyond), after the decay of the two -R = S 1 contributions. This long-lived signal corresponds to the absorption band of the deprotonated, ground-state photoproduct.

The results of the global analysis of the TA data (see methods in SI) are reported Figure 1g,h,i. The long time constants (yellow) were obtained from fluorescence decay measurements and fixed for the TA analysis. The short time constants of 0.6 ps and 2.2 ps for 1a, 0.43 ps for 1b at pH=5, and 0.5 ps and 4.25 ps for 1b at pH=11 characterize spectral relaxations likely due to vibrational and solvent relaxation. The 3-ps time constant for 1b at pH=5, is a time scale on which we may expect further vibrational and solvent relaxation in line with the 2.2-ps DAS obtained for 1a, but on this time scale, the rise of the 640 nm SE band of the deprotonated form ( -R = ) is clearly observed. On the 22 ps time constant, we observe the complete decay of the initial ESA band at 470 nm, as well as the decay of the initial SE and rise of the subsequent ESA both around 540 -570 nm. Hence, both the 3 ps and 22 ps time constants are associated to the ESPT reaction kinetics. The DAS associated to the infinite time constant is similar to the steady-state absorption spectrum measured at pH=11, confirming its assignment to the deprotonated ground-state. Therefore, reprotonation of the ground-state is significantly slower than 4 ns, which is the maximum accessible time delay in our TA experiment. We next report on the excited-state dynamics of 2a and 2b. For 2a, where two methoxy groups prevent deprotonation in S 0 or S 1 , the absorption and emission maxima are at 367 nm and 445 nm, respectively (Figure 2a). For 2b, only one methoxy group is present to lock the "phenol" moity -i.e. methoxyphenyl -such that the enol-keto conversion may occur. At pH=5, the absorption spectrum is maximum at 367 nm and corresponds to a mixture of both tautomers which indeed coexist at equilibrium in the ground state. Upon excitation at 400nm, the fraction of light absorbed by the enol and keto forms are 66% and and 33%, respectively. [START_REF] Rebarz | Deciphering the Protonation and Tautomeric Equilibria of Firefly Oxyluciferin by Molecular Engineering and Multivariate Curve Resolution[END_REF] Only the enol moiety can undergo an ESPT. At pH=11 instead, the enolate form is pure in the ground state with an absorption band (415 nm) characterizing the phenol-enolate form. The 2b emission maximum is 555 nm for both pH values.

The TA spectra for 2a are characterized by two ESA bands centered at 475 nm and 675 nm (Figure 2d), the former one overcoming -and therefore masking -the SE signal expected in the same spectral range. The 675 nm ESA will next be used as a specific signature of the phenol-enol form in S 1 and is denoted R hereafter. For 2b at pH=5, the same two ESA bands as in 2a (R signature) are observed at early times, and are replaced after several tens of ps by the S 1 signatures SE of the phenol-enolate (R -), observed at pH=11 (Figure 2f). We note here already, that since both tautomers are present in the ground state, we should also expect to observe in the 2b TA map the R = signature as illustrated in Figure 1d (remember that 2b cannot deprotonate on the phenol moiety, like 1a). However, while the R = signature is not easily identified by visual inspection of figure 2e, it will clearly arise from its quantitative analysis and corresponding DAS as discussed below.

The DAS resulting from global analysis are displayed in Figure 2g, h andi. Here again, the shortest time constants of 1.6 ps for 2a, 0.3 ps for 2b at pH=5 and 0.2 ps and 2.1 ps for 2b at pH=11 are attributed to intramolecular and solvent relaxations, and the longest time constants (yellow) are those obtained from fluorescence decay measurements (Table S2). The remaining 15 ps and 630 ps components for 2b at pH=5 are not present at pH=11. The 15 ps DAS is clearly associated to the phenol-enol to phenol-enolate ESPT, because its spectral shape reveals the decay of the 675 nm ESA band characterizing R, and the rise of the R-SE band around 580 nm. Finally, the 630 ps DAS (Figure 2h, grey vurve) displays -below 600nm -a spectral shape similar to that of the 930 ps DAS characterizing the decay of the phenol-keto (R = ) signature as identified with 1a (Figure 1g), in full line with our expectations above since both tautomers are excited at 400 nm. This analysis is confirmed by time-resolved fluorescence spectroscopy of 2b at pH=5, which also reveals a 630 ps lifetime -significantly different from the 930 ps one measured for 1a -together with a 7.9 ns time constant associated to the decay of the phenol-enolate form. As we will further argue below for the case of OxyLH 2 , we propose here that the decrease in the lifetime of the phenol-keto species in 2b with respect to 1a is due to the keto to enol conversion, which is an additional decay channel for 2b, as compared to 1a where the cyclopropyl group prevents it. We thus infer the rate constant of the S 1 keto to enol conversion of 2b as being (2ns) - After identifying the spectral signatures and kinetics of 1a and 2b, we conclude this work by discussing the photoluminescence pathway of OxyLH 2 at pH=5. TA spectroscopy data are displayed in Figure 3. At pH=5, OxyLH 2 is a 30%/70% keto/enol mixture, characterized by an absorption maximum at 371 nm, and a dual emission spectrum with a dominating band centered at 555 nm and a weaker one at 450 nm (Figure 3a). Upon 400 nm excitation of both tautomers, the TA data (Figure 3b) reveals the 675 nm ESA band characterizing the S 1 signature of the phenol-enol form (R) at early time delays. On the few-10-ps time scale, this signature disappears to give rise to a red (640 nm) SE while the high energy ESA red-shifts from 475 nm to ~520 nm. This indicates the formation of phenolate-keto ( -R = ) form, as observed in Figure 1e, produced upon direct excitation of the phenol-keto form already present in S 0 . Finally, the SE band broadens down to below 600 nm on the few-100-ps time scale and remains long-lived, which is the signature identified for the phenol-enolate (R-, Figure 2e) formed via ESPT upon direct excitation of the phenol-enol form.

The quantitative analysis of the TA data is reported in Figure 3c. The longest lifetime of 7.9 ns was determined by TCSPC fluorescence spectroscopy. The spectral shape of the 7.9 ns DAS is thought to correspond to the sum of the phenol-enolate ESA (7.9 ns DAS in Figure 2i) and the ground state absorption of the phenolate-keto observed for 1b ("inf" DAS in Figure 2h).

The 590 ps DAS has a spectral shape recalling the 630 ps DAS observed in Figure 1h and is thus assigned to the phenolate-keto ( -R = ) decay. The 21 ps DAS component (Figure 3c) likely accounts for both ESPT reactions occurring on similar time scales. The shortest time constant (0.2 ps) and corresponding DAS are assigned to spectral relaxations due to intramolecular and/or solvent relaxations. Similarly to the experiments performed on compounds 1b and 2b in bsic conditions, the excited state dynamics of oxyluciferin at pH=11 (Figure S2) is described by a single species displaying a fluorescence lifetime of 6.2 ns significantly different from the phenol-enolate lifetime (7.9 ns). This contribution is associated to the dianion OxyL 2-(i.e. phenolate-enolate) which was shown to be the only ground state species in basic condition [START_REF] Ghose | Emission Properties of Oxyluciferin and Its Derivatives in Water: Revealing the Nature of the Emissive Species in Firefly Bioluminescence[END_REF] , and does not contribute to the photoluminescence pathway in acidic conditions.

We next wonder if the S 1 keto to enol conversion observed with 2b may also occur for OxyLH 2 at pH 5. We were best able to address this question based on fluorescence spectroscopy after shifting the excitation wavelength to 430 nm, where only the phenol-keto form absorbs in the initial tautomer mixture (Figure S3). The measured fluorescence decays and emission spectrum clearly evidence the contribution of both phenolate-keto and phenol-enolate forms (Figure S5), demonstrating the excited state phenol-keto to phenol-enol conversion. Both forms are also rapidly converted, through an ESPT reaction, into the phenolate-keto and phenolenolate anions, respectively. More precisely, the relative amplitudes associated to the decay components of the phenolate-keto (0.62 ns) and of the phenol-enolate (7.9 ns) -see Figure S5a -allow us to infer the phenol-keto to phenol-enol conversion rate k1, on the basis of the global photoluminescence Scheme 5 and corresponding rate equations solved numerically (see SI). Independently, the relative weight of both anion emission spectra in the OxyLH2 steady-state fluorescence (Figure S5b) can be analyzed (see SI) to infer k1 as well. Both the analyses of time-resolved and steady-state emission data reveal the same value of k1 = (60 ps) In particular, the characteristic spectral signatures of the model compounds were used to disentangle the contributions from the phenol-enol and phenol-keto forms in the complex photophysics of OxyLH 2 , including site-specific ESPT reactions and their kinetics. In addition, Scheme 5: Photoluminescence Pathways of Oxyluciferin in Aqueous Solution at pH 5 experiments with red-shifted excitation were performed to characterize the excited-state conversion of OxyLH 2 from the phenol-keto to phenol-enol forms. All these results were used to fully unravel the complex photoluminescence pathways of OxyLH 2 at pH=5 by providing the excited-state reaction timescales (Scheme 5). Though these results do not directly apply to the photoreactivity within the luciferase active pocket, they demonstrate the role played by the pH on the excited-state photophysics, and the key role of specific hydrogen bonds between the chromophore and the protein binding pocket for controlling the bioluminescence spectrum via the tuning of excited-state proton transfer or tautomerism reaction kinetics. They also suggest that the same strategy could be used by incorporating these synthetic compounds in the natural binding pocket of Luciferase in order to unravel the photoluminescence mechanism of the oxyluciferin-luciferase complex. This work is in progress in our labs.

and 4 - 48 . 1 :

 4481 Scheme 1: Possible ground-state chemical forms of OxyLH 2 in solution.

  Still, the ground state keto-enol tautomerism results in the coexistence of both phenol-keto (Scheme 2 a) and phenol-enol (Scheme 2b) tautomers for the OxyLH 2 at pH=5 as previously reported.[START_REF] Ghose | Emission Properties of Oxyluciferin and Its Derivatives in Water: Revealing the Nature of the Emissive Species in Firefly Bioluminescence[END_REF]

Scheme 2 :

 2 Scheme 2: Model compounds used to mimic various forms of OxyLH2.

Figure 1 :

 1 Figure 1 : Excited-state signatures of the phenol-keto analogues. TA spectroscopy of 1a for all pH values (right) and 1b at pH=5 (middle) and pH=11 (left). (a), (b) and (c) are steady-state absorption (green) and emission (blue) spectra, (d), (e) and (f) are 2D maps, false-color representations of the transient absorption (ΔA) signal as a function of probe wavelength and pump-probe delay. Positive (red) and negative (blue) contributions are Excited State Absorptions (ESA) and Stimulated emissions (SE), respectively. (g), (h) and (i) are the Decay Associated Spectra (DAS) obtained from global analysis (see SI for details).

Scheme 3 :

 3 Scheme 3 : Photoluminescence pathway of the phenol-keto species. Scheme 3 summarizes the photoluminescence pathway of the phenol-keto form and the

Figure 2 :

 2 Figure 2 : Excited-state signatures of the phenol-enol analogues. TA spectroscopy of 2a for all pH values (right) and 2b at pH=5 (middle) and pH=11 (left). (a), (b) and (c) are steadystate absorption (green) and emission (blue) spectra, (d), ( e) and (f) are 2D map of the transient absorption (ΔA) signal as a function of probe wavelength and pump-probe delay. Positive contributions (red) are the Excited State Absorption (ESA) and the negative contributions (blue) corresponds to the Stimulated Emission (SE). (g), (h) and (i) are the DAS obtained after the global analysis.

1Scheme 4 :

 4 Scheme 4 : Photoluminescence pathway of the phenol-enol species

Figure 3 :

 3 Figure 3: Excited-state signatures of OxyLH 2 measured at pH 5. (a) Steady-state absorption (green) and emission (blue) spectra, (b) 2D map of the transient absorption (ΔA) signal as a function of probe wavelength and pump-probe delay. Positive contributions (red) are the ESA and the negative contributions (blue) correspond to SE. (c) DAS obtained after global analysis.

  -1 . All time constants associated to the kinetics of the complex photoluminescence pathway of OxyLH 2 determined in this work are summarized in Scheme 5. In conclusion, spectrally-resolved femtosecond transient absorption experiments and TCSPC fluorescence decay measurements were used to characterize the excited-state dynamics of OxyLH2 and its analogues as a function of the pH in water solution. With the help of derivatives where deprotonations or keto-enol tautomerization are blocked, it was possible to determine for the first time the characteristic time constants associated to the excited-state reactions. For the phenol-keto and phenol-enol model compounds, the measured ESPT time scales are on the order of 15-20 ps. Moreover, the DAS analysis performed on the model compounds allowed us to identify their specific S 1 spectral signatures. The results obtained with these compounds were used to interpret the dynamics observed on the natural emitter: OxyLH 2 .

Acknowledegments

This work was supported by the Agence Nationale de la Recherche (BIOLUM ANR-16-CE29-0013).

Supporting Information Available

Experimental details; fluorescence decays and singular transient measured at pH=5 and pH=11; table containing all the measured lifetimes; description of the model used to determine the keto to enol excited state conversion rate.


Francesco Fogliano

Benjamin Besga

Antoine Reigue

Laure Mercier De Lépinay

Philip Heringlake

Clément Gouriou

Eric Eyraud

Wolfgang Wernsdorfer

Benjamin Pigeau

Olivier Arcizet

Clement Gouriou

Ultrasensitive nano-optomechanical force sensor operated at dilution temperatures

come

Introduction-Operating force probes at low temperatures enables novel physical explorations in a quieter environment, with an increased force sensitivity granted by the reduction of their thermal noise which ultimately limits their performances. Since the invention of the atomic force microscope, the sensitivity of force sensors was greatly enhanced by using nanomechanical force probes. This enabled mechanical detection of ultraweak interactions such as the force exerted by single electronic spins [START_REF] Rugar | Single spin detection by magnetic resonance force microscopy[END_REF] or molecules [START_REF] Ganzhorn | Strong spin-phonon coupling between a single-molecule magnet and a carbon nanotube nanoelectromechanical system[END_REF] and investigations of fundamental processes in condensed matter such as the interaction of a nanoresonator to a superconducting qubit [START_REF] Lahaye | Nanomechanical measurements of a superconducting qubit[END_REF]. At cryogenic temperatures, the reduced thermal bath fluctuations often comes along with a decrease of mechanical damping rates which further improves the probe sensitivity. All those benefits can be fully exploited only if the vibrations of the nanomechanical force sensors can be read out without increasing their noise temperature.

This challenge becomes excessively difficult with ultrasensitive nanomechanical force sensors due to the desired decoupling from their mechanical support which is a general condition to achieve larger quality factors and to the extreme aspect ratios of the commonly employed ultrasensitive geometries in the form of nanotubes, graphene or nanowires [START_REF] Moser | Ultrasensitive force detection with a nanotube mechanical resonator[END_REF][START_REF] Chaste | A nanomechanical mass sensor with yoctogram resolution[END_REF][START_REF] Moser | Nanotube mechanical resonators with quality factors of up to 5 million[END_REF][START_REF] Weber | Force sensitivity of multilayer graphene optomechanical devices[END_REF], but also trapped particles [START_REF] Li | Millikelvin cooling of an optically trapped microsphere in vacuum[END_REF][START_REF] Kiesel | Cavity cooling of an optically levitated submicron particle[END_REF][START_REF] Gieseler | Subkelvin parametric feedback cooling of a laser-trapped nanoparticle[END_REF][START_REF] Ranjit | Zeptonewton force sensing with nanospheres in an optical lattice[END_REF]. The overall reduction of the heat conductance at low temperatures, which even accelerates below 100 mK renders this objective even more difficult. In this perspective, the choice of the readout probe is decisive and one should aims at minimizing its impact on the force sensor. Several approaches making use of electron based readout schemes were already implemented, using atomic quantum point contact [START_REF] Poggio | An off-board quantum point contact as a sensitive detector of cantilever motion[END_REF], SET [START_REF] Lahaye | Approaching the quantum limit of a nanomechanical resonator[END_REF][START_REF] Lassagne | Coupling mechanics to charge transport in carbon nanotube mechanical resonators[END_REF], squids [START_REF] Etaki | Motion detection of a micromechanical resonator embedded in a d.c. squid[END_REF][START_REF] Usenko | A superconducting quantum interference device based read-out of a subattonewton force sensor operating at millikelvin temperatures[END_REF], MW cavities [START_REF] Regal | Measuring nanomechanical motion with a microwave cavity interferometer[END_REF] or propagating electron-beam [START_REF] Nigues | Dynamical backaction cooling with free electrons[END_REF][START_REF] Siria | Electron beam detection of a nanotube scanning force microscope[END_REF]. However the spatial integration of the readout apparatus imposes serious geometrical constraints on the physics that can be investigated with such probes and in general does not allow preserving a scanning probe capacity.

In the optical domain, successful developments were realized by employing the toolbox of cavity optomechanics [START_REF] Anetsberger | Near-field cavity optomechanics with nanomechanical oscillators[END_REF][START_REF] Gavartin | A hybrid onchip optomechanical transducer for ultrasensitive force measurements[END_REF][START_REF] Meenehan | Silicon optomechanical crystal resonator at millikelvin temperatures[END_REF][START_REF] Macdonald | Optical microscope and tapered fiber coupling apparatus for a dilution refrigerator[END_REF][START_REF] Zhong | A millikelvin all-fiber cavity optomechanical apparatus for merging with ultra-cold atoms in a hybrid quantum system[END_REF], making use of an enhanced readout efficiency enabled by the large cavity finesse. However, a natural drawback of this optical multi-path strategy is to increase the overall system absorption, which has rendered difficult the observation of the oscillator thermal noise thermalized at sub-100 mK temperatures. In those approaches, low phonon number occupancies were obtained by exploiting dynamical backaction cooling, which however does not necessarily help increasing the bare force sensitivity of the system since the active cooling process simultaneously reduces the mechanical susceptibility of the oscillator. Several optical detection schemes making use of cavityfree optical interferometric readout were employed to probe ultrasensitive force sensors at low temperatures, in particular in the field of MRFM [START_REF] Mamin | Sub-attonewton force detection at millikelvin temperatures[END_REF][START_REF] Bleszynski-Jayich | Noise thermometry and electron thermometry of a sample-on-cantilever system below 1kelvin[END_REF][START_REF] Tao | Ultrasensitive mechanical detection of magnetic moment using a commercial disk drive write head[END_REF], but the thermal noise of the probe could not be measured below an effective noise temperature of approx 300 mK. Despite the low light powers employed, the residual light absorption has thus for the moment prevented existing experiments from reaching the sub-100 mK temperature regime. This is however an extremely interesting objective since -beyond increasing the force sensitivity of the nano-optomechanical force probes-it permits investigating the intrinsic mechanical, thermal and physical properties of the systems in their lowest excitation state and gives access to a new physical explorations.

Here we demonstrate the possibility to use a cavity free optomechanical readout scheme to probe the vibrations of an ultrasensitive nanomechanical force probe, a suspended silicon carbide nanowire, thermalized to the base temperature of a dilution fridge. We achieve force sensitivities in the 40 zN/Hz 1/2 range and a sensitivity to force field gradients of 20 fN/m in 40 s, a record for nanomechanical scanning force probes [START_REF] Usenko | A superconducting quantum interference device based read-out of a subattonewton force sensor operating at millikelvin temperatures[END_REF]. In our approach, the displacements of the nanowire vibrating extremity are readout using large numerical aperture optics enabling interferometric readout, designed for dilution temperatures. This lateral readout scheme provides an exceptional readout stability and preserves the bottom access to the nanowire vibrating extremity, thus maintaining a scanning probe capacity [START_REF] Nichol | Nanomechanical detection of nuclear magnetic resonance using a silicon nanowire oscillator[END_REF][START_REF] Gloppe | Bidimensional nano-optomechanics and topological backaction in a non-conservative radiation force field[END_REF][START_REF] Pigeau | Observation of a phononic mollow triplet in a multimode hybrid spin-nanomechanical system[END_REF][START_REF] Rossi | Vectorial scanning force microscopy using a nanowire sensor[END_REF][START_REF] Mercier De Lépinay | A universal and ultrasensitive vectorial nanomechanical sensor for imaging 2D force field[END_REF][START_REF] Mercier De Lépinay | Eigenmode orthogonality breaking and anomalous dynamics in multimode nano-optomechanical systems under non-reciprocal coupling[END_REF][START_REF] Braakman | Force sensing with nanowire cantilevers[END_REF][START_REF] Fogliano | Cavity nano-optomechanics in the ultrastrong coupling regime with ultrasensitive force sensors[END_REF][START_REF] Mattiat | Nanowire magnetic force sensors fabricated by focused-electron-beam-induced deposition[END_REF]. Part of the challenge resides in the thermalization of those ultra large aspect ratios nanomechanical force probes (length over diameter up to 2000) which presents thermal resistances already above 10 11 K/W at 10 K, while our measurements suggests that it even increases by 3 orders of magnitude at sub-100 mK temperatures. This requires operating at ultra-low photon fluxes, in a regime where all standard continuous detectors are totally blinded by their dark noise. Instead, we have developed a methodology based on avalanche single photon counters operated in the Geiger ("click") mode featuring ultra-low dark count rates in the attowatt range, while our measurements are realized with less than one photon detected per mechanical period. The demonstrated capacity to probe the thermal noise of our nanomechanical force probes allows the investigation of their thermal and mechanical properties in this unexplored temperature regime with minimal external perturbation. This approach opens the road towards vectorial imaging of ultra-weak force fields at dilution temperatures, with applications in condensed matter physics, in MRFM explorations down to the single quantum spin level [START_REF] Rugar | Single spin detection by magnetic resonance force microscopy[END_REF][START_REF] Pigeau | Observation of a phononic mollow triplet in a multimode hybrid spin-nanomechanical system[END_REF], or in cavity nano-optomechanics in the single photon regime [START_REF] Fogliano | Cavity nano-optomechanics in the ultrastrong coupling regime with ultrasensitive force sensors[END_REF].

The experiment -The nanomechanical force probes employed in this work are silicon carbide nanowires, see Fig. 1a, suspended at the extremity of a sharp tungsten tip. The samples employed, named A,B and C are 330, 216 and 245 µ m long respectively, with diameters of 300, 175 and conical from 240 to 120 nm (at the vibrating extremity), oscillating at 3.94, 5.84 and 11.6 kHz. They are attached over tenth of microns on the side of a high purity sharp tungsten tip using a carbon glue and annealed at 700 C, to increase the quality factors above ≈ 10 000 at room temperature. The tip is set in a highly conductive copper support and squeezed using an hydraulic press to ensure an optimal thermal contact and thermally connected to the mixing chamber of the dilution fridge using soft copper links. The vibrations of the nanowire are interferometrically readout by optical means as explained below. The experiment (see Fig. 1bd) is mounted on the cold plate of a table-top wet dilution cryostat adapted to the requirements of ultrasensitive optomechanical experiments such as low injection pressure and reduced vibrations. A suspension apparatus featuring a 2 Hz cut off frequency allows us to efficiently reduce the residual vibration noise originating from the boiling of the helium mixture in the still, down to a negligible level. The minimum mixing chamber temperature is of 20 ± 2 mK, while the mechanically decoupled experimental platform reaches a sample temperature of 27 ± 3 mK in operating conditions. It can be independently heated up to 500 mK without perturbing the dilution regime using a deported resistive heater. The objectives are micro-positioned using piezo-electric 3D scanning-stepping stages while the sample is mounted on an XYZ piezo scanner offering 9 × 2 × 9 µm scanning window, at 4K and below, sufficient to explore the waist area.

Optomechanical readout in the photon counting regime -The interferometric objectives (see Fig. 1ce) were specifically developed for low temperature purpose. The probe light (He-Ne laser, 632.8 nm) is focussed in a monomode fiber, carried into the cryostat and to the cold plate where it is collimated and focused using two aspheric lenses, the front one featuring a 0.72 numerical aperture. This produces a 500 nm waist at a working distance of 3 mm with an overall transmission around 92%. About 4 % of the incident light is reflected on the fiber output face inside the fibred objective and interfere on the detector with the light reflected from the nanowire which is channeled back into the fiber. This defines an ultra-stable cryogenic interferometer (with a fringe stability of a few nm over days without active stabilization), featuring a relatively large spatial contrast for the nanowires employed (see Fig. 1f andSI).

Moving the nanowire extremity in the optical waist allows us to spatially map the interference readout pattern Φ R (r) measured in the reflection channel, as shown in Fig. 1f. The expected λ/2 axial periodicity is clearly visible and serves to calibrate the piezo displacements at different temperatures, which are typically reduced by a factor of 4 along the horizontal XZ axes between 300 K and 20 mK. Due to the nanowire subwavelength-sized-diameter, this also reveals the optical properties of the focused light beam, and in particular the wavefronts structure and possible deviations from the paraxial approximation. Due to internal optical (Mie) resonances, the optical contrast achieved (see Fig. 2a) varies with the nanowire diameter, the probe wavelength and its polarization, see SI, but can be found close to unity which allows us to operate close to the dark fringe to increase the signal to background ratio (see below and SI). To avoid optical heating of the nanowires through residual absorption, we will see that it is necessary to operate at ultralow (sub-pW) optical powers, where the nanowire induced intensity fluctuations are likely to be hidden by the dark noise of standard continuous photodetectors. We circumvented this limitation by employing single photon counters, avalanche photodiodes operated in Geiger mode featuring an approx. 50 cts/s dark count rate, corresponding to approx. 15 attowatts in the visible and a quantum efficiency around 60%. The reflected signal recorded on the APD is thus constituted of a sequence of single photon pulses (see Fig. 2b), which can be either counted on photon counters to determine the mean photon flux (in cts/s) averaged over a measurement gate time, or analysed with time-stamping devices to acquire the full temporal statistics or with spectrum and network analysers to measure the nanowire thermal noise spectra or its response to an external driving force. As sketched in Fig. 2ab, when the nanowire extremity is positioned on the slope of the interference pattern Φ R (r), its transverse vibrations δr(t) around the rest position r 0 are dynamically encoded as temporal variations of the reflected photon flux Φ R (r 0 + δr(t)) according to: δΦ R (t) = ∇Φ R | r0 • δr(t). This provides a projective measurement channel of the nanowire vibrations δr β (t) = e β • δr(t), projected on a normalized measurement vector defined by

e β ≡ ∇Φ R | r0 /| ∇Φ R | r0 |.
The latter is measured using a routine computing the local tangent plane to the surface Φ R (r) as illustrated in Fig. 1f (see SI). The calibration of the spectral measurements performed with avalanche detectors is realized in comparison to the calibrated mechanical noise spectra obtained using standard photodiodes and well established calibration methods [START_REF] Gloppe | Bidimensional nano-optomechanics and topological backaction in a non-conservative radiation force field[END_REF][START_REF] Mercier De Lépinay | A universal and ultrasensitive vectorial nanomechanical sensor for imaging 2D force field[END_REF], following a methodology exposed in the SI. We determine in particular the conversion coefficient η = 3.98 × 10 8 V/(cts/s), which depends on the pulse shape and electrical impedances. It enables the conversion of the noise spectral density S V [Ω] measured on a spectrum analyzer into a projected displacement noise spectral density: (see Fig. 2). In the case of a perfectly dark fringe, it is possible to gain up to 3 dB on the signal to shot noise ratio, which can be appreciable at low temperatures.

S δr β [Ω] = S V [Ω]/(η 2 | ∇Φ R | r0 | 2 ), that
Noise thermometry -The following measurements were realized on nanowire A (330 µm long, 300 nm diameter), featuring an effective mass of 16 pg and a quality factor around 10 4 at 300 K. A typical thermal noise spectrum obtained at room temperature is shown in Fig. 2c. Despite the low optical power employed, 5 pW of injected light (measured at the fiber input), the 2 fundamental transverse eigenmodes emerge with a large dynamics (37 dB) above a flat shot-noise limited background S shot δr β . The mechanical thermal noise spectra can be fitted with the expression:

S δr β [Ω] = m=1,2 2Γ m k B T nw m M eff (e m • e β ) 2 (Ω 2 m -Ω 2 ) 2 + Γ 2 m Ω 2 + S shot δr β
to extract the nanowire noise temperature (T nw m ), where the other fitting parameters are the shot noise level, mechanical frequencies Ω 1,2 /2π, damping rates Γ 1,2 and eigenmode orientations e 1,2 (shown in the inset of Fig. 2c). The nanowire effective mass M eff was determined using the above expression after having verified the proper thermalization of the nanowire and the absence of optical heating or optical backaction [START_REF] Gloppe | Bidimensional nano-optomechanics and topological backaction in a non-conservative radiation force field[END_REF][START_REF] Mercier De Lépinay | Eigenmode orthogonality breaking and anomalous dynamics in multimode nano-optomechanical systems under non-reciprocal coupling[END_REF] on the system, which are both largely negligible at room temperature as long as the injected optical power remains lower than a few µW. The typical variability on the measured T nw /M eff ratio is around 5 %, and mainly depends on the imprecision of the measurement vector, which thus requires a careful estimation. At ultra-low photon fluxes the larger relative fluctuations in the measured photon counts lead to longer averaging times (tens of seconds) to reach a sufficient precision on the measurement vector. During the cool down phase, we need to compensate for thermal drifts (tens of µm) by tracking the nanowire position and maintaining it in the waist area using the stepper motors supporting the objectives. The optical interference pattern remains unchanged at cryogenic temperatures (besides the change in the piezo expansion coefficient). Fig. 3a shows the thermal noise spectra obtained for an intermediate temperature of 780 mK. The same fitting procedure permits to verify that the eigenmodes' orientations remain unchanged at cryogenic temperatures, while the quality factors are increased by a factor of 10. The effective mass is also unchanged within our experimental precision meaning that the eigenmodes longitudinal spatial profile is not altered, as expected for a singly clamped nanowire. Fig. 3b shows the thermal noise spectrum obtained at a sample temperature of 27 mK for an injected power of 100 fW. In that situation, the collected optical flux amounts to a few kcts/s, comparable to the mechanical frequency, meaning that this measurement is realized with only approx 1 photon collected per mechanical period. Despite this ultralow flux, it is still possible to preserve a signal to shot noise ratio slightly above unity. Those measurements are realized with a resolution bandwidth of 30 mHz and averaged for 30-60 minutes. Due to the modest signal to background ratio obtained, it can be delicate to properly estimate the nanowire quality factor, but it can be better evaluated using driven measurements (see below). To evaluate the corresponding imprecision in the noise temperature, we fit the data by testing a ±10% variation of the damping rate around the best fit value. As a result, we estimate the effective temperature of the nanowire vibration noise at a level of 32 ± 2 mK, for a sample temperature of 27 ± 3 mK. Achieving a noise temperature comparable to the cryostat base temperature requires minimizing the optical absorption (as discussed below) and other sources of heat impinging on the sample, but also taking a special care to minimize electrical and mechanical parasitic noises, which can drive the nanowire to an artificially large noise temperature. The electrical signals carried down to the piezo stages have to be strongly filtered (1.6 Hz bandwidth) and if possible grounded to avoid unwanted electrically driven vibrations of the nanowire support. To mitigate vibrational noise, the cryostat is mechanically decoupled from the building and the gas handling system (see SI), but we initially suffered from the acoustic noise generated by the circulation of the helium mixture (400 mbar injection pressure). This caused a vibration noise of the nanowire support of a few pm/Hz 1/2 at 4 kHz, which is relatively weak, but sufficient to resonantly drive the ultrasensitive nanowire, to an effective noise temperature in the 0.2 -4 K range depending on the circulation conditions (red point in Fig. 3c). Running a measurement sequence in which the circulation was stopped for a short duration (1 hour), sufficient to acquire meaningful spectra (circles in Fig 3c ), evidenced the impact of the circulation noise and noise temperatures down to 40 mK could be measured. We thus developed the suspension mechanism (see Fig. 1) with a cut-off frequency around 2 Hz, which allowed mitigating the circulation noise to a negligible level. Noise measurements conducted at different cryostat temperatures are shown in Fig. 3c and demonstrate the proper thermalization of the nanowire to the platform temperature.

Mechanical properties -The temperature dependence of the nanowire mechanical properties are shown in Fig. 4. They presents large similarities with the universal properties observed on resonators made of amorphous materials [START_REF] Enss | Low-Temperature Physics[END_REF][START_REF] Arcizet | Cryogenic properties of optomechanical silica microcavities[END_REF]. The relative mechanical frequency shift increases logarithmically at low temperatures and presents an an inversion above 700 mK. Also, the quality factor presents a minimum around 80 K and reaches a plateau below 10 K (the absence of quality factor improvement below 1 K is likely due to limiting clamping losses). However if the trends look similar, the observed magnitude is smaller. In particular the quality factors observed on the plateau are around 80 000, about 30 times larger than the universal value for amorphous materials of approx. 2500. Similarly the relative frequency shifts follows ∆Ω m /Ω m ≈ C log(T /T 0 ), with a constant C ≈ 2.5 × 10 -6 , which is 2 orders of magnitude smaller than the universal value, which is directly connected to the density of structural defects involved. A simple in- b, c Evolution of the relative frequency shifts and mechanical damping rates (mode 2) below 10 K. The open circles are obtained at minimal optical powers (sub pW) while varying the cryostat temperature. The full disks represent the frequency shifts and damping rates measured for increasing optical powers (from 100 fW to 10 nW, cryostat set at 20 mK), as a function of the measured effective noise temperature (after subtraction of a 30 mK of artificial noise contribution). The dashed line in b corresponds to ∆f2/f2 = 2.5 × 10 -6 ln(T /100mK).

terpretation could be that only a part of the nanowire behaves as an amorphous material, which is in agreement with the few nanometers oxide crust covering their surface, as observed in TEM imaging. The temperature dependence of the mechanical frequency is found to be a very good indicator of the nanowire internal temperature and can be used to localize regions of minimum absorption on the nanowire surface. We note that if the nanowire is artificially driven by a parasitic force noise, this will cause an increased mechanical power dissipated by mechanical friction in the nanowire. However it remains largely negligible ( Γ m k B T eff ≈ yW for 1 K noise temperature) compared to the injected optical powers. This explains why, prior to the shielding of mechanical and electric noises, a nanowire could be found with a frequency indicating a good thermalization even at the lowest cryogenic temperatures despite presenting a large artificial excess of vibration noise.

Optical heating -The above measurements were conducted at minimal optical powers to avoid optical heating through residual light absorption. Depositing heat at the extremity of the nanowire while measuring the induced noise temperature increase can be used [START_REF] Geitner | Low thermal fluctuations in a system heated out of equilibrium[END_REF] to investigate the thermal properties of the nanowire at dilution temperatures. Figure 3d shows the nanowire noise temperature measured for increasing optical powers, spanning over 4 orders of magnitude up to 0.6 nW, causing a non-linear temperature increase up to 650 mK with 3 different regimes. A rapid increase, scaling as P 1/2 , is observed below 100 mK, it flattens to approx. P 1/5 between 100 and 200 mK and accelerates again as P 1/2 above 200 mK. All those regimes present a sub-linear dependency with the injected optical power, a consequence of the increase of the nanowire thermal conductance with temperature. A conductance scaling as T µ will produce a P 1/(1+µ) optical heating trend (see SI). Similar behaviors were observed on several nanowires (see SI) and we note that the heating efficiency varies with the laser position on the nanowire which can be attributed to local variations in the absorption coefficient A abs (possibly due to contaminants). We also systematically observed that positioning the readout laser closer to the nanowire extremity (within 2 µm) also largely increases (x5 typically) the laser heating efficiency. This is attributed to a wave guiding mechanism which increases the amount of light guided inside the nanowire that can be subsequently absorbed. For larger optical powers, around 100 nW, the vibration noise temperature can be raised up to 10 K, see SI. Those measurements can be exploited to deduce the variations of the nanowire conductance K(T ) along the optically induced temperature increase. First, as shown in Fig. 4 one can notice that heating the nanowire with the cryostat at low optical power or with the laser generates the same evolution of mechanical properties with the cryostat or noise temperatures respectively [START_REF] Geitner | Low thermal fluctuations in a system heated out of equilibrium[END_REF]. In the limit where the phonon mean free path remains small compared to the nanowire length, which occurs in principle above 150 mK, see SI, a temperature can be defined quasi-continuously along the nanowire. When the conductance strongly increases with temperature, the temperature gradients in the nanowire are largely localized close to the supporting tip, which is assumed to be thermalized to the cryostat. The internal temperature profile is then expected to be almost homogeneous within the vibrating part of the nanowire and in first approximation, can be assimilated to the measured thermal noise temperature. This explains the good agreement between both measurements in Fig. 4. One can then adjust the optical heating curves to extract the thermal conductance, exploiting the relation K(T )/A abs = (dT nw /dP 0 ) -1 or an equivalent integral formulation given in the SI. We assume here that the optical absorption coefficient A abs does not vary with temperature and determine its value by setting the nanowire conductance at 10 K to the one given by the Casimir model, which accounts for the fact that the phonon mean free path is limited by incoherent reflections on the nanowire diameter, see SI. Having K(10 K) = 3.5 pW/K, we obtain A abs = 70 ppm. Since the probe laser wavelength is significantly larger than the optical bandgap of 3C silicon carbide (515 nm), it is likely that the optical absorption is dominated by local contaminants. The estimated conductance/absorption ratio, see SI, is reduced by 4 orders of magnitude at 30 mK, down to 0.4 fW/K. This extremely weak magnitude explains the rapid laser heating induced by the probe laser and the difficulty to observe a nanowire thermalized to the cryostat temperature, which required operating with sub-pW injected optical power. According to the mean free path analysis exposed in the SI, below 150 mK the heat propagation becomes ballistic over the entire nanowire length, a regime which will be the topic of future investigations.

Force sensing -The mechanical response of the nanomechanical force probes was tested under external drive by means of time-modulated piezo, electrostatic or optical actuation. We measured that they linearly respond to the external drives, up to a relatively large force magnitudes, of a few aN, see Fig. 5ab, which produce displacement amplitudes in excess of 10 nm. The absence of significant mechanical frequency shifts for increasing actuation strength suggests that the actuation mechanism does not heat up the nanowire. In order to investigate this question in greater details, we studied the nanowire noise temperature in presence of external actuation by resonantly driving one of its fundamental eigenmode, while realizing noise thermometry along the second perpendicular eigenmode, as sketched in Fig. 5c. We found that it was possible to observe the 11-kHz nanowire (sample C) thermalized down to 47 mK without suffering from additional unwanted heating due to the actuation mechanism, despite resonantly driving the first mode to a 3 nm amplitude. Those measurements also allowed to report the lowest thermal force noise measured, see Fig. 5de, around 40 zN/ √ Hz when thermalized at 47 mK. This value remains rather large compared to the one reported in single ions [START_REF] Biercuk | Ultrasensitive detection of force and displacement using trapped ions[END_REF][START_REF] Knünz | Injection locking of a trapped-ion phonon laser[END_REF], nanospheres [START_REF] Ranjit | Zeptonewton force sensing with nanospheres in an optical lattice[END_REF]or nanotube [START_REF] Moser | Nanotube mechanical resonators with quality factors of up to 5 million[END_REF] experiments, it however represents a record in force sensitivity for scanning force sensors, even if the quality factors (90 000) were still limited by amorphouslike damping and clamping losses. To put this number in perspective, it corresponds to the Coulomb Force between 2 electrons 70 µm apart. The above observations also present an important practical interest for ultrasensitive vectorial force field sensing [START_REF] Rossi | Vectorial scanning force microscopy using a nanowire sensor[END_REF][START_REF] Mercier De Lépinay | A universal and ultrasensitive vectorial nanomechanical sensor for imaging 2D force field[END_REF][START_REF] Mercier De Lépinay | Eigenmode orthogonality breaking and anomalous dynamics in multimode nano-optomechanical systems under non-reciprocal coupling[END_REF] since the nanowires can be resonantly driven up to large oscillation amplitudes, providing a large SNR on the readout channels, without seing their low noise properties being degraded. The frequency stability of the nanowires was investigated under similar driving conditions (see SI). We measured relative r.m.s. frequency deviations around 4 × 10 -8 over hours and a relative Allan deviation of 5 × 10 -11 for gate times of τ = 2π/Γ m . This intrinsic stability sets a limit on the smallest lateral force gradients which can be detected through the frequency shifts and eigenmode rotations they produce [START_REF] Mercier De Lépinay | A universal and ultrasensitive vectorial nanomechanical sensor for imaging 2D force field[END_REF], to a level of approx. 1 fN/m (see SI). This exceptionally faint magnitude is comparable to the maximum force gradient (2 fN/m) produced by the static radiation pressure force exerted by the 100 fW readout laser (0.6 zN varying over 300 nm laterally) [START_REF] Gloppe | Bidimensional nano-optomechanics and topological backaction in a non-conservative radiation force field[END_REF], underlying the importance to operate with ultra low readout powers in order to mitigate the probe back-action and preserve the ultimate performances of the ultrasensitive nano-optomechanical force probe.

Conclusions -We have shown the possibility to readout the vibrations of ultrasensitive nanomechanical force probes down to the base temperature of a dilution fridge, providing record force sensitivity for a scanning probe force sensor. We used readout techniques operating in the photon counting regime, were less than a pW is injected in the system and less than one photon is detected per mechanical period in order to prevent heating via residual light absorption and reduce the probe back-action. Their exceptional force sensitivity combined with a certain versatility opens the road towards vectorial imaging of weak forces in condensed matter systems, in proximity force measurements or experiments at dilution temperature aiming at exploring the single photon regime of cavity nano-optomechanics [START_REF] Fogliano | Cavity nano-optomechanics in the ultrastrong coupling regime with ultrasensitive force sensors[END_REF] or the spin qubit-nanoresonator interaction [START_REF] Pigeau | Observation of a phononic mollow triplet in a multimode hybrid spin-nanomechanical system[END_REF][START_REF] Arcizet | A single nitrogen-vacancy defect coupled to a nanomechanical oscillator[END_REF][START_REF] Lee | Topical review: spins and mechanics in diamond[END_REF]. Further investigations will also aim at exploring the onset at low temperatures of the ballistic regime of heat transport along the nanowires [START_REF] Anghel | Behavior of the phonon gas in restricted geometries at low temperatures[END_REF]. In particular, measurements based on pump-probe responses with excitation and detection lasers located at different positions along the nanowire [START_REF] Rosa | Experimental investigation of dynamic photo-thermal effect[END_REF][START_REF] Rivière | Optomechanical sideband cooling of a micromechanical oscillator close to the quantum ground state[END_REF][START_REF] Schwarz | Deviation from the normal mode expansion in a coupled graphene-nanomechanical system[END_REF][START_REF] Morell | Optomechanical measurement of thermal transport in two-dimensional mose2 lattices[END_REF] will help exploring those transitions.

FIG. 1 .

 1 FIG. 1.Experimental setup. a SEM and optical images of the silicon carbide nanowires of extremely large aspect ratios employed in this work. b,c Sketch and image of the experiment mounted on the cold plate of the dilution fridge: the light is fiber guided to the suspended cryogenic experimental platform, and focused on the nanomechanical oscillator under investigation. d Close up of the optical apparatus also showing a fraction of the focused light scattered by the nanowire. e Sketch of the cryogenic interferometric readout. Part of the light is reflected on the output face of the monomode fiber and interfere with the light back-scattered by the nanowire. f Moving the nanowire in the waist area, generates a very contrasted reflectivity map, where the phase curvature of the focused laser beam can be clearly identified.

FIG. 2 .

 2 FIG. 2. Nano-optomechanical readout in the photon counting regime. a When the nanowire is placed at a position presenting a large spatial variations of the reflected flux, its position fluctuations are converted into a large flux modulation (b). The SNR can be improved by working in the lower part of the interference slope, to reduce the shot noise level. c Typical thermal noise spectrum (nanowire A) obtained at room temperature with ≈ 5 pW of injected light, used to determine the effective mass (16 pg) and the eigenmodes orientations (inset) of both nanowire transverse fundamental eigen modes.

FIG. 3 .

 3 FIG.3. Noise thermometry.a Calibrated thermal noise spectrum obtained on nanowire A for an experiment temperature of 780 mK. The eigenmode orientations and effective mass are unchanged, while the quality factor is increased to ≈ 80000 b Similar measurement obtained for an injected power of 100 fW at 27 mK. c Vibration noise temperature as a function of the experiment temperature. Different measurements are shown, with suspensions (star/squares) and without while stopping (circles) or not (red circle) the mixture injection, using optical powers in the 100-300 fW range. The lowest noise temperatures obtained are around 32 ± 2 mK, for a sample temperature of 27mK and a base temperature of 20 mK. d Dependence of the nanowire noise temperature measured for increasing injected optical powers while maintaining the cryostat at its minimal temperature. The dashed lines are indicative power laws of 1/2, 1/5 and 1/2, to illustrate the different regimes of sub-linear heating rates observed.

FIG. 4 .

 4 FIG. 4. Mechanical properties. a Temperature dependence of the eigenfrequencies of the 5.8 kHz nanowire (B). b, c Evolution of the relative frequency shifts and mechanical damping rates (mode 2) below 10 K. The open circles are obtained at minimal optical powers (sub pW) while varying the cryostat temperature. The full disks represent the frequency shifts and damping rates measured for increasing optical powers (from 100 fW to 10 nW, cryostat set at 20 mK), as a function of the measured effective noise temperature (after subtraction of a 30 mK of artificial noise contribution). The dashed line in b corresponds to ∆f2/f2 = 2.5 × 10 -6 ln(T /100mK).

FIG. 5 .

 5 FIG. 5. Force sensing. a Response measurements obtained for increasing piezo actuation voltage. The resonant driven amplitude as well are reported in b.The linearity of the system is well verified, with driving force amplitudes spanning from 59 zN to 2.9 aN. c Sketch of the measurement configuration realized on a 11-kHz nanowire: mode 1 is resonantly driven to an amplitude of 1.5 nm (1.8 aN drive amplitude), while the noise temperature is simultaneously measured on mode 2. The red and black trajectories illustrate the driven and non driven situations. d Dependence of the force noise of eigenmode 2 for increasing optical readout powers, while resonantly driving mode 1. The dashed line is a P 1/2 opt guide line. The noise temperatures are indicated for 2 points in that plot, see SI. e Corresponding thermal noise spectrum obtained using 750 fW, featuring a 50 mK noise temperature corresponding to a 40 zN/Hz 1/2 force sensitivity.

Lett.19, 3143-3150 (2019).

Acknowledgments-We warmly thank N. Roch, F. Balestro, C. Winkelmann, J. Renard and L. Marty for in house cryogenic developments and experimental assistance, the PNEC group at ILM, O. Bourgeois and E. Collin for fruitful discussions as well as J.P. Poizat, A. Auffeves, G. Bachelier, J. Jarreau, C. Hoarau, C. Felix and D. Lepoittevin. F.F. acknowledges funding from the LANEF (ANR-10-LABX-51-01). This project is supported by the French National Research Agency (JCJC-2016 CE09-2016-QCForce, SinPhoCOM, LANEF framework (ANR-10-LABX-51-01, project CryOptics and "Investissements d'avenir program (ANR-15-IDEX-02, project CARTOF), and by the European Research Council under the EU's Horizon 2020 research and innovation programme (AttoZepto 820033 project).


Rabah Azzouz

Viviana Contreras Moreno

Clara Herasme-Grullon

Vincent Levacher

Lionel Estel

Alain Ledoux

Salim Derrouiche

Francis Marsais

Laurent Bischoff
email: laurent.bischoff@univ-rouen.fr

Viviana Contreras Moreno

Clara Herasme- Grullon

Efficient Conversion of Epoxides into Carbonates with CO2 and a Single Organocatalyst: Laboratory and Kilogram-Scale Experiments

Keywords: carbon dioxide, pyridines, organocatalyst, cyclic carbonate, epoxides epoxides, cyclic carbonates, carbon dioxide, organocatalysis, aminopyridine

come

Increasingly over recent years, efforts have been made to reduce the industrial production of carbon dioxide and thus avoid its effects on climate change. Besides the capture followed by geological or ocean storage of CO 2 , 1 several different products arising from its utilisation have been described. For instance, carbon dioxide can be reduced to produce formic acid derivatives, 2 carbon monoxide or methanol, 3 products that are highly-valuable energetic materials, since formic acid can further release hydrogen and methanol can be used directly as a fuel. In the present study, we were interested in the preparation of cyclic carbonates, via incorporation of carbon dioxide into epoxides. Cyclic carbonates such as ethylene or propylene carbonate are known as industrial solvents and, from a general viewpoint, dialkyl carbonates are high-value starting materials for the synthesis of polycarbonates through transesterification with diols.

Due to the high chemical stability of carbon dioxide, its direct esterification with methanol involves high energy input, requiring high pressures and temperatures to yield dimethylcarbonates. 4 The latter are precursors of diphenyl carbonate, a useful surrogate for phosgene in the industrial preparation of polycarbonate materials. A representative example consists of the use of cerium oxide as a catalyst. Lewis acid These energetic considerations have led several groups to focus on the reaction of carbon dioxide with molecules of higher reactivity, thus avoiding the requirement for an external source of energy. For instance, unstable carbamates can be trapped with electrophilic reagents and thus consumed in a stoichiometric amount. 5 In particular, the reactivity of epoxides is interesting, since these incorporate both electrophilic carbon and subsequent nucleophilic oxygen atoms. Thus, in the presence of appropriate catalysts, CO 2 can be inserted into reactive epoxides to yield the corresponding cyclic carbonates, or, in some cases, directly provide polycarbonates. 6 For this purpose, several catalytic systems have been described over the last decade. 7 The main strategies explored to date are summarized in Figure 1. Dual activation is often required to achieve high conversions under atmospheric pressure. In the epoxide-activation strategy, opening with a halide and resulting formation of a transient halogeno (bromo and iodo) intermediate results in both the release of a nucleophilic alkoxide with a remaining C-X electrophilic bond. This preliminary opening pathway represents the most commonly encountered form of epoxide activation. However, when a halide ion-containing catalyst such as tetrabutylammonium bromide is used, either high pressures and temperatures are required, or the assistance of a Lewis acid is employed, in order to stabilize the strongly basic alkoxide ion before its reaction with carbon dioxide. This electrophilic activation and stabilization can be obtained either via H-bond formation from an alcohol moiety, or with a various metal catalysts. 8,9 Cooperative catalysis has been explored with both Lewis acid metal catalysis with halide as counter ion, and H-bond donors such as diphenolic ligands such as salophen. 9 Cationic, organic solvent-soluble phosphonium salts, bearing an ortho-phenol group for H-bond activation of the epoxide, and bromide counter ion have been described. 9b-c Hirose and co-workers 9d also used organocatalysts encompassing both an alcohol as Hbond donor and nucleophilic assistance by iodide. Nucleophilic organocatalysts are also an efficient way to catalyse epoxide opening. 10 Many catalytic systems can be supported on montmorillonite clay 11 or natural 12a or synthetic macromolecules, 12b . Most of these systems afford good results but often involve high costs, and structural or surface analyses to ensure reproducible reactions.

X - O - R 1 H O R 2 X X - O - R 1 M + X CO 2 CO 2 O R 1 X -O O O O R 1 O i-)
Another mechanistic strategy consists of activating CO 2 with strong nucleophiles such as amidines or guanidines; 13 even NHCs can be employed. 14 Unfortunately, strong nucleophiles also behave as fairly strong bases. Indeed, the reaction of CO 2 with DBU was thoroughly studied and shown to be particularly sensitive to moisture, enabling the transformation of CO 2 into hydrogencarbonate if water is present. 15 However, in the case of industrial combustion exhausts, the removal of water will add an energy-consuming step. In addition, the cost of these reagents is generally too high for industrial applications.

2-aminopyridine is a relatively inexpensive compound (ca 15$ / kg), easily accessible by the Chichibabin reaction of sodium amide with pyridine. 16 It can exist as both tautomeric forms, and, in contrast to 2-hydroxy and 2-thio pyridines, 2aminopyridine mostly exists mainly as the 2-amino-rather than 2-iminopyridine tautomer. 17 Having a pKa value of 6.9, it is less basic than DBU or deprotonated NHCs, thus avoiding its acidobasic reaction with carbon dioxide and concomitant formation of hydrogencarbonate salt. It has a moderate toxicity and a good chemical stability towards water and most organic solvents. Owing to the electron-withdrawing properties of the pyridine nucleus, the amino group has a limited nucleophilic character, thus protecting this moiety from alkylation with epoxides or airoxidation, as observed in the case of many amines.

N NH 2 CO 2 N + N O - O N N + O - O H O R N N + O O H H H O O R O N NH 2 H H O -

Scheme 1 Catalysis of epoxide carbonatation with aminopyridine

Experiments conducted in NMR solvents (CDCl 3 , DMSO or acetonitrile) provided no evidence of the formation of the CO 2 adduct with 2-aminopyridine. Thus, we believe that a limited amount of 2-amino-pyridine/CO 2 complex is present; nevertheless exhibiting sufficient reactivity to lead to the opening of the epoxide. Interestingly, when the reaction is carried out in a glass reactor, the formation of a precipitate is noticed when CO 2 under pressure is applied. This precipitate disappears during the course of the reaction. We believe that the sole pyridine nitrogen atom is not nucleophilic enough to react with CO 2 , as may occur in the case of DMAP or DBU. Thus, a hydrogen bond with the vicinal 2-amino group is required for carbamate stabilization. Indeed, as described in the literature, 18 co-catalysis using both a phenol as the H-bond donor and DMAP as the nucleophilic catalyst has been described. In our case, the catalyst can be responsible for both activations (see Scheme 1). This is supported by the fact that 2-dimethylaminopyridine proved to be barely reactive under the same conditions; whereas 2-methylaminopyridine remained an effective catalyst, underlining the role of the hydrogen bond with the NH 2 or NHMe substituent

The catalytic properties of 2-aminopyridine were first examined using epichlorohydrin as a model substrate, on a 5 mL scale. 20 Epichlorohydrin is a widely-used synthon, mostly prepared as a monomer for epoxy paints. The corresponding cyclic carbonate is a high-value intermediate, especially if considered as a monomer for highlyfunctionalized polycarbonates. This preliminary work was aimed at determining which catalyst amongst the 2aminopyridine analogues would be most suitable for this reaction. In this set of experiments, we used a large amount of catalyst, under 12-bar pressure of CO 2 , in order to exhibit the catalytic properties of the catalysts even with low activities.

These results clearly show the importance of the catalyst itself since, with no catalyst barely any reaction occurred. The hydrogen bond also seemed crucial, the lowest yield being obtained using 2-dimethylaminopyridine. 2-aminopyrimidine can also catalyse this reaction, though less efficiently. Further examinations of catalytic activities were only performed with N-H bearing catalysts.

Then the same reaction was conducted under 10 bar of CO 2 over 16 hrs, at various temperatures. We observed a rapid increase in reaction above 40°C; however we did not wish to use temperatures higher than 60°C in order to avoid side-reactions such as degradation of the epichlorohydrin. In the absence of catalyst, only traces of cyclic carbonate were observed. 2-Aminopyridine remained the most active catalyst, though 2-methylaminopyridine and 2-aminopyrimidine also gave good yields at 60°C. As an important feature towards the possibility of industrial development, we next examined the catalyst loading, still working at 60°C under 10 bars of CO 2 pressure. These results clearly indicate that there is no requirement for high concentrations of catalyst, good yields being obtained even at 5% loading although, as expected, low concentrations of catalyst decreased the yields. Surprisingly, 2-methylaminopyridine remained effective at 2.5 mol %; whereas 2-aminopyridine only afforded a modest 52 % yield under the same conditions. When decreasing to 1 % ratio, we still noticed catalytic activity, although with low yields, except with 2-aminopyrimidine, which did not catalyse the reaction in such a concentration.

Having these results in hand, we wished to extend the scope of this transformation. Amongst commercially-available, suitablysubstituted epoxides that would lead to useful monomers in polycarbonate chemistry, we focused on glycidol, glycidyl ethers, and phenyl-oxirane.

In each case, we revisited the reaction conditions, especially the catalyst loading, the reactivities of the epoxides exhibiting strong differences. Glycidol is an important chemical intermediate, its carbonate represents a particularly interesting monomer for polycarbonate synthesis. When we studied carbonate formation from glycidol, we observed an unexpected reactivity of glycidol even if no catalyst is present. This may be due to self-activation by means of hydrogen bonding with the alcohol moiety. Pleasingly, the reactivity proved higher in the presence of the catalyst, as listed in the following table. These results confirm the high catalytic activity of 2aminopyridine, which remains the most efficient catalyst in this reaction, with 87 % yield being obtained at room temperature with a 5% catalyst loading under 10 bar pressure of CO 2 .

Having these results in hand, we examined the reactivity of other commercially-available epoxides having sufficiently-high boiling points for reactions at 70-85°C. We focused on 2aminopyridine, which allowed the formation of carbonates from every epoxide. Surprisingly, phenyl oxirane only gave a moderate yield, while having a high propensity for epoxide opening. Bis-glycidyl ether also lead to moderate yield of dicarbonate (62%), along with monocarbonate. Other epoxides gave good yields of carbonate formation, especially allyl glycidyl ether, which provided the carbonate in quantitative yield. Finally, with the aim of validating future industrial development, we examined the reactivity of epichlorohydrin in the presence of CO 2 and 2-aminopyridine at 1 kg-scale. The current commercial methods allowing production of cyclic carbonates require temperatures above 150 °C and high pressures of CO 2 between 50 -80 bar. Using 2-aminopyridine as catalyst, we could reduce the operating conditions at laboratory scale. To verify the applicability of the reaction at large scale by only using an organo-catalyst, different operating conditions were evaluated and optimized to maximize the amount and the quality of the product obtained.

The reactions between CO 2 and epichlorohydrin (ECH) were carried out in a stainless-steel stirred-tank reactor (1.5 L) at a range of temperatures (50°C -110°C) and pressures below 5 bar. The temperature in the reactor was maintained constant thanks to a carrier fluid circulating through a jacket. The accuracy of the temperature regulation inside of the reactor was ±0.1°C.

Stirring allowed the temperature and composition of the liquid phase to be kept homogeneous and improved the mass transfer. A reservoir of gas allowed continuous feeding into the reactor. The amount of gas-feed was controlled by regulating the pressure inside the stirred tank. Before every reaction, a vacuum was established to eliminate air contained in the reactor. The reactions were carried out under solvent-free conditions. Reactions performed at lab scale showed good conversions and yields at temperatures above 50 °C and concentrations of catalyst lower than 10% mol. We observed the behaviour of the reaction at pilot scale, by evaluating temperatures of between 50 and 110 °C. The pressure of the reaction and catalyst concentration were fixed to 5 bar and 5% mol., respectively.

Figure 8 shows the conversion of epichlorohydrin and additional information is given in Figure 9, which demonstrates the effect of temperature on the concentration of carbonate produced over time. At temperatures lower than 60°C, the production of the carbonate was quite slow, reaction taking more than 6 hours with conversions lower than 30%. At temperatures above 70°C, conversions were higher than 80% with a reaction time below 5 hours. We observed significant induction periods at temperatures lower than 70°C. 21 Indeed, we detected an optimal operating point over 80°C at pilot-scale with a conversion and yield of around 90%, which was comparable to that obtained at laboratory scale. At temperatures higher than 90°C, we observed a decrease in carbonate formation, which could be explained by thermal degradation of the catalyst. The effect of CO 2 pressure on the reaction rate was observed at the optimal temperature of 80°C and 5.0% mol. of 2aminopyridine, the pressure of CO 2 into the reactor being varied from 1 to 5 bar. Figure 10 shows the variation of carbonate formation over time at these pressures. Using 2-aminopyridine as catalyst, a relatively insignificant effect of CO 2 pressure was observed on the production rate of carbonate. A similar effect has also been noticed, but at higher pressures when, below 150 bar, there was no effect on the formation rate of dimethyl carbonate from CO 2 and ethylene oxide. 19 The conversions and yields were similar to that obtained at 5 bar (~90%). Under these conditions, we could minimize the pressure of the reaction without any consequence on the reaction performance. According to the studies performed at lab scale, catalyst concentrations from 2.5 to 10% mol., could be used with success to obtain the cyclic carbonate at a temperature of 60°C. However, at pilot-scale, the reaction requires a higher temperature to obtain similar yields. According to our study, we observed an optimal conversion at 80°C. This temperature was used to study the performance of the catalyst. Two different concentrations of catalyst (5 and 7 mol %), were used during these reactions and the pressure of CO 2 was fixed at 5 bar. Figure 11 shows the evolution of carbonate concentration over time according to the two catalyst concentrations used. An insignificant variation on the dynamic behaviour of carbonate formation was detected during the reaction. However, a slight difference was observed for the induction period. The concentration of carbonate was slightly lower when the concentration of catalyst was 7% mol. The yield of the reaction decreased to around 6%, but the overall conversion of epichlorohydrin remained relatively stable. We propose some catalyst degradation could occur during the last phases of the reaction, possibly due to the high temperature (80°C) and the higher concentration of catalyst (7 mol%). This result showed that, at pilot-scale, the temperature had an impact more significant on the reaction performance compared to the catalyst concentration at the conditions studied. Under appropriate conditions, we could use a relatively low concentration of catalyst and obtain good yields and conversions.

This whole set of experiments allow us to conclude that the laboratory-scale reaction of epichlorohydrin with carbon dioxide can be carried out with high reliability on a pilot-scale, thus paving the way towards an industrial process. In terms of sustainable chemistry, this constitutes an additional example of synthesis of cyclic carbonates from epoxides, with a very simple and readily-available organocatalyst, exhibiting low basicity and avoiding hydrogen carbonate formation. Importantly, carbonates having both reactive moieties such as glycidyl or epichlorohydrin carbonates, available through this phosgenefree preparation, are of great interest in the manufacturing of polymers. 7.36-7.22(m, 2H), 7.06-6.92(m, 1H), 6.91-6.85(m, 2H), 5.06-4.95(m, 1H), 4.65-4.56(m, 1H), 4.55-4.46(m, 1H), 4.22(dd, J = 10.5, 4.0Hz, 1H), 4.12 (dd, J = 10.5, 3.5Hz, 1H); 13 C NMR (75 MHz,CDCl3): 157.8,155.0,129.7,122.0,114.6,74.4,66.9,66.3;HRMS: 194.0581 4.88 (m, 2H), 4.49-4.53 (m, 2H), 4.30-4.41 (m, 2H), 3.73-3.87 (m, 4H); 13 C NMR (75 MHz,CDCl3) δ 154.8,133.5,117.6,75.0,72.4,68.7,66.

F

Figure 1

 1 Figure 1 Different modes of CO 2 activation

Figure 2

 2 Figure 2 Catalyst comparison (isolated yields)

Figure 3

 3 Figure 3 Role of the temperature

Figure 4

 4 Figure 4 Catalyst loading

Figure 5

 5 Figure 5 Carbonatation of glycidol

Figure 6

 6 Figure 6 Other carbonates prepared under 20 bar CO 2 , 10% 2aminopyridine

Figure 7

 7 Figure 7 Stirred reactor diagram. T: temperature -P: Pressure C: chamber -j: jacket -r: reactor

Figure 8 Figure 9

 89 Figure 8 Effect of pressure on carbonate production over time at 80°C bar and 5 mol% of 2-aminopyridine

Figure 10

 10 Figure 10 Effect of pressure on carbonate production over time at 80°C and 5 mol% of 2-aminopyridine

Figure 11

 11 Figure 11 Effect of catalyst concentration on carbonate production over time at 80°Cand 5 bar CO 2

  1b: pale yellow liquid; 7.73 g obtained (87 %) from 5 mL (75.2 mmol) glycidol and 354 mg (3.8 mmol) 2-aminopyridine, under 5 bar CO 2 at 25°C during 16 hrs; 1 H NMR (300 MHz, CDCl 3 ) 5.04 (1H, brs), 4.78-4.81 (1H, m), 4.46-4.55 (2H, m), 4.01 (1H, dd J= 3.2, 13 Hz), 3.72 (1H, dd J= 3.5, 13 Hz); 13 C NMR (75 MHz, CDCl3): 155.2, 75.0, 65.8, 61.6 1c: plae yellow liquid; 3.30 g obtained (46 %) from 5 mL (43.7 mmol) glycidol and 410 mg (4.4 mmol) 2-aminopyridine, under 20 bar CO 2 at 75°C during 16 hrs; 1 H NMR (300 MHz, CDCl 3 ): 7.42-7.37(m, 2H), 7.37-7.30(m, 3H), 5.70(t, 1H, J=7.9 Hz), 4.82(t, 1H, J=8.4 Hz), 4.37(t, 1H J=8.4 Hz); 13 C NMR (75 MHz, CDCl3): 155.0,135.9, 129.7, 129.2, 126.0, 76.8, 71.2 1d: pale brown solid; 6.84 g obtained (96 %) from 5 mL (36.6 mmol) phenyl glycidyl ether and 345 mg (3.7 mmol) 2-aminopyridine, under 20 bar CO 2 at 85°C during 16 hrs; 1 H NMR (300 MHz, CDCl 3 ):

  Colourless liquid; 6.72 g obtained (100 %) from 5 mL (42.5 mmol) glycidol and mg (mmol) 2-aminopyridine, under 20 bar CO 2 at 75°C during 16 hrs; 1 H NMR (300 MHz, CDCl 3 ) δ 5.71-5.83 (m, 1H), 5.13-5.21 (m, 2H), 4.71-4.81 (m, 1H), 4.43 (t, J = 8.4 MHz, 1H), 4.28-4.32 (m, 1H), 3.92-3.97(m, 2H), 3.49-3.64 (m, 2H); 13 C NMR (75 MHz, CDCl3) δ 154.9, 133.6, 117.8, 75.0

  Colourless liquid; 7.68 g obtained (82 %) from 5 mL (43 mmol) diglycidyl ether and 405 mg (4.3 mmol) 2-aminopyridine, under 20 bar CO 2 at 85°C during 16 hrs; 1 H NMR (300 MHz, CDCl 3 ) δ 4.81-

2

 2

  diylbis(oxy))bis(methylene))bis(1,3-dioxolan-2-one): Colourless liquid; 7.68 g obtained (82 %) from 5 mL (43 mmol) diglycidyl ether and 405 mg (4.3 mmol) 2-aminopyridine, under 20 bar CO 2 at 85°C during 16 hrs

Table 1

 1 Influence of the temperature with different catalysts

	T°C	2-amino-pyridine % a	2-methylamino	2-amino-
			pyridine % a	pyrimidine % a
	25	8	5	0
	40	25	28	25
	60	90	81	66
	a: isolated yields

Table 2

 2 Yields obtained with various catalyst concentrations

	Mol %	2-amino-	2-methylamino-	2-amino-
	catalyst	pyridine %	pyridine %	pyrimidine %
	10	90	81	67

Table 3

 3 Synthesis of glycidyl carbonate under various conditions

	catalyst	no	2-amino-	2-methyl	2-amino-
			pyridine	aminopyridine	pyrimidine
	20 bar, 50% cat. 45°C	60	80	70	83
	20 bar, 10% cat. mol., 45°C	19	90	80	78
	10 bar, 5% cat., 40°C	1.2	80	80	76
	10 bars, 5% cat., 25°C	36	87	78	78

Efficient conversion of epoxides into carbonates with CO 2 and a single organocatalyst: laboratory and kg-scale experiments

  An open glass Schlenk tube was charged with epichlorhydrin (5 mL, 61.8 mmol), 2-aminopyridine (580 mg, 6.18 mmol) and a magnetic stir bar. It was placed in a stainless-steel autoclave, then pressurized with carbon dioxide to 10 bar pressure. It was heated under stirring at 60°C (internal temperature) for 16 hrs. After cooling, the carbonate was purified by filtration through a short pad of silica, eluting with diethyl ether, followed by evaporation of It is placed in a stainless-steel autoclave, then pressurized with carbon dioxide under 10 bar pressure. It is heated under stirring at 60°C (internal temperature) during 16 hrs. After cooling, the carbonate can be purified by filtration through a short pad of silica and eluting with diethyl ether followed by evaporation of the solvent to yield 7.58 g of carbonate (90 %).

	Synlett															Letter / Cluster / New Tools
	Supplementary information for:						the solvent to yield 7.58 g of carbonate (90 %). 4-(chloromethyl)-1,3-dioxolan-2-one 1a: pale-yellow liquid; 1 H NMR (300 MHz,
												CDCl 3 ) δ 4.95-5.05 (m, 1H), 4.59 (t, J = 8.4 Hz, 1H), 4.38-4.44 (m,
												1H), 3.71-3.80(m, 2H); 13 C NMR (75 MHz, CDCl3) δ 154.6, 74.6,
												67.0, 44.2
	Rabah Azzouz, Viviana Contreras Moreno, Clara Herasme-Grullon, Vincent Levacher, Lionel Estel, Alain
	Ledoux, Salim Derrouiche, Francis Marsais, Laurent Bischoff
	Experimental procedure: the carbonatation of epichlorhydrine is illustrative:
	An open glass Schlenk tube is charged with 5 mL (61.8 mmol) epichlorhydrine, 580 mg (6.18 mmol) 2-
	aminopyridine and a magnetic stir bar. 4-(chloromethyl)-1,3-dioxolan-2-one 1a: light-yellow liquid liquid; 1 H NMR (300 MHz, CDCl 3 ) δ 4.94-
	o r 5.02 (m, 1H), 4.56 (t, J = 8.4 Hz, 1H), 4.34-4.39 (m, 1H), 3.63-3.82(m, 2H); 13 C NMR (75 MHz, CDCl3) δ F 154.6, 74.6, 67.0, 44.2 o r Jun28-2013 AR-132-B 3.65 3.66 3.69 3.71 3.75 3.77 3.79 3.81 4.35 4.37 4.52 4.55 4.55 4.58 4.96 4.98
							P P
								e e r e e r
										R e v R e v
											i e w i e w
										1.00	1.03	1.02	2.08
	9.5	9.0	8.5	8.0	7.5	7.0	6.5	6.0	5.5	5.0	4.5	4.0	3.5	3.0	2.5	2.0	1.5	1.0	0.5
										f1 (ppm)
	Template for SYNLETT © Thieme Stuttgart • New York	2019-11-25									page 7 of 7

Georg Thieme Publishers KG, Rüdigerstraße 14, 70469 Stuttgart, Germany Manuscript Submitted to Synlett

Acknowledgment

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Funding Information

This work has been partially supported by INSA Rouen, Rouen University, CNRS, EFRD, European INTERREG IV A France (Channel) and Labex SynOrg (ANR-11-LABX-0029). Rabah Azzouz is grateful to FEDER BIOFLUORG and Viviana Contreras Moreno is grateful to Veolia Recherche et Innovation for financial support.

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