A reliable and automated synthesis of 6-[18F]fluoro-L-DOPA and the clinical application on the imaging of congenital hyperinsulinism of infants

6-[18F]fluoro-L-DOPA is a radiotracer widely used in the diagnosis of a range of diseases, including neuro-oncology, endocrinology, and Parkinson’s disease. To meet the rapidly growing clinical need for this radioactive compound, this study reports an optimized radiosynthesis for this molecule, which proved to be highly reliable and compatible with different types of automated radiosynthesizers. Moreover, with 6-[18F]fluoro-L-DOPA, the PET/CT imaging of a total of 23 patients has been conducted, further demonstrating this radiotracer as a clinically valuable reagent to diagnose congenital hyperinsulinism (CHI) of infants in a non-invasive manner and, more importantly, localize the exact lesion on pancreas. Graphical abstract Graphical abstract


Introduction
Positron emission tomography (PET), which allows imaging the physiological processes of human being in a noninvasive manner, has become a highly important technique for disease diagnosis [1,2]. The implementation of PET imaging relies on radioactive PET tracers, positronemitting radionuclides-labeling bioactive molecules or radiopharmaceuticals. And thus a reliable and automated synthesis of PET tracers has been critical for PET imaging.
6-[ 18 F]fluoro-L-DOPA [3], since its first clinical application in the imaging of dopaminergic pathways of human brain [4], has been used to report a wide variety of diseases [5], including neuro-oncology [6,7], endocrinology [8], and Parkinson's disease [9,10]. As a result, a range of methods have been developed for this valuable radiotracer [11,12]. The first synthesis of radioactive L-DOPA (5-[ 18 F]fluoro-L-DOPA) was accomplished by Firnau and co-workers in 1973 using diazonium fluoroborate compound as precursor [13]. The same lab later reported the synthesis of this radiotracer by the reaction of L-DOPA and [ 18 F]F 2 , which afforded only a mixture of 2-, and 5-[ 18 F]fluoro-L-DOPA] [14]. Benzaldehydes containing a leaving group, such as nitro [15] and quaternary ammonium salt [16,17], have also been employed as radiofluorination precursors and 6-[ 18 F]fluoro-L-DOPA was obtained after a few steps. Nevertheless, the requirement of a multiple-step chemical transformations of radioactive molecules makes these approaches less feasible in automated radiosynthesis. Enantiomerically pure organostannane [18,19], organosilane [20], or organomercury [21], has been reported to give 6-[ 18 F]fluoro-L-DOPA in a two-step process upon treatment with electrophilic [ 18 F]F 2 or [ 18 F]AcOF. Nevertheless, these electrophilic reagents are less accessible and specialized settings are requisite in these processes. Enantiomerically pure diaryliodonium salt, on the other hand, allows the synthesis of 6-[ 18 F]fluoro-L-DOPA with nucleophilic [ 18 F] KF, but in poor radiochemical yield (RCY) [22].
In 2014, the Gouverneur lab developed a coppermediated radiofluorination of BPin precursor with [ 18 F]KF as fluorination source [23]. On the basis of this approach, 6-[ 18 F]fluoro-L-DOPA could be readily synthesized in two steps from precursor 1a (Scheme 1) [23,24]. Soon after that, aryl-and vinylboronicacids were reported to be compatible with the copper-mediated radiofluorination [25]. In spite of these tremendous progresses, to meet the rapidly increasing clinical need for 6-[ 18 F]fluoro-L-DOPA in oncology, neurology, and endocrinology, a highly reliable, time-saving, and automated synthesizer-compatible radiosynthetic approach for this tracer is still in high demand. Herein, we report a copper-promoted automated synthesis of 6-[ 18 F]fluoro-L-DOPA with high reliability and, more importantly, the clinical applications of this radiotracer in the diagnosis of congenital hyperinsulinism (CHI) of infants, which, to the best of our knowledge, is the first clinical investigation of 6-[ 18 F]fluoro-L-DOPA in the diagnosis of CHI in China.

Results and discussion
We commenced our investigation by testing the coppermediated radiofluorination of BPin-substituted L-DOPA (1a) [23]. Following this protocol, we managed to obtain clinically useful amount of 6-[ 18 F]fluoro-L-DOPA, though in lower radiochemical yield compared to original study. Nevertheless, the use of highly corrosive hydroiodic acid (57%) in the final deprotection reaction resulted in significant damage on the sealing ring of automated synthesizer (Fig. 1a); replacement of this part was needed after a few attempts of syntheses. Moreover, the injection loop of HPLC was blocked occasionally during the purification of this radioactive molecule with RP-HPLC. We reasoned this is likely due to the formation of water-insoluble iodine during the final deprotection, which might be resulted from the oxidation of hydroiodic acid by oxygen or other oxidants. As the radiosynthesis of tracer is a highly timesensitive process, the frequent occurrence of these issues significantly jeopardizes the reliability of this 18 F-labeling approach.
To tackle these challenges, we then moved to employ BPin-substituted L-DOPA 1b as precursor, in which methoxymethyl (MOM, R 1 ) and tert-butyl (R 2 ) were selected as protecting groups. During the course of our study, a similar synthetic approach has been reported [26,27]. We conceived the use of this more acid-sensitive precursor should allow the final deprotection using hydrochloric acid, instead of problematic hydroiodic acid (Scheme 1).
As shown in Scheme 2, precursor 1b was readily synthesized from commercially available L-DOPA by a 6-step process [28]. With this precursor, we then conducted the standard copper-mediated radiofluorination and resulted in the 18   Nonetheless, further testing of this copper-mediated protocol revealed the synthetic yields fluctuated greatly, ranging from 1.7 to 8.7% RCY (decay uncorrected, Table 1). This promoted us to conduct further investigations to improve this radiosynthetic process. We soon realized the fluctuation of yield might associate with the salt in the eluent for 18 F -. As a result, we replaced potassium carbonate with potassium oxalate in the eluent for 18 Fand the yields were boosted to 13.0 ± 3.3% RCY (n = 10, decay uncorrected, Table 1). The whole process, including radiosynthesis and purification, took~85 min and, most of the time, the yields were higher than 10% (RCY, decay uncorrected). Upon analysis by HPLC (Fig. 2), the radiochemical purity of 6-[ 18 F]fluoro-L-DOPA was higher than 99%, which meets the clinical need for this important PET radiotracer.
To further assess the compatibility of this radioflurination protocol, we then examined it on two types of commercial automated radiosynthesizers: RNplus (Synthra GmbH Company, Germany) and PET-MF-2V-IT-1 (Beijing PET Technology Co., Ltd, China) (Fig. 3). After a number of tests, both synthesizers gave desired radiotracer in comparable yields, demonstrating the high compatibility of this radiosynthetic method.
CHI, the inappropriate secretion of insulin by the pancreatic β-cells, is among the major causes for severe hypoglycemia in infants [29]. Rapid diagnosis of CHI is of high importance because the presence of abnormal level of insulin may lead to seizure and significant brain damage or even death [30]. PET imaging with 6-[ 18 F]fluoro-L-DOPA as radiotracer has been reported as an important approach to diagnose CHI with high accuracy [31][32][33]. Having confirmed purity of 6-[ 18 F]fluoro-L-DOPA and the reliability of this automated synthesis protocol, we next turned our attention to the diagnosis of CHI in infants or young kids using this radiotracer.
Upon approval by the institutional review board of Huashan Hospital (HIRB), Fudan University, the first patient tested with 6-[ 18 F]fluoro-L-DOPA is an infant girl at the age of 9 months, who had repeated episodes of hypoglycemia since birth and a significant increase in serum insulin (25.1 IU/mL). However, neither magnetic resonance imaging nor computed tomography (CT) scan found the exact lesions on the patient (Fig. 4). The patient was thus administered with 1.2 mCi of 6-[ 18 F]fluoro-L-DOPA and scanned with PET/CT at 60 min' post-injection. As shown in Fig. 4, we observed strong radiation signal from the head of patient's pancreas, indicating significantly higher uptake of L-DOPA by these pancreatic β-cells. On the basis of these data, this patient was diagnosed to have focal forms of CHI (FoCHI). Fortunately, upon selective resection of pancreatic tissue (2 cm) proximal to the uncinated process by laparoscopic surgery, the patient recovered to normal level of blood glucose at the second day after surgery. Encouraged by these results, a total of 23 children (13 boys and 10 girls) diagnosed with CHI were tested with 6-[ 18 F] fluoro-L-DOPA. These patients were at an average age of 15.4 ± 21.3 months, ranging from 2 to 78 months ( Table 2). All of these patients under fasting state showed: (1) insulin was still secreted abnormally even when the blood glucose was lower than 2.6 mmol/L; (2) the blood glucose increased more than 1.5 mmol/L in the islet glucagon provocation test; (3) the adrenocortical hormone, growth hormone, and thyroid function were at normal level at hypoglycemia; (4) the tests of blood-and urine-ketone were negative; (5) the analysis of blood by LC-MS and urine by GC-MS were negative. Upon injection of 6-[ 18 F]fluoro-L-DOPA, all of these children showed strong radiation signal at pancreas in PET/CT images. As summarized in Table 1

General information
Unless otherwise stated, all chemicals were obtained from commercial sources and used without further purification. The 1 H and 13 C NMR spectra were taken on Bruker nuclear magnetic resonance spectrometer. Chemical shifts are reported as δ in units of parts per million (ppm) relative to internal standard ( 1 H NMR: SiMe 4 = 0.00 ppm). Data for 1 H NMR spectra are reported as follows: chemical shifts are reported as δ in units of parts per million (ppm) relative to tetramethylsilane (δ = 0, s); multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), or br (broadened); coupling constants are reported as a J value in Hertz (Hz); the number of protons (n) for a given resonance is indicated nH, and based on the spectral integration values. Highperformance liquid chromatography (HPLC) analysis and purification was performed on a waters HPLC system equipped with a SPD UV detector, a LC pump system, and a CBM BUS module. A Lablogic Scan-RAM radio-HPLC detector was used for the radioactive signal. Water containing 0.1% acetic acid/sodium acetate and 0.2 g/L ascorbic acid) was filtered before use as HPLC mobile phase.

Synthesis of precursor 1b
tert-Butyl (S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate (2) Under argon atmosphere, to a solution of L-DOPA (2.96 g, 15 mmol) in tert-butyl acetate (30 mL, 225 mmol) at 0 o C was added HClO 4 (70% in H 2 O, 1.9 mL, 22 mmol). The resulting mixture were stirred overnight, allowing the reaction temperature raise to room temperature. Upon completion of the reaction, water was added and the pH of solution was adjusted to 8 with a 10% aqueous solution of K 2 CO 3. The organic phase was separated and the aqueous phase was extracted with DCM (60 mL × 3). The combined organic layers were dried over Na 2 SO 4 and purified by chromatography on a silica gel column to afford titled compound as a yellow liquid (1.62 g, 42%). 1

Radiosynthesis of 6-[ 18 F]fluoro-L-DOPA
Noncarrier-added [ 18 F]fluoride was obtained via the 18 O (p,n) 18 F nuclear reaction on a RDS111 cyclotron using enriched H 2 18 O. A QMA cartridge was eluted to a reaction vessel with an aqueous solution of Kryptofix 222 and K 2 C 2 O 2 and the solvent was dried azeotropically at 110°C under N 2 . Acetonitrile (anhydrous, 2 mL) was added and dried at 110°C under N 2 . A solution of 1b (20 mg) and Cu(OTf) 2 (py) 4 (20 mg) in DMF (0.8 mL) were added and the mixture were heated to 120°C for 20 min. Then the reaction mixture were cooled down and diluted with water (8 mL). The resulting mixture were transfer to a C18 cartridge and washed with water (8 mL). The compounds in the C18 cartridge were thus eluted with acetone (3 mL) to another reaction vessel and the volatile solvent was removed by heating to 110°C under N 2 . HCl (aq. 6 M) was then added and the reaction solution was heated to 120°C for 20 min. Then was reaction mixture was cooled down and diluted with a NaOH (aq. 0.1 M, 5 mL). 6-[ 18 F]fluoro-L-DOPA was obtained after purification by HPLC on a C18 column with water containing 0.1% acetic acid/sodium acetate and 0.2 g/L ascorbic acid as eluent (flow rate = 5 mL/ min, tR = 8.9 min).

PET/CT scanning
This study was approved by the Institutional Review Board of Huashan Hospital (HIRB), Fudan University, China. All patients were administered intravenously 0.08-0.16 mCi/kg of [ 18 F]fluoro-L-DOPA was administered intravenously. A 10 min/bed abdominal static emission scan was acquired 60-70 min after injection with a PET/CT scanner (Siemens Biograph 64 HD PET/CT, Siemens, Germany). Attenuation correction was performed using a low-dose CT (30-40 mAs, 120 kV, Acq. 32 × 1.2 mm) before the emission scan. Following corrections for scatter, dead time, and random coincidences, PET images were reconstructed by TrueX+TOF with 4 iterations and 21 subsets, a Gaussian Filter (FWHM 4.0 mm).