Charlotte Collet
email: charlotte.collet@univ-lorraine.fr

Timothé Vucko

Julen Ariztia

Gilles Karcher

Nadia Pellegrini- Moïse

Sandrine Lamandé-Langle
email: sandrine.langle@univ-lorraine.fr

Fully Automated radiosynthesis of [ 18 F]Fluoro-C-glyco-c(RGDfC): Exploiting all the abilities of the AllInOne synthesizer †

Radiolabelled peptides are crucial tools for biomedical research purposes. Peptides are usually labeled with fluorine-18 via an indirect prosthetic group approach, which requires a complex multistep procedure not easily automatable. However, automation is a compulsory step of paramount importance to produce radiotracers with high reliability and minimal human involvement. Herein, we describe the fully automated radiosynthesis of [ 18 F]fluoro-C-glycoc(RDGfC) on an AllInOne (AIO) synthesizer, exploiting for the first time all the abilities of the AIO module. The radiotracer was prepared in high radiochemical purity via a challenging three-steps sequence in 140 minutes with a decay-corrected radiochemical yield of 3.6 ± 0.4%. Presenting a high stability and an improved hydrophilicity, [ 18 F]fluoro-C-glycoc(RDGfC) could become a valuable tool in PET imaging for cancer diagnosis. Furthermore, the described automated procedure is modular and could be adapted to other prosthetic groups and/or peptides with minimal changes to afford a large panel of 18 F-radiolabeled peptides.

Introduction

Positron Emission Tomography (PET) is the most sensitive imaging modality providing a specific view of biological processes and allowing an early diagnosis of various diseases. 1,2 Utilization of PET for molecular imaging relies on the availability of suitable radiotracers.

PET chemistry has therefore evolved to simplify complex chemical and radiochemical processes to aid in the development of new PET radiotracers Peptides are attracting increasing interest because of their in vivo therapeutic efficacy due to their favorable properties, which include high target specificity with low toxicity but also biocompatibility and biodegradability. [3][4][5] This has prompted researchers to develop radiolabeled peptides for PET. [6][7][8] For example, cyclic arginine-glycine-aspartic acid(RGD)-containing peptides have been the focus of intense investigations toward the development of PET radiotracers in cancer diagnosis because they bind with high specificity and affinity to α v β 3 integrin. [9][10][11][12][13][14][15] Several labeled cyclic RGD peptides have been investigated and some of them are nowadays employed in clinical use for PET imaging of angiogenesis in cancer. 11,12,[15][16][17][18] Fluorine-18 ( 18 F) is a commonly used radioelement for peptide radiolabeling because of its ease of production, low positron energy, high specific activity and appropriate half-life (t 1/2 = 109.8 min) compatible with multistep synthesis. 19,20 However, 18 F-radiolabeling of biomolecules, i.e. peptides, remains a challenge because its incorporation into organic compounds often requires basic conditions and high temperatures, incompatible with their sensitivity. Direct 18 F-fluorination of peptides in mild conditions is reported in few examples. [21][22][23][24][25][26] The most commonly used strategy for 18 F-fluorination of peptides adopt an indirect approach 27,28 involving a prosthetic group. In this strategy, a small molecule is easily radiolabeled by [ 18 F]fluoride and then conjugated in mild conditions with biomolecules of interest. Copper(I) catalyzed azide-alkyne cycloaddition (CuAAC), a click version of the Huisgen cycloaddition, 27,[29][START_REF] Chapleur | Click Chemistry in Glycoscience: New developments and strategies -Toward imaging glycotools by click coupling[END_REF][START_REF] Kettenbach | [END_REF][32] is widely applied in this approach. The production of 18 F-labeled peptides requires a multistep radiosynthesis developed by expert radiochemists.

A fully automated radiochemical sequence will enhance reproducibility and reliability, promoting [ 18 F]labeled peptide translation from pre-clinical to clinical studies. Automation is thus a crucial step in radiochemistry development to produce the final radiotracer with minimal human intervention. However, due to its complexity, fully or almost fully automated 18 F-labeled-peptide radiosynthesis via a prosthetic approach was only scarcely described. [33][34][35][36][37] Most of these examples have required some form of manual handling, for reformulation or final purification particularly. Indeed, multistep radiosyntheses are not easily automatable and only few synthesizers have the versatility to implement such sequence. Among the commercially available synthesizers, the AllInOne (AIO) module from Trasis ® could easily implement a large panel of radiochemical sequences thanks to two heaters and built-in HPLC system with two HPLC column positions.

Herein, we present a challenging fully automated radiosynthesis of [ 18 F]fluoro-C-glycoc(RDGfC) on an AllInOne synthesizer. A [ 18 F]fluoro-C-glycoside based prosthetic group was used for indirect peptide radiofluorination. It is worthy to note that introduction of glycoside on biomolecules i.e. peptides has often been shown to enhance their pharmacokinetic and in vivo clearance properties, [38][39][START_REF] Stromgaard | Pharmaceutical Formulation Development of Peptides and Proteins (2nd Edition)-Peptide and protein derivatives[END_REF] notably in the case of RGD derivatives. 15,[START_REF] Maschauer | [END_REF][42][43] This is of major interest, in particular for molecular imaging. The subsequent ligation by CuAAC of This multistep radiosynthesis exploits for the first time, to the best of our knowledge, all the abilities of the AIO module.

Experimental

Materials and methods

Solvents and liquid reagents were purified and dried according to recommended procedures.

Thin layer chromatography (TLC) analyses were performed using standard procedures on Kieselgel 60F254 plates (Merck). Compounds were visualized using UV light (254 nm) and a methanolic solution of sulfuric acid and charred. Column chromatography was performed on silica gel SI 60 (63-200 µm) (Merck). Purification of glycopeptide was achieved by gel filtration on LH20 using water as eluent. Melting points were determined with a Tottoli apparatus and are uncorrected. FTIR spectra were recorded on a Shimadzu IRAffinity-1, ATR PIKE Technologies model GladiAT (cm -1 ). Optical rotations were measured on an Anton-Paar MCP 300 polarimeter. Analytical High performance liquid chromatography (HPLC) analyses were run on a Waters system (2695eb pump, auto sampler injector, 2998 PDA detector and NaI detector from Berthold [Bad Wildbad, Germany]) controlled by the Empower Software (Orlando, FL, USA). Analyses were performed on a Vydac 218TP C18 (5 μm, 250 × 4.6 mm) from Grace with ACN/H 2 O/TFA mixture (proportions given in brackets) at 1 mL/min. Integrated HPLC system on AllInOne synthesizer was equipped with a radioactivity detector, Smartline UV detector 200 (Knauer, Berlin, Germany) at 254 nm and a HPLC pump P4.1.5 (Knauer, Berlin, Germany). Semi-preparative HPLC were performed on an Alltima C8 endcapped (5µm, 150×10 mm) from Grace (HPLC n°1) and on a Vydac 218TP C18 (5 µm, 250x10mm) from Grace (HPLC n°2) with ACN/H 2 O or ACN/H 2 O/TFA mixture (proportions given in brackets) at 4.5 or 3 mL/min respectively. 1 H, 13 C, and 19 F NMR spectra were recorded on a Bruker spectrometer DPX250 (250 MHz, 62.9 MHz and 235 MHz, respectively) or Avance III 400 (400 MHz and 100.6 MHz, respectively). For complete assignment of 1 H and 13 C signals, two-dimensional 1 H, 1 H COSY and 1 H, 13 C correlation spectra were recorded. Chemical shifts (δ) are given in parts per million relative to the solvent residual peak. For clarity, atom numbering starts from the end of the anomeric arm rather than resulting from the IUPAC naming of compounds (cf SI, fig S1). The following abbreviations are used for multiplicity of NMR signals: s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, b = broad signal and app = apparent multiplicity. The given J values refer to apparent multiplicities and do not represent the true coupling constants. Mass and high resolution mass spectrometry spectra were recorded on a Bruker microTOFQ apparatus and MALDI spectra on a Bruker Ultraflex apparatus. 0.9% Sodium chloride (NaCl) solution for injections was purchased from Fresenius Kabi ® , sterile water for injections (WFI) Macoflex N from Macopharma ® and sterile product vials from Cis-Bio international ® (Tec-Elu-5). 18 Oenriched water was supplied by CortecNet ® , Sep-Pak ® QMA-carbonate, silica and Oasis HLB from Waters Corporation ® and BD luer lock syringes from Dutscher ® . All the reagents and materials were used as received. No-carrier-added fluoride-18 was produced via the 18 O (p,n) 18 F nuclear reaction on a PET Trace cyclotron (GE). The bombardment was performed at 10 µA during 5 minutes to provide about 5 GBq of fluoride-18 delivered as a solution in 18 Oenriched water (1.6 mL). Radiosynthesis was carried out on an AIO module from Trasis ® .

Radiolabeling precursor and non radioactive prosthetic group reference syntheses

Synthesizer

The AllInOne synthesis module is a very versatile synthesizer equipped with two arrays of eighteen rotary actuators having multiple positions, two heaters and seven radiation detectors.

A built-in HPLC system with two HPLC column positions is integrated with UV detector and radioactivity detector. AIO operates with single use cassettes (structured with three-way valve manifolds) and reagent sets, facilitating GMP compliance and preventing crosscontamination. The parts available in bulk allow the assembly of these cassettes for research purpose on which various components can be plugged: spikes (for reagent vials), SPE cartridges, syringes or tubes. All these materials are compatible with acids, bases and organic solvents and are suitable for medical devices. The user-friendly software reproduces the picture of the cassette layout (Figure 1) and shows in real time all the movements of the machine during the radiotracer production.

Cassette preparation

Before the activity reception, the module performed automatically the internal and the cassette tests. Columns and eluents for the semi-preparative HPLC were installed for the 1 st HPLC (column: Alltima C8, eluent: ACN/H 2 O 55/45 v/v) and for the 2 nd HPLC (column: Vydac 218TP C18, eluent: ACN/H 2 O 17/83 v/v with 0.1% of TFA). The single use cassette was built from scratch with two arrays of three manifolds. Various components were plugged on the cassette as described Figure 1: TPX spikes, SPE cartridges, luer-lock syringes (BD) and silicone tubing equipped with male or female luer lock connector. Crimped glass vials of 50 mL (vials A, J and D), of 4 mL (vials B, C and K) and of 1.2 mL (vials E, F, G, H and I) were plugged on spikes. All materials were single use and were suitable for medical devices. The list of reagents and materials used for the automated procedure are described in Table 1.

Table 1 Preparation details for the radiosynthesis of [ 18 F]6 on the AIO synthesizer.

Position

Reagents or material Quantities

P2

Dry acetonitrile (ACN) / Vial A 10 mL

P3 K 222 /K 2 CO 3 (5.3 mg/1.3 mg) in ACN/H 2 O (8/2 v/v) / Vial B 1 mL P4-P5 Sep-Pak® Light QMA-carbonate 1 P6 Plunger (20 mL) / SA2 1 P8 Precursor 2 / Vial C 5 mg

P9

Syringe of 20 mL / SA3 1

P10-P11

Sep-Pak® silica cartridge 1

P12

Syringe of 10 mL / SA4 1 P13 WFI (Water for injection) Bag / Bag W 1

P15-P16

Cation exchange (Chelex®) cartridge 1 P21 0.9% Sodium chloride bag / Bag N 1

P22

Ethanol/NaCl 0.9% (5/5 v/v) / Vial K 2 mL

P23-P25

Oasis HLB cartridge 1

P27 ACN/H 2 O (55/45 v/v) / Vial D 10 mL P28 Cu(OAc) 2 in H 2 O (3.3 mol) / Vial I 0.3 mL P29 Propargylated c(RGDfC) 5 in H 2 O (0.5 µmol) / Vial H 0.3 mL P30 Sodium ascorbate in H 2 O (6.6 mol) / Vial G 0.3 mL  P31 NaOH 1M / Vial E 0.3 mL P33 HCl 1M / Vial F 0.3 mL P35 H 2 O Dilution Vial / Vial J 10 mL
The precursor 2 (vial C, P8) was diluted with 2 mL of dry ACN (vial A, P2) before activity reception. The module was then ready to receive activity for radiosynthesis.

Radiosynthesis

[ 18 F]6 was synthesized using an original and fully automated in-house reaction sequence described in Table 2.

Table 2 Reaction sequence for [ 18 F]6 radiosynthesis

Results and discussion

Non radioactive syntheses

The synthesis of C-glycoside based prosthetic group, i.e. non-radioactive reference and the corresponding radiolabeling precursor was first investigated. C-glycosidic compounds, thanks to their robust C-C link at pseudo-anomeric position, are resistant to acidic and enzymatic hydrolysis and are thus stable in vivo. 9,44,[START_REF]Levy Strategies towards C-glycosides in The Organic Chemistry of Sugars[END_REF] A C-glucoside derivative in beta configuration was designed with a spacer-arm bearing an azido group, necessary for the CuAAC. A three carbons spacer-arm has shown a good flexibility and steric arrangement in the final C-glyco-c(RGDfC), conferring an affinity in the same range than native c(RGDfC) toward  v  3 integrin. 9 This is of particular importance, because the introduction of prosthetic group on biomolecules can sometimes generate an affinity decrease.

Radiolabeling precursor 2 was easily obtained from 1 by reacting triflic anhydride with pyridine as base in 80% yield (Scheme 1). 9 Triflate was chosen as leaving group for its high reactivity. Triflated compounds could be less stable compared to tosylated compounds for example, but 2 was stable several weeks and even months at -20°C. The synthesis of the nonradioactive reference of C-glycoside based prosthetic group 4 was carried out starting from alcohol 1 in two steps, by fluorination with DAST followed by Zemplén deprotection (Scheme 1). 9

S-propargylation of the cysteine residue of c(RGDfC) was performed according to our described method 29 allowing to work with non protected peptides and regioselectively providing the S-propargyl-peptide derivative 5. Non-radioactive reference fluoro-C-glyco-c(RGDfC) 6 was prepared from 5 and C-glycoside reference 4 via CuAAC in good yield (69%). 9

Scheme 1 Synthesis of radiolabeling precursor 2, and non radioactive references 4 and 6.

Reagents and conditions: i) Tf 2 O, pyridine, -25°C to rt, 25 min, 80%; ii) DAST, diglyme, 110°C, 8 min, 61%; iii) Na°, dry MeOH, 25°C, 1h, quantitative; iv) 5, H 2 O, Cu(OAc) 2 , Na ascorbate, rt, 2h, 69%.

Radiochemistry development and automation

The development of the radiosynthesis of [ 18 F]6 required a multistep radiosynthesis, more precisely a two-pot three-steps synthesis. The AIO equipped with two reactors, two HPLC column positions and multiple possibilities of vial arrangements thanks to two arrays of 18 valves allows such radiosynthesis. Nevertheless, implementation of all the steps of this synthesis on the AIO was challenging and we have exploited, for the first time to the best of our knowledge, all the abilities of this module.

The fully automated process involved a two-pot three-step reaction: radiolabeling of the The different steps were symbolized with different luer lock ring colours on the layout (Figure 1). Vials with red luer lock rings were used for K[ 18 F]F-K 222 preparation, yellow luer lock rings for radiofluorination, green luer lock rings for deprotection, blue luer lock rings for coupling by CuAAC and white luer lock rings for reformulation.

Scheme 2 General strategy for [ 18 F]6 radiosynthesis

The non-carrier added ambitious three-step radiosynthesis of [ 18 F]F-C-glyco-c(RGDfC) started with classical preparation of K[ 18 F]F-K 222 complex. The first step was the 18 Fradiolabeling of precursor 2 in the reactor 1. By taking advantage of our previous work on glycoside based prosthetic groups, 9,10 in order to obtain a high radiolabeling rate and a better repeatability, different parameters such as the amount of precursor, the reaction duration and the temperature were screened upstream. The following conditions were found to be optimal for the 18 F-radiofluorination: 5 mg of precursor in 2 mL of dry acetonitrile and reaction of 5 min at 95°C. Before HPLC purification, a pre-purification of the mixture was performed by trapping unreacted 18 F-fluoride on a silica SPE cartridge. The eluted solution was then purified by semi-preparative HPLC to remove the precursor and potential related impurities.

Developments made with non radioactive derivatives have shown good separation of the radiolabeling precursor 2 (t R ~ 16 min) from the fluorinated acetylated C-glycoside 3 (t R ~ 7.5 min) on a C8 analytical column with acetonitrile/water 55/45 v/v as eluent at a flow rate of 1.0 mL/min. Purification of [ 18 F]fluoro-C-glycoside [ 18 F]3 was performed using these conditions by semi-preparative HPLC at a flow rate of 4.5 mL/min (Figure 2). This HPLC purification step is time consuming but could not be avoided. Indeed, precursor of radiolabeling 2 can not be separated from radiofluorinated compound [ 18 F]3 by simple SPE cartridge purification. This radiosynthesis is the first fully automated production of [ 18 F]radiolabeled peptide via a prosthetic approach in a three-step two-pot method including two HPLC purification steps.

Indeed, to date, automated batch production of [ 18 F]radiolabeled peptides via a prosthetic strategy has been scarcely reported due to its complexity. To our knowledge, the first example was proposed by Thonon et al. 36 using the FASTLab™ GE synthesis module. [ 18 F]SFB was synthesized via a one-or two-pot method and was purified by a tC18 cartridge. The [ 18 F]SFB was then coupled to RGD and the final radiotracer was purified by HPLC. [ 18 F]FPRGD 2 was thus produced in 130 minutes in 13 ± 3% decay corrected yield. However, this production was not fully automated as the RDG addition was manually carried out. Valdivia et al. 34 reported the automated radiosynthesis of As described above, HPLC purification of the prosthetic group could be circumvented by a simple cartridge purification. This technique is very time saving but is not applicable to every type of molecules. Only precursors and corresponding fluorinated compounds with very different polarities or solubilities can be separated by cartridge. In our case, the sugar-based prosthetic group was chosen for its hydrophilicity properties despite the purification inconvenience. Indeed, the triflated precursor 2 and the resulting fluorinated molecule [ 18 F]3

have close solubilities and polarities, explaining the compulsory HPLC purification. One pot fluorination and deprotection were first envisaged, however, the acetate removal lead to inseparable mixture of polyhydroxylated compounds (very close t R ). For this reason, it is mandatory to achieve the first HPLC purification of [ 18 F]3 prior to acetate deprotection.

Therefore, the following reactions (deprotection and CuAAC coupling) were both performed in reactor 2. This three-step synthesis including a deprotection step thus requires a two-pot sequence with two HPLC purifications. Consequently, this complex radiosynthesis ensures a maximal adaptability to a large panel of peptides and prosthetic groups.

Quality control and physico-chemical data

Quality control of [ 18 F]6 was performed on an aliquot of the final batch. The prepared radiotracer solution was clear and colourless with a pH around 7. Chemical and radiochemical purities were determined by analytical HPLC with a C18 column using radioactive and UV detection (Figure 4). UV chromatogram showed no by-products demonstrating good chemical purity. The identity of [ 18 F]6 (t R = 7.8 min) was confirmed by a second injection with the authentic non radioactive standard. The radiochemical purity was determined to be over 98%.

Molar activity can not be determined due to the poor UV absorbance of 6 (detection limit: 2 g/mL), indicating that A m is higher than 20.8 GBq/mol. in the same range than native c(RGDfC). 9 The improved hydrophilicity combined with adequate in vitro stability and high affinity toward  v  3 integrin make [ 18 F]6 suitable in PET imaging for cancer diagnosis.

Conclusion

The radiosynthesis of [ 18 F]fluoro-C-glyco-RGD conjugate has been fully automated on an AllInOne synthesizer via a prosthetic group approach. [ 18 F]fluoro-C-glyco-c(RGDfC) was obtained with an ambitious two-pots three-steps procedure with high radiochemical purity (≥98%) and satisfactory and reliable decay-corrected radiochemical yield of 3.6 ± 0.4%. The radiotracer shows an enhanced hydrophilicity, surely promoted by the C-glycoside prosthetic group, and appears therefore as a promising tool in PET imaging for cancer diagnosis. For the first time, all the abilities of the AIO synthesizer were exploited in this challenging fully automated multistep radiosynthesis. The described automation is modular and its implementation could be easily adapted to other prosthetic groups and/or peptides to afford a large panel of 18 F-radiolabeled peptides. Moreover, this fully automated method without any manual handling should contribute to the development of automation of complex radiosyntheses in the future.

[ 18 F

 18 ]fluoro-C-glycoside to a cyclic RGD-containing peptide generated [ 18 F]fluoro-C-glycoc(RGDfC) which could become an efficient radiotracer in PET imaging for cancer diagnosis.

4, 8 -

 8 anhydro-1-azido-1,2,3-trideoxy-5,6,7-tri-O-acetyl-D-glycero-D-gulo-nonitol (1), 4,8anhydro-1-azido-1,2,3-trideoxy-9-O-trifluoromethylsulfonate-5,6,7-tri-O-acetyl-D-glycero-Dgulo-nonitol (2, radiolabeling precursor), 4,8-anhydro-1-azido-1,2,3,9-tetradeoxy-9-fluoro-5,6,7-tri-O-acetyl-D-glycero-D-gulo-nonitol (3) and 4,8-anhydro-1-azido-1,2,3,9-tetradeoxy-9-fluoro-D-glycero-D-gulo-nonitol (4, non radioactive prosthetic group reference) were synthesized according to our previously reported methods. 9 Glycopeptide synthesis S-propargyl-c(RGDfC) 5 and cyclo[L-arginylglycyl-L--aspartyl-D-phenylalanyl-S-[[1-[1-(4,8-anhydro-5-fluoro-5,1,2,3-trideoxy-D-glycero-D-gulo-nonitol)]-1H-1,2,3-triazol-4-yl]methyl]-L-cysteinyl] 6 were synthesized according to our previously reported methods.9,29

Fig. 1

 1 Fig. 1 Schematic representation of the AIO module used for the preparation of [ 18 F]6.

Fluorination of prosthetic group precursor 2 1 -[ 18 F 6 1- 6 1- 18 F] 4 . 18 F] 4 . 18 F] 6 .[ 18 F] 4 ([ 18 F] 6 Approximately 5 . 6 In vitro stability in serum An aliquot of [ 18 F] 6 ( 5 . 5

 218661841841861841865618655 ]F -trapping on QMA carbonate cartridge 2 -[ 18 F]F -elution with eluent 3 -Azeotropic evaporation at 110°C for 10 min to give dry K[ 18 F]F-K 222 complex 4 -Addition of precursor 2 into the reactor 1 5 -[18 F]Radiofluorination at 95°C for 5 min 6 -Pre-purification on silica cartridge Purification of [ 18 F]3 on 1 st semi-preparative HPLC 1 -Injection of crude product on semi-preparative HPLC 2 -Collection of [ 18 F]3 into the reactor 2 Preparation of 18 F-fluoro-C-glycoside based prosthetic group [ 18 F]4 1 -Evaporation of mobile phase 2 -Saponification with 1M NaOH at 30°C for 9 min 3 -Neutralization with 1M HCl Preparation of the [ 18 F]F-C-glyco-c(RGDfC) [ 18 F]Addition of Na-ascorbate 2 -Addition of propargylated peptide 5 3 -Addition of Cu(OAc) 2 4 -Coupling at 55°C for 25 min Purification of the glycopeptide [ 18 F]Injection of crude product on 2 nd semi-preparative HPLC 2 -Collection of [ 18 F]6 into the dilution vial Formulation of [ 18 F]6 1 -Trapping of [ 18 F]6 on Oasis HLB cartridge 2 -Washing of the cartridge with WFI 3 -Elution with ethanol/NaCl 0.9% mixture then with NaCl 0.9% 4 -Recovering of [ 18 F]6 into the final product vial Preparation of K[ 18 F]F-K 222 complex. 18 F-fluoride (~ 5 GBq) in [ 18 O]water was recovered in the AIO synthesizer via the plunger (SA2, P6, vacuum -200 mbar) by suction and passed through a Sep-Pak ® light QMA-carbonate cartridge (P5-P4), where [ 18 F]fluoride was trapped and [ 18 O]water was collected for recycling (P1). The QMA-carbonate cartridge was then flushed with nitrogen gas flow during 40 seconds by combination of positive and negativepressure (pressure 400 mbar; vacuum -300 mbar). The trapped18 F-fluoride was eluted by suction from the QMA-carbonate cartridge into the reactor 1 (P7, vacuum -350 mbar) with the K 222 /K 2 CO 3 solution (vial B, P3, previously pressurized at 500 mbar). Solvent was removed under a stream of nitrogen gas flow (combination of pressure 15 to 500 mbar and vacuum -850 mbar) at 110°C during 10 minutes to give the dried K[ 18 F]F-K 222 complex. The reactor was then cooled at 75°C to perform the 18 F-radiolabeling.18 F-radiolabeling of the labeling precursor 2. The triflated precursor 2 (Vial C, P8) was pressurized (pressure 500 mbar) and was sucked into the reactor 1(P7, vacuum -800 mbar) containing the dried K[ 18 F]F-K 222 complex. Labeling was performed at 95°C for 5 min in a closed reactor thanks to pinch valves. After cooling at 30°C, the reaction mixture was passed through a Sep-pak ® plus silica 690 mg cartridge (P10-P11), to remove unreacted18 F-fluoride, and then recovered into SA4 (P12) containing 1.5 mL of WFI (Bag W). The reactor 1 and silica cartridge were rinsed with 1 mL of dry acetonitrile (Vial A, P2) taken with syringe 3 (P9). The eluted solution was recovered into syringe 4 (P12). HPLC loop was preconditioned with 8 mL of HPLC eluent (vial D, P27) via syringe 3 (P9). Crude product in SA4 was transferred into the injection loop of the semi-preparative HPLC and was purified on the first HPLC column (Alltima C8, solvent: ACN/H 2 O 55/45, 4.5 mL/min). [ 18 F]3 was collected (t R ~ 7.5 min) during approximately 40 seconds and recovered into the reactor 2 (P32-P34). Preparation of 18 F-fluoro-C-glycoside based prosthetic group [ HPLC solvent (~3 mL, reactor 2) was evaporated first gently by heating 15 min at 80°C under low pressure (vacuum -300 to -1000 mbar), and then under nitrogen gas flow during 1 minute by combination of positive and negative pressure (pressure 250 mbar; vacuum -500 mbar). After complete evaporation and cooling at 30°C, saponification of the 18 F-glycoside [ 18 F]3 was performed for 9 min at 30°C with an aqueous NaOH 1M solution (vial E, P31) added by suction (vacuum -500 mbar) after pressurization of the vial (pressure 500 mbar). Aqueous HCl 1M solution (vial F, P33) was then added by the same way to neutralize the solution containing [ 18 F]fluoro-C-glycoside based prosthetic group [ Preparation of the [ 18 F]F-C-glyco-c(RGDfC) [ To [ 18 F]fluoro prosthetic group reactor 2, ~850 MBq) were added by suction (vacuum -500 mbar), after pressurization of each vial (pressure 500 mbar), sodium ascorbate (vial G, P30), propargylated peptide 5 (vial H, P29) and Cu(OAc) 2 (vial I, P28). The solution was heated at 55°C during 25 min under a gentle nitrogen gaz flow (pressure 150 mbar; vacuum -500 mbar). No solvent evaporation occurred at this step. The elution lines and SA4 werewashed with WFI (10 mL, Bag W). The reaction mixture was then diluted with 3 mL of WFI (Bag W, taken with SA4), the solution was passed through a cartridge pre-charged with Chelex ® 100 resin (V15-V16) to remove copper, and was recovered in syringe 4 (P12). Crude product in SA4 was transferred into the injection loop of the semi-preparative HPLC and was purified on the 2 nd HPLC column (Vydac 218TP C18, solvent: ACN/H 2 O 17/83 v/v with 0.1% of TFA, 3 mL/min). The collected peak of [ 18 F]6 (t R ~ 15.0 min) was recovered into the dilution vial containing 10 mL of WFI (vial J, P35). To reformulate [ 18 F]6, the pressurized mixture (pressure 500 mbar) was trapped on an Oasis HLB cartridge (P25-P23, vacuum -100 mbar) by suction. The cartridge was flushed with nitrogen gas flow during 10 s by combination of positive and negative pressure (pressure 500 mbar; vacuum -300 mbar) and then washed with 7.5 mL of WFI (Bag W, taken with SA4). The product was eluted from the Oasis HLB cartridge with 0.7 mL of ethanol/NaCl 0.9% 50/50 v/v solution (vial K, P22) taken by SA4 (P12) and recovered into final product vial (P26). The cartridge was rinsed with 1,8 mL of NaCl 0.9% (Bag N, taken with SA4) pushed into final product vial (P26), and then flushed by nitrogen gas flow (pressure 500 mbar) during 10 seconds. was produced with a decay corrected (d.c.) radiochemical yield of 3.6 ± 0.4% (n = 12).Quality controlAn aliquot of final product [18 F]fluoro-C-glyco-c(RGDfC) [ 18 F]6 was injected on analytical HPLC (20 µL) for chemical and radiochemical purities determination. A second injection with the corresponding reference compound 6 confirmed the identity of [ 18 F]6. A Vydac 218TP C18 column was used in isocratic conditions; eluent: ACN/H 2 O 17/83 v/v with 0.1% of TFA, flow: 1.0 mL/min and UV detection (230 nm). Molar activity (A m ) was checked on analytical HPLC using a standard curve relating mass to UV absorbance. Radiochemical stabilities of the entire preparation of [ 18 F]6 were tested by HPLC at regular intervals during 120 min. The pH of the solution was determined by applying a drop onto a pH indicator strip and comparing the result with the provided scale. MBq of [ 18 F]6 was diluted in 2 mL of phosphate-buffered saline (pH 7.4, PBS). Five aliquots from this sample (50 L) were distributed into five separate Eppendorf tubes containing 500 L of 1-octanol and 450 L of PBS. Samples were vortexed for 1 min at room temperature and then centrifuged for 5 minutes at 4000 rpm. From each sample, 100 μL samples of both the organic and the PBS layer were transferred into plastic tubes, and the samples were counted with a gamma counter (2470 WIZARD ® , PerkinElmer). MBq each) was added to human serum (200 L, 5 samples) and incubated at 37 °C. Samples were collected at various interval times (0, 10, 30, 60 and 120 min) and precipitated using acetonitrile (200 μL). The samples were centrifuged at 4000 rpm for 5 min, and the supernatant layer was then analyzed by radio-HPLC (column: Vydac 218TP C18 eluted with ACN/H 2 O 17/83 v/v with 0.1% of TFA at 1.0 mL/min). The experiments were performed in triplicate.

precursor 2 , 18 F] 6 (

 2186 deprotection to afford the prosthetic group [ 18 F]4 and coupling with the cyclic RGD-containing peptide to give [ 18 F]fluoro-C-glyco-c(RGDfC) [ Scheme 2). This strategy involved two HPLC purification procedures and a formulation step.

Fig. 2 5 .

 25 Fig. 2 Semi-preparative HPLC purification of [ 18 F]3. (t R ~ 7.5 min, Alltima C8, ACN/H 2 O 55/45 v/v, 4.5 mL/min). Radioactivity (top) and UV (254 nm, bottom) traces are shown.

Fig. 3

 3 Fig. 3 Semi-preparative HPLC purification of [ 18 F]6 (t R ~ 15.0 min, Vydac 218TP C18, ACN/H 2 O 17/83 v/v with 0.1% of TFA, 3 mL/min). Radioactivity (top) and UV (254 nm, bottom) traces are shown.

[ 18 F

 18 ]FPyKYNE-c(RGDyK) performed in a dual reactor TRACERlab FX N Pro, (GE Healthcare). After semi-preparative HPLC, the radiotracer was manually reformulated. [ 18 F]FPyKYNE-c(RGDyK) was obtained via this semi-automated method in 12-18% decay corrected radiochemical yield in 125 minutes. More recently, Haskali et al.33 described the fully automated radiosyntheses (including two HPLC purifications and reformulation) of [ 18 F]FP-GalactoRGD, [ 18 F]FP-PRGD 2 and the sulfonated versions of these peptides. The 2-[ 18 F]fluoropropionate labeled RGD-peptides were obtained via a two-pot method in 90 to 105 minutes with non-decay corrected radiochemical yields of 4 to 7%. These radiosyntheses, including the two HPLC purifications (performed on the iPHASE Flexlab fix-part synthesizer) were fully automated, similarly to our sequence performed on the AIO. It is noteworthy that the AIO operates with single use cassettes, facilitating GMP compliance and preventing cross-contamination. Davis at al.35 reported a two-pot automated production of three radiolabeled peptides (YGGFL, cRGDyK, and Pyr-QKLGNQWAVGHLM) via coupling of 6-[ 18 F]fluoronicotinyl-2,3,5,6tetrafluorophenyl ester ([ 18 F]FPy-TFP) as prosthetic group. [ 18 F]FPy-TFP was purified with an Oasis MCX-Plus cartridge and the final purification of 18 F-radiolabeled peptides was not automated. The ELIXYS FLEX/CHEM® module allowed the semi-automated preparation of [ 18 F]FPy-peptides in 13-26% decay corrected radiochemical yields in 97 ± 3 minutes. Allott et al.37 proposed an improved fully automated radiosynthesis of [ 18 F]FET-βAG-TOCA using the GE FASTlab™ platform with a non-decay corrected radiochemical yield of 16.7 ± 0.6%. This elegant one-pot procedure cleverly uses SPE cartridges for purification of prosthetic group (2-[ 18 F]fluoroethylazide) and also for CuAAC coupling (HLB SPE cartridge). HPLC purification with biocompatible mobile phase enabled the bypass of reformulation step but was not yet optimal in terms of purity and A m .

Fig. 4 .

 4 Fig. 4. Analytical HPLC profile of [ 18 F]6. Retention time of [ 18 F]6: 7.8 min; radiochemical purity of [ 18 F]6 was ≥ 98%. Conditions: Vydac 218TP C18 column eluted with ACN/H 2 O

Acknowledgments

This work was supported by Nancyclotep. We acknowledge The Ministère de l'Enseignement Supérieur et de la Recherche for PhD funding to T. Vucko. We thank R. Filosa for technical assistance.

Conflict of Interest

There are no conflicts to declare.


Thomas Fix
email: tfix@unistra.fr

Guy Schmerber

Jean Luc Rehspringer

Mircea V Rastei

Stéphane Roques

Jeremy Bartringer

Abdelilah Slaoui

J.-L Rehspringer

Insights on hexagonal TbMnO3 for optoelectronic applications: From powders to thin films

Keywords: thin films, oxide materials, photoconductivity and photovoltaics

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

Introduction

Ferroelectric photovoltaics (FPV) are different from conventional pn junction solar cells because the photogenerated electron hole pairs are separated by the ferroelectric polarization originating from the polar noncentrosymmetry of the structure. This can lead to open circuit voltage values (Voc) much higher than the bandgap of the materials [START_REF] Yang | Above-bandgap voltages from ferroelectric photovoltaic devices[END_REF] and to the possibility of switching the direction of the photocurrent by applying an electric field pulse to the device [START_REF] Nechache | Bandgap tuning of multiferroic oxide solar cells[END_REF]. The main limitation of ferroelectric photovoltaics has been the high bandgap of such ferroelectric materials. A prototypical example is BiFeO3 with a bandgap up to 2.7 eV [START_REF] Alexe | Tip-enhanced photovoltaic effects in bismuth ferrite[END_REF][START_REF] Belhadi | Conduction mechanism and switchable photovoltaic effect in (111) oriented BiFe0.95Mn0.05O3 thin film[END_REF]. A solution was provided by considering a double perovskite Bi2FeCrO6 (BFCO) in which the bandgap could be reduced from 2.6 to 1.9 eV [START_REF] Nechache | Bandgap tuning of multiferroic oxide solar cells[END_REF][START_REF] Quattropani | Band-Gap Tuning in Ferroelectric Bi2FeCrO6 Double Perovskite Thin Films[END_REF][START_REF] Quattropani | Tuning photovoltaic response in Bi2FeCrO6 films by ferroelectric poling[END_REF] simply by controlling the Fe/Cr order. Along the same line, other materials are being investigated such a KBiFe2O5 (with a bandgap ranging between 1.6-2.3 eV) [START_REF] Fix | Investigation of KBiFe2O5 as a Photovoltaic Absorber[END_REF]. Still, the efficiency record of FPV solar cells remains with BFCO with a power conversion efficiency of 8.1 % [START_REF] Nechache | Bandgap tuning of multiferroic oxide solar cells[END_REF]. This value corresponds to the efficiency record of oxide based solar cells, shared with Cu2O [START_REF] Fix | Oxide and Ferroelectric Solar Cells[END_REF][START_REF] Pérez-Tomás | Metal Oxides in Photovoltaics: All-Oxide, Ferroic, and Perovskite Solar Cells[END_REF].

In a previous study by Han et al., hexagonal manganite thin films were proposed as a ferroelectric solar cell absorber, namely h-LuMnO3 and h-YMnO3 [START_REF] Han | Switchable Photovoltaic Effects in Hexagonal Manganite Thin Films Having Narrow Band Gaps[END_REF]. It was found that the photocurrent was reversibly switchable, with an open circuit voltage Voc up to 0.71 V and a short circuit current density Jsc up to 0.55 mA/cm², leading to a maximum conversion efficiency of around 0.1 % [START_REF] Han | Switchable Photovoltaic Effects in Hexagonal Manganite Thin Films Having Narrow Band Gaps[END_REF]. The latter value was two orders of magnitude higher than a similar study with h-TmFeO3 and h-LuFeO3 absorbers [START_REF] Han | Switchable ferroelectric photovoltaic effects in epitaxial h-RFeO3 thin films[END_REF].

In this work, inspired by a theoretical article by Y. Tsymbal et al. [START_REF] Huang | Hexagonal rare-earth manganites as promising photovoltaics and light polarizers[END_REF], we focus on hexagonal rare-earth manganites (h-RMnO3 where R is a rare earth ion), and in particular on h-TbMnO3 (h-TMO). These materials have received a lot of interest due to their improper structural ferroelectricity and multiferroic properties, that differ from the orthorhombic TMO (o-TMO) phase. o-TMO is indeed a spin-driven ferroelectric perovskite [START_REF] Li | Hexagonal rare-earth manganites and ferrites: a review of improper ferroelectricity, magnetoelectric coupling, and unusual domain walls[END_REF][START_REF] Kimura | Magnetic control of ferroelectric polarization[END_REF][START_REF] Shimamoto | Tuning the multiferroic mechanisms of TbMnO3 by epitaxial strain[END_REF][START_REF] Glavic | High quality TbMnO3 films deposited on YAlO3[END_REF] and it was proposed as an ultraviolet photodetector [START_REF] Jin | Favorable ultraviolet photoelectric effects in TbMnO3/Nb-SrTiO3 heterostructures[END_REF]. In h-TMO ferroelectricity originates from the rotation and tilt of the MnO5 polyhedra [START_REF] Kim | Room-Temperature Ferroelectricity in Hexagonal TbMnO3 Thin Films[END_REF]. Orthorhombic rare-earth manganites such as o-TMO display ferroelectric ordering at low temperature, below 100 K (41 K for o-TMO films) [START_REF] Shimamoto | Tuning the multiferroic mechanisms of TbMnO3 by epitaxial strain[END_REF]. The orthorhombic phases are generally obtained in the bulk when the rareearth radius is large (R = La, Pr, Nd, Sm, Eu, Gd, Tb, Dy). In contrast, for smaller radius (R = Y, Ho, Er, Yb, Lu) the hexagonal structure is obtained and ferroelectricity can reach 600-1000 K [START_REF] Kim | Room-Temperature Ferroelectricity in Hexagonal TbMnO3 Thin Films[END_REF]. However the h-TMO (R = Tb) phase can be grown even if unfavourable thanks to the concept of epitaxial stabilisation using appropriate substrate such as yttria-stabilized zirconia (YSZ) (111) [START_REF] Mandal | Strain-driven structure-ferroelectricity relationship in hexagonal TbMnO3 films[END_REF] or buffer layers such as Pt(111)//Al2O3 (0001) [START_REF] Kim | Room-Temperature Ferroelectricity in Hexagonal TbMnO3 Thin Films[END_REF][START_REF] Lee | Formation of hexagonal phase of TbMnO3 thin film and its multiferroic properties[END_REF]. The growth of this material is complex due to the fact that the orthorhombic phase of TMO is more favourable and generally obtained. However the h-TMO material has been shown with a large remnant ferroelectric polarization (8 µC/cm²) at room temperature, making it a potential candidate for FPV [START_REF] Kim | Room-Temperature Ferroelectricity in Hexagonal TbMnO3 Thin Films[END_REF]. In addition, h-TMO offers a direct bandgap of about 1.4 eV [START_REF] Huang | Hexagonal rare-earth manganites as promising photovoltaics and light polarizers[END_REF], which is almost the ideal value for a single bandgap photovoltaic absorber, together with an absorption coefficient (for polarization perpendicular to the c direction) almost two times larger than CdTe in the visible [START_REF] Huang | Hexagonal rare-earth manganites as promising photovoltaics and light polarizers[END_REF]. For a h-TMO thickness of 0.5 µm, the maximum photovoltaic energy conversion efficiency was calculated to be 32 %, compared to 27 % for CdTe and 2 % for BaTiO3 [START_REF] Huang | Hexagonal rare-earth manganites as promising photovoltaics and light polarizers[END_REF]. These predictions are very promising and the aim of the current work is to confront these calculations to experiments.

In the present work we first provide a simple guide on the deposition of h-TMO by pulsed laser deposition (PLD). A systematic investigation of doping effects of TMO powders and films by different elements such as In, Lu, Gd, Sm, Nd, Y, is presented. We also describe the potential of h-TMO as a photovoltaic absorber, by demonstrating significant values of surface photovoltage effects.

Experimental procedure

The TMO and doped TMO powders were prepared as follows. For pure TMO, first Tb2O3 powders (8.070 g) were poured in nitric acid (~50 ml) and heated, while stirring with a magnetic bar until a colourless solution was obtained. Then, manganese (II) acetate tetrahydrate (10.812 g) was poured in deionized water (~100 ml). The two solutions were poured into one beaker. Citric acid (15.4 g) is dissolved in the solution together with ethylene glycol (5 g, using the Pecchini method). The excess of water is evaporated and a viscous liquid without precipitate appears. This compound is pre-calcinated in a furnace at 350 °C for two hours, mechanically ground in a mortar, and further calcinated at 750 °C for 1 h in air. The obtained powder is ground again, pressed into a pellet and sintered at 850°C for 1 h in air. For the doped TMO powders, the dopants were introduced in the required quantities in the manganese (II) acetate tetrahydrate solution in the form of lutetium (III) nitrate hydrate, gadolinium (III) nitrate hexahydrate, samarium (III) nitrate hexahydrate, neodymium (III) nitrate hexahydrate, and yttrium (III) nitrate hexahydrate, respectively. For indium doping, the required In metal quantity was poured in nitric acid and heated at 60 °C for one hour until disappearance of the metal. The solution was then poured into the solution of the manganese (II) acetate tetrahydrate. The pictures of the powders are shown in the supplementary information (SI, Figure S1).

The substrates used for deposition were Al2O3 (0001) one side polished (ALO) (insulating, thickness 0.5 mm). A 4-6 nm thick Pt layer was deposited at 450 °C in high vacuum on ALO (0001). The PLD process was performed using a KrF laser (248 nm), for the Pt target with a 10 Hz repetition rate and a laser fluence of around 5-6 J/cm² ; for the TMO targets with a 4-10 Hz repetition rate and a laser fluence of around 1-2 J/cm 2 . The deposition of h-TMO was carried out at 750 °C under an oxygen pressure of 10 -2 mbar. h-TMO film thicknesses were in the 40-52 nm range. The as-grown films were cooled down under the same atmosphere at a rate of 10°C/min until 300°C, below this temperature it was naturally cooled down until room temperature. Around 30 samples were deposited for the study. The pictures of the films are shown in the supplementary information (SI, Figure S1).For the fabrication of solar cells, a mask was placed on the h-TMO films with openings of 1 mm². Indium tin oxide (ITO) was deposited by PLD through the mask at 250 °C in 10 -2 mbar O2. Doped TMO powders were investigated with a Bruker-AXS D8 Advance X-ray diffractometer with Cu Kα1 radiation (40 kV, 40 mA, 0.154056 nm). Lattice parameters of the powders were refined using the FullProf Suite. The crystalline structure of the TMO films was analyzed by X-ray diffraction using a Rigaku SmartLab diffractometer equipped with a monochromatic source delivering a Cu Kα1 incident beam (45 kV, 200 mA, 0.154056 nm). X-ray θ-2θ and reflectivity were performed in order to look for the film structure, check the presence of spurious phases, and determine the Pt film thickness (see SI Figure S2). Scanning electron microscopy (SEM) was performed with a Gemini 500-ZEISS apparatus and the stoichiometry was checked by Energy-dispersive X-ray spectroscopy (EDS) (see SI Figure S3). The optical properties of the undoped and doped h-TMO films were investigated by spectroscopic ellipsometry using a HORIBA Uvisel Lt M200 FGMS (210-880 nm) apparatus. An anisotropic uniaxial layer was inserted on top of the Pt//Al2O3 structure to simulate the undoped and doped h-TMO films.

The birefringence of the films had to be considered in the models. The dispersion formula for both the ordinary (o) and extraordinary (e) components were based on the Adachi-New Forouhi model [START_REF] Yoshikawa | Optical constants of ZnO[END_REF]. The goodness-of-fit values (Chi Square χ²) were always better than 0.25. The spectroscopic ellipsometry analysis was also used to determine the TMO films thicknesses (see SI Figure S4). The ferroelectric properties were investigated by the Piezoresponse Force Microscopy (PFM) technique using an atomic force microscope (AFM). The tip was a 0.01-0.025 Ω•cm antimony n-doped Si covered with conductive diamond. A 1000-4000 mV ac voltage was applied to the sample at 40 kHz using the VPFM mode.

Surface photovoltage (SPV) measurements were performed with a halogen source with calibrated illumination with a single point Kelvin Probe system by KP Technology (KP020). The tip provided was in Au with a 2 mm diameter. For photovoltaic measurements, current-voltage (I-V) characteristics were carried out at room temperature using an Oriel VeraSol-2 AAA Solar Simulator in AM1.5G conditions and a Keithley 2461 SourceMeter. Contacts were made either directly with a tungsten tip on TMO or by depositing 1 mm² indium tin oxide (ITO) contacts.

Results and discussion

Because h-TMO films were found difficult to grow as the hexagonal phase is not the most favourable, we studied doping of TMO powders with different elements (M = In, Lu, Gd, Sm, Nd, Y) in order to stabilise the hexagonal phase against the orthorhombic one. The different compositions of MxTb1-xMnO3 prepared are summarised in Table 1. For In, Lu and Y a value of x=0.20 was kept for comparison between the dopants. For Gd, Sm, Nd, because of the large size of the atoms, x was kept at 0.10. Figure 1 shows the XRD pattern of the different powders. It can be seen that for all the compositions shown, the characteristic peaks of o-TMO were preserved. In some cases, a parasitic phase of TbMn2O5 is observed in addition to the main o-TMO phase. For In0.40Tb0.60MnO3 and Lu0.40Tb0.60MnO3, the diffractogram did not match either o-TMO or h-TMO (see SI Figure S5). For all the other compositions, no sign of h-TMO could be detected but only o-TMO. Table 1 shows the lattice parameters extracted from Rietveld refinements, the volume V of the unit cell and the average atomic radius calculated as V 1/3 . The ion-oxygen distance is indicated for each dopant element [22,23]. First it can be seen that going from pure TMO to In0.15Tb0.85MnO3 and In0.20Tb0.80MnO3 the volume or average atomic radius is gradually decreasing. The effect looks much less pronounced with Lu, Gd, Sm, Y doping as V 1/3 stays in the 6.1 Å range. Nd doping shows a reduction of V and V 1/3 even more pronounced than for In in In0.15Tb0.85MnO3. These results suggest than In and Nd are inserted in the TMO matrix while Lu, Gd, Sm, Y have low influence on the cell size. Two of the dopants were thus selected for fabrication of doped TMO targets, namely In, because of its large effect on V, and Y, because YMnO3 is easily synthesized in the hexagonal phase [START_REF] Kim | Room-Temperature Ferroelectricity in Hexagonal TbMnO3 Thin Films[END_REF] and is a known ferroelectric material [START_REF] Pang | Preparation of epitaxial hexagonal YMnO3 thin films and observation of ferroelectric vortex domains[END_REF]. Three aspects must be investigated with In-TMO and Y-TMO films, first the impact of the dopants on the formation of the hexagonal TMO phase, second the impact on the ferroelectric properties of the films and third the impact on the optical and photovoltaic properties.

Films of TbMnO3 (TMO), In0.20Tb0.80MnO3 (In-TMO), Y0.20Tb0.80MnO3 (Y-TMO) and YMnO3 (YMO) were then grown by PLD on Pt//ALO (0001). Pt is grown epitaxially by PLD and plays two key roles. The first one is that it is used as a conductive electrode for further ferroelectric and photovoltaic measurements. The second is that as shown in [START_REF] Kim | Room-Temperature Ferroelectricity in Hexagonal TbMnO3 Thin Films[END_REF][START_REF] Lee | Formation of hexagonal phase of TbMnO3 thin film and its multiferroic properties[END_REF][START_REF] Lee | Epitaxial stabilization of a new multiferroic hexagonal phase of TbMnO3 thin films[END_REF] the in-plane symmetry of the Pt layer is rotated by 30° in respect to the ALO (0001) substrate, leading to a Pt layer with hexagonal structure that is well adapted to h-TMO. Figure 2a shows the XRD pattern of the films grown at 10 Hz laser repetition rate and 2 J/cm² fluence. It can be seen that for all the films, Pt is grown epitaxially as demonstrated by the presence of the Pt (111) and (222) reflections. As opposed to o-TMO, h-TMO presents a distinct (0002) peak at around 2θ=15.4° [START_REF] Lee | Formation of hexagonal phase of TbMnO3 thin film and its multiferroic properties[END_REF]. It can be seen that the YMnO3 is present only in the hexagonal phase, while for pure TMO and In-TMO both hexagonal and orthorhombic phases can be observed. Y-TMO does not show any distinct peak for the TMO phases. TMO films were grown at higher temperature (900 °C) at 10 Hz repetition rate and no improvement was observed.

Figure 2b shows the XRD pattern of films grown this time with 4 Hz repetition rate and 1 J/cm² fluence.

Here, for pure TMO, In-TMO and Y-TMO only the hexagonal phase can be observed. This shows that by reducing the laser repetition rate and the laser fluence, thereby giving more time for growth kinetics after nucleation, the hexagonal phase can be obtained. The epitaxial relationship of the films is

[1000]TMO(0001) / [2 -1 -1]Pt(111) // [2 1 -3 0]ALO(0001) as reported in [START_REF] Lee | Epitaxial stabilization of a new multiferroic hexagonal phase of TbMnO3 thin films[END_REF]. Figure 2c shows a zoom in the 2θ region from 28 to 45°. It can be seen that while the Pt (111) and ALO (0006) are precisely in the same position, the position of the h-In-TMO (004) peak is noticeably shifted by about 0.2° to the left of the h-TMO (004) peak. The corresponding c lattice parameter is 11.463 Å for h-TMO and 11.531 Å for h-In-TMO. The former is an excellent match with previous values [START_REF] Lee | Epitaxial stabilization of a new multiferroic hexagonal phase of TbMnO3 thin films[END_REF] and the latter means that c is increased by 0.6 % with In doping which is a sign that In is inserted in the h-TMO matrix as it was the case for the o-TMO powders.

The optical properties of the films were investigated by spectroscopic ellipsometry as discussed in the experimental section. As a matter of fact, calculations for pure h-TMO in [START_REF] Huang | Hexagonal rare-earth manganites as promising photovoltaics and light polarizers[END_REF] indicate extraordinary optical linear dichroism and linear birefringence properties. Figure 3a shows the ordinary αo (light polarization in the ab plane) and extraordinary αe (light polarization along the c axis) absorption coefficients for h-TMO, h-In-TMO, h-Y-TMO films and for the h-TMO calculation [START_REF] Huang | Hexagonal rare-earth manganites as promising photovoltaics and light polarizers[END_REF]. It can be observed that between experimental and calculated h-TMO absorption coefficients the absorption band edge is less than 0.4 eV apart so that globally the match is globally satisfactory. It is well known that calculations cannot predict precisely the bandgap of materials. Only the complex shape above 2 eV for the ordinary contribution is not visible in the experimental data. The predicted high αo absorption plateau of around 10 5 cm -1 is also present. The doped h-TMO films provide similar properties to pure h-TMO, apart from the extraordinary contribution of h-In-TMO that has a band edge 0.6 eV higher. Indoping causes a stronger modification of lattice parameters than Y-doping, as evidenced in Figure 2c.

It was also the case for o-TMO as shown in Table 1. Lattice parameters changes are well known to modify the bandgap properties [START_REF] Fix | Effects of strain relaxation on the electronic properties of epitaxial Sr2FeMoO6 grown by pulsed laser deposition on SrTiO3 (001)[END_REF] and therefore the absorption coefficients. The Tauc plots extracted from αo are shown in Figure 3b. A direct bandgap of 1.55, 1.61 and 1.60 eV is observed for h-TMO, h-In-TMO and h-Y-TMO respectively, which is a good match with previously reported values [START_REF] Choi | Electronic structures of hexagonal RMnO3 (R=Gd, Tb, Dy, and Ho) thin films: Optical spectroscopy and first-principles calculations[END_REF]. The high absorption coefficient 10 5 cm -1 , the values of the bandgap of about 1.6 eV, and the low thicknesses of the films can be compared to similar values for another material in the two dimensional form, MoSSe, that is envisaged as a water-splitting photocatalyst [START_REF] Ma | A Janus MoSSe monolayer: a potential wide solar-spectrum water-splitting photocatalyst with a low carrier recombination rate[END_REF][START_REF] Ma | Transition Metal-Functionalized Janus MoSSe Monolayer: A Magnetic and Efficient Single-Atom Photocatalyst for Water Splitting Applications[END_REF].

In the next part, we report on the electrical properties of the PLD films. PFM was performed to investigate the possible ferroelectricity of the films. It was found that the h-TMO, h-In-TMO and h-Y-TMO did not provide any clear ferroelectric loop with a well-defined hysteresis nor poling images indicating ferroelectricity. Only the h-YMO film showed ferroelectric characteristics, as shown in Figure 4, but with a very low hysteresis voltage (see SI Figure S6 for the other samples). The first report on ferroelectricity in h-TMO by Lee et al. found a Curie temperature well below room temperature, around 60 K, based on dielectric and electrical measurements [START_REF] Lee | Epitaxial stabilization of a new multiferroic hexagonal phase of TbMnO3 thin films[END_REF]. Eight years later Kim et al.

performed PFM on h-TMO at room temperature and by heating up to 180 °C, while hysteretic PFM loops were observed [START_REF] Kim | Room-Temperature Ferroelectricity in Hexagonal TbMnO3 Thin Films[END_REF]. In the present case we do not observe any sign of ferroelectricity at room temperature, except for h-YMO. The surface of the films was found very flat with root-mean-square (RMS) roughness below 0.6 nm over 0.25 µm² (see SI Figure S7). I-V characteristics under 1 sun showed that the short-circuit current ISC of the structures was below the detection limit of the equipment (below 0.1 nA). The I-V characteristics were not showing any rectification but rather a tunnelling behaviour. This indicates that the films are extremely resistive which may account for the absence of a short-circuit current ISC. Voltage pulses up to ±10 V did not provide any change on the ISC.

Another powerful technique that was used to investigate the photovoltaic properties is the surface photovoltage (SPV). It is based on a Kelvin probe setup where the sample is illuminated with a white light while monitoring the contact potential difference (CPD). Figure 5a shows such a measurement for the different devices. The Pt electrode is connected to the ground while an Au tip is approached to the surface of the manganite films. The h-TMO and In-TMO show no response to light, while h-Y-TMO shows a better response than h-YMO. This means that both h-Y-TMO and h-YMO provide a photovoltaic response. The SPV signal determined as SPV = CPD(light)-CPD(dark) is shown in Figure 5b as a function of illumination of the halogen light source and confirms the observation of the photovoltaic behaviour. In this solar cell configuration, the SPV signal is related to the open-circuit voltage which is found to be the highest for the h-Y-TMO sample with about 80 mV at 20 mW/cm². This corresponds to a relatively large area measurement as the tip has a 2 mm diameter. Therefore it is clear that doping has a large impact on the photovoltaic properties of h-TMO and that the properties of h-YMO can be surpassed with h-Y-TMO which may encourage further research.

Conclusions

Powders of doped TMO were investigated to encourage the formation of an hexagonal TMO phase. In and Nd incorporation were found to greatly reduce the TMO cell size without leading to a hexagonal phase. Hexagonal films of TMO, In-TMO, Y-TMO and YMO were obtained thanks to epitaxial stabilisation on Pt (111), by carefully adjusting the PLD laser repetition rate to 4 Hz. The optical properties namely the absorption coefficient and the bandgap were determined for TMO, In-TMO, and Y-TMO films. Birefringence was found in these films. The absorption coefficient was about 10 5 cm -1 at 2 eV for the TMO and doped TMO films and the direct bandgap in the 1.5-1.6 eV range (ordinary contribution). Concerning the ferroelectric properties of the films at room temperature, only the h-YMO films showed a ferroelectric-like hysteretic behaviour by PFM. I-V measurements under 1 sun showed the absence of any measurable and switchable ISC. Tunnelling-like I-V curves were obtained and the high resistivity observed may account for the low ISC observed. Still, the h-Y-TMO and h-YMO films provide a photovoltaic response as observed by SPV which is a sign that incorporation of Y has a beneficial effect on the PV properties. Further work on h-TMO for PV and photoferroelectric applications should focus on the increase of conductivity of the materials for example with additional dopants.

Table 1: a,b,c lattice parameters obtained from Rietveld refinement for the different powders synthesised and from the ICDD TMO reference. The volume of the cell, the average atomic radius and the ion-oxygen distance from [22,23] [23] are also indicated. The error bar on a,b,c is ± 0.005 Å.

Figure 1 :

 1 Figure 1: Room temperature θ-2θ XRD pattern showing the different powders measured and the ICDD references.

Figure 2 :

 2 Figure 2: Room temperature θ-2θ XRD patterns showing the different films grown on Pt//Al2O3 (0001) at a) 10 Hz b) 4Hz laser repetition rate during PLD growth and c) is a zoom of b).

Figure 3 :

 3 Figure 3: a) Absorption coefficient vs. photon energy for the different layers as calculated from spectroscopic ellipsometry. b) Corresponding Tauc's plot assuming a direct bandgap.

Figure 4 :

 4 Figure 4: PFM phase and amplitude hysteresis loops at room temperature as a function of the tip bias voltage for a) h-TbMnO3 and b) h-YMnO3 films.

Figure 5 :

 5 Figure 5: a) Contact potential difference (CPD) from the Kelvin probe as a function of time alternating between dark and halogen visible light. b) Surface photovoltage (SPV) from the Kelvin probe under variable halogen visible light illumination.

Acknowledgments

The authors thank the XRD and SEM platforms of IPCMS including M. Lenertz and C. Leuvrey, and the staff of the C 3 Fab platform of ICube. This work was partly carried out under the framework of the FERROPV project supported by the French Agence Nationale de la Recherche (ANR) under the reference ANR-16-CE050002-01.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Floriane Mangin

Charlotte Collet

Valérie Jouan-Hureaux

Fatiha Maskali

Emilie Roeder

Julien Pierson

Katalin Selmeczi

Pierre-Yves Marie

Cédric Boura

Nadia Pellegrini-Moïse
email: nadia.pellegrini@univ-lorraine.fr

Sandrine Lamandé-Langle
email: sandrine.langle@univ-lorraine.fr

Synthesis of a DOTA-C-glyco bifunctional chelating agent and preliminary in vitro and in vivo study of [ 68 Ga]Ga-DOTA-C-glyco-RGD

The design of bifunctional chelating agents (BFCA) allowing straightforward radiometal labelling of biomolecules is a current challenge. We report herein the development of a bifunctional chelating agent based on a DOTA chelator linked to a C-glycosyl compound, taking advantage of the robustness and hydrophilicity of this type of carbohydrate derivatives. This new BFCA was coupled with success by CuAAC with c(RGDfK) for α v β 3 integrins targeting. As attested by in vitro evaluation, the conjugate DOTA-C-glyco-c(RGDfC) demonstrated high affinity for  v  3 integrins (IC 50 of 42 nM). [

68 Ga]Ga-DOTA-C-glyco-c(RGDfK) was radiosynthesized straightforwardly and showed high hydrophilic property (logD 7.4 = -3.71) and in vitro stability (> 120min).

Preliminary in vivo PET study of U87MG engrafted mice gave evidence of an interesting tumor-to-non-target area ratio. All these data indicate that [ 68 Ga]Ga-DOTA-C-glyco-c(RGDfK) allows monitoring of α v β 3 expression and could thus be used for cancer diagnosis. The DOTA-C-glycoside BFCA reported here could also be used with various ligands and chelating other (radio)metals opening a broad scope of applications in imaging modalities and therapy.

Peptides can be radiolabelled with fluorine-18 ( 18 F) by addition of a fluorinated prosthetic group or with radiometals (gallium-68, copper-64 or zirconium-89) through a bifunctional chelating agent (BFCA).

1,9,10

However, this structural modification can disrupt the metabolic profile and/or decrease the affinity of the biomolecule for its biological target, which is a major drawback, in particular for molecular imaging. In light of this, it is worth noting that the introduction of a carbohydrate moiety on biomolecules, such as peptides, has often been shown to enhance their pharmacokinetic and in vivo clearance properties. 5,11-13 18 F-glycopeptides, obtained by attachment of a radiofluorinated carbohydrate entity on peptides for an application in PET imaging have been a subject of interest in our group for several years. In particular, RGD peptide derivatives are selective for α v β 3 integrins, which are important cell adhesion receptors involved in angiogenic processes allowing cancer diagnosis. 4,14,15 Various 18 F-glyco-RGD containing an O-or a C-glycosidic moiety conjugated with a RGD peptide derivative have been designed, synthetized and their automated radiosynthesis has been developed. 4,5,16,17 However, 18 F-radiolabelling of biomolecules remains a challenge because it requires time consuming multistep automated radiosyntheses. The use of radiometals allows straightforward peptides radiolabelling by simple metal coordination via a chelating agent previously introduced on the peptide. Furthermore, 68 Ga-labelled peptides are attracting increasing interest in PET diagnostics because 68 Ga is easily produced with a [ 68 Ge]/[ 68 Ga] generator thus not

requiring a biomedical cyclotron, and its short half-life of 68 min reduces the radiation burden to the patient.

 For example, the side arm has been derivatized with different polyethylene glycol moieties (PEG), amino acids or aliphatic chains displaying functional group like amine, carboxylic acid or azide for subsequent coupling with targeting molecules. 27,29-31 In close link with our interests in PET radiotracers, we have designed an original strategy for 68 Ga-labelling of peptides and we have implemented this method for the preparation of 68 Ga-labelled glyco-RGD. A new bifunctional chelating agent named DOTA-C-glyco was elaborated by linking the common polyazamacrocycle DOTA and a functional carbohydrate moiety. This latter bears an azido-arm which enables the conjugation of biological vectors of interest, by Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) with a propargylated peptide such as propargylated c(RGDfK). The ability of the synthesized DOTA-C-glyco-c(RGDfK) to target integrins was evaluated in vitro by a solid-phase binding assay. 68 Ga-labelling was carried out and PET imaging was implemented to assess the performance of this new 68 Ga-labelled radiotracer for cancer diagnosis.

Results

Chemistry

Introduction

Peptides are an important class of ligands and have emerged in molecular imaging and nuclear medicine due to their advantageous properties, which include high target specificity and selectivity, low toxicity, biocompatibility and biodegradability [1][2][3] In particular, radiolabelled peptides or proteins are suitable for Positron Emission Tomography (PET) 1,[4][5][6][7][8] as new diagnostic agents for preclinical and clinical research and are today considered as fully "druggable" moieties.

The construction of DOTA-C-glycopeptide requires the preliminary synthesis of the DOTA-C-glyco bifunctional chelating agent. We first investigated the preparation of a DOTA moiety bearing an amino functional arm for the conjugation with the C-glycoside. To this end, N-(2-benzyloxycarbonylaminoethyl)bromoacetamide 1 was chosen (Scheme 1). 32 The commercially available protected tri-tert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7triacetate (DO3A(t-BuO)) 2 was N-alkylated with 1 using K 2 CO 3 and few drops of dimethylformamide in dry acetonitrile. The removal of the Cbz protecting group by catalytic hydrogenolysis was carried out under H 2 (atmospheric pressure) with Pd/C-10% (10% w/w) in methanol to furnish compound 4 in quantitative yield.

32 Scheme 1. DOTA derivative synthesis. Reagents and conditions: (i) K 2 CO 3 , DMF, dry acetonitrile, rt, 12h, 52%;

(ii) H 2 , Pd/C-10% (10% w/w), MeOH, rt, 5h, quantitative.

The synthesis of the glycosidic derivative was then carried out taking advantage of our precedent work on the synthesis of fluoro-C-glyco-RGD. 4 Interestingly, the robust C-C link at the anomeric position displays a high resistance to acidic and enzymatic hydrolysis, conferring an improved in vivo stability to C-glycosyl compounds compared to O-glycosides. 4,33 A C-glycoside moiety with a 3-carbon spacer-arm bearing an azido group for the CuAAC reaction was thus investigated. The pseudo-anomeric position was chosen for the azido propyl arm introduction given the high reactivity of this position. A protection/deprotection strategy allowed us to generate a reactive primary hydroxyl group and its tosylated counterpart in position 6 (sugar numbering) for DOTA coupling. Azido-C-glyco derivative 7 was synthesized as previously described (Scheme 2). 4 Briefly, 1-Oacetyl-2,3,4,6-tetra-O-benzyl-D-glucopyranose 5 was stereoselectively converted with allyl bromide into β-Callylglucopyranoside subsequently treated with 9-borabicyclo(3.3.1)nonane (9-BBN) to furnish the resulting alcohol. The latter was then converted to bromide derivative 6 via a bromination reaction followed by hydrogenolysis of the benzyl ether protecting groups under H 2 atmosphere. Compound 6 was reacted with sodium azide, underwent a selective tritylation of the primary alcohol, then peracetylation and trityl group removal provided compound 7. 4 Lastly, tosylated compound 8 was obtained in 52% yield after reaction of 7

with tosyl chloride, triethylamine and a catalytic amount of dimethylaminopyridine (DMAP). The introduction of the peptide on DOTA-C-glyco tool 11 was performed site-specifically by CuAAC with alkynemodified c(RGDfK) 12. 34 The reaction proceeded efficiently under very mild conditions in a water/tert-butanol solution (1/1 v/v) using copper(II) acetate (1.5 equivalents) and sodium ascorbate (3 equivalents) as reducing agent (Scheme 3). A stoichiometric quantity of copper(II) acetate was required instead of the catalytic amounts usually employed, because copper ions were chelated by the DOTA macrocycle. Furthermore, the removal of complexed Cu 2+ from the cage proved tricky. Indeed, the classical strategy using a chelating resin (Chelex®) has failed. In our case, the best solution was the addition of 6 equivalents of sodium sulfide nonahydrate leading to the precipitation of copper(II) as monosulfide. 35 After filtration on a Celite pad and freeze-drying, purification by steric exclusion gel filtration was performed affording DOTA-C-glyco-c(RGDfK) 13 in a high yield of 72%. High resolution mass spectrometry as well as 1D and 2D NMR data confirmed the structure of the conjugate.

The gallium(III) complex of compound 13, (Ga)13, was also synthetized in order to evaluate its in vitro affinity for α v β 3 integrin. For the complexation reaction, 1 equivalent of gallium(III) nitrate was added to a solution of compound 13 in water and the mixture was heated for 20 min at 110 °C. After freeze-drying, Ga-DOTA-C-glycoc(RGDfK) (Ga) 13

Integrin α v β 3 binding assay

The affinity of compounds 13 and (Ga)13 for α v β 3 integrin was evaluated by a solid-phase binding assay (Fig. 1).

The results showed that the IC 50 values of 13 and (Ga)13 (77 and 45 nM respectively), although slightly lower than the reference positive controls Echistatin (4 nM) and c(RGDfK) (16 nM), remain in the nanomolar range.

These data demonstrate a high affinity of 13 and (Ga) 13 for the α v β 3 integrin. were calculated using one-site fit log-IC 50 non-linear analysis regression (graphpad prism).

Radiochemistry

DOTA-C-glyco-c(RGDfK) 13 was radiolabelled with gallium-68 on a MiniAllInOne (Trasis ® ) synthesiser (Scheme 4). Different parameters such as amount of 13, reaction time and temperature were screened. The optimal amount of compound 13 was found to be 50 g in 1 mL of 0.7 M acetate solution (pH = 7.9). As commonly described for 68 Ga-DOTA radiolabelling, a temperature of 110°C was required to reach maximum gallium-68 chelation for a reaction time of 10 min (86% determined by HPLC analyses). Formulation of the resulting

PET Imaging

A series of dynamic images (coronal sections) were obtained up to 120 min after the injection of [ 68 Ga]13 on whole-body PET images of U87MG engrafted mice (Fig. 2a). α v β 3 integrins, which are targeted by RGDcontaining tracers, are highly expressed in high-grade gliomas in which the magnitude of this expression has been associated with the potential for tumor invasion, angiogenesis, and metastasis. Whole-body image of the second hour of PET recordings is depicted in Fig. 3a, providing an illustration of the potential of [ 68 Ga]13 for tumor imaging. The Figure 3b shows the mean standard value uptakes within various organs of reference (heart, brain, kidney, liver), within the tumor and within a non-target area (contralateral area). The Figure 3c displays the uptake ratios of the tumor versus the area cited above. These data were computed for the second hour of PET recording and showed the radiotracer profile. High radioactivity accumulation was observed in the kidneys, showing a raised clearance of the tracer during the second hour.

Moreover, the tumor activity from [ 68 Ga]13, which likely corresponds to integrin binding, was found two-fold higher than that of the non-target region (0.21 ± 0.04 vs. 0.10 ± 0.01; p<0.05 for SUVmean of tumor and non-target area respectively), and approximatively four times higher than that of normal brain (0.06 ± 0.01), giving high tumor-to-non-target area (2.20 ± 0.45) and tumor-to-brain (3.90 ± 0.10) ratios.

Discussion

The development of new diagnostic/therapeutic tools for molecular imaging like PET or molecular radiotherapy, combining high affinity toward receptor, high in vivo stability and optimal pharmaco-kinetic profile remains a daunting challenge. Bioavailability is another crucial characteristic, especially in the context of PET imaging relying on radioisotopes with short half-lives. In this paper, we have designed an original DOTA-Cglyco-c(RGDfK) containing a C-glycosyl moiety bearing an azido spacer-arm. Having a stable and nonhydrolysable C-C link at the pseudo-anomeric position, C-glycosyl compounds are key components for the construction of stable glycoconjugates for various in vivo applications. 4,33,[START_REF] Levy | Strategies towards C-glycosides in The Organic Chemistry of Sugars[END_REF] Besides, the DOTA-C-glycoside tool bears an azide group to ensure CuAAC conjugation with biological ligands. This strategy allows a site-specific conjugation to a large panel of ligands previously derivatized with an alkyne function. Previous works on C-glyco-RGD derivatives showed adequate flexibility and ideal steric arrangement with a three-carbon spacerarm, conferring them similar affinities toward  v  3 integrin than the native RGD. 4 Given the importance of maintaining the highest affinity, an analogous C-glycosyl compound was chosen for the design of the tracer. We conjugated with success the DOTA-C-glycosyl derivative on alkyne-modified c(RGDfK) peptide for  v  3 integrin targeting. Compared to common DOTA-c(RGDfK), [START_REF] Decristoforo | [END_REF]40 the C-glycosyl moiety with a long spacer arm afforded a distance between DOTA and the peptide, which is obviously favourable for binding. The gallium(III) complexed counterpart of this DOTA-glycopeptide was also successfully synthesized.

In vitro evaluation and comparison of derivative DOTA-C-glyco-c(RGDfK) 13 were carried out with the gold standard integrin inhibitor Echistatin and with native c(RGDfK). To be as close as possible to the 68 Garadiolabelled form injected for PET imaging, Ga-DOTA-C-glyco-c(RGDfK) (Ga) 13 was also evaluated in the same experimental protocol. In many cases, the addition of reporting moieties like fluorophores or chelating agents affects the binding affinity of the modified biomolecules. In our case, compounds (Ga) 13 and 13 presented a slight decrease of affinity compared to Echistatin and native RGD, however, remaining in the same nanomolar range. These data showed that the Ga-DOTA complex and the carbohydrate linker did not significantly impede the affinity of c(RGDfK) peptide for α v β 3 integrin. The binding affinity of (Ga)13 is slightly greater compared to compound 13. However, both IC 50 are in the same range (42 nM vs. 75 nM for (Ga)13 and 13 respectively). to 60 min after tracer injection. [43][44][45][46] This avoids an early high level of nonspecific activity, possibly due to the circulating blood tracers, and thus prevents overestimation of integrin density. The 120 min time activity curves of biodistribution of [ 68 Ga]13 showed a higher uptake of the tracer in the tumor than in the non-target area, suggesting a specific uptake of [ 68 Ga]13 by α v β 3 integrin in glioblastoma. These integrins, highly expressed in high-grade gliomas, are known to be poorly expressed within the different tissue components of normal brain.

Conclusion

In this study, we reported for the first time the synthesis of DOTA-C-glycosyl bifunctional chelating agent Furthermore, the possibility of using others ligands and (radio)metals opens a broad scope of applications in imaging modalities and therapy.

Experimental section

General information

Solvents and liquid reagents were purified and dried according to recommended procedures.

Experimental procedures and physico-chemical characterizations

Chemistry

Compounds 1, 32 2, 32 4, 32 6, 4 7
4 and 12 34 were prepared according to previously described methods (see ESI).

Integrin α v β 3 binding assay

The affinity of compounds for integrin protein was evaluated in terms of the half maximal inhibitory concentration (IC 50 values) through a solid-phase assay as previously described by Tobias G. Kapp and al. 47 Briefly, the surface of Maxisorp microplates (NUNC, Thermoficher scientific, France) was coated with 1 µg/ml human vitronectin (Bio-techne, France), overnight at +4°C. The non-specific sites were blocked with TSB buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM MnCl 2 , pH 7,5, 1% BSA) (Sigma-Aldrich, France) for 1 h at 37°C. Binding of compounds was assessed using 2 µg/mL integrin α v β 3 (Bio-techne, France) in the presence of dilution series of compounds or two reference molecules: c(RGDfK) (Bachem, Germany) and echistatin (Bio-techne, France), as positive control. After 1 h incubation at room temperature, the plates were washed and the amount of bound integrin was stained by incubation with 2 µg/ml mouse anti-human CD51/61 (BD Pharmingen, France) and 1 µg/ml anti-mouse IgG horseradish peroxidase conjugate antibody (Bio-techne, France). The enzymatic reaction was allowed in the dark by addition of substrate (Bio-techne, France) and stopped after 10 min by the addition of H 2 SO 4 (Stop Solution, Bio-techne, France). The optical densities were measured at 450 nm. Values were blank subtracted and results were expressed as a mean of relative absorbance percentage to wells containing only integrin αvβ3 of triplicate measurements. Affinities were estimated as IC 50 values (i.e. the concentration of compounds that displaced 50 % of integrin binding) calculated using one-site fit log-IC 50 non-linear analysis regression of GraphPad Prism 6 software (v 6.05, USA).

Radiochemistry

The 68 Ga-labelling of 13 was performed using a mAIO synthesizer (Trasis, Ans, Belgium) with removable cassettes. The

Fig. 1 .

 1 Fig. 1. Solid-phase binding assay. Effect of 13 (dark) and (Ga)13 (grey) on the binding of α v  3 integrin onto vitronectin compared with two reference molecules: c(RGDfK) (red) and Echistatin (orange). IC 50 values (nM)

[Scheme 4 . 68 Ga] 13 . 68 Ga] 13 .

 468136813 Scheme 4. Radiosynthesis of [ 68 Ga]13. Reagents and conditions: (i) [ 68 Ga]GaCl 3 , AcONa (0.7 M) 110°C, 10 min,

36 , 37 68 Ga] 13 68 Ga] 13 ,

 363768136813 Corresponding time-activity curves for tumor and non-target area (contralateral area) showed that the uptake of [ increased faster in the tumor than in the non-target area (Fig.2b), resulting in an enhancement of the tumor-to-non-target area ratio during 120 min of dynamic analysis (Fig.2c). Otherwise, [ showed a fast renal clearance resulting in particularly high activities in the kidneys and bladder.

Fig. 2 .

 2 Fig. 2. (a) Dynamic PET images displayed through coronal sections and recorded during 120 min following [ 68 Ga]13 injection in U87MG engrafted mice (n=3). Color scale is expressed in SUV values. The tumors are indicated with white arrows, the kidneys with yellow arrows and the bladder with red arrows. (b) Corresponding time-activity curves for mean SUV from tumor (black square) and from non-target area (white circle), as well as for tumor-to-non-target area ratio (black triangles) (c).

Fig 3 .

 3 Fig 3. (a) Whole-body image of the 2 nd hour of PET recordings after [ 68 Ga]13 injection. (b) Results of SUVmean values obtained at the 2 nd hour of PET recording for tumors and various organs, after the injection of [ 68 Ga]13. (c) Comparison of tumor-to-organ ratio for activities measured during the 2 nd hour of PET recording. *: p<0.05The tumor is indicated with white arrows and the color scale is expressed in SUV values (n = 3).

68 Ga] 13 . 68 Ga] 13 .

 68136813 was synthesized straightforwardly in high radiochemical yields and purity. Incorporation of gallium-68 into the DOTA requires heating to 110°C, however, despite this weakness, this chelator was chosen for its versatility towards other metal ions. Some other chelator like the acyclic HBED-CC or the NOTA require lower energy for complex formation. However, HBED-CC forms different diastereomers upon complexation of gallium, which is undesirable because different isomers may have different pharmacological profiles. Whereas NOTA does not present the optimal number of coordination site for the chelation of other metals used in medical imaging and molecular radiotherapy. 41,42 Experimental partition coefficient (-3.71) demonstrated excellent hydrophilic property for [ This value is slightly lower than those of commonly used [ 68 Ga]Ga-DOTA-c(RGDfK) (around -3.43) 40 indicating an improved hydrophilicity which could be attributed to the carbohydrate moiety. A 120 min dynamic PET imaging with [ 68 Ga]13 showed a slow increasing uptake of the tracer in the tumor and non-target area up to 60 min post injection of [ This observation is in accordance with previous literature on RGD-containing tracers, leading to the current recommendation of delaying PET recording for 30

(

  BFCA) and its conjugation to c(RGDfK) peptide using CuAAC. The design of the DOTA-C-glyco BFCA exploited the hydrophilicity and the robustness of C-glycosyl moiety. In vitro binding of DOTA-C-glyco-c(RGDfK) and its gallium(III) complexed counterpart, to α v β 3 integrin showed that conjugation of DOTA-C-glycoside to c(RGDfK) did not affect its affinity. [ 68 Ga]Ga-DOTA-C-glyco-c(RGDfK) [ 68 Ga]13 was radiosynthesized straightforwardly and showed high hydrophilic property (logD 7.4 = -3.71) and in vitro stability (> 120 min). Tumor-to-non-target area ratio obtained by in vivo PET study in U87MG engrafted mice, demonstrated that [ 68 Ga]Ga-DOTA-C-glycoc(RGDfK) could be a potential radiotracer for cancer diagnosis by PET imaging. As reported in this study, the DOTA-C-glycoside BFCA is a versatile tool, successfully employed for 68 Ga-radiolabelling of c(RGDfK).

1 ) 1 H 13 C 7 (Compound 10 .

 1113710 O-acetyl-9-O-tosyl-D-glycero-D-gulo-nonitol (8). To a solution of 4,8-anhydro-1-azido-1,2,3-tridesoxy-5,6,7-tri-O-acetyl-D-glycero-D-gulo-nonitol (424 mg, 1.14 mmol) in dry DCM (10 mL) tosyl chloride (658.4 mg, 3.42 mmol), DMAP (66 mg, 0.57 mmol) and triethylamine (0.63 mL, 4.54 mmol) were added under argon. The reaction was stirred overnight at 30°C. The mixture was concentrated under reduced pressure and purified on by flash column chromatography (silica gel, Cyclohexane/EtOAc 6/4) to afford 8 (607 mg). Yield 52%. White solid. Mp: 78-80°C; [α] D : +12.0 (c 0.09; EtOH); IR film (, cm -: 2102, 1738, 1599, 1368, 1256, 1227, 1188, 1173, 1119, 1098, 1032; NMR (400 MHz, CDCl 3 ): δ 1.36-1.46 (m, 1H, H3a), 1.51-1.63 (m, 2H, H2a and H3b), 1.68-1.78 (m, 1H, H2b), 1.98 (s, 3H, CH 3 ), 1.99 (s, 3H, CH 3 ), 2.03 (s, 3H, CH 3 ), 2.46 (s, 3H, CH 3 ), 3.20-3.31 (m, 2H, J 1,2a = J 1,2b = 6.5 Hz, H1), 3.38 (bddd, 1H, J 4,5 = 9.5 Hz, J 4,3b = 9.0 Hz, J 4,3a = 2.5 Hz, H4), 3.66 (ddd, 1H, J 8,7 = 9.0 Hz, J 8,9b = 6.0 Hz, J 8,9a = 3.0 Hz, H8), 4.02 (dd, 1H, J 9a,9b = 11.0 Hz, H9a), 4.09 (dd, 1H, H9b), 4.80 (app t, 1H, J 4,5 = J 5,6 = 9.5 Hz, H5), 4.89 (app t, 1H, J 7,6 = J 8,7 = 9.0 Hz, H7), 5.13 (app t, 1H, H6), 7.35 (d, 2H, J = 8.0 Hz, Ar), 7.78 (d, 2H, J = 8.0 Hz, Ar). NMR (100.6 MHz, CDCl 3 ): δ 20.CH 3 ), 20.7 (CH 3 ), 20.8 (CH 3 ), 21.8 (CH 3 ), 24.6 (C2), 28.4 (C3), 51.2 (C1), 68.1 (C9), 69.0 (C7), 71.7 (C5), 74.1 (C6), 75.5 (C8), 77.4 (C4), 128.2 (2C Ar ), 130.0 (2C Ar ), 132.7 (C q ), 145.3 (C q ), 169.6 (C=O), 169.7 (C=O), 170.4 (C=O). HRMS (ESI, C 22 H 29 N 3 O 10 SNa: [M + Na] + ) calcd 550.1471, found 550.1493. To a solution of 4,8-anhydro-1-azido-1,2,3-trideoxy-5,6,7-tri-O-acetyl-D-glycero-D-gulononitol 7 (75 mg, 0.2 mmol) in dioxane (1.4 mL), was added under argon, N,N-carbonyldiimidazole (38.9 mg, 0.24 mmol). The mixture was stirred at room temperature until completion of the reaction (TLC monitoring), then was concentrated under reduced pressure. The residue was diluted in a cyclohexane/EtOAc 1/

  Galiapharm; Eckert & Ziegler Europe) was eluted with 5 mL of a 0.1 M HCl solution. 13 (50 μg, 34 nmol) previously solubilized in 1 mL of acetate buffer solution (0.7 M), was added to the eluted solution. The mixture was heated at 110°C during 10 min. The resulting product, [ 68 Ga]13, was trapped on a HLB Oasis cartridge. The product was eluted from the cartridge with 0.6 mL of ethanol. The cartridge was rinsed with 5 mL of NaCl 0.9%. [

  TrCl, DMAP, pyridine, 48h, 45°C. 2) Ac 2 O, 0°C to rt, 2h, 62%; (vi) TFA (20% v) in CH 2 Cl 2 , 0°C, 10 min, 73%; (vii) Tosyl chloride, DMAP cat., triethylamine, CH 2 Cl 2 , 12h, 30°C, 52%. Unfortunately, the reaction did not occur and the starting materials were recovered even after modification of various parameters such as the nature of the solvent (acetonitrile or DMF), temperature (30 or 80°C), reaction time and stoichiometry. A second strategy based on the formation of a carbamate bond has been explored (Scheme 3). Reaction of 7 with N,Ncarbonyldiimidazol (CDI) was performed in dioxane to give intermediate 9 (62%). The imidazolylcarbonyl derivative 9 was treated with compound 4 in the presence of triethylamine to afford the protected DOTAglycoconjugate 10 in 53% yield. Deprotection of tert-butyl ester and acetyl protecting groups with an aqueous solution of 4N HCl at room temperature during 4 hours led to compound 11 quantitatively.

Scheme 2. C-glycoside derivative synthesis. Reagents and conditions: (i) TMSI, CH 2 Cl 2 , 0°C, 1h, then AllylMgBr, 0°C to rt, 3h, 90%; (ii) 1) 9-BBN, THF, Δ, 4h; 2) EtOH, NaOH, H 2 O 2 , 12h, 89%; (iii) PPh 3 , CBr 4 , CH 2 Cl 2 , 0°C, 1h, 90%;

(iv) Pd(OH) 2 /C (20% w/w), THF/H 2 O (5/1 v/v), H 2 , 33 psi, 13h, 93%; (v) NaN 3 , acetone/water (5/1 v/v), reflux, 12h, 95%; (vi) 1) Having in hand DOTA derivative 4 and C-glycoside compounds 7 and 8, their coupling was examined. We first attempted a N-alkylation of 4 with tosylated compound 8. Scheme 3. DOTA-C-glyco and DOTA-C-glyco-c(RGDfK) synthesis. Reagents and conditions: (i) N,Ncarbonyldiimidazole, dioxane, rt, 62%. (ii) 4, triethylamine, dry THF, rt, 12h, 53%. (iii) H 2 O, 4N HCl, rt, 4h, quantitative. (iv) 1) H 2 O/tert-butanol 1/1 v/v, sodium ascorbate, Cu(OAc) 2, rt, 5h. 2) Na 2 S.9H 2 O, rt, 15 min, 72%.

(v) Ga(NO 3 ) 3 , H 2 O, 110°C, 20 min.

  The synthesized DOTA-C-glycosyl bifunctional chelating agent is a versatile tool. In this study, it has been studied and illustrated with gallium-68 labelling of an RGD peptide derivative, however this BFCA could be used with others biological ligands like peptides, proteins or antibodies and for other medical applications. Indeed, DOTA is widely used in the field of medical imaging and molecular radiotherapy because it chelates efficiently a

	large panel of (radio)metals. Hydrophilic BFCA 11 could be potentially suitable for different imaging modalities
	and therapy, such as PET (	68 Ga,	64 Cu), SPECT (	99m Tc,	111 In) and MRI (Gd). Besides, targeted cancer therapy might
	also be possible with DOTA-C-glycoside 11 labelled with	177 Lu,	90 Y or	186 Re. DOTA-C-glyco could also be
	employed in theranostics enabling concomitant diagnostic and radiotherapy by labelling with diagnostic and
	therapeutic radioisotopes.
	These considerations likely explain the high tumor-to-normal brain ratio documented herein for [

68

Ga]13, i.e., 3.90 ± 0.10 for this mouse model. These data combined with high in vitro stability and affinity toward  v  3 integrin makes [ 68 Ga]13 a potential radiotracer for cancer diagnosis.

  MHz and 235 MHz, respectively) or DRX400 (400 MHz and 100.6 MHz, respectively) spectrometers on the NMR Platform of the Jean Barriol Institute (Université de Lorraine, Nancy, France). For complete assignment of Chemical shifts (δ) are given in parts per million relative to the solvent residual peak. For clarity, atom numbering starts from the end of the anomeric arm rather than resulting from the IUPAC naming of compounds (see ESI Fig S1). The following abbreviations are used for multiplicity of NMR signals: s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, b = broad signal and app = apparent multiplicity. The given J values refer to apparent multiplicities and do not represent the true coupling constants. High resolution ESI-MS spectra were recorded on a BrukerDaltonics microTOFQ apparatus provided by the MassLor Platform (Université de Lorraine, Nancy, France). Radiosynthesis was carried out on a miniAllInOne (miniAIO) module from Trasis®. Female immunodeficient (nu/nu) NMRI mice were purchased from Janvier Laboratories. PET recordings were obtained with a camera dedicated to small animal studies (Inveon, Siemens Preclinical Solutions, Knoxville, USA).

		1 H and	13 C signals,
	two-dimensional C correlation spectra were recorded. 68 1 H, 1 H COSY and 1 H, 13 GaCl 3 was eluted from a 68 Ge/ 68 Ga generator (Galliapharm; Eckert & Ziegler Europe) with 5 mL of a 0.1 N HCl
	solution.
			Thin layer
	chromatography (TLC) analyses were performed using standard procedures on Kieselgel 60F254 plates (Merck,
	Kenilworth, NJ, USA). Compounds were visualized using UV light (254 nm), ninhydrin and/or a methanolic
	solution of sulfuric acid and charred. Column chromatography was performed on silica gel SI 60 (63-200 µm)
	(Merck). c(RGDfK) was purchased from Bachem (Germany) with >95% purity. Purification of RGD-conjugate was
	achieved by gel filtration on LH20 using water as eluent. Melting points were determined with a Tottoli
	apparatus and are uncorrected. FTIR spectra were recorded on a Shimadzu IRAffinity-1, ATR PIKE Technologies
	model GladiAT (cm	-1 ). Optical rotations were measured on an Anton-Paar MCP 300 polarimeter. Analytical High
	Performance Liquid Chromatography (HPLC) analyses were run on a Waters system (2695eb pump, auto

sampler injector, 2998 PDA detector, 2424 ELSD detector, and NaI detector from Berthold [Bad Wildbad, Germany]) controlled by the Empower Software (Orlando, FL, USA). Analyses were performed on a Vydac 218TP C18 (5 μm, 250 × 4.6 mm) from Grace with ACN/H 2 O/TFA mixture (proportions given in brackets) at 1 mL/min. 1 H, 13 C, and 19 F NMR spectra were recorded on a Bruker DPX250 (250 MHz, 62.9

  1 v/v solution (5 mL), filtered on a silica gel pad and evaporated under reduced pressure to obtain intermediate 9 as a colorless oil (57.8 mg, 62%). To a solution of 9 in dry THF (2 mL), 4 (110.8 mg, 0.18 mmol) and triethylamine (167 µL, 1.2 mmol) were added. The reaction mixture was stirred overnight at room temperature. After evaporation under reduced pressure, the crude product was purified by LH20 chromatography with MeOH as eluant, then by flash chromatography (silica, CH 2 Cl 2 /MeOH 9/1) to afford compound 10 (64.5 mg). Yield 53%. Ar ), 128.8 (2C Ar ), 129.2 (2C Ar ), 136.0 (C qAr ), 146.3 (C27), 155.0 (C=N), 157.0 (C=O carb ), 171.3 (C=O), 172.5 (C=O), 172.6 (C=O), 173.4 (C=O), 173.6 (C=O), 174.3 (C=O), 174.4 (C=O), 175.8 (C=O), 176.2 (C=O), 177.9 (C=O), 178.0 (C=O). HRMS (ESI, C 60 H 95 N 18 O 21 K: [M + K] 2+ ) calcd 721.3275, found 721.3256.

	(m, 2H, H33a, H2a), 1.51-1.58 (m, 2H, H42), 1.58-1.75 (m, 2H, H33b, H3a), 1.78-1.95 (m, 2H, H2b, H41a), 1.94-
	2.15 (m, 2H, H41b, H3b), 2.56 (dd, 1H, J 38a,38b = 16.5 Hz, J 38a,37 = 7.0 Hz, H38a), 2.65 (bt, 2H, J 29,28 = 7.0 Hz, H29),
	2.70 (dd, 1H, J 38b,37 = 7.0 Hz, H38b), 2.93-3.05 (m, 4H, H28, H30), 3.07-3.36 (m, 26H, H5, H39, H43, H4, H12,
	H11, 16H cyclen), 3.37-3.56 (m, 15H, H1, CH 2 Bn, H7, H6, H14, H17, H20, H23), 3.61 (dd, 1H, J 9a,9b = 11.5 Hz, J 9a,8
	= 7.5 Hz, H9a), 3.67 (dd, 1H, J 9a,8 = 4.5 Hz, H9b), 3.80-3.92 (m, 3H, H8, H34), 4.50 (d, 1H, J = 14.0 Hz, CH 2 Bn),
	Yellow solid. Mp: 99-101°C; [α] D : + 3.6 (c 0.05; EtOH); IR film (, cm -1 ): 2976, 2816, 2093, 1722, 1668, 1520, 4.43 (app t, 1H, J = 7.0 Hz, H40), 4.56-4.63 (m, 1H, H35), 4,67-4.75 (m, 1H, H37), 7.23-7.29 (m, 3H, H Ar ), 7.31-
	1368, 1221, 1155, 1105, 1030. 7.38 (m, 2H, H Ar ), 7.83 (bs, 1H, H26). 1 H NMR (400 MHz, DMSO-d6): δ 1.36-1.43 (m, 1H, H3a), 1.45 (s, 18H, tBu), 1.46 13 C NMR (100.6 MHz, D 2 O): δ 21.2 (C28), 22.4 (C32), 24.2 (C2), 27.3 , 27.4 ,
	(s, 9H, tBu), 1.54-1.64 (m, 2H, H2), 1.65-1.74 (m, 1H, H3b), 1.93 (s, 3H, CH 3 ), 1.98 (s, 3H, CH 3 ), 2.00 (s, 3H, CH 3 ), 27.6, 27.6 (C41, C42, C31, C3), 30.0 (C33), 35.3 (C29), 37.8 (2C, C39, C30), 38.9 (3C, C38, C12, C11), 40.5 (C43),
	2.24-2.41 (m, 8H, CH 2 cyclen), 2.54-2.70 (m, 8H, CH 2 cyclen), 3.00-3.21 (m, 12H, H11, H12, H14, H17, H20, H23), 43.8 (C36CH 2 Bn), 47.8-48.7 (8C, C-cyclen), 50.0 (2C, C40, C1), 50.8 (C37), 55.2 (C35 or C34), 55.5 (C34 or C35),
	3.33 (t, 2H, J 1,2a = J 1,2b = 6.5 Hz, H1), 3.64 (b ddd, 1H, J 8,7 = 10.0 Hz, J 8,9b = 7.5 Hz, J 8,9a = 2.5 Hz, H4), 3.80 (b ddd, 58.5-59.0 (4C, C14, C17, C20, C23), 63.2 (C9), 72.0 (C7), 72.1 (C8), 73.4 (C5), 77.2 (C6), 78.8 (C4), 123.3 (C26),
	1H, J 8,7 = 8.0 Hz, J 8,9b = 5.0 Hz, J 8,9a = 3.0 Hz, H8), 4.02 (dd, 1H, J 9a,9b = 12.0 Hz, J 9a,8 = 3.0 Hz, H9a), 4.07 (dd, 1H, 127.2 (C
	J 9b,8 = 5.0 Hz, H9b), 4.71 (app t, 1H, J 7,6 = 9.5 Hz, H5), 4.88 (dd, 1H, J 5,6 = 9.5 Hz, H7), 5.17 (app t, 1H, H6), 6.87
	(bs, 1H, NH), 7.93 (bs, 1H, NH).	13 C NMR (100.6 MHz, DMSO-d6): δ = 20.7 (CH 3 ), 20.7 (CH 3 ), 20.8 (CH 3 ), 24.6 (C2),
	27.9 (3CH 3 , tBu), 28.0 (6CH 3 , tBu), 28.3 (C3), 39.7 (C12), 40.4 (C11), 51.3 (C1), 55.7 (8C, C-cyclen), 56.1 (4C, C14,
	C17, C20, C23), 63.6 (C9), 69.6 (C7), 72.0 (C5), 74.6 (C6), 76.0 (C8), 76.9 (C4), 82.0 (6C qtBu ), 82.0 (3C qtBu ), 156.4
	(C=O carb ), 169.8 (C=O), 169.8 (C=O), 170.3 (C=O), 172.0 (C=O), 172.3 (3C=O). HRMS (ESI, C 46 H 80 N 9 O 16 : [M + H] + )
	calcd 1014.5723, found 1014.5741.
		Compound 11. To a suspension of compound 10 (73 mg; 0.072 mmol) in water (1.13 mL) was added
	HCl 37% (375 µL). The reaction mixture was stirred at room temperature for 4h and evaporated under reduce
	pressure using MeOH as co-solvent to afford 11 (51.7 mg). Yield quantitative. Yellow solid. Mp: 93-95°C; [α] D : -
	7.8 (c 0.03; EtOH); IR film (, cm	-1 ): 3260, 3082, 2932, 2864, 2095, 1722, 1674, 1539, 1385, 1352, 1211, 1084;	1 H
	NMR (400 MHz, D 2 O): δ 1.44-1.57 (m, 1H, H3a), 1.64-1.76 (m, 1H, H2a), 1.76-1.86 (m, 1H, H2a), 1.88-1.98 (m,
	1H, H3b), 3.22 (appt, 1H, J 5,4 = J 5,6 = 8.5 Hz, H5), 3.24-3.60 (m, 25H, H4, H7, H12, H11, H6, H14, 16Hcyclen ), 3.37
	(t, 2H, J 1,2a = J 1,2b = 7.0 Hz, H1), 3.53-3.61 (m, 1H, H8), 3.73-4.00 (m, 6H, H17, H20, H23), 4.24 (dd, 1H, J 9a,9b =
	11.5 Hz, J 9a,8 = 6.5 Hz, H9a), 4.37 (bd, 1H, H9b).	13 C NMR (100.6 MHz, D 2 O): δ 24.2 (C2), 27.8 (C3), 39.3 (C12),
	39.6 (C11), 44.4-50.4 (8C, C-cyclen), 50.9 (C1), 55.0 (4C, C14, C17, C20, C23), 64.0 (C9), 69.9 (C7), 73.4 (C5), 77.2
	(C6), 77.4 (C8), 79.1 (C4), 158.4 (C=O carb ), 175.3 (C=O), 175.6 (3C=O). HRMS (ESI, C 28 H 49 N 9 O 13 Na: [M + Na] + )
	calcd 742.3348, found 742.3252; (ESI, C 28 H 48 N 9 O 13 Na 2 : [M -H + 2Na]	+ ) calcd 764.3167, found 764.3066.
		Compound 13. To a solution of azido derivative 11 (4.3 mg, 0.006 mmol) diluted in 0.6 mL of water,
	were added at room temperature a solution of c(RGDfK) derivative 12 (4.8 mg, 0.006 mmol) diluted in 1mL of
	tert-butanol, a solution of sodium ascorbate (0.018 mmol) in 0.2 mL of water and a solution of Cu(OAc) 2 (0.009
	mmol) in 0.2 mL of water. The solution was stirred 5 h at room temperature, then Na 2 S.9H 2 O (8.5 mg, 0.036
	mmol)	was	added.	The	mixture	was	stirred	15	min.
										51

Black precipitate appeared immediately, the mixture was filtered on a Celite pad, then freeze-dried.

Purification was achieved on Sephadex LH20 resin. Elution with water provided pure compound 13 (6 mg). Yield 72%. White foam. 1 H NMR (400 MHz, D 2 O): δ 0.72-0.86 (m, 2H, H32), 1.17-1.29 (m, 2H, H31), 1.38-1.

Acknowledgment

This work was supported by NancycloTEP. We thank F. Dupire and S. Adach for technical assistance. We gratefully acknowledge the financial support of the "Contrat de Plan Etat-Région" (CPER) from French government and Grand Est Regional Council.

Animal models and experimental plan

All protocols were approved by the Lorraine Ethics Committee N°68 according to Guidelines of Animal Care and Use (APAFIS#25133-2020041509493813). Tumor xenografts were obtained in mice flanks as described previously. 44

Author contributions

FlM with the participation of SLL and NPM synthesized all the non-radioactive compounds (1 to 13) and completed the NMR analysis of these compounds. KS synthesized (Ga)13, completed the mass spectra analysis and co-wrote the manuscript. VJH with the participation of CB performed in vitro integrin binding assay and they co-wrote the manuscript. CC with the participation of SLL and NPM performed the radionuclide-labelling of 13 and wrote radiochemistry section. FM and CB co-wrote the ethical request for project authorization. JP and CB provided the animal models. ER with the collaboration of JP and FM performed the experimental studies in mice. FM performed images analysis on the small animal PET. FM and PYM co-wrote the manuscript.

SLL and NPM designed the study and wrote the manuscript. All authors read and approved the final manuscript.

Conflicts of interest

There are no conflicts to declare.