Light-Triggered Radiochemical Synthesis: A Novel 18F-Labelling Strategy Using Photoinducible Click Reaction to Prepare PET Imaging Probes

Novel probe development for positron emission tomography (PET) is leading to expanding the scope of molecular imaging. To begin responding to challenges, several biomaterials such as natural products and small molecules, peptides, engineered proteins including affibodies, and antibodies have been used in the development of targeted molecular imaging probes. To prepare radiotracers, a few bioactive materials are unique challenges to radiolabelling because of their complex structure, poor stability, poor solubility in aqueous or chemical organic solutions, and sensitivity to temperature and nonphysiological pH. To overcome these challenges, we developed a new radiolabelling strategy based on photoactivated 1,3-dipolar cycloaddition between alkene dipolarophile and tetrazole moiety containing compounds. Herein, we describe a light-triggered radiochemical synthesis via photoactivated click reaction to prepare 18F-radiolabelled PET tracers using small molecular and RGD peptide.


Introduction
Molecular imaging probes provide better understanding of fundamental pathways to monitor biochemical changes in vivo.
ey are important for diagnosis, monitoring of therapeutic response, and drug development [1,2]. PET is an attractive nuclear medicine technique that serves as a noninvasive and functional imaging modality at picomolar levels in vivo with excellent sensitivity based on positron-emitting radionuclide [3,4]. PET scan information can be used to assess biological processes in vivo during early stages of various diseases, including cancer, heart disease, and dementia in Alzheimer's disease and Parkinson's disease. It is also important to assess response to chemotherapy or radiotherapy in various malignancies [5,6]. Several radiopharmaceuticals targeting specific diseases have been developed. Among various PET tracers, FDG (2-deoxy-2-[ 18 F]fluoro-D-glucose) is the most commonly used one in nuclear medicine and molecular cellular biology. FDG was discovered approximately 40 years ago [7]. It has led to a new medical paradigm involving more accurate diagnosis via functional information through quantitative analysis in fields of oncology, neuroscience, and cardiology. Several studies on 18 F-radiolabelled targeting radiotracers for disease monitoring via PET images have been reported. Fluorine-18 has a relatively long half-life (T 1/2 � 109.8 min) and low energy (0.635 MeV) that permits PET imaging protocols with a duration up to 6 h and short positron linear range in tissue due to low positron energy, resulting in high sensitivity in PET imaging [8,9]. However, 18 F-incorporation into biotargeting vectors can be challenging because it must be performed rapidly and efficiently under mild radiolabelling conditions due to short half-life of the radioisotope and regioselectivity labelling of high specificity with acceptable radiochemical yield [10]. To overcome these obstacles, 18 F-radiolabelling strategies using 18 F-prosthetic groups such as N-succinimidyl-4-[ 18 [16,17], and N-(4-[ 18 F]fluorobenzyl)-2-bromoacetamide ([ 18 F]FBBA) [18,19] have been introduced for labelling of amine and sulfhydryl functionalities of sensitive biomolecules, including small molecules, peptides, and proteins. Click chemistry, copper (I)-catalyzed Huisgen 1,3-dipolar cycloaddition, is still one of the attractive approaches to prepare targeted molecular imaging probes for PET by radiolabelling with small molecules, peptides, and proteins. A number of novel radiotracers have been reported through the click chemistry, including copper-free approaches to avoid toxicity to humans who are susceptible to even low levels of copper [20][21][22][23][24][25][26][27]. Recently, Lin and coworkers have reported that advanced photoinducible 1,3-dipolar cycloaddition reactions show extremely rapid reaction rate and high regioselectivity of desired product without opposite regioisomer under photoactivated mild condition [28][29][30]. is has stimulated efforts to further develop 18 F-radiolabelling strategy to create a new labelling platform using photoactivation by UV radiation. Herein, we report a new strategy that utilizes photoactivated click chemistry between 18 F-labelled terminal alkene dipolarophile and tetrazole moiety compound under radiation using UV light source for a biocompatible and mild reaction with significantly high radiochemical yield and molar activity of desired radiolabelled product for PET imaging.

Results and Discussion
First, to determine whether photoinducible click reaction with radiolabelled compound could be used as a novel approach to prepare radiopharmaceuticals, we examined photoactivated radiolabelling between 18 F-radiolabelled compound [ 18 F]2 and 2,5-diaryl tetrazole compound 3 under mild condition using handheld 302 nm UV lamp. For feasibility study of radiochemical synthesis using photoinducible reaction, we chose a 2-(allyloxy)ethanol as a terminal alkene moiety. It is commercially available. It readily undergoes a catalyst-free cycloaddition reaction with photoinduced nitrile imine from tetrazole compound. To prepare 18 F-radiolabelled compound [ 18 F]2, trimethylammonium triflate precursor 1 was prepared from 4-(dimethylamino)benzoyl chloride and conjugated with 2-(allyloxy)ethanol followed by conversion of the dimethylamino functional group using methyl triflate at room temperature. Synthesis of 2,5-diaryl tetrazole compound 3 was performed using a previously reported procedure [11]. Radiolabelling of precursor 1 was carried out with K[ 18 (Figure 1). Increasing irradiation time to 5-30 min of 302 nm UV lamp resulted in the maximum radiochemical yield at room temperature. In order to investigate the radiochemical yield with reaction time, the best result was obtained at 30 min under the same reaction condition with the time difference of 5 to 30 min. Heating conditions were not considered for the application of temperature-sensitive proteins or peptides. Results indicated that the radiochemical synthesis through photoinducible reaction could be performed under mild conditions, which was possible for both large and small molecules. To evaluate the wavelength effect of light source, we carried out optimized experiment with tetrazole compound 3 and [ 18 F]2 under irradiation of different light sources such as halogen and red LED (720 nm) at room temperature. e desired product [ 18 F]4 was obtained when 302 nm UV light was employed. However, no reaction was observed when halogen or red LED was employed ( Figure 2). Of various light sources, only 302 nm UV led to a good conversion to intermediate nitrile imine when 2,5-diaryl tetrazole compound 3 was used. ese results demonstrated that photoactivated click reaction between 2-(allyloxy)ethyl-4-[ 18 F] fluorobenzoate [ 18 F]2 and nitrile imine was efficient under irradiation with 302 nm UV. Of various factors investigated for photoactivated click reaction, UV wavelength was found to be the most important factor to synthesize the desired product. We aimed to develop water-soluble mass materials such as peptides or proteins as imaging agents. Hence, we subsequently applied the method to radiolabel cyclic RGD peptide and evaluated tumor targeting ability of integrin α v β 3 as a molecular imaging probe. Integrin α v β 3 is associated with angiogenic endothelial as well as tumor cells, including cancers of the prostate, skin, ovary, kidney, lung, and breast. RGD peptides can specifically and strongly bind to integrin α v β 3 [31,32]. Cyclic RGDyK peptide was selected as targeting material due to its high binding affinity to tumor cells, small size peptide, and available description in the literature on tumor studies. We performed conjugation with 2,5-diaryl tetrazole benzoic acid (5) to form 2,5-diaryl tetrazole-RGD (7) by reaction of NH 2 -Lys-RGD with N-hydroxysuccinimide active ester of 2,5-diaryl tetrazole (Scheme 2). It was purified with HPLC. For radiochemical synthesis of the desired 18 F-labelled RGD peptide ([ 18 F]8), photoactivated click reaction was performed between 2,5-diaryl tetrazole-RGD peptide (7) and 2-(allyloxy)ethyl-4-[ 18 F]fluorobenzoate ([ 18 F]4) using 302 nm UV lamp for irradiation at room temperature. After 20 min of reaction, the crude mixture was purified with HPLC.
Identity of the radiolabelled product ([ 18 F]8) was confirmed by HPLC retention time after coinjection with authentic nonradioactive compound 8. Radiochemical synthesis of [ 18 F]8 was successfully carried out by photoactivation. Radiochemical yield from [ 18 F]fluoride was 10-12% (n � 4) by two-step onepot reaction. e desired product showed excellent purity with adequate molar activities (20-72 GBq/μmol) in a total synthesis time of 95 min (including HPLC purification and reformulation). Based on these results, introducing a 2-(allyloxy)ethyl-4-[ 18 F]fluorobenzoate ([ 18 F]2) tag into peptide is suitable and effective due to the use of aqueous reaction media at room temperature and high chemoselectivity without requiring toxic metal catalyst. Biologically, the uptake of [ 18 F]8 by U87MG tumor cell was increased in a time-dependent manner, plateauing between 60 and 120 min. Furthermore, U87MG cell blocking study using nonradiolabelled c(RGDyK) peptide showed cell uptake inhibition indicative of specific binding of [ 18 F]8 to integrin α v β 3 expressed in U87MG glioblastoma cells ( Figure 3).
In vivo PET imaging following intravenous injection of [ 18 F]8 (4.66 MBq tail-vein injected) to RR1022 tumor-bearing SD rats showed tumor uptake of [ 18 F]8 with renal clearance of 18 F-activity and high tumor-to-background contrast (tumor/muscle � 53.5 and 76.2 at 1 h and 2 h, resp.). No significant defluorination from [ 18 F]8 uptake at bone was observed up to 2 h after injection. Furthermore, inhibition study showed that the excess amount (10 mg/kg) of c(RGDyK) peptide significantly blocked [ 18 F]8 uptake in the tumor (tumor/muscle � 8.2 and 6.3 at 1 h and 2 h, resp.) ( Figure 4). ese in vivo imaging studies indicated that 18 F-labelled RGD peptide via photoinduced 1,3-dipolar cycloaddition could successfully visualize tumor in vivo through integrin α v β 3 .

Conclusion
Our results suggest that 18 F-labelled RGD([ 18 F]8) radiotracer is useful for monitoring tumor response in angiogenesis research. e development of novel radiolabelling method for diagnosis of various diseases including cancer has benefit for mild conditions as an important approach to obtain accurate imaging. Photoinduced 1,3-dipolar cycloaddition using 18 Fradioisotope is an efficient radiolabelling strategy to prepare molecular imaging probes. It could be applied as a bioorthogonal approach for chemical modification in biomedical research. Further study is required on the application of such photoinducible radiolabelling strategy based on its high availability and excellent chemoselectivity.

Synthesis of 2-(Allyloxy)ethyl 4-(dimethylmino)benzoate.
2-Allyloxyethanol (2.0 mL, 18.7 mmol) was added to 4-N,Ndimethylaminobenzoyl chloride (0.5 g, 2.72 mmol) and triethylamine (0.76 mL, 5.44 mmol) in 10 mL of dichloromethane in a flame-dried flask with nitrogen stream. e reaction mixture was stirred for two hours at room temperature. e reaction mixture was then diluted with 50 mL of ethyl acetate and 30 ml of water. e product of organic layer was washed twice with 30 mL of water, and the organic layer was washed with 50 mL of brine. e organic layer was dried over sodium sulfate, evaporated, and purified with flash column chromatography (ethyl acetate : hexane � 1 : 5) to obtain a yellow oil product with yield of 55% (373.7 mg). 1 (1). To a solution of 2-(allyloxy) ethyl 4-(dimethylamino)benzoate (0.3 g, 1.20 mmol) dissolved in 2 mL of dichloromethane, methyl trifluoromethanesulfonate (0.27 mL, 2.40 mmol) was added. e mixture was stirred for two hours with nitrogen stream at room temperature. e reaction mixture was evaporated, and the resulting crude mixture was dissolved in 0.5 mL of ethanol. e product was crystallized with 100 mL of diethyl ether and dried to obtain a compound with a yield of 38% (122 mg). 1 18 F-labelled desired compound showed a yield of 79% on radio-TLC using 1 : 5 mixture of ethyl acetate-hexane as developing solvent. We used crude mixture of [ 18 F]2 in the next step for photoinducible click reaction without purification.

Synthesis of 2-(Allyloxy)ethyl 4-Fluorobenzoate (2).
Synthesis of 2-(allyloxy)ethyl 4-fluorobenzoate was carried out according to the published method of Vaidyanathan et al. [33] with slight modifications. Briefly, 2-allyloxyethanol (0.76 ml, 0.71 mmol) was added to a mixture of 4-fluorobenzoic acid (0.1 g, 0.71 mmol), DCC (0.15 g, 0.71 mmol), and DMAP (0.6 mg, 8.0 mmol) in 4 mL of ethyl acetate with a N 2 stream in a flask dried with a heat gun. e reaction mixture was stirred at room temperature overnight, and a white precipitate was filtrated out. After removing the precipitate, the residual reaction mixture was evaporated and purified by flash column chromatography (ethyl acetate : hexane � 1 : 5), resulting in a colorless oil with a yield of 42% (67.6 mg). 1H NMR (600 MHz, CDCl 3 ) data were δ (ppm) � 8.10 (m, 2H), 7.13 (t, J � 8.6 Hz, 2H), 5 (3). For the preparation of phenylsulfonylhydrazone [29], p-anisaldehyde (0.68 g, 5 mmol) was dissolved in 50 mL of ethanol and mixed with benzensulfonylhydrazide (0.86 g, 25 mmol). e mixture was stirred at room temperature for  30 min. After addition of 100 mL water, a white precipitate formed. It was separated with a filter. Diazonium salt solution was prepared by adding NaNO 2 (0.35 g, 5 mmol) into 2 mL of water. It was dropped into cooled aniline (0.47 g, 5.0 mmol) dissolved in 8 mL of water/ethanol (1 : 1) and 1.3 mL of concentrated hydrochloric acid (∼36%). Phenylsulfonylhydrazone was dissolved in 30 mL of pyridine. Diazonium salt was then added dropwise with stirring at −10°C. A red precipitate formed after addition of 250 mL of 3NHCl. e precipitate was then collected and extracted with chloroform and water. e organic layer was dried and subjected to flash column chromatography on a silica gel (dichloromethane : ethyl acetate � 50 : 1). A red solid was obtained with a yield of 30% (382 mg).1H NMR (600 MHz, CDCl 3 ) data were δ (ppm)  (4). Photoinducible 1,3-dipolar cycloaddition was performed between 2 (0.1 g, 0.4 mmol) and 3 (0.18 g, 0.79 mmol) in 16 mL of mixture of acetonitrile/PBS (50/50). e reaction mixture was irradiated with 302 nm UV lamp for 5 min, 10 min, 20 min, 30 min, 1 h, and 2 h with stirring at room temperature. After that, the mixture was extracted with chloroform and water. e organic layer was then dried. e crude mixture was then purified by flash column chromatography on silica gel (dichloromethane : ethyl acetate � 50 :1) to obtain a product with a yield of 63% (112.  e reaction mixture was irradiated with 302 nm UV lamp for 30 min with stirring. For purification, the reaction mixture was immediately loaded into RP-HPLC (A � 0.1% TFA in water/B � 0.1% TFA in acetonitrile, 254 nm, 3.0 mL/min) followed by gradient purification (isocratic flow with 10% B for 5 min, gradient increase from 10% → 100% B for 25 min, and maintaining the flow with 100% B for 10 min). Retention time of the desired compound was 27 min. RP-HPLC was performed for the collected peak for identification using nonlabelled standard compound. Decay-corrected radiochemical yield of [ 18 F]4 was 36% including HPLC purification and synthesis time was 58 min.

Synthesis of 4-(2-Phenyl-2H-tetrazol-5-yl)benzoate (5).
Compound 5 was prepared by a previously reported procedure [29]. To a flask containing compound 4-formylbenzoic acid (2.25 g, 15.0 mmol), ethanol (50 mL) was added. Benzoylsulfonohydrazide (2.58 g, 75.0 mmol) was added to the above solution. A white precipitate formed after addition of 150 mL water. It was collected in a funnel. e white solid was dissolved in 90 mL pyridine to give solution A. A solution of NaNO 2 (1.04 g, 15.0 mmol) in 6 mL water was added dropwise to a cooled mixture of aniline (1.40 g, 15.0 mmol) dissolved in 24 mL water-ethanol (1 : 1) and 4 mL concentrated HCl to give solution B. Solution B was added slowly to solution A cooled with an ice bath. e reaction mixture was then extracted with ethyl acetate (3 × 30 mL (6). N-hydroxysuccinimide (NHS, 86.5 mg, 0.75 mmol) was added to a mixture of 1-ethyl-3-3-dimethylaminopropylcarbodiimide (EDC, 116.7 mg, 0.75 mmol) and 4-(2-phenyl-2H-tetrazole-5-yl)-benzoic acid (100 mg, 0.38 mmol) in 5 mL acetonitrile followed by incubation at room temperature overnight with a stream of N 2 gas. e reaction mixture was then diluted with 100 mL of CH 2 Cl 2 and water. e organic layer was washed three times with 100 mL of water followed by wash with 100 mL of brine once. e organic layer was dried over sodium sulfate and then evaporated. e resulting crude mixture was purified with flash column chromatography (ethyl acetate : hexane � 1 : 1). An orange powder was obtained with a yield of 21.9% (30 mg (7). Cyclic Arg-Gly-Asp-D-Tyr-Lys (cRGDyK, 5.8 mg, 0.006 mmol) was Contrast Media & Molecular Imaging added to a mixture of 6 (5 mg, 0.01 mmol) and N, N′diisopropylethylamine -(DIPEA, 1.8 mg, 0.01 mmol) in 1 mL of dimethylformamide (DMF) followed by incubation at room temperature for two hours with a stream of N 2 gas. e mixture was purified using RP-HPLC. Flow rate was set at 2.5 ml/min. e mobile phase consisted of solvent A (0.1% trifluoroacetic acid in water) and solvent B (0.1% trifluoroacetic acid in acetonitrile). Gradient details were as follows: 4.13. Tumor Cell Uptake Assay. U87MG human glioma cells were maintained and subcultured every other day in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in 5% CO 2 and 95% air environment. e cells were seeded into 24-well plates at density of 5 × 10 4 cells/well and cultured overnight. e cells were rinsed once with PBS followed by addition of [ 18 F]8 (∼0.33 MBq/well) or RGD (100 μM/well) to cultured wells in quadruplicate. After incubation at 37°C for 5, 15, 30, 60, and 120 min, cells were rinsed twice with cold PBS and harvested after treatment with TrypLE. e cells were collected in measurement tubes for counting. Finally, radioactivity of the cells was measured using a gamma counter. All experiments were performed in quadruplicate. Results are expressed as mean ± SD. 4.14. In Vivo Experiments 4.14.1. Tumor Models. All experimental protocols with animals were performed in compliance with the policies and procedures of the Institutional Animal Care and Use Committee of Chonbuk National University (Jeonju, Korea). Four male SD rats were purchased from Orient-Bio (Seoul, Korea) at 13 weeks of age. ey were injected subcutaneously (s.c.) in the right flank with 1 × 10 7 RR1022 fibrosarcoma cells suspended in 150 µL RPMI 1640 medium. When tumors reached 0.8-1.0 cm in diameter (7 d after inoculation), rats were used for microPET imaging experiment.

MicroPET Studies.
MicroPET scans and image data analysis were performed using a Biograph TruePoint 40 PET/CT scanner (Siemens Medical Solutions, Knoxville, TN, USA). Rat bearing RR1022 tumor was tail-vein injected with 5.1 MBq of [ 18 F]8 under zoletil anesthesia (mg/kg). Ten-minute static PET images were then acquired at two time points (1 h and 2 h) after injection. CT scan was obtained first by a continuous spiral technique (120 kVp, 200 Ma, 0.5 s rotation time). A PET scan was then acquired in 3-dimensional mode at 15 minutes per bed position. Obtained PET data were reconstructed iteratively using an ordered-subset expectation maximization algorithm. Initial CT data were used for attenuation correction. For receptor-blocking experiment, c(RGDyK) (10 mg/kg) was coinjected with 5.4 MBq of [ 18 F]8 to RR1022 tumor rat. At 1 h and 2 h after injection, ten-minute static microPET scans were acquired. Assessment of tracer distribution in tumor tissue was expressed as tumor-to-muscle (T/M) ratio, dividing the mean activity within the ROI of the tumor by the mean activity within thigh muscle ROI.

Conflicts of Interest
e authors declare no conflicts of interest.