Clickable C-Glycosyl Scaffold for the Development of a Dual Fluorescent and [18F]fluorinated Cyanine-Containing Probe and Preliminary In Vitro/Vivo Evaluation by Fluorescence Imaging

Considering the individual characteristics of positron emission tomography (PET) and optical imaging (OI) in terms of sensitivity, spatial resolution, and tissue penetration, the development of dual imaging agents for bimodal PET/OI imaging is a growing field. A current major breakthrough in this field is the design of monomolecular agent displaying both a radioisotope for PET and a fluorescent dye for OI. We took advantage of the multifunctionalities allowed by a clickable C-glycosyl scaffold to gather the different elements. We describe, for the first time, the synthesis of a cyanine-based dual PET/OI imaging probe based on a versatile synthetic strategy and its direct radiofluorination via [18F]F-C bond formation. The non-radioactive dual imaging probe coupled with two c(RGDfK) peptides was evaluated in vitro and in vivo in fluorescence imaging. The binding on αvβ3 integrin (IC50 = 16 nM) demonstrated the efficiency of the dimeric structure and PEG linkers in maintaining the affinity. In vivo fluorescence imaging of U-87 MG engrafted nude mice showed a high tumor uptake (40- and 100-fold increase for orthotopic and ectopic brain tumors, respectively, compared to healthy brain). In vitro and in vivo evaluations and resection of the ectopic tumor demonstrated the potential of the conjugate in glioblastoma cancer diagnosis and image-guided surgery.


Introduction
As a pillar of diagnosis and patient care, molecular imaging is a field of great interest. Early diagnosis could avoid morbidity and medical expenses. Moreover, molecular imaging allows the observation of in vitro or in vivo cellular and molecular processes [1,2]. Specificity of molecular probes for biomarkers or receptors is obviously the corner stone of molecular imaging and is most of the time achieved by vector targeting. Molecular imaging techniques, such as positron emission tomography (PET) [3,4], single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and optical imaging (OI) [5,6], have improved over the years. However, these techniques also display limitations in spatial and temporal resolution or sensitivity. The trend for the last ten years has been the development of bimodal imaging approaches, with the most expanded combinations being PET/MRI and PET/OI. PET/MRI displays high penetrability, but the pitfall

Synthetic Strategy: A Clickable Scaffold
The corner stone of the synthetic strategy is a platform obtained by the modifications of a C-glycosyl derivative that is conveniently substituted to enable the introduction of the key elements at different stages. The main idea is to use copper-catalyzed alkyne-azide cycloaddition (CuAAC) and apply it for fluorophore introduction and in the bioconjugation step [33,34]. To this end, a clickable scaffold was designed with free or temporarily masked triple bonds which could be activated at an appropriate time. Therefore, a γ-Dribonolactone was selected as the starting compound since this sugar configuration allows an extended spatial distribution of the different arms, i.e., upper and lower face of the central core. A multi-step synthetic strategy provided access to the non-radioactive dual probe (with a 19 F) and to the radiolabeling precursor (bearing a leaving group) for 18 Fradiolabeling.

Scaffold Synthesis
The central core was obtained from a sugar γ-lactone being transformed into a Cglycosyl derivative. Among the numerous synthetic methods to prepare C-glycosyl derivatives, we have developed and used for several years the Wittig reaction on sugar γ-lactones, which gives an efficient access to functionalized C-glycosylidene compounds (commonly called exo-glycals), and their subsequent stereoselective double bond reduction (Scheme 1) [27,35]. Thus, starting from a commercially available D-ribonolactone 1, the Cglycosyl compound 4 was obtained by a three-step synthetic sequence involving hydroxyls protection, Wittig reaction with a methoxycarbonyl phosphorane (Ph3P = CHCO2Me), and stereoselective hydrogenation [29,31,32]. Zemplen reaction on compound 4 led to the 7-hydroxy derivative 5, which is a key intermediate for the introduction of the non-radioactive fluorine atom ( 19 F) or for the introduction of the leaving group of the precursor which will undergo a nucleophilic substitution during 18

Synthetic Strategy: A Clickable Scaffold
The corner stone of the synthetic strategy is a platform obtained by the modifications of a C-glycosyl derivative that is conveniently substituted to enable the introduction of the key elements at different stages. The main idea is to use copper-catalyzed alkyne-azide cycloaddition (CuAAC) and apply it for fluorophore introduction and in the bioconjugation step [33,34]. To this end, a clickable scaffold was designed with free or temporarily masked triple bonds which could be activated at an appropriate time. Therefore, a γ-D-ribonolactone was selected as the starting compound since this sugar configuration allows an extended spatial distribution of the different arms, i.e., upper and lower face of the central core. A multi-step synthetic strategy provided access to the non-radioactive dual probe (with a 19 F) and to the radiolabeling precursor (bearing a leaving group) for 18 F-radiolabeling.

Scaffold Synthesis
The central core was obtained from a sugar γ-lactone being transformed into a C-glycosyl derivative. Among the numerous synthetic methods to prepare C-glycosyl derivatives, we have developed and used for several years the Wittig reaction on sugar γ-lactones, which gives an efficient access to functionalized C-glycosylidene compounds (commonly called exo-glycals), and their subsequent stereoselective double bond reduction (Scheme 1) [27,35]. Thus, starting from a commercially available D-ribonolactone 1, the C-glycosyl compound 4 was obtained by a three-step synthetic sequence involving hydroxyls protection, Wittig reaction with a methoxycarbonyl phosphorane (Ph 3 P = CHCO 2 Me), and stereoselective hydrogenation [29,31,32]. Zemplen reaction on compound 4 led to the 7-hydroxy derivative 5, which is a key intermediate for the introduction of the nonradioactive fluorine atom ( 19 F) or for the introduction of the leaving group of the precursor which will undergo a nucleophilic substitution during 18 F-radiolabeling (Scheme 2).
Compound 5 was reacted with diethylaminosulfur trifluoride (DAST) for fluorination and the targeted 7-fluoro derivative 6 was obtained at 85% yield. The strategy based on sequential functionalization required the 7-hydroxyl protection prior to its activation with a leaving group. Thus, a silyl protecting group, which is stable under the envisioned experimental conditions and compatible with 4,5-isopropylidene, was selected. The TBDPS ether 7 was obtained at 95% yield via the reaction of 5 with TBDPSCl and imidazole in DMF. The reduction of the methyl ester on compounds 6 and 7 performed by LiAlH 4 in THF led, respectively, to 8 and 9 in high yields. The resulting primary hydroxyl of compound 8 was etherified by reaction with propargyl bromide in the presence of sodium hydride (3 eq.) in DMF and led to 10 at 95% yield. The same reaction performed on silyl ether 9 required optimization. The amount of NaH (3.0, 1.2 and 0.9 eq.), the solvent (DMF, acetone or THF), and the reaction duration of deprotonation were screened and the optimal yield of 11 (70%) was obtained in 16 h with 1.2 eq. of NaH and 3.0 eq. of propargyl bromide in THF. Compound 5 was reacted with diethylaminosulfur trifluoride (DAST) for fluorination and the targeted 7-fluoro derivative 6 was obtained at 85% yield. The strategy based on sequential functionalization required the 7-hydroxyl protection prior to its activation with a leaving group. Thus, a silyl protecting group, which is stable under the envisioned experimental conditions and compatible with 4,5-isopropylidene, was selected. The TBDPS ether 7 was obtained at 95% yield via the reaction of 5 with TBDPSCl and imidazole in DMF. The reduction of the methyl ester on compounds 6 and 7 performed by LiAlH4 in THF led, respectively, to 8 and 9 in high yields. The resulting primary hydroxyl of compound 8 was etherified by reaction with propargyl bromide in the presence of sodium hydride (3 eq.) in DMF and led to 10 at 95% yield. The same reaction performed on silyl ether 9 required optimization. The amount of NaH (3.0, 1.2 and 0.9 eq.), the solvent (DMF, Compound 5 was reacted with diethylaminosulfur trifluoride (DAST) for fluorination and the targeted 7-fluoro derivative 6 was obtained at 85% yield. The strategy based on sequential functionalization required the 7-hydroxyl protection prior to its activation with a leaving group. Thus, a silyl protecting group, which is stable under the envisioned experimental conditions and compatible with 4,5-isopropylidene, was selected. The TBDPS ether 7 was obtained at 95% yield via the reaction of 5 with TBDPSCl and imidazole in DMF. The reduction of the methyl ester on compounds 6 and 7 performed by LiAlH4  Considering the advantages of CuAAC [33,34], we next planned to introduce two other propargyl groups on positions 4 and 5 (see numbering on Scheme 1). These two protected alkynes were used for the introduction of two peptide vectors in a subsequent step. The removal of the 4,5-isopropylidene was easily performed on compound 10 by treatment with a TFA/H 2 O mixture and led to compound 12 in a nearly quantitative yield (84%). Looking at the potential lability of TBDPS in acidic medium, attention was required when the reaction was performed on compound 11. Indeed, the use of TFA/H 2 O mixture did not permit us to obtain 13 in a sufficient yield since a significant amount of 4,5,7-deprotected compound was formed (observed by TLC). Taking advantage of our experience in this type of selective deprotection, we opted for AcOH in H 2 O at 80 • C that provided compound 13 at 54% yield. It should be noted that the formation of approximatively 10% of 4,5,7deprotected compound could not be avoided and 15% of compound 11 were still remaining in the crude mixture. This selective deprotection was obviously a crucial point in this synthetic strategy, and compound 13 was obtained in a moderate but sufficient yield to proceed further with the synthesis. After careful purification using a silica gel column chromatography, compounds 12 and 13 were etherified with TIPS-propargyl bromide and NaH in DMF or THF, and the corresponding ethers 14 and 15 were obtained at 94% and 78% yields, respectively.
The cyanine derivative 16 was previously obtained by coupling the commercially available cyanine-5-NHS with the 3-azidopropylamine in DMF (Scheme 2). Cyanine 16 was engaged in CuAAC reaction, and cycloadducts 17 and 18 were obtained in excellent yields after purification using a column chromatography. The temporary TIPS and both TBDPS protecting groups were efficiently removed by tetrabutylammonium fluoride, leading to compounds 19 and 20.

Peptide Functionalization and Coupling
At this stage, two parallel syntheses were carried out for the radiolabeling precursor and the non-radioactive compound coupled with RGD derivatives. c(RGDfK) was previously derivatized on the N-ε-amine lysine side chain with two different NHS-activated spacers bearing a terminal azido group for CuAAC (Scheme 3). A hexyl linker and a PEG 4 linker were chosen as they enable flexibility, and the PEG 4 linker was intended to increase the hydrophilic character of the final construct. The resulting azido cyclic peptides 21 and 22 were reacted with compound 19 in a mixture of ACN/H 2 O (Scheme 3). For CuAAC with two cyclopeptides, particular attention must be paid to the equivalent numbers of reactants. Indeed, catalytic amounts of copper sulfate and sodium ascorbate were not sufficient and 3.0 and 7.5 eq. were, respectively, used to provide cycloadducts in good yields. Compounds 23 and 24 were purified using a size-exclusion chromatography (Sephadex LH20) and were obtained at 42 and 62% yields, respectively, and successfully characterized by HRMS. Their in vitro and in vivo biological properties were then evaluated. The fluorescent monomeric derivative c(RGDfK)-Cy5 25, i.e., the compound linked to the cyanine-5 directly on the N-ε of the lysine residue, was required for in vitro evaluation and was obtained at 59% yield by coupling c(RGDfK) with Cy5-NHS in DMF (Scheme 4).

Precursor of Radiolabeling and 18 F-Radiolabeling
The 7-O-activated compound was the key intermediate for nucleophilic radiolabeling with 18 F. In order to avoid competition with other nucleophilic anions from the reaction mixture, the iodide counter anion of the cyanine indolium was removed and replaced by a triflate group, a non-nucleophilic anion. The anionic metathesis was carried out using an Oasis ® MCX cartridge (Waters, Milford, MA, USA) with a solution of silver triflate (0.2 M) (Scheme 5). The anion exchange was confirmed using a 1 H NMR spectroscopy, revealing some variation in the chemical shifts of polymethine chain protons H-27, H-25, and H-29 of cyanine (δ = 6.91, 6.61, and 6.21 ppm for compound 20, 6.78, 6.29, and 6.24 ppm for compound 26, see ESI for atom numbering). The 19 F NMR also showed the signal of trifluoromethyl group (see ESI). Regarding the synthesis of the labeling precursor, we first planned to carry out the preparation of the 7-trifluoromethylsulfonate derivative. Despite careful attention toward the experimental conditions, the recovery of the expected 7-Otriflate compound was not possible. We moved on to the 7-O-methylsulfonate derivative, which was obtained by reaction with methyl sulfonate anhydride in dichloromethane. The expected mesylate 27 was obtained at 38% yield, which is satisfactory given the tedious and careful column chromatography purification required for cyanine-based compounds.

Precursor of Radiolabeling and 18 F-Radiolabeling
The 7-O-activated compound was the key intermediate for nucleophilic radiolabeling with 18 F. In order to avoid competition with other nucleophilic anions from the reaction mixture, the iodide counter anion of the cyanine indolium was removed and replaced by a triflate group, a non-nucleophilic anion. The anionic metathesis was carried out using an Oasis ® MCX cartridge (Waters, Milford, MA, USA) with a solution of silver triflate (0.2 M) (Scheme 5). The anion exchange was confirmed using a 1 H NMR spectroscopy, revealing some variation in the chemical shifts of polymethine chain protons H-27, H-25, and H-29 of cyanine (δ = 6.91, 6.61, and 6.21 ppm for compound 20, 6.78, 6.29, and 6.24 ppm for compound 26, see ESI for atom numbering). The 19 F NMR also showed the signal of trifluoromethyl group (see ESI). Regarding the synthesis of the labeling precursor, we first planned to carry out the preparation of the 7-trifluoromethylsulfonate derivative. Despite careful attention toward the experimental conditions, the recovery of the expected 7-Otriflate compound was not possible. We moved on to the 7-O-methylsulfonate derivative,

Precursor of Radiolabeling and 18 F-Radiolabeling
The 7-O-activated compound was the key intermediate for nucleophilic radiolabeling with 18 F. In order to avoid competition with other nucleophilic anions from the reaction mixture, the iodide counter anion of the cyanine indolium was removed and replaced by a triflate group, a non-nucleophilic anion. The anionic metathesis was carried out using an Oasis ® MCX cartridge (Waters, Milford, MA, USA) with a solution of silver triflate (0.2 M) (Scheme 5). The anion exchange was confirmed using a 1 H NMR spectroscopy, revealing some variation in the chemical shifts of polymethine chain protons H-27, H-25, and H-29 of cyanine (δ = 6.91, 6.61, and 6.21 ppm for compound 20, 6.78, 6.29, and 6.24 ppm for compound 26, see ESI for atom numbering). The 19 F NMR also showed the signal of trifluoromethyl group (see ESI). Regarding the synthesis of the labeling precursor, we first planned to carry out the preparation of the 7-trifluoromethylsulfonate derivative. Despite careful attention toward the experimental conditions, the recovery of the expected 7-Otriflate compound was not possible. We moved on to the 7-O-methylsulfonate derivative, 18 F-radiobeling of 27 was then investigated using both AllInOne (AIO) and TracerLab FxFN synthesizers with the classical method of radiofluorination using the K[ 18 F]F-K 222 complex (Scheme 5). After the optimization of the K 222 /K 2 CO 3 quantities and ratios, the best conditions were 15 mg/1.3 mg in 8/2 acetonitrile/water (v/v) for AIO and 12 mg/2 mg in 7/3 acetonitrile/water (v/v) for Tracerlab FxFN, respectively, with a 10 min reaction in both cases. The identity of [ 18 F]19 was confirmed using an analytical radio-HPLC by comparison with the non-radioactive compound 19, both having identical retention times (see ESI Figures S1 and S2). The 18 F-radiolabeling of 27 was reproducible using the two different synthesizers, and decay-corrected radiochemical yields (RCY) of 13 and 11% as determined by HPLC analyses were obtained with AIO and TracerLab FxFN, best conditions were 15 mg/1.3 mg in 8/2 acetonitrile/water (v/v) for AIO and 12 mg/2 mg in 7/3 acetonitrile/water (v/v) for Tracerlab FxFN, respectively, with a 10 min reaction in both cases. The identity of [ 18 F]19 was confirmed using an analytical radio-HPLC by comparison with the non-radioactive compound 19, both having identical retention times (see ESI Figures S1 and S2). The 18 F-radiolabeling of 27 was reproducible using the two different synthesizers, and decay-corrected radiochemical yields (RCY) of 13 and 11% as determined by HPLC analyses were obtained with AIO and TracerLab FxFN, respectively. These results establish the proof of concept of direct 18 F-radiolabeling of a cyanine-containing precursor via a [ 18 F]F-C bond formation.

In Vitro Biological Evaluation
Integrins are cell surface receptors involved in many physiological and pathological processes [37,38]. The most important member of this receptor family is the αvβ3 integrin, which is involved in blood vessel formation (angiogenesis) and is overexpressed in several cancer types (melanoma, glioma, ovarian, and breast cancers). Therefore, visualizing αvβ3 expression is obviously of great interest and the development of αvβ3 imaging agents is still a concern in the field of molecular imaging. RGD peptide derivatives and, in particular, cyclic c(RGDfK) show high αvβ3 affinity in vitro and receptor-specific tumor uptake in vivo [39,40].

In Vitro Biological Evaluation
Integrins are cell surface receptors involved in many physiological and pathological processes [37,38]. The most important member of this receptor family is the α v β 3 integrin, which is involved in blood vessel formation (angiogenesis) and is overexpressed in several cancer types (melanoma, glioma, ovarian, and breast cancers). Therefore, visualizing α v β 3 expression is obviously of great interest and the development of α v β 3 imaging agents is still a concern in the field of molecular imaging. RGD peptide derivatives and, in particular, cyclic c(RGDfK) show high α v β 3 affinity in vitro and receptor-specific tumor uptake in vivo [39,40].
The affinity of compounds 23 and 24 for α v β 3 integrin was evaluated in the presence of a coated vitronectine (reference ligand) at different concentrations (1.6 nM-20 µM, solid-phase binding assay), and the IC 50 values were determined ( Figure 2). These data demonstrate a high affinity toward α v β 3 integrin for both compounds (IC 50 of 10 and 16 nM for 23 and 24, respectively). The positive control compound c(RGDfK) showed an IC 50 value of 40 nM, while the fluorescent monomeric reference compound c(RGDfK)-Cy5 (25) showed an IC 50 value of 8542 nM, which is not surprising considering the steric hindrance induced by the cyanine moiety. The negative control compound 19 did not inhibit the binding of α v β 3 integrin to vitronectin. Cellular uptake was evaluated using a confocal microscopy on U-87 MG spheroids after 1, 4, and 24 h of exposure with conjugates 23 and 24 and c(RGDfK)-Cy5 (25) at 1 µM. The fluorescence signal appeared to be stable over time and the distribution of the compounds was homogeneous over all cells and throughout the spheroids ( Figure 3A). These three compounds were localized rapidly in the cell cytoplasm due to receptor internalization after ligand binding. As expected from photophysical properties, the fluorescence signal was higher for compound 24 compared to 23. Compound 23 showed a better cellular uptake probably due to its higher hydrophobicity introduced by hexyl linker. The amount of compounds seemed to decrease slightly at 4 h but stayed relatively stable until 24 h and represented 25.8, 14.7, and 28.6 pmoles/10 6 cells, respectively, for compounds 23, 24 and 25. No significant differences were observed between the different compounds, suggesting that the number of c(RGDfK) did not seem to affect cell incorporation (compare compounds 23 and 24 to compound 25 in Figure 3B).
The affinity of compounds 23 and 24 for αvβ3 integrin was evaluated of a coated vitronectine (reference ligand) at different concentrations (1 solid-phase binding assay), and the IC50 values were determined (Figure demonstrate a high affinity toward αvβ3 integrin for both compounds (IC nM for 23 and 24, respectively). The positive control compound c(RGDfK) value of 40 nM, while the fluorescent monomeric reference compound c(RG showed an IC50 value of 8542 nM, which is not surprising considering the s induced by the cyanine moiety. The negative control compound 19 did binding of αvβ3 integrin to vitronectin. Cellular uptake was evaluated using a confocal microscopy on U-87 after 1, 4, and 24 h of exposure with conjugates 23 and 24 and c(RGDfK)-C The fluorescence signal appeared to be stable over time and the distribut pounds was homogeneous over all cells and throughout the spheroids (Fig three compounds were localized rapidly in the cell cytoplasm due to recep tion after ligand binding. As expected from photophysical properties, th signal was higher for compound 24 compared to 23. Compound 23 showe lar uptake probably due to its higher hydrophobicity introduced by he amount of compounds seemed to decrease slightly at 4 h but stayed relativ 24 h and represented 25.8, 14.7, and 28.6 pmoles/10 6 cells, respectively, for 24 and 25. No significant differences were observed between the differe suggesting that the number of c(RGDfK) did not seem to affect cell incor pare compounds 23 and 24 to compound 25 in Figure 3B).

In Vivo Fluorescence Imaging
In order to evaluate the in vivo biodistribution and tumor uptake of compound 24, which has the best photophysical and solubility properties in comparison to compound 23, mice bearing ectopic or orthotopic brain tumors were imaged in NIRF using a fluorescent small animal imager. Compound 24 showed a more rapid tumor uptake at 1 h post intravenous administration with a maximum fluorescence signal at 4 h (ectopic tumor, Figure 4A) compared to compound 25 uptake. The fluorescence signal of compound 24 decreased slightly after 6 h, but it was significantly high until 24 h; the elimination clearly seems to occur via urinary tract and liver metabolism ( Figure 4B). The healthy brain did not fix either compound 24 or compound 25. For compound 24, the orthotopic tumor was largely identifiable and a high tumor uptake was observed for both tumors with ratios of 40 and 100 between the orthotopic and ectopic brain tumors and the healthy brain, respectively, whereas a lesser tumor uptake was observed for compound 25 ( Figure 4C).
In order to assess the possibility of using compounds 24 or 25 for extemporaneous tumor cell identification during anatomo-pathology analysis, the ectopic tumors were harvested 24 h after administration and observed using confocal imaging ( Figure 5). The whole tumors showed more pronounced staining with compound 24 ( Figure 5A) compared to compound 25 ( Figure 5B). Moreover, compound 24 showed a clearer staining of all tumor cells at high resolution ( Figure 5C) compared to compound 25 ( Figure 5D).
Because they allow the early detection of pathologies, participation in patient care, and provision of theranostic tools, molecular imaging and, more specifically, bimodal imaging are research fields in strong expansion. Currently, the main trend is the development of molecular imaging agents targeting the specific biomarkers of a pathology. This contribution aims at designing and synthesizing a monomolecular [ 18 F]F-C cyanine-containing dual PET/OI imaging probe. The non-radioactive dual probe was conjugated to two RGD derivatives targeting α v β 3 integrin, and the resulting conjugates were evaluated in vitro and in vivo. amount of compounds seemed to decrease slightly at 4 h but stayed relatively stab 24 h and represented 25.8, 14.7, and 28.6 pmoles/10 6 cells, respectively, for compou 24 and 25. No significant differences were observed between the different comp suggesting that the number of c(RGDfK) did not seem to affect cell incorporation pare compounds 23 and 24 to compound 25 in Figure 3B).

In Vivo Fluorescence Imaging
In order to evaluate the in vivo biodistribution and tumor uptake of compound which has the best photophysical and solubility properties in comparison to compo 23, mice bearing ectopic or orthotopic brain tumors were imaged in NIRF using a fluo cent small animal imager. Compound 24 showed a more rapid tumor uptake at 1 h intravenous administration with a maximum fluorescence signal at 4 h (ectopic tu Figure 4A) compared to compound 25 uptake. The fluorescence signal of compoun decreased slightly after 6 h, but it was significantly high until 24 h; the elimination cle seems to occur via urinary tract and liver metabolism ( Figure 4B). The healthy brain not fix either compound 24 or compound 25. For compound 24, the orthotopic tumor largely identifiable and a high tumor uptake was observed for both tumors with rati 40 and 100 between the orthotopic and ectopic brain tumors and the healthy brain, res tively, whereas a lesser tumor uptake was observed for compound 25 ( Figure 4C). In order to assess the possibility of using compounds 24 or 25 for extemporan tumor cell identification during anatomo-pathology analysis, the ectopic tumors w harvested 24 h after administration and observed using confocal imaging ( Figure 5). whole tumors showed more pronounced staining with compound 24 ( Figure 5A) c pared to compound 25 ( Figure 5B). Moreover, compound 24 showed a clearer stainin all tumor cells at high resolution ( Figure 5C) compared to compound 25 ( Figure 5D). Because they allow the early detection of pathologies, participation in patie and provision of theranostic tools, molecular imaging and, more specifically, bimo aging are research fields in strong expansion. Currently, the main trend is the d ment of molecular imaging agents targeting the specific biomarkers of a patholo contribution aims at designing and synthesizing a monomolecular [ 18 F]F-C cyan taining dual PET/OI imaging probe. The non-radioactive dual probe was conjug two RGD derivatives targeting αvβ3 integrin, and the resulting conjugates were ev in vitro and in vivo.
As mentioned in the introduction, the direct radiolabeling of a cyanine-con compound via a [ 18 F]F-C bond formation has not yet been reported, except for one ular example via SNAr [22]. Strategically, if a peptide vector is introduced at an ear in the synthetic sequence, the entire synthesis has to be repeated for any new vecto limits the versatility of the approach [24,23,25]. On the other hand, conjugating tor(s) to a structure already carrying the key elements (i.e., the [ 18 F]F-C cyanine-bas imaging probe) in the last step drastically increases the versatility. Besides, severa must be taken into consideration during the radiosynthesis, such as reaction con (organic solvent, high temperature), automation of the radiosynthesis, and potent tivity of polyene moiety of cyanine [41,42]. These specifications strongly advoca late introduction of the vector in the synthesis scheme as well. In this work, we p for the first time, the synthesis of a [ 18 F]F-C cyanine-based dual PET/OI imagin suitable for a late-stage vector grafting. To face this challenge, we selected a C-g moiety that offers sequential functionalization possibilities thanks to its polyhydro structure. Furthermore, the C-C bond at the anomeric position displays acidic an matic hydrolysis resistance, conferring an improved in vivo stability compared to cosides [43][44][45]. The considered synthetic pathway employed orthogonal click re [33,34] to achieve linkage of the fluorescent entity in the first step and conjugation c(RGDfK) vectors in a second step. According to our previous works on [ 18  As mentioned in the introduction, the direct radiolabeling of a cyanine-containing compound via a [ 18 F]F-C bond formation has not yet been reported, except for one particular example via SNAr [22]. Strategically, if a peptide vector is introduced at an early stage in the synthetic sequence, the entire synthesis has to be repeated for any new vector, which limits the versatility of the approach [23][24][25]. On the other hand, conjugating the vector(s) to a structure already carrying the key elements (i.e., the [ 18 F]F-C cyanine-based dual imaging probe) in the last step drastically increases the versatility. Besides, several factors must be taken into consideration during the radiosynthesis, such as reaction conditions (organic solvent, high temperature), automation of the radiosynthesis, and potential reactivity of polyene moiety of cyanine [41,42]. These specifications strongly advocate for a late introduction of the vector in the synthesis scheme as well. In this work, we propose, for the first time, the synthesis of a [ 18 F]F-C cyanine-based dual PET/OI imaging probe suitable for a late-stage vector grafting. To face this challenge, we selected a C-glycosyl moiety that offers sequential functionalization possibilities thanks to its polyhydroxylated structure. Furthermore, the C-C bond at the anomeric position displays acidic and enzymatic hydrolysis resistance, conferring an improved in vivo stability compared to O-glycosides [43][44][45]. The considered synthetic pathway employed orthogonal click reactions [33,34] to achieve linkage of the fluorescent entity in the first step and conjugation of two c(RGDfK) vectors in a second step. According to our previous works on [ 18 F]F-glycosyl tracers for PET imaging, the introduction of the 18 F is ensured by a nucleophilic substitution on a mesylate derivative introduced on a primary hydroxyl. The 18 F-radiolabelling step was successfully performed in standard fluorine activation conditions (K 222 /K 2 CO 3 ) and decay-corrected RCY of 13% and 11% were obtained using two different synthesizers (AIO and TracerLab FxFN, respectively). This constitutes the proof of concept of the direct radiolabeling of a cyanine-containing compound via [ 18 F]F-C bond formation.
Imaging modalities, such as TEP and OI, require proper detection moieties (radioisotope for PET and fluorescent dye for OI), which must be linked to the targeting ligand. This can induce structural modifications of the vector and affect its binding and, consequently, its affinity for the receptor. Small molecules and peptides are the most impacted by the potential steric hindrance induced by imaging moieties. A way to overcome this limitation is to incorporate linkers long enough to ensure spacing between the ligand and the imaging entities. Another way is to enhance the binding by grafting multiple copies of a ligand on a central core (multivalent effect). Indeed, an increase of receptor binding affinity has previously been observed when more than one RGD peptide is grafted [39,[46][47][48]. In this work, we exploited both the use of long linkers and the grafting of several peptide vectors to enhance binding and tumor uptake. Two c(RGDfK) were, thus, successfully introduced through the two available positions on the central C-glycosidic moiety. This approach appeared fruitful with IC 50 of 10 and 16 nM for dimers 23 and 24, respectively. These values are noticeably below the IC 50 of c(RGDfK)-Cy5 25 (8542 nM) and in the same range of, if not slightly lower than, the one obtained for c(RGDfK), demonstrating that sufficiently long linkers and divalency are effective in enhancing ligand-target interactions. The nature of the two linkers, i.e., hexyl for 23 and (PEG) 4 for 24, did not have a significant impact on the binding. Nevertheless, it seems that due to its hydrophobicity (hexyl linker), compound 23 was internalized by cells in a greater extent than compound 24. This lower internalization was largely compensated by the fluorescence properties of compound 24, enabling the easy imaging of cells by confocal microscopy. Moreover, the fluorescence signal could be followed for up to 24 h for both compounds 23 and 24. The poor solubility of compound 23 in aqueous solution is a pitfall for its in vivo evaluation, explaining the choice of compound 24 for in vivo fluorescence imaging. The ectopic tumor uptake of compound 24 was maximum at 4 h post intravenous administration; this was in contrary to the tumor uptake of c(RGDfK)-Cy5 25, which decreased rapidly after its administration. PEG modification of compound 24 allowed a better tumoral distribution with a probable elimination via urinary tract and liver metabolism [49]. Interestingly, the healthy brain tissue did not fix compound 24, but a high staining of the orthotopic brain tumor was observed after 24 h post administration. All biological evaluations highlighted the efficiency of the dimeric-PEG structure 24 to rapidly accumulate in the orthotopic tumor, with a noticeable uptake and a fluorescence signal 40-fold higher in the tumor compared to the healthy brain.

General Information
The solvents and liquid reagents were purified and dried according to recommended procedures. Cy5-NHS was purchased from CHEMFORASE and Oasis ® MCX cartridges from Waters (Milford, MA, USA). Thin layer chromatography (TLC) analyses were performed using standard procedures on Kieselgel 60F254 plates (Merck, Kenilworth, NJ, USA). The compounds were visualized using UV light (254 nm), with ninhydrin and/or a methanolic solution of sulfuric acid, and charred. Flash column chromatography was performed using a Puriflash (Interchim, Montluçon, France). c(RGDfK) was purchased from Bachem (Budendorf, Switzerland) with >95% purity. The purification of RGD-conjugates was achieved using size-exclusion chromatography on Sephadex LH20 with methanol as the eluent. FTIR spectra were recorded using a Shimadzu IRAffinity-1, ATR PIKE Technologies model GladiAT (cm −1 ) (Cottonwood, WI, USA). Optical rotations were measured using an Anton-Paar MCP 300 polarimeter (Graz, Austria). 1 H, 13 C and 19 F NMR spectra were recorded using a Bruker Avance III (400 MHz, 100.6 MHz, and 376 MHz, respectively, Billerica, MA, USA) on the NMR Platform of the Jean Barriol Institute (Université de Lorraine, Nancy, France). For the 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. The following abbreviations are used for the multiplicity of NMR signals: s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, b = broad signal, and app = apparent multiplicity. Atom numbering used in NMR attribution signals is provided on copies of NMR spectra (see ESI). The given J values refer to apparent multiplicities and do not represent the true coupling constants. High resolution ESI-MS spectra were recorded using a Bruker Daltonics microTOFQ apparatus provided by the mass spectrometry MassLor platform of Université de Lorraine. UV-vis spectra were recorded using a PerkinElmer Lambda1050 spectrophotometer (Waltham, MA, USA), and room temperature fluorescence emission spectra were recorded using a Fluorolog-3 spectrofluorometer from Horiba Scientific (Kyoto, Japan). All the spectroscopic measurements were performed using the PhotoNS spectroscopic platform of the L2CM Laboratory. No-carrier-added fluoride-18 was produced via the 18 O (p,n) 18 F nuclear reaction using a PET Trace cyclotron (GE) or Cyclone-18/9 cyclotron (18 MeV proton beam, IBA). For the PET Trace cyclotron, the bombardment was performed at 10 µA for 5 min to provide about 2 GBq of fluoride-18 delivered as a solution in 18 O-enriched water (1.6 mL). In the case of Cyclone-18/9 cyclotron, the bombardment was performed at 6.2 µA for 14 min to provide about 17 GBq of fluoride-18 delivered as a solution in 18 O-enriched water (2.0 mL) Radiosynthesis was performed using an AIO module from Trasis ® or a TracerLab FxFN from General Electric ® (GE, Boston, MA, USA). Analytical High-Performance Liquid Chromatography (HPLC) analyses were performed using a Waters system (2695eb pump, auto sampler injector and 2998 PDA detector) coupled to a radioHPLC detector (Herm LB500 with NaI from Berthold, Bad Wildbad, Germany) controlled by the Empower Software (Orlando, FL, USA) or using a Waters Alliance 2690 (UV spectrophotometer (Photodiode Array Detector, Waters 996 (Waters)) and a Berthold LB509 radioactivity detector). The analyses were performed using a Luna PFP column (5 µm, 150 × 4.6 mm) from Phenomenex (Torrance, CA, USA) with ACN/H 2 O (v/v 60/40) eluant and 0.1% of TFA at 1 mL/min (Waters system) or at 1.5 mL/min (Waters Alliance 2690). U.V. detection at λ = 650 nm.

3,6-anhydro-2-deoxy-4,5-O-(1-methylethylidene)-7-fluoro-D-ribo-heptanoic acid methyl ester 6
To a solution of 5 (100 mg, 0.40 mmol) in diglyme (3 mL), 110 µL of DAST (2 eq., 0.81 mmol) were added dropwise at 0 • C under an inert atmosphere. The reaction mixture was stirred for 30 min at 0 • C and 1 h 30 at 110 • C. After cooling at room temperature, the mixture was neutralized by the addition of a saturated solution of NaHCO 3 (10 mL), and the solvent was removed under vacuum. The residue was solubilized in water, the aqueous layer was extracted with CH 2 Cl 2 (2 × 20 mL), and the organic layer was dried over MgSO 4 . The solvent was removed under vacuum, and the crude product was purified using a flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 60/40) to afford compound 6. Yield: 85% as a colorless oil, To a solution of compound 5 (1 g, 4.05 mmol) in dry DMF (10 mL), a solution of 1.58 mL of tert-butyldiphenylsilyl chloride (1.5 eq., 6.07 mmol) and 496 mg of imidazole (1.8 eq., 7.29 mmol) in dry DMF (3mL) was added dropwise at 0 • C under an inert atmosphere. The reaction was stirred overnight at room temperature, and the mixture was then diluted with water (50 mL) and extracted with EtOAc (3 × 80 mL). The organic layer was dried over MgSO 4 and filtered, and the solvent was removed under vacuum. The crude product was To a solution of 7 (92 mg, 0.37 mmol, 1.0 eq.) in dry THF (8 mL), 42 mg of LiAlH 4 (1.11 mmol, 3.0 eq.) was added at 0 • C under an inert atmosphere, and the mixture was stirred at room temperature for 3 h. The reaction was quenched with the addition of water, and the mixture was filtered off using a Celite ® pad. The organic solvent was removed under reduced pressure, and the aqueous layer was extracted with CH 2 Cl 2 (3 × 50 mL). The combined organic layers were dried over MgSO 4 , and the solvent was removed under vacuum. The crude product was purified using a flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 50/50) to afford compound 8. Yield: 82% as yellowish oil, To a suspension of NaH 60% in mineral oil (150 mg, 3.91 mmol, 2.0 eq.) in dry DMF (4 mL), a solution of 431 mg of 8 (0.48 mmol, 1.0 eq.) in dry DMF (4 mL) was added at 0 • C under an inert atmosphere. After 1 h, 654 µL of propargyl bromide with 80% in toluen (5.88 mmol, 3.0 eq.) was added, and the mixture was stirred at room temperature for 16 h. The reaction was quenched with the addition of an aqueous saturated solution of NH 4 Cl. The solvent was removed under reduced pressure, and the resultant residue was dissolved in water. The aqueous layer was extracted with CH 2 Cl 2 (3 × 50 mL). The organic layer was dried over MgSO 4 , and the solvent was removed under vacuum. The crude product was purified using a flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 50/50) to afford compound 10 Yield: 95% as yellowish oil. To a suspension of NaH 60% in mineral oil (27 mg, 0.66 mmol, 1.2 eq.) in dry THF (1 mL), a solution of 250 mg of 9 (0.55 mmol, 1.0 eq.) in dry THF (2 mL) was added at 0 • C under an inert atmosphere. After 10 min, 183 µL of propargyl bromide 80% in toluene (1.64 mmol, 3.0 eq.) was added, and the mixture was stirred at room temperature for 6 h. The reaction was quenched with the addition of an aqueous saturated solution of NH 4 Cl. The solvent was removed under reduced pressure, and the residue was dissolved in water. The aqueous layer was extracted with CH 2 Cl 2 (3 × 50 mL). The organic layer was dried over MgSO 4 , and the solvent was removed under vacuum. The crude product was purified using a flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 60/40) to afford compound 11. Yield: 70% as colorless oil,

Compound 13
A solution of 11 (70 mg, 0.14 mmol, 1.0 eq.) in AcOH 80% in water (1.5 mL) was stirred at 80 • C for 4 h. The mixture was cooled at 0 • C, and the reaction wash quenched with the addition of water (5 mL) and solid NaHCO 3 until pH = 7. The aqueous layer was extracted with CH 2 Cl 2 (3 × 10 mL), the combined organic layers were dried over MgSO 4 , and the solvent was removed under vacuum. The crude product was purified using a flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 60/40) to afford compound 13. Yield: 54% colorless oil, To a suspension of NaH 60% in mineral oil (88 mg, 2.20 mmol, 3.0 eq.) in dry DMF (1 mL), a solution of 160 mg of 12 (0.73 mmol, 1.0 eq.) in dry DMF (3 mL) was added at 0 • C under an inert atmosphere. After 45 min, 1.2 g of 3-bromo-1-(triisopropylsilyl)-1propyne (4.38 mmol, 6.0 eq.) was added, and the mixture was stirred at room temperature for 5 h. The reaction was quenched with the addition of an aqueous saturated solution of NH 4 Cl. The solvent was removed under vacuum, and the residue was dissolved in water. The aqueous layer was extracted with CH 2 Cl 2 (3 × 50 mL). The organic layer was dried over MgSO 4 , and the solvent was removed under vacuum. The crude product was purified using a flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 50/50) to afford compound 12. Yield: 94% as yellowish oil, R f = 0.59 (Cycl/EtOAc: To a suspension of NaH 60% in mineral oil (12 mg, 0.29 mmol, 2.2 eq.) in dry THF (1 mL), a solution of 60 mg of 13 (0.13 mmol, 1.0 eq.) in dry THF (1 mL) was added at 0 • C under an inert atmosphere. After 10 min, 218 mg of 3-bromo-1-(triisopropylsilyl)-1-propyne (0.79 mmol, 6.0 eq.) was added, and the mixture was stirred at room temperature for 5 h. The reaction was quenched with the addition of an aqueous saturated solution of NH 4 Cl. The organic solvent was removed under reduced pressure. The aqueous layer was extracted with CH 2 Cl 2 (3 × 50 mL). The organic layer was dried over MgSO 4 , and the solvent was removed under vacuum. The crude product was purified using a flash chromatography on silica gel (eluent: cyclohexane/EtOAc 100/0 to 50/50)

Compound 26
A solution of 20 (9 mg, 9.14 µmol) in ACN (300 µL) was diluted in water (60 mL). The obtained solution was passed through a series of 4 Oasis ® MCX cartridges to trap the compound. The cartridges were washed with 100 mL of water (until pH = 7). The product was eluted with a mixture of NaOTf (0.2 M)/ACN (1/9) (100 mL), and the solvent was complex. Radiolabeling was performed at 95 • C for 10 min in a closed reactor thanks to pinch valves. After cooling at 30 • C, the reaction mixture was pushed into the final product vial. [ 18 F]19 was produced with a decay-corrected radiochemical yield of 13%, as determined using radio-HPLC analyses.

Absorption and Fluorescence Measurements
Absorption spectra were recorded in diluted solution (µM) in an aqueous PBS buffer (0.01 M, pH 7.4). Fluorescence quantum yields Φ fluo were measured in diluted solutions with an absorbance lower than 0.1 using the following equation: where Φ fluo is the fluorescence quantum yield; Grad is the gradient from the plot of integrated fluorescence intensity vs. absorbance; n the refractive index of the solvent; and the subscripts x and ref denote sample and reference. The fluorescence quantum yields of 23 and 24 were measured relative to the commercial Cy5.Cl in PBS for which Φ fluo,ref = 0.13 [36]. The excitation of the reference and sample compounds was performed at the same wavelength (λ ex = 640 nm).

Integrin α v β 3 Binding Assay
The affinity of the compounds for integrin protein was evaluated in terms of the half maximal inhibitory concentration (IC 50 values) using a solid-phase assay, as previously described by Tobias G. Kapp et al. [52]. Briefly, the surface of Maxisorp microplates (NUNC, ThermoFicher Scientific, Paris, France) was coated with 1 µg/mL human vitronectin (Biotechne, Lille, France) overnight at +4 • C. The non-specific sites were blocked with a 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, Saint-Quentin-Fallavier, France) for 1 h at 37 • C. The binding of the compounds was assessed using 2 µg/mL integrin α v β 3 (Bio-techne, Lille, France) in the presence of the serial dilutions of the compounds or the reference compound c(RGDfK) 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 of mouse anti-human CD51/61 (BD Pharmingen, Paris, France) and 1 µg/mL of anti-mouse IgG horseradish peroxidase conjugate antibody (Bio-techne, France). The enzymatic reaction was carried out in the dark by the addition of the enzyme substrate (Bio-techne, France) and stopped Affinities were estimated from 3 independent series performed in duplicate as IC 50 values (i.e., the concentration of the compounds that displaced 50% of integrin binding calculated using one-site fit log-IC 50 non-linear analysis regression using the GraphPad Prism 6 software (v 6.05, USA)).
U-87 MG cells were seeded in 12 multi-well plates at 10 × 10 4 cells/cm 2 and cultivated for 48 h. The old culture medium was discarded, and the cells were exposed to 1-10 µM of compound for 1, 4, or 24 h. After 3 washes, the U-87MG cells were then detached from their support by trypsination, centrifuged for 10 min at 300 g, and suspended in 1 mL of HBSS. An aliquot of cell suspension was taken for numeration (TC20, Biorad. Hercules, CA, USA), and the rest of the cells were centrifuged. Afterwards, the pellet was resuspended in DMSO and sonicated in a water bath for 10 min to lyse the cells and solubilize the Cy5 conjugates. The fluorescence signals of Cy5 in the samples were measured in duplicate at an excitation wavelength of 645/9 nm and an emission wavelength of 680/20 nm (Tecan Infinite M200 Pro spectrofluorometer, Tecan, Männedorf, Switzerland). The fluorescence signals of known concentrations of Cy5-conjugates diluted in DMSO were used to draw a standard calibration curve. The concentration of Cy5 present in the samples (nM) was determined from the linear regression analysis of the standard calibration curve (Equation (1)). The cell count (number of cells per mL) was used to calculate the number of cells present in the samples (number of cell per 850 µL). The results of the cellular uptake were expressed as the concentration of Cy5 (nM) incorporated per one million of cells (Equation (2)).

Animal Models and NIRF Imaging
Animal experiments were performed in accordance with the protocols approved by the French Ministry of Research after a review by the local animal protection and use committee (APAFIS# 30902). For ectopic tumor biodistribution, a U-87 MG tumor model was established by subcutaneous injection of U-87 MG cells (2 × 10 6 in 100 µL of 5% glucose solution) into the front right flank of female athymic nude mice (Charles Rivers, Wilmington, MA, USA). The mice were subjected to imaging studies when the tumor volume reached 500 mm 3 (3-4 wk after inoculation) at 1, 4, 6, and 24 h after the intravenous administration of the compounds 24 and 25 at 0.5 nmoles in 100 µL of water for injections mixed with ethanol (20% EtOH/80% water). For brain tumor imaging, U-87 MG spheroids of approximatively 500 µm diameter were implanted in the cortex of the right hemisphere and imaged 24 h after the administration of 0.5 nmoles of the compound 24 or 25. In the same manner, major organs were harvested and subjected to small animal NIRF imaging (Fluorvivo, Indec Biosystem, Los Altos, CA, USA). A customized filter set (excitation, 510-550 nm; emission, 630-690 nm) was used for data acquisition. All fluorescence images were acquired with a 1-s exposure. The fluorescence intensity of each tissue was measured after subtraction of the background signal from an ROI of the same size and shape drawn over an area without any tissue using imageJ.

Conclusions
In this contribution, we developed a clickable C-glycosyl compound as central scaffold for the multistep synthesis of a cyanine-based dual PET/OI imaging probe. The radiofluorination was successfully performed by nucleophilic substitution of a mesylate leaving group, establishing the first direct radiofluorination of a cyanine-containing compound via the formation of a [ 18 F]F-C bond. Two c(RGDfK) peptides were coupled to the two remaining positions of the glycosyl scaffold, and the resulting dimeric structures retained substantial affinity toward α v β 3 with IC 50 of 10 and 16 nM for compounds 23 and 24, respectively. The original strategy reported in this paper permits the synthesis of a PET/OI dual probe which can be conjugated to any peptide of interest in the last step, allowing the versatility and imaging of a wide range of relevant biological targets. In vivo fluorescence imaging on U-87 MG engrafted nude mice displayed an orthotopic tumor accumulation with a fluorescence signal 40-fold higher in the tumor than in the healthy brain, as well as a high ectopic tumor uptake (ratio of 100 to 1 compared to the healthy brain). The ectopic tumor was resected, and confocal imaging of the tumor sections allowed the identification of tumor cells at high resolution. These preliminary results highlight the potential of compound 24 for glioblastoma cancer diagnosis using PET and image-guided surgery using OI. PET/OI bimodal imaging experiments will be reported in due course, opening a broad scope of clinical applications.