Stefan Michiels

Fabrice Andre

Sandra Demaria

Damien Drubay

Roberto Salgado

Sherene Loi
email: sherene.loi@petermac.org

Jerome Lemonnier

Magali Lacroix-Triki

Giancarlo Pruneri

Andrea Vingiani

Sylvia Adams

Prudence A Francis

Heikki Joensuu

Maria Vittoria Dieci

Sunil Badve

Robert Gray

Elisabetta Munzone

Christos Sotiriou

Pirkko Liisa Kellokumpu-Lehtinen

Kathryn Gray

Fabrice André

Carsten Denkert

Martine Piccart

Elvire Roblin

Tumor infiltrating lymphocyte stratification of prognostic staging of early-stage triple negative breast cancer

INTRODUCTION

The importance of integrating biomarkers into the TNM staging has been emphasized in the 8 th Edition of the American Joint Committee on Cancer (AJCC) Staging System (www.cancerstaging. org). Prognostic biomarkers are becoming increasingly important given that many low early-stage breast cancer patients have excellent outcomes with standard of care treatments. The clinical guidelines for systemic treatment in early-stage TNBC are not consistent. The evidence for the integration of biomarkers into the TNM staging system is mostly retrospective and with limited prospective data available [START_REF] Murthy | Incorporation of clinical and biological factors improves prognostication and reflects contemporary clinical practice[END_REF] . In the case of multigene panels, their clinical validity for defining prognosis of particular subgroups of patients had been shown in several retrospective analyses and these were routinely used and incorporated into national (NCCN and ASCO) and international (ESMO, St Gallen) treatment guidelines far before prospective data became available. In the 8 th edition of the AJCC, histologic grading, molecular subtype (luminal, HER2-positive, and triple negative [TN]) and multigene panels/genomic signatures were included into staging assessments. Whilst pathological and clinical stage remain a valuable aspect of the staging process and are strongly prognostic, we highlight here the evidence that supports Tumor Infiltrating Lymphocytes (TILs) as a biologic biomarker which improves discrimination over prognostic pathological and clinical staging for early stage TNBC.

A recent pooled analysis provides the relevant work for integrating TIL quantity into clinical staging for early-stage TNBC (JCO 2019) [START_REF] Loi | Tumor-infiltrating lymphocytes and prognosis: a pooled individual patient analysis of early-stage triple-negative breast cancers[END_REF] . These data were from 2148 patients enrolled onto eight prospective clinical trials as well as one large institutional cohort. All patients had received anthracyclines with or without taxanes based regimens in the adjuvant setting. The median follow-up was 6.6 years. In the current study, using the Pathological and Clinical Prognostic Stage Groups from the AJJC 8th edition Cancer Staging Manual (for definitions hereof, see footnote*), three endpoints were calculated for each Stage group: invasive disease-free survival (iDFS), distant disease-free survival (D-DFS) as well as overall survival (OS). At a cut-point of 30% or more, TIL scores are found to strongly up and downstage traditional pathological-staging in this large data set (Fig. 1, Supp Table 1). This suggest that clinical and research studies on TNBC should take TILs into account in addition to stage, as patients with stage II TNBC and high TILs have a better outcome than patients with stage I and low TILs. Importantly, histological grade is not prognostic in this pooled analysis. At the established prognostic cut-off of 30%, trained pathologists have a high pairwise concordance rate up to 0.93 using both core-biopsies as well as full sections, indicating that TILs are a reliable biomarker that can be used in daily practice and in research and clinical trial settings [START_REF] Kos | Pitfalls in assessing stromal tumor infiltrating lymphocytes (sTILs) in breast cancer[END_REF] . This compares favorably to the reproducibility of other commonly used morphological features in breast cancer practice [START_REF] Gilchrist | Interobserver reproducibility of histopathological features in stage II breast cancer[END_REF] . Similarly, adding TILs to clinical staging is shown to up-and downstage prognostic groups which also take subtype into consideration (Fig. 2; Supp Table 2) though we note the small numbers in some of the subgroups (ie Stage IIA).

Risk assessment models meeting the AJCC criteria and included in the 8 th Edition were Adjuvant Online and PREDICT-Plus. We have developed a prognostic model for early-stage disease that includes age, number of positive lymph nodes, tumor size, grade, and stromal TIL percentage. This prognostic model is available on the website of the International Immuno-Oncology Biomarker Working Group (www.tilsinbreastcancer.org). This prognostic model could be used in routine patient care as well as future trial designs.

The inclusion of immune biomarkers is timely given reporting of phase III registration trials of PD-1/PD-L1 targeting agents in early-stage TNBC. In the recently reported KEYNOTE-522 study (NCT03036488) [START_REF] Schmid | Pembrolizumab for early triple-negative breast cancer[END_REF] , pembrolizumab, a PD-1 inhibitor, was found to significantly but moderately increase the rates of pathological complete response (pCR) and significantly improve event-free 1 and2.

survival (EFS) when added to a neoadjuvant regimen of anthracycline, taxanes and carboplatin in patients with stage II and III TNBC. Similar magnitude of EFS improvements have been reported in smaller studies [START_REF] Mittendorf | Neoadjuvant atezolizumab in combination with sequential nab-paclitaxel and anthracycline-based chemotherapy versus placebo and chemotherapy in patients with early-stage triple-negative breast cancer (IMpassion031): a randomised, double-blind, phase 3 trial[END_REF][START_REF] Loibl | Durvalumab improves long-term outcome in TNBC: results from the phase II randomized GeparNUEVO study investigating neodjuvant durvalumab in addition to an anthracycline/taxane based neoadjuvant chemotherapy in early triple-negative breast cancer (TNBC)[END_REF] . PD-L1 protein is known to be highly expressed on the breast cancer TIL and correlations between both markers have been observed [START_REF] Loi | Relationship between tumor infiltrating lymphocyte (TIL) levels and response to pembrolizumab (pembro) in metastatic triple-negative breast cancer (mTNBC): Results from KEYNOTE-086[END_REF] . In the KEYNOTE-522 study, patients designated as PD-L1 positive by the PD-L1 IHC 22C3 pharmdDx assay (Agilent Technologies, Carpinteria, CA, USA) were reported to have higher rates of pCR in both pembrolizumab and control arms across all TNM stages. An important treatment by PD-L1 interaction was not observed, in contrast to late-stage disease [START_REF] Loi | Abstract PD5-03: relationship between tumor-infiltrating lymphocytes (TILs) and outcomes in the KEYNOTE-119 study of pembrolizumab vs chemotherapy for previously treated metastatic triple-negative breast cancer (mTNBC)[END_REF][START_REF] Schmid | Atezolizumab and nab-paclitaxel in advanced triple-negative breast cancer[END_REF] . In any case, the increasing incorporation of PD-1 inhibition into the treatment of both early-and late stage TNBC further reinforces the need for routine evaluation of TIL in all TNBC patients, particularly given discordances in PD-L1 IHC assays. One could also speculate that patients with high expression of both immune biomarkers, i.e. those "immune enriched" could be strong candidates for future treatment optimization (for example, reduced cytotoxic chemotherapy) [START_REF] Gonzalez-Ericsson | The path to a better biomarker: application of a risk management framework for the implementation of PD-L1 and TILs as immunooncology biomarkers in breast cancer clinical trials and daily practice[END_REF] .

METHODS

This is a follow-up analysis of the Loi et al 2019 study [START_REF] Loi | Tumor-infiltrating lymphocytes and prognosis: a pooled individual patient analysis of early-stage triple-negative breast cancers[END_REF] . The study protocol of this project was approved by the local institutional review committee at Gustave Roussy in November 2014 and authorized by the French National Committee on Computing and Liberty. Identified studies were prospective randomized clinical trials or large retrospective hospital series that evaluated the prognostic associations of TILs in patients who were diagnosed with early-stage TNBC treated with anthracycline-based chemotherapy with or without taxane in the adjuvant setting using the same prespecified method for reading TILS (according to International Immuno-Oncology Biomarker Working Group guidelines). TNBC was defined by estrogen receptor (ER), progesterone receptor (PR), and HER2 negativity as outlined in Loi et al. (ref).

The prespecified primary end point was invasive disease-free survival (iDFS), defined as the date of first invasive recurrence, or second primary or death from any cause. Patients still alive without an event of interest were censored at the date of the last visit. Distant disease-free survival (D-DFS) is defined as the date of first distant recurrence or death from any cause. Patients still alive without an event of interest were censored at the date of the last visit. Overall survival (OS) was defined as the date of death from any cause. Patients still alive were censored at the date of the last visit. We used the Kaplan-Meier method to establish survival curves according to AJCC staging categories. The Kaplan-Meier estimates are provided together with bootstrap confidence intervals (1000 samples).

Fig. 1

 1 Fig. 1 Kaplan-Meier curves of overall survival of triple-negative breast cancer patients treated with anthracycline-based chemotherapy with or without taxanes, according to pathological prognostic stage and TILs. Pathological Prognostic Stage is assigned stage for patients who have surgical resection as the initial treatment of their cancer before receipt of any systemic or radiation therapy. It is based on clinical information, biomarker data, and findings from surgery and resected tissue. The 5-year estimated overall survival values (5-Yr OS) are provided together with bootstrap confidence intervals.

Fig. 2

 2 Fig.2Kaplan-Meier curves of overall survival of triple-negative breast cancer patients treated with anthracycline-based chemotherapy with or without taxanes, according to clinical prognostic stage and TILs. Clinical prognostic stage assigned stage for all patients based on history, physical examination, imaging studies performed (but not required) and relevant biopsies. Clinical prognostic stage is determined by T, N, M, tumor grade, as well as subtype information using human epidermal growth factor receptor (HER2), estrogen receptor (ER), and progesterone receptor (PR)-status. Supplementary Tables1 and 2.

npj Breast Cancer (2022) 3 Published in partnership with the Breast Cancer Research Foundation 1234567890():,;

Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2022) 3

npj Breast Cancer (2022) 3Published in partnership with the Breast Cancer Research Foundation

ACKNOWLEDGEMENTS S.L. is supported by National Breast Cancer Foundation of Australia (NBCF) and the Breast Cancer Research Foundation (BCRF), NY, USA. RS is supported by the Breast Cancer Research Foundation (BCRF), NY, US. M.L.-T., J.L., F.A. and S.M. are supported by ANR and CGI (RHU MyPROBE and ANR-17-RHUS-0008).

DATA AVAILABILITY

Trial data can be made available by contacting the individual trial investigators of the trials included in this project, pending individual sponsors approval. The corresponding author of the paper can redirect requests. A specific data transfer agreement between the individual sponsors of the trials and the researcher may be requested. This data transfer agreement details both parts responsibilities to ensure the required level of data integrity and legal and ethical obligations.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

AUTHOR CONTRIBUTIONS

Conception and design: S.L., S.M. and R.S. Provision of study materials or patients: all. Collection and assembly of data: all. Data analysis and interpretation: all. Manuscript writing and final approval: all.

COMPETING INTERESTS ADDITIONAL INFORMATION

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41523-021-00362-1.

Correspondence and requests for materials should be addressed to Sherene Loi.

Reprints and permission information is available at http://www.nature.com/ reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Electron-photon temporal correlations in electron energy loss (EELS) and cathodoluminescence (CL) spectroscopies have recently been used to measure the relative quantum efficiency of materials. This combined spectroscopy, named Cathodoluminescence excitation spectroscopy (CLE), allows the identification of excitation and decay channels which are hidden in average measurements. Here, we demonstrate that CLE can also be used to measure excitations' decay time. In addition, the decay time as a function of the excitation energy is accessed, as the energy for each electronphoton pair is probed. We used two well-known insulating materials to characterize this technique, nanodiamonds with NV 0 defects and h-BN with 4.1 eV defects. Both also exhibit marked transition radiations, whose extremely short decay times can be used to characterize the instrumental response function. It is found to be typically 2 ns, in agreement with the expected limit of the EELS detector temporal resolution.

The measured lifetimes of NV 0 centers in diamond nanoparticles (20 to 40 ns) and 4.1 eV defect in h-BN flakes (< 2 ns) match those reported previously. a) luiz.galvao-tizei@universite-paris-saclay.fr

The specific pathway a system takes to return to its ground state following an optical excitation reveals details about its internal electronic structure [START_REF] Hill | Observation of excitonic Rydberg states in monolayer MoS 2 and WS 2 by photoluminescence excitation spectroscopy[END_REF][START_REF] Roquelet | Quantum efficiency of energy transfer in noncovalent carbon nanotube/porphyrin compounds[END_REF][START_REF] Beha | Optimum photoluminescence excitation and recharging cycle of single nitrogen-vacancy centers in ultrapure diamond[END_REF][START_REF] Museur | Defect-related photoluminescence of hexagonal boron nitride[END_REF][START_REF] Brunner | Photoluminescence from a single GaAs/AlGaAs quantum dot[END_REF] . A key quantity in this type of spectroscopy is the excitation lifetime (the time for its decay). It is defined as the time, τ , by which a single excitation has probability 1/e of having decayed. In typical timedependent optical spectroscopy experiments, photons are used as the excitation source and the decay trace of the generated photoluminescence provides a measurement of τ . Electrons can also be used for spectroscopy in the optical range [START_REF] García De Abajo | Optical excitations in electron microscopy[END_REF][START_REF] Losquin | Unveiling nanometer scale extinc-tion and scattering phenomena through combined electron energy loss spectroscopy and cathodoluminescence measurements[END_REF][START_REF] Polman | Electron-beam spectroscopy for nanophotonics[END_REF] . Among the possible available techniques, the two mostly used in the optical range are electron energy loss spectroscopy (EELS) which provides a local measurement of optical extinction 7 ; and cathodoluminescence (CL) which is equivalent to off-resonance photoluminescence [START_REF] Mahfoud | Cathodoluminescence in a scanning transmission electron microscope: A nanometer-scale counterpart of photoluminescence for the study of II-VI quantum dots[END_REF] , which can also be used for lifetime measurements [START_REF] Merano | Probing carrier dynamics in nanostructures by picosecond cathodoluminescence[END_REF][START_REF] Meuret | Lifetime measurements well below the optical diffraction limit[END_REF][START_REF] Liu | Exciton dynamics at a single dislocation in GaN probed by picosecond time-resolved cathodoluminescence[END_REF][START_REF] Meuret | Time-resolved cathodoluminescence in an ultrafast transmission electron microscope[END_REF][START_REF] Kim | Cathodoluminescence in ultrafast electron microscopy[END_REF] . Experiments with electrons have the added benefit of high spatial resolution, down to the nanometer range [START_REF] Zagonel | Nanometer scale spectral imaging of quantum emitters in nanowires and its correlation to their atomically resolved structure[END_REF] , and the possibility of exciting non-optical transitions due to the larger momentum carried by electrons [START_REF] García De Abajo | Optical excitations in electron microscopy[END_REF] . Electron excitation is broadband in energy, which can be a benefit, as easy excitation in the far ultra-violet is possible, and a penalty, as the excitation energy cannot be controlled. This penalty can be mitigated by temporal correlation experiments in which the energy lost by each electron leading to a photon emission is measured [START_REF] Feist | Cavity-mediated electron-photon pairs[END_REF][START_REF] Varkentina | Cathodoluminescence excitation spectroscopy: Nanoscale imaging of excitation pathways[END_REF] , a technique coined as Cathodoluminescence Excitation spectroscopy (CLE) [START_REF] Varkentina | Cathodoluminescence excitation spectroscopy: Nanoscale imaging of excitation pathways[END_REF] as a reference to its photonic counterpart, photoluminescence excitation spectroscopy.

Concerning only electron spectroscopies, lifetimes have been measured using essentially two methods. The first one uses pulsed electron sources [START_REF] Merano | Probing carrier dynamics in nanostructures by picosecond cathodoluminescence[END_REF] . In these the electron arrival time on the sample (excitation time) is controlled by the emission time (triggered by a laser [START_REF] Bostanjoglo | Nanosecond electron microscopes[END_REF][START_REF] Arbouet | Ultrafast transmission electron microscopy: historical development, instrumentation, and applications[END_REF] or by a fast beam blanker [START_REF] Arbouet | Ultrafast transmission electron microscopy: historical development, instrumentation, and applications[END_REF][START_REF] Bostanjoglo | Ultrasonically induced magnetic reversals observed by stro-boscopic electron microscopy[END_REF] . In short, the time delay between a photon emission (CL) and the excitation event is measured, constructing a decay trace [START_REF] Merano | Probing carrier dynamics in nanostructures by picosecond cathodoluminescence[END_REF] . Lifetime measurements with pulsed electron beams are a more straightforward and efficient way of measuring excitation dynamics. However, this is depreciated by the technical burden of using a pulsed electron gun, well illustrated by the fact that time-resolved CL in a TEM has just recently been demonstrated [START_REF] Meuret | Time-resolved cathodoluminescence in an ultrafast transmission electron microscope[END_REF][START_REF] Kim | Cathodoluminescence in ultrafast electron microscopy[END_REF] . In the second method, light intensity interferometry using an Hanbury Brown and Twiss interferometer [START_REF] Brown | A test of a new type of stellar interferometer on sirius[END_REF] is used in an electron microscope [START_REF] Tizei | Spatially resolved quantum nano-optics of single photons using an electron microscope[END_REF] to measure the temporal width of photon bunches emitted by each electron impact, which has been proven to be directly related to the lifetime [START_REF] Meuret | Photon bunching in cathodoluminescence[END_REF] . CL light intensity interferometry has been applied to the measurement of lifetimes at sub-20 nm spatial resolution [START_REF] Meuret | Lifetime measurements well below the optical diffraction limit[END_REF] and, in conjunction with CL hyperspectral imaging, to measure the local excitation and emission efficiencies [START_REF] Meuret | Nanoscale relative emission efficiency mapping using cathodoluminescence g (2) imaging[END_REF][START_REF] Finot | Surface recombinations in III-nitride micro-LEDs probed by photon-correlation cathodoluminescence[END_REF] . CLE appears as an interesting extension of such approaches, as it could add to the ability to link absorption, emission and dynamics information.

Here we show that temporal correlations between electron energy loss (EELS) and photon emission (CL) events provide a measurement of decay traces. We studied two materials: diamond and hexagonal boron nitride (h-BN). To estimate the instrument response function (IRF), we first focused on transition radiation (TR). TR photons are emitted when a fast electron traverses a dielectric interfaces and it occurs at energies in the gap of semi-conductors. Its lifetime is known to be much smaller than the measured 2 ns IRF. The decay time of CL emission from NV 0 centers in diamonds nanoparticles was measured to vary between 20 and 40 ns, in agreement with previous measured values using light intensity interferometry [START_REF] Lourenço-Martins | Probing plasmon-NV 0 coupling at the nanometer scale with photons and fast electrons[END_REF] . Finally, the decay time of 4.1 eV defects in h-BN were measured to be barely distinguishable to the IRF, in agreement with previous reports [START_REF] Meuret | Lifetime measurements well below the optical diffraction limit[END_REF][START_REF] Bourrellier | Bright UV single photon emission at point defects in h-BN[END_REF] .

Experiments were performed on a VG HB501 STEM equipped with a cold field emission electron source, an Attolight Mönch light injection/collection system and an EELS spectrometer with an ASI Cheetah direct electron detector Fig. 1(a). This detector is based on the Timepix3 detector from the Medipix3 consortium, which has recently been used for temporally resolved EELS experiments [START_REF] Feist | Cavity-mediated electron-photon pairs[END_REF][START_REF] Varkentina | Cathodoluminescence excitation spectroscopy: Nanoscale imaging of excitation pathways[END_REF][START_REF] Jannis | Coincidence detection of EELS and EDX spectral events in the electron microscope[END_REF][START_REF] Auad | Event-based hyperspectral eels: towards nanosecond temporal resolution[END_REF] . The detector used has 4 chips, aligned in a 4x1 array with 4x(256x256) pixels. In addition, its electronics is equipped with two external time-to-digital-converters. Visible range photons were detected using a photomultiplier tube (PMT H10682-210 single photon counting head from Hamamatsu). The combination of these two time-resolved detectors allows the detection of the time delay between electron scattering and photon detection events as a function of electron energy (Fig. 1(b)), as described in detail later. Defects in two materials were studied: NV 0 in diamond and 4.1 eV defects in hexagonal boron nitride (h-BN). In accordance to the emission energy of the corresponding defects, for the diamond experiments a long pass wavelength filter at 532 nm was used, while for the h-BN a short pass wavelength filter at 532 nm was used, unless otherwise noted. 60 keV electrons were focused on a 1 nm-wide probe which was raster scanned on a sample cooled down to 150 K. Electron scattering at high angles (> 80 mrad) allows the formation of high angle annular dark field images, which have an intensity proportional to the projected atomic number, Z, and are used for further beam positioning on areas of interest (Fig. 1(c-d)). Electron beam convergence and collection angles were 7.5 and 10 mrad. 75-150 nm wide diamond nanoparticles containing a large number (> 100 NV 0 centers) were single crystals produced by proton irradiation of diamond nanoparticles [START_REF] Yu | Bright fluorescent nanodiamonds: no photobleaching and low cytotoxicity[END_REF] (Fig.

1(c))

. h-BN flakes were produced by a high pressure and high temperature method [START_REF] Taniguchi | Synthesis of high-purity boron nitride single crystals under high pressure by using Ba-BN solvent[END_REF] and chemically exfoliated by sonication in isopropyl alcohol (Fig. 1(d)). The diamond nanoparticles and the thin h-BN flakes were supported on thin amorphous carbon membranes. The CL emission spectrum of these two samples are shown in the insets of Fig. 1(e-f) and in the supplementary material (SM) Fig. SI1. These emissions stem from NV 0 in diamond nanoparticles 3 and the 4.1 eV defect in the h-BN flakes [START_REF] Museur | Defect-related photoluminescence of hexagonal boron nitride[END_REF] , respectively. In addition to this, a A small but statistically significant variation of the NV 0 decay as a function of excitation energy is observed. Some of the statistical error bars are smaller than the plotted symbols.

broad emission due to TR occurs [START_REF] Ginzburg | Radiation of a uniformly moving electron due to its transition from one medium into another[END_REF] . In the diamond nanoparticles two other minor emissions are observed at 2.5 and 3.2 eV, which are cut by the 532 nm long pass filter (SM Fig. SI1).

For the h-BN flakes, a tail to higher energies was observed, which gives a background to the emission at 4.1 eV (SM Fig. SI1).

Electrons were detected after a magnetic prism which disperses them along one direction as a function of their energy, with an energy sampling between 0.050 and 1 eV per pixel on the detector. Therefore, for each electron its energy loss after scattering, E, and arrival time on the detector, t e , were measured. For photons, only their arrival time, t ν , was stored.

A search algorithm [START_REF] Varkentina | Cathodoluminescence excitation spectroscopy: Nanoscale imaging of excitation pathways[END_REF] was used to locate temporally correlated events, from which (E, ∆t) 2D histograms were constructed, with the time delay defined as ∆t = t e -t ν . In these histograms (Fig. 1(b) and 2(a,c)) correlated events appear around zero time delay while uncorrelated events appear at longer time delays. These later occur due to various noise sources (detector noise, external particles, such as cosmic rays and ambient photons) and detection losses. For example, if the photon in an electron-photon pair is not detected (for example, if it is emitted away from the collection mirror), the electron which generated it may appear as correlated to a photon from a later scattering event, giving rise to spurious correlations.

An integral of the (E, ∆t) 2D histograms for all energies results in a temporal profile which represent the rise and decay times of the photon emission probability of an excitation created by an electron inelastic scattering (Figs. 1(e-f) and 2(b-d)). The time between an electron inelastic event, when an excitation is created, and the first emitted photons may not be zero, leading to a non-zero rise time. For example, a high energy excited state might need to relax, before optical transitions are possible. However, these processes are faster (< 100 ps scale) than our temporal resolution. Following the maximum of the emission intensity, the probability of photon emission will decay, with a typical time scale given by the excitation lifetime. The data presented here was modeled by two exponential functions, from which rise and decay times were extracted. Solid line curves in Fig. 1(e-f) are fits using this model.

The impulse response function (IRF) is better approximated by a Gaussian curve. However, a model with a Gaussian and two exponential curves have too many free parameters and lead to inconsistent fits. The rise and decay time constant for our instrument response function (IRF) was 2 ns, which was estimated from the temporal profile of the transition radiation (TR). TR photons are emitted when a fast electron traverses a dielectric interfaces and it occurs at energies in the gap of semiconductors. Its lifetime is known to be much smaller than the measured 2 ns IRF [START_REF] Scheucher | Discrimination of coherent and incoherent cathodoluminescence using temporal photon correlations[END_REF] (blue curves in Fig. 1(e-f)). From the (E, ∆t) 2D histograms in Fig. 2(a-c) a CLE spectrum is calculated by summing all electrons leading to photon emission, that is, a projection along the time-delay axis (Fig. 2(b-d)); total EELS spectrum is the sum of all electrons scattered (at any time); and the relative quantum efficiency (rel. QE [START_REF] Varkentina | Cathodoluminescence excitation spectroscopy: Nanoscale imaging of excitation pathways[END_REF] ) is calculated by dividing the CLE spectrum by the total EELS spectrum, and gives an insight in the preferential electron energy losses responsible for photon emission.

As a function of electron energy loss (Figs. 2(a) and 4(a)), two contributions for diamonds are observed: i) between 2.0 and 5.0 eV a fast decay (white vertical lines); and ii) between 6.5 and 440.0 eV a slower decay, orange vertical lines). The corresponding decay profiles are shown in Figs. 2(b) and 4(a) (with the 2.0 to 5.0 eV decay curve in blue, marked TR). The fast decay at low electron energy losses is attributed to TR. The slow decay contribution is attributed to the decay of NV 0 centers (20 and 45 ns for the orange profiles in Fig. 2(c)).

In fact, for all energies above the band gap of diamond (≈ 5.5 eV) a similar decay trace is observed. For this reason, the energy integrated temporal profile (Fig. 1(e)) has the same long decay. This is expected, as the majority of the emission observed in these diamonds stem from NV 0 centers. For the h-BN only fast decays were observed (Figs. 2(c) and 4(c)). Different decay times were observed for NV 0 in diamond nanoparticles (between 20 and 40 ns in Fig. 1(e) and 3(a)) and the 4.1 eV defect in h-BN (2 ns in Fig. 1(f)). The data presented in Fig. 3 includes eighteen NV 0 and five 4.1 eV defects independent measurements. Similar decay times were measured when including electron losses up to core-hole excitations (Fig. 4).

The range of lifetimes measured for the NV 0 matches the values measured in CL experiments in the literature [START_REF] Meuret | Lifetime measurements well below the optical diffraction limit[END_REF][START_REF] Lourenço-Martins | Probing plasmon-NV 0 coupling at the nanometer scale with photons and fast electrons[END_REF][START_REF] Solà-Garcia | Electron-induced state conversion in diamond nv centers measured with pump-probe cathodoluminescence spectroscopy[END_REF] . A similar lifetime range has also been reported in photoluminescence [START_REF] Storteboom | Lifetime investigation of single nitrogen vacancy centres in nanodiamonds[END_REF] . The reduction in lifetime, which implies an emission rate decrease, was linked to the nanoparticle size, a direct consequence of the presence of an interface close to the emitting dipole [START_REF] Kreiter | Orientation dependence of fluorescence lifetimes near an interface[END_REF] . A nanoparticle size dependence has also been observed for NV 0 lifetime measured by photoluminescence 37,38 . For the 4.1 eV defect in h-BN the measured decay time is barely distinguishable from the IRF of our experiment. However, one can see that their decay traces are slightly longer than that of TR, consistent with the convolution of two decay times of 2 and 1 ns.

Similar decay traces of the NV 0 centers and the 4.1 eV defects are observed for losses up to 600 eV, which include core-electron excitation for the carbon, the boron and the nitrogen for h-BN K edges (Fig. 4(a-d). The boron and nitrogen core-electron excitations are not markedly visible in relative quantum efficiency curves (green in upper right inset), despite being visible in the EELS and CLE spectra (Fig. 4(d) upper left insets). The fine structure of the carbon K edge appears in the relative quantum efficiency curve for NV 0 centers in a diamond nanoparticle (Fig. 4(b)). Compared to previously reported data [START_REF] Varkentina | Cathodoluminescence excitation spectroscopy: Nanoscale imaging of excitation pathways[END_REF] , where no structure was detected in the relative quantum efficiency at core loss edge energies, two improvements were crucial: improved pixel-to-pixel temporal calibration of the TPX3 detector (more details in 39 ) and increased signal-to-noise ratio. The observation of a connection between core-electron excitations and photon emission gives hope to the observation of light emission from individual atoms with atomic resolution. In short, coreelectron transitions can be mapped down to the atomic scale [START_REF] Muller | Atomic-scale chemical imaging of composition and bonding by aberration-corrected microscopy[END_REF][START_REF] Kimoto | Element-selective imaging of atomic columns in a crystal using stem and eels[END_REF] , if they occur at sufficiently high energies. Rare earth atoms might be good candidates, given their atomically localized M absorption edges [START_REF] Tizei | Single atom spectroscopy: Decreased scattering delocalization at high energy losses, effects of atomic movement and x-ray fluorescence yield[END_REF] and photon emission under electron excitation. Shifts of the order of 1 or 2 ns of the "zero" time delay exists, which was defined as the peak of the temporal profile for the 2D histogram integrated for all energies. This can be seen in detail in Fig. 4d. These changes are comparable to the temporal sampling and resolution of the current TPX3 implementation for EELS and probably arise due to modal dispersion in the 3.0 ± 0.1 m long, 600 µm core, fiber 43 . With a better temporal resolution and a monomode fiber or free-space detection, one could first confirm if these shifts are physical. More interestingly, with a substantially improved temporal resolution (tens of ps) one could start to investigate the excitation dynamics of materials under electron irradiation and also increase the spatial resolution of CL hyperspectral maps, as already demonstrated for CL using pulsed electron sources 12 .

To conclude, a method for measuring excitation lifetimes is demonstrated, with a current decay time resolution of around 2 ns. For optimal conditions the IRF can be reduced to 1.6 ns. This is still a factor of 2 smaller than the optimal performance of Timepix3 detectors.

The reasons for this discrepancy is discussed in ref. 39 . In principle, a spatial resolution as good as that of CL experiments 15 is possible [START_REF] Varkentina | Cathodoluminescence excitation spectroscopy: Nanoscale imaging of excitation pathways[END_REF] . This method can be implemented in electron microscopes equipped with continuous electron sources, as described here, being compatible with current technologies of electron monochromation [START_REF] Krivanek | Vibrational spectroscopy in the electron microscope[END_REF] . The current temporal resolution is limited by the electron detector. Future upgrades with faster electron detectors (for example, the Timepix4 detector 45 ) will allow experiments in the sub-nanosecond temporal scale.

FIG. 1 .

 1 FIG. 1. (a) Sketch of the experiment: an electron microscope is used to produce a 1 nm wide beam of 60 keV electrons. An EELS spectrometer equipped with a magnetic prism measures the transmitted electrons energy spectrum. A parabolic mirror collects light generated by excitations' decay and couples it to a PMT outside the microscope vacuum using a single 3.0 ± 0.1 m long multimode 600 µm-core optical fiber. (b) 2D histograms, (E; ∆t), for NV 0 in diamond (above) and 4.1 eV defects in h-BN (below). Because of the shorter lifetimes for the 4.1 eV defects, coincident electron-photon pairs are less probable for longer time delays in comparison to NV 0 defects. (c-d) Annular dark field images of diamond nanoparticles and an h-BN flake, respectively, supported on an amorphous carbon membrane. (e-f) Temporal traces integrated for all energy losses, E, for a diamond nanoparticle and an h-BN flake, with their CL emission spectrum in the inset. The solid lines are a two exponential curve model fitted to the data.

FIG. 2 .

 2 FIG. 2. (a,c) (E, ∆t) 2D histograms between 1 and 60 eV for NV 0 centers in a diamond nanoparticle and 4.1 eV defects in an h-BN flake. Colored vertical dashed lines mark energy ranges where temporal profiles are integrated. (b,d) Temporal profiles extracted from (a,c), with the time axis set so decay occurs at positive times. The NV 0 decay is considerably longer than the system IRF. The decay of the 4.1 eV defects is only marginally longer than the IRF, shown in panel (d) and in Fig. 1(f). A diamond nanoparticle and an h-BN flake EELS spectra are shown in the left insets of (b,d). The corresponding relative quantum efficiency curves are shown in the right insets.

FIG. 3 .

 3 FIG. 3. (a-b) Decay times as a function of energy bins for eighteen NV 0 and five 4.1 eV defect independent measurements. (b) is the same plot as (a) with only the 4.1 eV defects in h-BN data.

FIG. 4 .

 4 FIG. 4. (a,c)(E, ∆t) 2D histograms between 1 and 400 eV for NV 0 centers in a diamond nanoparticle and 4.1 eV defects in an h-BN flake. Colored vertical dashed lines mark energy ranges where temporal profiles were integrated. (b,d) Temporal profiles extracted from (a,c). A diamond nanoparticle and an h-BN flake EELS spectra are shown in the left insets of (b,d). The colorscale in (a,c) is in a logarithmic scale. The corresponding relative quantum efficiency curves are shown in the right insets.

SUPPLEMENTARY MATERIAL

The supplementary material contain CL spectra of the two defects studied in this manuscript: NV 0 centers in diamond and 4.1 eV defects in h-BN.

ACKNOWLEDGEMENT

Data processing and availability

All data was processed using the following Python libraries: Numpy 1.23.5, Matplotlib 3.6.2, Scipy 1.10.0, Hyperspy 1.7.3 [START_REF] De La Peña | hyperspy/hyperspy: Release v1[END_REF] . The raw data processing code is available in Zenodo [START_REF] Auad | Timestem/tp3 tools: Release v1.0.0[END_REF] .

All published data are available at the Zenodo repository with the following DOI: 10.5281/zenodo.8090907.

I. SUPPLEMENTARY MATERIAL

A. Cathodoluminescence spectra with full energy range


Manon Louis

Alex Talbot

Diana Lamaa

Antoine Sallustrau

Davide Audisio

Frédéric Taran

A sydnone-based route to access indazolo[2,3-a]quinoxaline derivatives

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A sydnone-based route to access indazolo[2,3-a]quinoxaline derivatives

Manon Louis, 1∫ Alex Talbot, 1∫ Diana Lamaa, [START_REF] Chandrasekhar | Sydnones: a brief review[END_REF] Antoine Sallustrau, [START_REF] Chandrasekhar | Sydnones: a brief review[END_REF] Davide Audisio, 1 Frédéric Taran 1 * 1 Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SCBM, 91191 Gif-sur-Yvette, France ∫ These authors contributed equally to this work ABSTRACT: A new synthetic route to indazolo [2,3-a]quinoxaline derivatives is described. The strategy is based on the 1,3dipolar cycloaddition of arynes and quinoxaline-sydnone derivatives, as a key step. These polyaromatic sydnones were prepared through a copper-promoted intramolecular cyclisation of the C-4 position of sydnones on imines.

Planar polyaromatic quinoxaline derivatives, especially those fused with nitrogen-rich five-membered heterocycles are important pharmacophores displaying interesting biological properties, which received special attention from the pharmaceutical industry. 1 Among this family, indazolo [2,3-a]quinoxaline derivatives, bearing a fused pyrazine-pyrazole bicyclic aromatic core, represent an important class of anti-tumor drugs 2 due to their ability to intercalate between the base pairs of doublestranded DNA. [START_REF] Decuypère | Sydnone-alkyne cycloaddition: applications in synthesis and bioconjugation[END_REF] However, despite their pharmacological interest, only a few procedures for their preparation are described in the literature. To our knowledge, only two synthetic approaches to indazolo [2,3-a]quinoxalines have been reported to date to give access to these derivatives (Scheme 1).

Scheme 1. Synthetic routes to indazolo[2,3a]quinoxalines.

The first one involves a C3-functionalization of 2Hindazoles via three different ways: I 2 -mediated sp 2 C-Hactivation in presence of benzylamines, 4 Pd(II)catalyzed isocyanide insertion 5 or intramolecular cyclisation of imine intermediates, followed by oxidation with DDQ. 6 The second is based on a visible-light induced radical coupling of ortho-substituted aryl-isocyanides and radicals generated from phenyliodine(III) dicarboxylate reagents, affording these polyaromatic structures in mild conditions (Scheme 1). 7 Herein, we describe a novel synthetic method giving direct access to indazolo [2,3-a]quinoxaline derivatives from tricyclic sydnones. This strategy is complementary to the state of the art and might provide alternative solutions to medicinal chemistry programs.

Polycyclic sydnones have been found interesting substrates to react with arynes to construct polyaromatic products through [3+2] cycloadditions. 8 We postulated that similar strategy would be possible for the construction of indazolo [2,3-a]quinoxalines from reaction of benzyne derivatives with tricyclic nitrogen-containing sydnones (Scheme 1). However, as such sydnone patterns have not been described, a suitable synthetic access was investigated. The C-4 position of sydnones is well-known to undergo facile C-H activation in cross-coupling reactions. Deshydrogenative Suzuki 9 and Heck 10 type reactions are notably described to functionalize sydnones by Pd-catalyzed C-H activation of the C-4 position. This position is also easily functionalisable by reaction with various electrophiles after abstraction of the proton in position C-4 with strong bases. 11 Based on these reports, we hypothesized that access to 4 would be possible via intramolecular cyclisation of imines 2, obtained either from azido-sydnone 1a by Staudinger aza-Wittig reaction or from amino-sydnone 1b. Under optimal conditions, 2 may react as an internal electrophile to form a new sixmembered ring intermediate 3, which should afford sydnone 4 upon oxidative rearomatisation (Scheme 2).

Scheme 2. Proposed synthetic route to polycyclic sydnones 4.

Azido-sydnone 1a was easily obtained in 4 steps from aniline by slight adjustments from literature protocols. 12 Notably, the procedure was easily scaled-up, and 2.9 grams of 1a could be isolated. Staudinger reaction with PPh 3 in mixture of THF/water afforded 1b in a good yield (Scheme 3).

Scheme 3. Synthesis of sydnones 1a and 1b from aniline.

Having sydnones 1a and 1b in our hands, we tested our synthetic strategy with ethyl 4-formylbenzoate used as a model activated aldehyde. Unfortunately, all attempts to form imine derivatives from sydnone 1b were unsuccessful. On the contrary, the Staudinger aza-Wittig reaction starting from azide 1a was successful but very slow. Excess of aldehyde and the use of nucleophilic phosphines such as PhPMe 2 were necessary to form the expected imine 2a. This imine was found not stable under purification conditions and was used in situ for the next cyclisation step. We therefore screened a series of one-pot conditions using PhPMe 2 for the first step and several Lewis or Brönsted acids to activate the imine function and favor the nucleophilic attack of the C-4 position of the sydnone leading to sydnone 4a by intramolecular cyclisation (Table 1). Among the conditions explored, only Cu(OTf) 2 was found efficient when used in excess (entries 7 and 8). Under optimized conditions, 2 equivalents of Cu(OTf) 2 at room temperature in acetonitrile allowed complete conversion of imine 2a into sydnone 4a, which was isolated in 81% yield. It is worth mentioning that during our investigations, no traces of the non-aromatic intermediate 3 was observed. We then turned our attention to evaluating the scope in the construction of other tricyclic sydnones 4 by varying the structure of the aldehyde starting reactant (Scheme 4).

Scheme 4. Scope of the reaction.

Overall, this methodology demonstrated good functionalgroup tolerance, thus enabling preparation of a range of polycyclic sydnones in moderate to good yields starting from aromatic (both electron rich and poor) and aliphatic aldehydes (4d, 4j). Vinyl aldehydes, in the contrary, were found not suitable substrates for this reaction. For example, only 5% yield of sydnone 4h were obtained from cinnamaldehyde.

We were then interested in looking at the reactivity of these new sydnones as dipoles for cycloaddition reactions with alkynes. Sydnones are well-known to undergo efficient thermal cycloaddition reactions with terminal or disubstituted alkynes at elevated temperatures or under milder conditions through Cu-catalysis. [START_REF] Audisio | For reviews in the field see : a) Karine Porte, Margaux Riomet, Carlotta Figliola[END_REF] They are also known to react smoothly with strained alkynes such as cyclooctynes 14 and arynes. 15 Unfortunately, polyaromatic sydnones 4 were found inactive toward either terminal, activated di-substituted alkynes such as dimethyl acetylenedicarboxylate (DMAD) or cyclooctynes such as bicyclo-[6.1.0]-nonyne (BCN) and dibenzocyclooctynes (DBCO) even at elevated temperatures. We thus focused our attention to cycloaddition reactions with aryne derivatives starting from the Kobayashi precursor 16 in presence of TBAF and were please to observe a smooth reaction took place at room temperature, affording the desired indazolo [2,3-a]quinoxalines 5 in moderate to good yields (Scheme 5). In conclusion, we have developed a new synthetic route to indazolo [2,3-a]quinoxaline derivatives based on the cycloaddition of arynes with polycyclic sydnones. These new sydnones were obtained in good yields upon intramolecular cyclisation of imines at the C-4 position of sydnones through Cu(II) assistance. We think this new synthetic route complete advantageously the only few methods actually available to construct these nitrogencontaining polyaromatic compounds that find several interesting biological properties.

ASSOCIATED CONTENT Supporting Information

Synthetic procedure and Analytical data of all compounds

  Scheme 5. Synthesis of indazolo[2,3-a]quinoxalines 5 from sydnones 4. See supporting information for experimental details.

Table 1 . Optimization of the reaction. a crude yields determined by LCMS. b isolated yields

 1

	Entry MLn or H+	Solvent	Time T°C	4a
	1	CH3SO3H -2 equiv.	CH3CN	24 h	25 °C 22% a
	2	AgOTf -0.2 equiv.	CH3CN	24 h	25 °C n.d.
	3	Sc(OTf)3 -0.2 equiv.	CH3CN	24 h	25 °C n.d.
	4	Cu(OTf)2 -0.2 equiv.	CH3CN	24 h	25 °C n.d.
	6	Cu(OTf)2 -0.6 equiv.	CH3CN	24 h	25 °C 29% b
	7	Cu(OTf)2 -1.1 equiv.	CH3CN	24 h	25 °C 68% b
	8	Cu(OTf)2 -2 equiv.	CH3CN	14 h	25 °C 81% b

See for example: a) A. A. Kalinin, V. A. Mamedov, Chem. Heterocycl. Compd.

2011, 46, 1423 -1442; b) V. A. Mamedov,A. A. Kalinin, Chem. Heterocycl. Compd. 2010, 46, 641 -664.

ACKNOWLEDGMENT

This work was supported by the French Research National Agency (ANR-20-CE44-0013-01) and CEA. The authors thank Sabrina Lebrequier, Amélie Goudet and David-Alexandre Buisson for excellent analytical support.

AUTHOR INFORMATION

Corresponding Author * E-mail: frederic.taran@cea.fr; ORCID : 0000-0001-5461-329X

Author Contributions

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

Notes

The authors declare no competing financial interest.


Dr Minghao Feng

Dr Léa Madegard

Margaux Riomet

Dr Manon Louis

Dr Pier Alexandre Champagne

Dr Grégory Pieters

Dr Davide Audisio

Frédéric Taran
email: frederic.taran@cea.fr

Selective Chlorination of Iminosydnones for Fast Release of Amide, Sulfonamide and Urea-Containing Drugs

Herein, we describe a methodology for iminosydnone chlorination and we demonstrate the high beneficial effect of this modification on the reactivity of these mesoionic dipoles in strain-promoted cycloaddition reactions. Exploiting their reaction with cyclooctynes, we used these new iminosydnones for bioorthogonal release of amide, urea and sulfonamide containing drugs. Notably, drugs containing a terminal amide function were released for the first time with good kinetic constants.

The use of prodrugs -caged bioactive substances whose activities can be restored in vivo -is a well-established strategy that has led to the marketing of several pharmaceutical products. 1 Prodrugs not only improve the solubility, 2 permeability 3 and biostability 4 of drugs, but also allow their release in specific tissues. Conventional prodrug activation strategies typically rely on physiological characteristics of pathological cells, such as pH and redox variations or the presence of an over-expressed enzyme. Upon activation, the active parent drug is released and can then exert the desired pharmacological effect. 5 However, healthy and pathological tissues are often poorly differentiable, resulting in a nonselective activation of prodrugs, therefore decreasing their efficiency at reducing undesired side-effects. 6 An emerging alternative strategy is the use of selective, bioorthogonal reactions, which can be triggered on demand where the disease is taking place. 7 Bioorthogonal click-and-release reactions appear as particularly suitable for such prodrug strategies. 8 In this approach, a bioorthogonal function is attached to a key part of the drug in order to mask its original activity. Addition of the complementary bioorthogonal trigger then allows the decaging of the drug, restoring its biological activity. The most representative examples include Staudinger-type reductive cleavage reactions, 9 transition-metal-catalysed deallylation 10 or depropargylation 11 and the inverse electron demand Diels-Alder reaction (iEDDA, Fig. 1a) between tetrazines (Tz) and trans-cyclooctenes (TCO). 12 The latter is considered as the most effective among all reported decaging reactions both in terms of kinetics and biocompatibility. The use of this reaction for prodrug development has been pioneered in 2013 by Robillard and co-workers who reported the bioorthogonal release of the drug Doxorubicin upon reaction of a Tz with a TCO bearing a carbamate in allylic position. 13 Since then, several key developments have been reached notably by Weissleder 14 and Robillard 15 to improve the efficiency of the iEDDA reaction for drug release. These efforts Please do not adjust margins Please do not adjust margins recently led to the development of a TCO-doxorubicin prodrug, currently under clinical trials for solid tumor therapy in human patients. 16 First used to release NH 2 -containing drugs, iEDDA was extended in 2016 to the release of alcohols by the group of Bernardes using a vinyl ether/Tz pair 17 and to phenols release from TCO-ethers as described in 2018 by Robillard 18 (Fig. 1a). As of today, decaging technologies were mostly focused on the release of alcohols, phenols and amines, either directly or with the intermediate of a self-immolative linker. In 2017, our group reported a new bioorthogonal click and release reaction involving iminosydnones and cyclooctynes (SPICC: Strain-Promoted Iminosydnone-Cyclooctyne Cycloaddition, Fig. 1b), which allows the release of ureas, carbamates and sulfonamides. 19 This reaction was later used to release Celecoxib, a sulfonamide-containing anti-inflammatory drug. 20 While the SPICC reaction has the major advantage of being quantitative, its moderate kinetics represent a limitation for its applicability. Herein, we demonstrate that the selective chlorination at position C-4 of iminosydnones significantly boosts their reactivity towards cyclooctynes and allows the rapid release of bioactive substances containing a terminal amide function for which no bioorthogonal prodrug strategy was possible until now (Fig. 1b).

N N O N S R a) iEDDA R' R N N N N R O OH O O NH O O R' N N N N R' R NH O O N N N N R R R'
Our group previously showed that halogenation at position C-4 of sydnones, another family of mesoionics, dramatically enhanced the kinetics of their cycloaddition with cyclooctynes. 21 Computational studies carried out by the group of Houk revealed that the origin of this halogen effect is due to lowering the distortion energy of sydnones. 22 We later confirmed this halogen effect on iminosydnones, where their bromination improved the kinetics of the SPICC reaction by one order of magnitude. 23 Motivated by this remarkable effect, we turned our attention to the incorporation of more electronegative halogen atoms. Unfortunately, all attempts to fluorinate iminosydnones in position 4 were unsuccessful, probably due to the apparent instability of the expected fluoroiminosydnones. We then tested various chlorinating conditions on iminosydnone IS1, used as the model substrate (Scheme 1a). We first began our investigation with N-chlorosuccinimide 1, and trichlorocyanuric acid 2 but unfortunately with no success. The desired chlorinated product could finally be observed when 1,3-dichloro-5,5-dimethylhydantoin 3 was used, albeit in low yield and along with some of remaining unreacted iminosydnone. Unfortunately, rapid degradation happened when a higher stoichiometry of 3 was used (entry 2, Scheme 1a). Palau'chlor 24 4 gave an encouraging 73% yield after 7 hours of reaction at room temperature (entry 3, Scheme 1a), however, separation of the desired chlorinated iminosydnone from the polar by-products proved challenging. Finally, tBuOCl 5 was found to be the most efficient and practical reagent affording 83% of IS1' in only 15 min at room temperature (entry 7, Scheme 1a). With these optimised conditions in hands, we further explored the tolerance and selectivity of our chlorination method. To this end, an array of iminosydnones IS2-15 were prepared using established protocols. 25 Carbamoyl and urea derivatives of iminosydnones were prepared in one step by N-exocyclic functionalisation involving the addition of strong electrophiles (activated esters, chloroformates, or isocyanates) on 6 under slightly basic conditions or the addition of alcohols and amines on 7 (see ESI for details). The chlorination protocol was found quite general and afforded a variety of 4-chloro-iminosydnones in good yields (Scheme 1b). The presence of amino-acids, alcohols, phenols, carbohydrate and heterocyclic moieties did not affect the reaction. No additional chlorination was observed even in the case of substrates bearing electron-rich aromatic sites (IS10, 11, 13 and 15), showing that position C-4 of the iminosydnone core displays a particular reactivity towards chlorination. The chemoselectivity of this reaction allowed us to successfully perform the chlorination of iminosydnones carrying biologically relevant compounds such as Estradiol (hormone, IS10), Naproxen (nonsteroidal anti-inflammatory drug, IS11), Progabide (GABA analogue, IS13), Carbamazepine (antiepileptic drug, IS14) and Apixaban (anticoagulant drug, IS15). The chlorination reaction was also effective on sulfonamide

ChemComm Accepted Manuscript

Published on 01 July 2022. Downloaded by CEA Saclay on 7/1/2022 7:46:59 AM.

View Article Online DOI: 10.1039/D2CC02784D

Please do not adjust margins Please do not adjust margins compounds as shown by the preparation of prodrug IS17' of the COX-2 inhibitor Celecoxib (Scheme 1c). Kinetic studies were then carried out to evaluate the aptitude of chlorinated iminosydnones to react with cyclooctynes. We selected a series of iminosydnones bearing amide, urea, carbamate and sulfonamide functions at position N-6 and compared the rate constants of the non-chlorinated versus chlorinated compounds with dibenzocyclooctyne (DBCO). As shown in Scheme 2, the effect of the chlorination on the reaction rate is quite significant, as it induced an increase of several orders of magnitude regardless of the iminosydnone substitution pattern.

H 2 N R N N O N O NHFmoc t BuO2C
EtO2C The most spectacular effect was observed for iminosydnones bearing an exocyclic amide function (IS1 and IS3). While the parent compounds (X = H) are almost unreactive, the corresponding chloro-iminosydnones (IS1' and IS3') react smoothly with rate constants k > 1 M -1 .sec -1 at room temperature. These rate constants are in the same range as the well-established strain promoted azide-alkyne cycloaddition (SPAAC) reaction and support the use of chloro-iminosydnones as masking for bioactive compounds containing terminal amide functions. The beneficial influence of the chlorine atom was also observed for iminosydnones bearing carbamate and sulfonamide moieties (Scheme 2), inducing a rate increase of the SPICC reaction by more than 100 (Scheme 2, IS8 vs IS8', IS16 vs IS16'). The chlorine effect was also significant on the urea derivative IS7, but to a lesser extent. Notably, 3-N-alkyl iminosydnone IS6' was also effective in the reaction with DBCO whereas its non-chlorinated analogue IS6 was completely unreactive. We also confirmed the beneficial impact of bromination at position C-4 of iminosydnones although it is slightly lower than chlorination (compare IS3', 7' and 8' with IS3'', 7'' and 8'' in scheme 2). To better understand the origin of the chlorine atom effect on the kinetics, we performed a theoretical study using DFT calculations at the M06-2X/6-31+G(d,p)/CPCM(H 2 O) level of theory (Fig. 2, see ESI for computational details). Using two model iminosydnones mIS1 and mIS2 (Fig. 2a), the transition structures (TS) and activation free energies (ΔG ⱡ water ) for their reactions with a DBCO model were computed. As the cycloaddition step is rate-determining and irreversible in similar systems, we compared the corresponding [3+2] TSs for the two systems. 26 The ΔG ⱡ water for the chlorinated iminosydnone mIS2 (20.5 kcal mol -1 ) was found to be significantly lower than the one for mIS1 (23.0 kcal mol -1 ). This free energy difference of 2.5 kcal mol -1 results into an almost 100-fold increase in rate at room temperature due to chlorination, which is in line with the experimentally-observed rate enhancement. As already demonstrated by Houk and co-workers in the case of sydnones, 22 the origin of the beneficial halogen effects on cycloaddition kinetics could be due to the lower distortion energy of the 4-chloro-iminosydnone compared with the nonhalogenated structure. In addition, we also found that the presence of the halogen atom on C-4 prevents the iminosydnone from adopting a low-energy conformation with an intramolecular C-H…O interaction (Fig. 2a), and as such chlorinated iminosydnones might be less stabilized. Overall, these theoretical and experimental results demonstrated that the chlorination of iminosydnones accelerates the SPICC reaction to a degree that allows its use for drug decaging applications. To this end, we investigated the bioorthogonal release of the drugs Carbamazepine, Apixaban and Celecoxib from their corresponding prodrugs IS14', IS15' and IS17' (Fig. 3). We first checked the stability of these prodrugs and found t 1/2 > 7 days in PBS (see ESI). Decaging was then carefully monitored at 37 °C in PBS buffer by following both the disappearance of the starting 4-chloro-iminosydnones and the appearance of the drugs released through the retro-Diels-Alder (rDA) step of the reaction. As expected, all drugs were rapidly released upon addition of DBCO. Importantly, according to HPLC analysis, the SPICC reactions were quantitative (> 98% of drug release) and perfectly selective, as no byproduct was observed. The beneficial influence of the chlorine atom can be highlighted by the rate constant obtained with compound IS17' which allows a 100 times faster release of Celecoxib compared to published data. 20 These results validate the approach of using chloroiminosydnones as masking agents for drugs containing terminal amide, urea or sulfonamide drugs. Please do not adjust margins Please do not adjust margins

IS1 : X = H, k < 0.001 M -1 .s -1 IS1' : X = Cl, k = 3.9 M -1 .s -1 N N O N O EtO2C IS3: X = H, k < 0.001 M -1 .s -1 IS3': X = Cl, k = 2.3 M -1 .s -1 IS3'' : X = Br, k = 1.3 M -1 .s -1 N N O N O OtBu Me IS8: X = H, k = 0.001 M -1 .s -1 IS8': X = Cl, k = 3.0 M -1 .s -1 IS8'' : X = Br, k = 1.7 M -1 .s -1 N N O N O H N EtO2C IS7 : X = H, k = 2.8 M -1 .s -1 IS7

ChemComm Accepted Manuscript

In summary, we have developed a general method for iminosydnones chlorination and have demonstrated the beneficial impact of this modification on the SPICC reaction. 4chloro-iminosydnones were thus found particularly reactive leading to a fast click and release reaction with cyclooctynes. These mesoionic compounds can be employed to temporarily mask terminal amide, urea or sulfonamide functional groups in bioactive substances. This approach can be used for the development of new prodrugs but also in the design of new drug delivery technologies such as antibody-drug conjugates (ADCs), offering additional tools for chemical biology.

Notes and references

Fig. 1 .

 1 Fig. 1. Drug release after bioorthogonal click and release reactions.

  Scheme 1. a) Optimisation of iminosydnone chlorination. b) and c) scope of the chlorination reaction. All yields are isolated yields.

3 MScheme 2 .

 32 Scheme 2. Halogen effect on the SPICC reaction. Rate constants k were determined by monitoring the reaction between IS and DBCO in 0.01M PBS/DMSO (80/20) at 25 °C; PBS: Phosphate buffered-saline.

Fig. 2 .

 2 Fig. 2. Theoretical study on the SPICC reaction. a) Mechanism of the reaction and transition structure involving mIS1 (TS 1d ), b) Mechanism of the reaction and transition structure involving mIS2 (TS 2b ), with a DBCO model (M06-2X/6-31+G(d,p)/CPCM(H 2 O) level of theory). Only one pyrazole regioisomer was considered for clarity reasons.

Fig. 3 .

 3 Fig. 3. Release of drugs upon SPICC reactions. a) reactions and rate constants values, b) drug release as function of time after addition of DBCO, c) chromatogram after 1 h reaction of IS15' with DBCO. Reaction conditions: [IS] = 30 µM, [DBCO] = 45 µM, 37 °C in 0.01M PBS containing 20% DMSO. Internal standard = 3-fluoro-4-nitrophenol.

ChemComm Accepted Manuscript


Myssipsa Mehraz
email: myssipsa.mehraz@imt-atlantique.fr

François Gallée
email: francois.gallee@imt-atlantique.fr

Impact of measurement parameters on antenna radiation pattern reconstruction using phaseless iterative technique

Keywords: Nearfield measurements, Antenna characterization, Iterative Fourier Technique, Phaseless techniques, Radiation pattern reconstruction Introduction (Heading 1)

This study focuses on the impact of planar nearfield measurement parameters, such as plane size, sampling step and the distance between the Antenna Under Test (AUT) and the measurement plane. The main objective of this work is to determine the optimal near-field measurement parameters that achieve an assessment of far-field (FF) radiation pattern accuracy versus measurement duration.

I. INTRODUCTION

Near-field antenna characterization methods offer an alternative to direct far-field measurements but often necessitate considerable data acquisition and both amplitude and phase measurements for subsequent near-to-far-field transformations. Phaseless techniques have emerged as a practical alternative to these traditional methods and offer several advantages, such as the ability to eliminate the need of expensive equipment and highly precise positioning systems [START_REF] Razavi | Resilience to Probe-Positioning Errors in Planar Phaseless Near-Field Measurements[END_REF]. Additionally, they hold the promise of simplifying OTA (Over-The-Air) for devices without direct access to the antenna. The most commonly used technique in this type of measurement is called the Iterative Fourier Technique (IFT) [START_REF] Yaghjian | Upper-bound errors in far-field antenna parameters determined from planar near-field measurements : PART 1: analysis[END_REF], which is based on propagating the measured amplitude between two surfaces, located at different distances from the antenna aperture using plane wave spectrum in order to reconstruct the phase and then apply a traditional near-field to far-field transformation.

The parameters that the user must define for a standard planar near-field measurement are the size of the measurement plane, the distance separating the Antenna Under Test (AUT) and the measurement plane, and the sampling step. In theory, for complete and precise reconstruction, the measurement plane should be infinite in size, and the sampling step should be as small as possible to capture all variations in the electric field on this plane. In practice, the plane is truncated from infinity, and the sampling step is calculated while respecting Nyquist's condition. Furthermore, the required separation distance between the AUT and the measurement plane is determined by the reactive field radiated. The Phaseless Iterative Fourier Technique (IFT)starts with an initial guess of phase and amplitude across the antenna's aperture. Then, it propagates this initial distribution towards Plane 1 using Plane Wave Spectrum (PWS) theory. Here, the propagated amplitude is replaced with the actual measured one, while the phase is retained. This process repeats forward (from Plane 1 to Plane 2) and backward (from Plane 2 to Plane 1), updating the propagated amplitude with the real measurements on each plane. The phase is adjusted on each iteration and it keeps iterating until a predefined error rate or when a maximum number of iterations is reached.

The parameters addressed in this study for planar nearfield measurement using phaseless IFT method are the size of the measurement plane, the distance separating the Antenna Under Test (AUT) and the measurement plane, the sampling step. The main objective of this work is to contribute to the specification of near-field measurement parameters that achieve an assessment of far-field (FF) radiation pattern accuracy versus measurement duration.

II. DEFINE OPTIMAL MEASUREMENT PARAMETERS

A. Measurement plane size and distance for the AUT

Firstly, to establish the optimal near-field measurement parameters regarding the desired radiation pattern cutoff angle, a criterion (Fig. 2) has been empirically established through extensive measurements [START_REF] Yaghjian | Upper-bound errors in far-field antenna parameters determined from planar near-field measurements : PART 1: analysis[END_REF] and substantiated through theoretical analysis [START_REF] Newell | Planar near-field measurements on high performance array antennas[END_REF]. It could be used to determine the minimum size of the scan plane for a given desired angular region of coverage and the distance d between the aperture of the antenna and the measurement plane. The curves on the fig. 3 show that this criterion is very restrictive but necessary if very small measurement error is wanted over a large angle range. In our case, to optimise the size of the measurement, an amplitude range from the maximum amplitude will be defined, for example (30dB). Outside, the measurement plane size could be increase by filling null value.

The optimal distance d of the measurement planes from the antenna aperture should be in the Rayleigh zone to avoid the reactive where strong coupling effect between the AUT and the probe could disturb the measurement. Our test bench "caronchip" is presented on the fig. 4. On the fig. 5, the evolution of the electric field amplitude measured and simulated above a rectangular horn antenna at 96GHz along vertical axis, allows firstly to validate your test bench and from the antenna aperture size to define the optimal distance of the measurement planes. The first plane will be located at the end of the reactive in order to minimise the measurement plane size. In the case of the horn antenna, the distances of measurement planes should be selected between 24λ and 64λ. Spatial sampling Once the measurement plane size and the distance are optimized, the measurement duration depend on the number of measurement point. The general rule is to fix a half wavelength spatial sampling without angle restriction. It is possible to increase the spatial sampling but with an impact about the angle range and the goal is to evaluate it.

The spatial sampling effect could be study from the theory of PWS [START_REF] Yaghjian | Upper-bound errors in far-field antenna parameters determined from planar near-field measurements : PART 1: analysis[END_REF]. The electric field is written as follows :

E x, y, z 1 4π F k , k x̂" F k , k ŷ $% &% " F ' k , k ẑ) e &+,-. $-/ $0-1 2 &-. 2 &-/ 2 '3 dk dk 2 With ,k x " k y " 0k 1 k k z3 k. r 3
And k 1 2π λ 4

The radiation condition in 3D requires that for z≥0, the relation between wavenumbers should ensure:

k ' 9 k k k : j k k k : if k " k > k otherwise D 5
The method of stationary phase is used to derive easy-touse formula far field calculations.

∂k. r ∂k ∂k. r ∂k 0 6

The solution to ( 5) is:

k k 1 sinθcosφ 7 k k 1 sinθsinφ 8
θ and ϕ represent the angles associated with the spherical coordinate system: θ is the elevation angle, and ϕ is the azimuth angle.

The wavenumbers kx and ky can be written in the Cartesian domain as:

k 2πm MΔ 9 k 2πn NΔ 10
With Δx and Δy the sampling steps following respectively the x and y-axis. M and N are the maximum number of samples. In addition, m and n index to the positions in the scan plane as:

M 2 > m > M 2 1 N 2 > n > N 2 2
Let us now deduce the sampling step from the previous equations. We'll show the development only for ky as the calculation is the same for kx.

By replace in (6) T 90° for the far-field E plane, the maximum farfield cutoff V WW XYZ is obtained for

k [\] _. 3 . θ ``[\] sin 1 a k bcd k 1 e 4 Thus θ ``[\] sin 1 a λ 2Δ e
The curves Fig 6 . illustrates the spatial sampling step as a function of the desired far-field cutoff angle respectively when there's no aliasing. 6) and obtained angle from y axis Plane Wave Spectra in E plane with a maximum error of 0.1dB. The difference between supposed and obtained azimuthal angles is due to aliasing after Fourier transform in NF to FF algorithm that depends on the crossing point between the central spectrum and the first spectra repeated to the left, and right of it. During aliasing, some of the secondary spectra overlap with the main spectrum, resulting in distortions of the reconstructed spectrum as shown in Fig 13 . An assessment of the aliasing impact depending on the electric field distribution should be taking into account to avoid or control the error.

III. IFT METHOD VALIDATION

The IFT (Iterative Fourier Transform) method allows measurements to be made in amplitude only and then reconstructs the phase through an iterative process between the two measurement surfaces.

Before starting this iterative process, it's crucial to choose an initial distribution of amplitude and phase across the antenna aperture. While amplitude is usually set to 1 across all points on the aperture special attention is needed when setting the initial phase, as it impacts whether the iterative algorithm successfully converges to a global solution and influences the convergence time. Three main methods were developed for IFT initial guess and presented in [START_REF] Li | Investigation of the Iterative Fourier Technique for Phaseless Planar Near-Field Antenna Measurements[END_REF]. In our study, we use the "Basic Approach" for the initial guess that defines constant phase and ones for amplitudes across the antenna aperture. To validate the implementation of the IFT, Fig. 11 and Fig. 12 show the magnitude and phase correctly reconstructed with only near field planes amplitude data taken from FEKO simulatio, with a maximum in the plane center of 26.3128 dBV/m for theoretical plane and 26.2815 dBV/m for reconstructed one. depicts the relationship between the amplitude error and phase difference according to the electric field magnitude between theoretical and reconstructed amplitudes and phases. It is evident that the error is concentrated in regions with low field magnitude and allow to validate the measurement. These plots could be very interesting to define a criterion during the iterative process of the ITF. The measured radiation pattern computed with a spatial sampling of 0.64 and compare to the simulated radiation pattern in E plane is showed in the Fig. 14. The blue and red curves are the measurement at two distances plane measurement. In this plane, the aliasing error is present. This can be explained by uniform distribution of the field in the antenna aperture. In the H plane, Fig. 15, there is no aliasing error because the electric filed distribution in this plane is a cosinus distribution. The difference between the simulated and the measurement is due to the measurement test bench sensitivity.

IV. CONCLUSION AND PERSPECTIVES

In this paper, the impact of the nearfield antenna measurement parameters has been studied with the definition of primary rules to optimize the measurement duration according to the angle range and the error. Depending on the expected antenna parameters, for example, 3dB beamwidth, first side lobes level, it's possible to increase the spatial sampling to reduce the measurement duration

The future work will be to test these different criteria from different antenna radiation pattern to validate them and propose technics like interpolation or non-uniform mesh of measurement points.

Fig. 1 .

 1 Fig. 1. Phaseless measurement principle

Fig. 2 . 1 Fig. 3 .

 213 Fig. 2. Critical cutoff angle (θc)

Fig. 4 .

 4 Fig. 4. "Caronchip" test bench

Fig. 6 .

 6 Fig. 6. Sampling step function of desired FF cutoff angle Fig 7. and 8. Shows respectively radiation pattern for the E and H plane of a 96 GHz horn antenna, computed using near-field data simulated in FEKO for various sampling steps computed. Plane size is about 256 λ

Fig. 9 .

 9 Fig. 9. Difference between reference PWS for 0.5 lambda step and for greater steps -E PLANE

Fig. 10 .

 10 Fig. 10. Illustation of aliasing error due to overlapping of offset spectra (C.NEWELL, 1988)

Fig. 11 .

 11 Fig. 11. theoretical (left) and reconstructed (right) magnitude using ITF d1 = 24λ d2 = 45λ

Fig. 12 .

 12 Fig. 12. Theoretical phase (left) and reconstructed phase (right) using ITF Fig 13.depicts the relationship between the amplitude error and phase difference according to the electric field magnitude between theoretical and reconstructed amplitudes and phases. It is evident that the error is concentrated in regions with low field magnitude and allow to validate the measurement. These plots could be very interesting to define a criterion during the iterative process of the ITF.

Fig. 13 .

 13 Fig. 13. Amplitude and phase difference between theoretical and reconstructed field

Fig. 14 .

 14 Fig. 14. Measured (Blue and red) and simulated (green) radiation pattern in the E plane.

Fig. 15 .

 15 Fig. 15. Measured (Blue) and simulated (red) radiation pattern in the E plane.

Table 1 .

 1 shows a comparison between calculated cutoff angle from (fig.

TABLE I .

 I COMPARISON BETWEEN SUPPOSED CUTOFF ANGLE AND OBTAINED ONE

	NF Sampling step	E PLANE
		Supposed	Obtained
	0.64 λ	51.37°	32°
	0.8 λ	38.68°	19°
	0.96 λ	31.88°	10°
	1 λ	30°	10°


Kim Anh Nguyen

Emilie Lesur

Maxime Ribéraud

Antoine Sallustrau

Davide Audisio

Frédéric Taran
email: frederic.taran@cea.fr

Sydnone-Cyanines as Clickable Probes for Fluorescent Labelling

Keywords: Click chemistry, Mesoionics, Iminosydnones, Cyanine

come

Introduction

Cyanines are major family of dyes for in vivo imaging applications due to their outstanding photophysical properties, excellent biocompatibility, and low toxicity to living systems. [START_REF] Sun | Recent Development of Chemosensors Based on Cyanine Platforms[END_REF] Among them, near-infrared fluorescence heptamethine cyanine (Cy7) dyes are of particular interest because of their ability to generate strong fluorescence emission at the range of 700 to 1000 nm, offering the possibility of biomedical imaging, especially in the field of fluorescence-guided tumor surgery. [START_REF] Frangioni | In vivo near-infrared fluorescence imaging[END_REF] In addition, some Cy7 have been reported to display photosensitizing properties, which could be potentially exploited in photodynamic therapy for cancer treatments [START_REF] Tan | I NIR heptamethine dye with intrinsic cancer targeting, imaging and photosensitizing properties[END_REF] and their ability to bioaccumulate in tumors has been mainly used to deliver anticancer drugs. [START_REF] Choi | Heptamethine Cyanine Dye Mediated Drug Delivery: Hype or Hope[END_REF] All of these applications require to connect the Cy7 to the desired ligands, drugs or biomolecules, a task which could be challenging. Click chemistry is the ideal solution to link two compounds together under smooth conditions and thus has been widely used to attach cyanines to small as well as large molecules such as antibodies. [START_REF] Huang | Molecular optical imaging probes for early diagnosis of drug-induced acute kidney injury[END_REF] The main clickable Cy7 described in literature are derivatized with azide or alkyne functions to allow copper-catalyzed [START_REF] Wang | NIR Scaffold Bearing Three Handles for Biocompatible Sequential Click Installation of Multiple Functional Arms[END_REF] or strained promoted [START_REF] Ornelas | Combining Aminocyanine Dyes with Polyamide Dendrons: A PromisingStrategy for Imaging in the Near-Infrared Region[END_REF] azide-alkyne cycloaddition click reactions (CuAAC, SPAAC respectively, Scheme 1) or with tetrazines to allow fast coupling with trans-cyclooctenes (iEDDA, Scheme 1). [START_REF] Zhang | Bioorthogonally activatable cyanine dye with torsion-induced disaggregation for in vivo tumor imaging[END_REF] Scheme 1. Examples of known clickable Cy7, Cy7-sydnones envisaged in this work.

Besides these click reactions, the strained promoted sydnone-cycloalkyne cycloaddition (SPSAC) [START_REF] Wallace | Strain-promoted sydnone bicyclo-[6.1.0]-nonyne cycloaddition[END_REF] appears as an interesting alternative to connect Cy7 to cyclooctyne-containing (bio)molecules. In the present article, we describe the synthesis and properties of Cy7-sydnone derivatives that may complete the choice of available click tools to prepare Cy7 conjugates.

Results and Discussion

Based on our own expertise in sydnone chemistry, [START_REF] Specklin | One-Pot Synthesis of 1,4-disubstitued Pyrazoles from Arylglycines via Copper-Catalyzed Sydnone-Alkyne Cycloaddition Reaction[END_REF] we designed four new clickable Cy7: 4, 5, 6 and 7 taking advantage of the well-known ability of the chloride on the polyene core of Cy7 to undergo Pd-catalyzed cross-coupling reactions (Scheme 2). [START_REF] Lee | Heptamethine cyanine dyes with a robust C-C bond at the central position of the chromophore[END_REF] At first, we carried out the synthesis of pinacolboronate-sydnones 3a and 3b in 4 steps starting from meta-or paraiodoaniline. Reaction of the anilines in the presence of glyoxylic acid and sodium cyanoborohydride allowed smooth reductive amination at room temperature in methanol leading to phenylglycines 1a and 1b in 99% and 48% yield respectively (Scheme 2). 4-iodophenyl-sydnones 2a and 2b were obtained in 62 and 75% yields by a two-steps one-pot procedure involving nitrosylation of the secondary amine function by tert-butyl nitrite followed by cyclisation in the presence of trifluoroacetic anhydride. Subsequent palladium cross-coupling between iodo-sydnones 2a and 2b with pinacolborane afforded the expected pinacolboronate-sydnones 3a and 3b. These products were found not stable enough to be stored and were thus used directly in the next step after rapid filtration on celite. The sydnone moiety was incorporated on Cy7 scaffold via a Pd-catalyzed Suzuki reaction between 3a and 3b and the chloride on the polyene scaffold of three different Cy7. Four clickable Cy7 were thus obtained in low to high yields (Scheme 2). Once these cyanine-sydnone probes synthesised, we characterised their physico-optical properties in organic solvent and in aqueous media (Table 1). At the outset, we were aware that the hydrophobicity of the heptamethine cyanine scaffold may cause potential aggregation in aqueous media, a phenomenon which is minimised by the presence of hydrophilic sulfonate moieties and which can be further influenced by the presence of the sydnone moiety. In fact, cyanines 4 and 5, the most hydrophobic among the four cyaninesydnone synthesised, were poorly soluble and formed aggregates in water.

On the other hand, they displayed strong fluorescence properties in organic solvents. For solubility reasons, compound 5 was solubilized in DMSO, while others were solubilized in methanol. In addition, compound 5 showed the lowest Stock shift among the four cyanines tested, the highest (60 nm) being obtained with cyanine 7 in organo-aqueous media (Table 1).

Additional experiments were carried out in other solvents (see supporting information); the high molar attenuation coefficients are in the common range of cyanine derivatives. We then studied the click reactions between cyanines 4-7 with two different cyclooctynes, the bicyclononyne BCN and the dibenzocyclooctyne DBCO.

Cyclooctynes were added to a solution of cyanine-sydnone and the reaction mixture was stirred for 4 hours with DBCO or overnight with BCN at room temperature to reach completion (Scheme ). LC/MS analysis of all crude reactions proved complete conversion of the cyanine-sydnones 4-7 into the corresponding cyanine-pyrazoles 8-11, and no byproduct was detected. The optical properties of the clicked products resulting from the SPSAC click reactions were then studied to compare them to those of the starting sydnones (Table 2). In order to do so, the cyanine-pyrazoles were solubilised in the same solvents as those used to characterise the cyaninesydnones precursors. The results showed that the optical properties of the cyanine-pyrazoles 9, 10 and 11 are almost identical to those of the corresponding cyaninesydnones 5, 6 and 7 independently from the cyclooctyne used for the click reaction (see supporting information). On the contrary, cyanine-sydnone 4

and cyanine-pyrazole 8a presented quite different fluorescent properties in MeOH resulting in a significant turn-on effect after click reaction. At 825 nm, the fluorescence is 12 times higher for cyanine-pyrazole than for cyaninesydnone and at 850 nm it is 67 times higher. The absorbance and fluorescence spectra of both compounds are indicated in Figure 1.

Surprisingly, this turn-on phenomenon is much lower when the reaction was carried out with DBCO as pyrazole 8b presented similar optical properties as sydnone 4. Furthermore, the turn-on effect observed with 8a is limited to organic solvent as shown in Table 2 indicating possible positive solvatochromism of pyrazole 8a. Hydrophobicity of the cyanine 4 and DBCO may induce aggregation in organo-aqueous solvents explaining the absence of turn on effect in that case.

Conclusions

In this work we described the synthesis, reactivity and physico-optical properties of four Cy7 derivatised with clickable sydnones. All of these clickable cyanines reacted smoothly with cyclooctynes to afford the corresponding pyrazoles quantitatively at room temperature and with good kinetics. These compounds represent valuable additional biorthogonal tools permitting the attachment of cyanine moieties into complex molecules or biomolecules.

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Clickable sydnone-cyanine derivatives allow fast and quantitative coupling of Cy7 scaffold to molecules or biomolecules.

Scheme 2 .

 2 Scheme 2. Synthesis of Sydnone-Cyanine 4, 5, 6 and 7.

Scheme 3 .

 3 Scheme 3. SPSAC reactions between compounds 4, 5, 6 or 7 and cyclooctynes DBCO and BCN. Reactions were carried out with 1.05 equiv of cyclooctyne at room temperature for 4h with DBCO or overnight with BCN, and analyzed by LC-MS.

Figure 1 .Scheme 3 .

 13 Figure 1. Absorbance and fluorescence excitation and emission spectra of sydnone 4 and pyrazole 8a. Absorbance and fluorescence spectra were obtained in MeOH with 1 µM and 0.1 µM concentrations of compounds respectively. We were then interested in the kinetics of the reaction between the cyanine-sydnone 4 and the two cyclooctynes BCN and DBCO. Kinetics of click reactions are indeed of prime importance to allow coupling at low concentrations. SPSAC reactions of 4 with BCN and DBCO were monitored by HPLC. Their kinetics is of order 2 following the equation: ln ( [𝑆𝑦𝑑] [𝑐𝑦𝑐𝑙𝑜𝑜𝑐𝑡𝑦𝑛𝑒] ) [𝑆𝑦𝑑] 0 -[𝑐𝑦𝑐𝑙𝑜𝑜𝑐𝑡𝑦𝑛𝑒] 0 = 𝑘𝑡 + 𝑎 As expected, DBCO was more than 30 times more reactive than BCN (Scheme 5). With a kinetic constant of 18.2 mol.L -1 .s -1 , DBCO afforded a fast SPSAC reaction, which will allow easy attachment of Cy7 on biomolecules even when used at low concentrations. Reaction with BCN is in the same range as SPAAC and can be also useful if concentrations of reactants are not below 1 µM.

Table 1 .

 1 Physico-optical properties of cyanine-sydnones (Cy7-Syd).

	Cy7-Syd	Solvent	exc (nm)	em (nm)	 (L.mol -1 .cm -1 )
	4	MeOH	768	781	212000
		PBS/MeOH	aggregate	aggregate	-
	5	DMSO	820	823	207000
		PBS/DMSO	aggregate	aggregate	-
	6	MeOH	771	785	270000
		PBS/MeOH	764	777	-
	7	MeOH	771	785	172000
		PBS/MeOH	763	823	-

PBS : Phosphate Buffer (0.1 M); 1% of organic solvent were used.

Table 2 .

 2 Physico-optical properties of cyanine-pyrazoles (Cy7-Pyr).

	SPSAC	Cy7-Pyr	Solvent	exc	em	Turn-on
	4 + BCN	8a	MeOH	762	825	12.3
				762	850	67
	4 + DBCO	8b	MeOH	763	777	1.1
	5 + BCN	9a	DMSO	816	823	1.3
	5 + DBCO	9b	DMSO	806	823	1.7
	6 + BCN	10a	MeOH	763	777	1.5
			PBS/MeOH	758	771	1.4
	6 + DBCO	10b	MeOH	764	779	1.4
			PBS/MeOH	758	771	1.3
	7 + BCN	11a	MeOH	765	779	1.6
			PBS/MeOH	760	772	1.3

Acknowledgements

We are very grateful to Amélie Goudet, Sabrina Lebrequier and David-Alexandre Buisson for their excellent analytical support.

Supplementary Material

Supporting information for this article is available on the WWW unde http://dx.doi.org/10.1002/MS-number. Complete analytical data of compounds, kinetics, chemical and optical characterization of cyanine probes.

Author Contribution Statement

Kim Anh Nguyen -performed the majority of the experiments described in this manuscript.

Emilie Lesur -completed the experiments, summarized and formatted all the results, write a part of the manuscript.

Maxime Ribéraud -participated to the writing of the manuscript.

Antoine Sallustrau -completed the experiments.

Davide Audisio -supervised the experimental work and participated to the writing of the manuscript.

Frédéric Taran -had the original idea of the paper, supervised the experimental work and participated to the writing of the manuscript.