{"paper_id":"4dc4a78d-40bc-4d24-83c5-ebe91c450f72","body_text":"Automated Radiosynthesis of 2-[18F]BPA for PET-based Planning of Boron Neutron Capture Therapy (BNCT): Rational Precursor Design, Radiofluorination, and Characterization of Methodology | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Automated Radiosynthesis of 2-[18F]BPA for PET-based Planning of Boron Neutron Capture Therapy (BNCT): Rational Precursor Design, Radiofluorination, and Characterization of Methodology Vincenzo Paolillo, Cong-Dat Pham, Robert Ta, Dimitra K. Georgiou, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8289343/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Mar, 2026 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted 5 You are reading this latest preprint version Abstract Background: Boron neutron capture therapy relies on the selective accumulation of boron-containing compounds in tumor tissue, making accurate quantification of boron distribution essential for effective treatment planning. The amino acid analog boronophenylalanine is widely used as a boron delivery agent, yet direct assessment of its biodistribution remains challenging. A fluorine-18 labeled analog, 2-fluoro-boronophenylalanine, offers the potential to visualize and quantify uptake through positron emission tomography. However, reported radiosynthetic methods often suffer from low radiochemical yield, complex workflows, and limited compatibility with automated production platforms. The aim of this study was to design a stable precursor suitable for nucleophilic fluorination, develop a fully automated single-reactor radiosynthesis, and characterize the resulting tracer to support both preclinical use and future clinical translation. Results: A rationally protected precursor incorporating tert-butyloxycarbonyl and pinacol ester groups was synthesized and isolated with high chemical and enantiomeric purity. Using this precursor, an automated single-pot radiosynthesis was implemented on a commercial synthesis module employing copper-mediated nucleophilic fluorination followed by acidic hydrolysis. Across eight production runs, the method yielded 2-fluoro-boronophenylalanine with non-decay-corrected radiochemical yields of 3–5 percent and a total synthesis time of approximately 60–70 minutes. Radiochemical purity consistently exceeded 98 percent, and the molar activity at the end of synthesis ranged from 85 to 120 gigabecquerels per micromole. The final formulation remained chemically and radiochemically stable for at least four hours at room temperature, and residual solvent levels were within accepted safety limits. Analytical and chiral chromatographic assessments confirmed product identity, purity, and retention of stereochemical configuration. Conclusions: This study establishes a practical and fully automated radiosynthetic approach for producing 2-fluoro-boronophenylalanine using a single-reactor nucleophilic fluorination strategy. The method overcomes key limitations of electrophilic fluorination and multi-pot workflows, provides high radiochemical purity and suitable molar activity, and is compatible with commercially available synthesis equipment. These features support routine preclinical application and position the method for future current good manufacturing practice adaptation to enable clinical use in boron neutron capture therapy planning. 2-[18F]BPA Radiosynthesis Boron Neutron Capture Therapy Copper-mediated fluorination PET tracer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Boron neutron capture therapy (BNCT) is a binary therapeutic strategy that relies on the selective accumulation of boron-containing compounds in tumor cells, followed by targeted irradiation with low-energy neutrons 1 . Among boron delivery agents, p-boronophenylalanine (BPA) has demonstrated clinical relevance due to its preferential uptake in malignant cells 2 . However, the success of BNCT depends critically on the ability to quantify BPA biodistribution in patients prior to treatment. Positron emission tomography (PET) with a fluorine-18 labeled analog, 2-[18F]BPA, offers a powerful tool to support patient selection, treatment planning, and therapy monitoring 3 . Despite this potential, existing radiosynthetic methods for 2-[18F]BPA are limited by poor yields, low molar activity, and workflows that are often incompatible with clinical good manufacturing practice (cGMP). Several reported approaches rely on electrophilic fluorination 4 , 5 , 6 , which is low-yielding and operationally challenging. Another study 7 , 8 described automated nucleophilic fluorination using a two-pot strategy, which limits its reproducibility on the more common single-reactor automated synthesis modules. To address these limitations, we sought to: (i) design and synthesize a rationally protected precursor for nucleophilic fluorination, (ii) develop a fully automated radiosynthetic method using copper-mediated fluorination on a commercial synthesis platform, and (iii) characterize the resulting radiotracer for radiochemical purity, molar activity, and stability to support preclinical applications and potential cGMP translation. Materials and Methods General The automated radiosynthesis of 2-[ 18 F]BPA using GE TRACERlab™ FXFN was performed inside the lead-shielded COMECER hot cell. The Cryptand-222 (Kryptofix® [2.2.2]) (Prod. # 800) and the preconditioned QMA light cartridges (Prod. # K-920) were purchased from ABX GmbH (Radeberg, Germany). Deionized water was obtained through a Milli-Q water (18 MΩ•cm) taken from a Millipore Milli-Q Integral 5 water purification system. All the other reagents were purchased from MilliporeSigma (St. Louis, MO) The ethanol 200 PROOF was obtained from Pharmco. Nitrogen and argon gas used primarily in drying and transferring of solutions were provided through Matheson Tri-gas. The automation synthesis on the TRACERlab™ FXFN module was controlled by the TRACERLab FX software. The Alumina N Plus Light Cartridge (Part # WAT023561) and tC18 Plus Short Cartridge (Part # WAT036810) were acquired through Waters (Milford, MA). Purification was performed using a Phenomenex Luna C18 column (250 mm Å~ 4.6 mm, 5 um). The Kryptofix stock solution was prepared, in-house, with ratio of Cryptand-222 (10 mg/ml) and potassium carbonate (6 mg/ml) in methanol. Both BPA precursor (Fig. 1 ) and FBPA reference standard are produced in-house, as shown below. Precursor Synthesis Overview A multi-step synthetic route was designed to generate a BPA precursor stabilized by tert-butyloxycarbonyl (Boc) protection of the amino group and a pinacol ester to stabilize the boronic acid moiety. This precursor was tailored for copper-mediated [ 18 F]fluorination. The precursor was synthesized through a multi-step route (Scheme 1 ) involving Boc protection of the amine group and pinacol ester formation of the boronic acid moiety to enhance stability during coupling and fluorination. Key intermediates were purified by column chromatography, and their identity confirmed by NMR and MS. Enantiomeric purity was assessed by chiral HPLC (Fig. 1 ). Procedure of Preparation of Intermediate 3 To a solution of N-(diphenylmethylene)glycerine tert-butyl ester 1 (4 g, 13.54 mmol), 2,4-dibromo-1-(bromomethyl)benzene 2 (4.45 g, 13.54 mmol) and tetrabutylammonium bromide (TBAB) (43.66 mg, 0.135 mmol) in 30 mL toluene, KOH (10 g, 0.178 mmol) in 8 mL H2O was added. Then the mixture was stirred at room temperature for 12 h and was monitored by thin layer chromatography (TLC) (hexanes:EtOAc = 20:1). After completion of the reaction, the mixture was diluted with 20 mL EtOAc and extracted with EtOAc (20 mL x 2). The combined organic layers were washed with brine (30 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/EtOAc = 100:1 to 20:1). Yield: 3.64 g (91%) 1H NMR (300 MHz, CDCl3): δ = 7.62 (3H, m), 7.35 (7H, m), 7.11 (1H, d, J = 7.82 Hz), 6.70 (2H, J = 6.75 Hz), 4.34 (1H, dd, J1 = 4.07 Hz, J2 = 9.53 Hz), 3.42 (1H, dd, J1 = 4.06 Hz, J2 = 13.40 Hz), 3.21 (1H, dd, J1 = 9.59 Hz, J2 = 13.59 Hz), 1.49 (9H, s). MS (ESI+): m/z = 544.2 [M + H]+. Procedure of preparation of intermediate 4 To a solution of compound 3 (1.2 g, 2.21 mmol) in 2.8 mL THF citric acid (1.27 g, 6.63 mmol) in 4.8 mL H2O was added and stirred for 12 h. Then Na2CO3 (1.17 g, 11.04 mmol) in 6 mL H2O and Boc2O (530.26 mg, 2.43 mmol) was added to the mixture and stirred for 4 h and is monitored by TLC (hexane:EtOAc = 10:1) until compound 3 was consumed completely. The reaction mixture was extracted with EtOAc (8 mL x 2). The combined organic layers were washed with (8 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/EtOAc = 100:1 to 10:1). Yield: 787.9 mg (74.4%). 1H NMR (300 MHz, CDCl3): δ = 7.75 (1H, d, J = 1.82 Hz), 7.37 (1H, dd, J1 = 1.81 Hz, J2 = 8.15 Hz), 7.14 (1H, d, J = 8.15 Hz), 5.06 (1H, d, J = 7.80 Hz), 4.51 (1H, m), 3.24 (1H, dd, J1 = 5.87 Hz, J2 = 13.84 Hz), 3.01 (1H, m), 1.43 (9H, s), 1.38 (9H, s). MS (ESI+): m/z = 480.1 [M + H]+. Procedure of Preparation of Intermediate 4a and 4b The compound 4 (787.9 mg, 0.747 mmol) was separated by multiple rounds of chiral chromatography (loading concentration 20–30 mg/ml) to obtain the enantiomers 4a and 4b. An isocratic gradient of 62% MeCN + 0.1% TFA (phase A) and 38% H2O + 0.1% TFA (phase B) was used as the mobile phase (Fig. 1 and Fig. 2 ). Compound 4a was eluted at -18.5-20 min and 4b at 21–22 min (flowrate 10 ml/min). Compound 4a: Yield: (48.1%). 1H NMR (300 MHz, CDCl3): δ = 7.74 (1H, d, J = 1.81 Hz), 7.38 (1H, dd, J1 = 1.81 Hz, J2 = 8.15 Hz), 7.13 (1H, d, J = 8.15 Hz), 5.07 (1H, d, J = 7.80 Hz), 4.53 (1H, m), 3.25 (1H, dd, J1 = 5.87 Hz, J2 = 13.84 Hz), 3.02 (1H, m), 1.44 (9H, s), 1.39 (9H, s). MS (ESI+): m/z = 480.1 [M + H]+. Compound 4b: Yield: (45.8%). 1H NMR (300 MHz, CDCl3): δ = 7.73 (1H, d, J = 1.81 Hz), 7.39 (1H, dd, J1 = 1.81 Hz, J2 = 8.15 Hz), 7.14 (1H, d, J = 8.15 Hz), 5.06 (1H, d, J = 7.80 Hz), 4.53 (1H, m), 3.24 (1H, dd, J1 = 5.87 Hz, J2 = 13.84 Hz), 3.01 (1H, m), 1.44 (9H, s), 1.38 (9H, s). MS (ESI+): m/z = 480.1 [M + H]+. Enantiomeric excess: Compound 4a ~ 92%, compound 4b ~ 97%. Procedure of Preparation of Precursors P1a and P1b. A mixture of compound 4a (124 mg, 0.259 mmol), B 2 Pin 2 (329 mg, 1.3 mmol), AcOK (101.74 mg, 1.04 mmol), Pd(dppf)Cl 2 (18.91 mg, 0.026 µmol) in 1.5 mL dioxane was degassed and purged with Ar gas for 3 times. Then the mixture was stirred at 90°C for 1 h under argon atmosphere. The crude reaction mixture was filtered and diluted with 14 mL H 2 O and extracted with EtOAc (7 mL x 3). The organic phase was washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated. The combined crude product was purified by preparative RP C18 HPLC with water and MeCN mixture as mobile phase. The precursor was also prepared using the same procedure from compound P1a: compound 4b (117 mg, 0.244 mmol), B 2 Pin 2 (310.27 mg, 1.22 mmol), AcOK (95.93 mg, 0.977 mmol), Pd(dppf)Cl 2 (17.83 mg, 0.024 µmol). P1a yield: 106.9 mg (71%); P1b yield: 91.20 mg (67%). ( 1 H NMR (300 MHz, CDCl 3 ): δ = 8.16 (1H, s), 7.76 (1H, dd, J 1 = 1.39 Hz, J 2 = 7.71 Hz), 7.22 (1H, dd, J = 7.71 Hz), 5.82 (1H, d, J = 8.28 Hz), 4.13 (1H, m), 3.13 (2H, m), 1.39 (9H, s), 1.30 (12H, s), 1.25 (12H, s), 1.19 (9H, s). MS (ESI + ): m/z = 596.6 [M + Na] + . P1b: 1 H NMR (300 MHz, CDCl 3 ): δ = 8.15 (1H, s), 7.75 (1H, dd, J 1 = 1.39 Hz, J 2 = 7.71 Hz), 7.21 (1H, dd, J = 7.71 Hz), 5.81 (1H, d, J = 8.28 Hz), 4.15 (1H, m), 3.13 (2H, m), 1.39 (9H, s), 1.31 (12H, s), 1.26 (12H, s), 1.20 (9H, s). MS (ESI + ): m/z = 596.6 [M + Na] + . Procedure of Preparation of the Precursors P2a and P2b A mixture of compound P1a (106.9 mg, 0.186 mmol), DMAP (24.83 mg, 0.203 mmol) and Boc 2 O (134.29 mg, 0.615 mmol) were dissolved in 30 mL anhydrous MeCN in a round-bottom flask stirred at room temperature for 24 h and monitored by TLC (hexane:EtOAc = 4:1) until the consumption of compound P1a was complete. The solvent of mixture was distilled under reduced pressure and purified with RP C18 HPLC (MeCN + 0.1% TFA 90–100%, H 2 O + TFA 0.1%, 10 − 0%, eluted at 16.7 min, flow rate 10 ml/min). The fraction of compound P2a and P2b were collected, neutralized with saturated aqueous Na 2 CO 3 , extracted with 2x 50 ml dichloromethane, evaporated under reduced pressure, and repurified with silica gel chromatography (hexane:EtOAc = 4:1) to afford P2a 45 mg (36%). The compound P2b was also prepared using the same procedure from compound P1b: P1b (91.20 mg, 0.132 mmol), Boc 2 O (95.27 mg, 0.436 mmol), DMAP (17.61 mg, 0.144 mmol). Yield: 37 mg (31%). P2a: 1 H NMR (300 MHz, CDCl 3 ): δ = 8.13 (1H, s), 7.67 (1H, dd, J 1 = 1.21 Hz, J 2 = 7.51 Hz), 6.96 (1H, d, J = 7.59 Hz), 5.15 (1H, d, J = 3.82 Hz), 3.89 (1H, d, J = 3.88 Hz), 3.06 (2H, dd, J 1 = 13.30 Hz, J 2 = 11.36 Hz), 1.42 (9H, s), 1.27 (42H, s). MS (ESI + ): m/z = 696.7 [M + Na] + . P2b: 1 H NMR (300 MHz, CDCl 3 ): δ = 8.13 (1H, s), 7.67 (1H, dd, J 1 = 1.21 Hz, J 2 = 7.51 Hz), 6.97 (1H, d, J = 7.59 Hz), 5.13 (1H, d, J = 3.82 Hz), 3.89 (1H, d, J = 3.88 Hz), 3.07 (2H, dd, J 1 = 13.31 Hz, J 2 = 11.36 Hz), 1.41 (9H, s), 1.26 (42H, s). MS (ESI + ): m/z = 696.7 [M + Na] + . P2b as the precursor with the naturally occurring ( S )-configuration was used in the next step for radiofluorination. Figure 3 represents the chiral HPLC chromatograms showing enantiomeric excess of intermediates. Automated Radiosynthesis of 2-[F]BPA The automatic radiosynthesis of 2-[ 18 F]BPA is produced by copper-mediated nucleophilic substitution of aryl boronic ester precursor on a GE Tracerlab FX FN module, Scheme 2 . The automatic radiosynthesis, including the nucleophilic radiolabeling of the precursor and the acidic hydrolysis of the radiolabeled intermediate, was performed with a single-pot reactor. The list of the reagents and their corresponding vials in the Tracerlab are shown in Table 1 . Table 1 Material and reagent list used in the radiosynthesis of 2-[ 18 F]BPA via TRACERlab™ FXFN module. Item # Reagents or consumables 1 Potassium Carbonate/Kryptofix QMA Elution Solution,1.0 mL 2 Methanol, 0.6 mL 3 BPA precursor (10-15mg), Cu(OTf) 2 Py 4 (20 mg) dissolved in DMA/nBuOH/pyridine (800,100,100 µl), 1.0 mL 4 6M HCl, 0.6 mL 5 HPLC MP, 1.5 mL 6 Pre-conditioned QMA light Sep-Pak cartridge, 1 cartridge 7 Ion exchange cartridge/Glass membrane filter assembly, 1 set 8 Final Product Vial, 1 vial The [ 18 F]Fluoride was produced by irradiating 2.5 mL of enriched [ 18 O]H2O with 60 µAh beam current from the 16.5 MeV GE PETrace cyclotron. The [ 18 F]Fluoride was separated from the [ 18 O]H2O and capture on the preconditioned QMA light cartridge then eluted with 1 ml of the Kryptofix solution. After drying the Kryptofix/[ 18 F]KF solution by heating at 100 ºC under vacuum and nitrogen gas flow, the precursor (15 mg) and Cu(OTf) 2 Py 4 (20 mg) dissolved in DMA/nBuOH/pyridine (800,100,100 µl) were added to the reactor and heated at 120 ºC for 25 minutes. After the completion of the labeling step, the reactor was cooled to 40 ºC and 0.6 mL of 6 M HCl was added to the reaction mixture. Hydrolysis occurs as the reactor is heated at 130 ºC for 15 minutes. Purification was achieved using semi-preparative HPLC (Fig. 6 ) with isocratic elution (0.1% acetic acid/1% methanol, 4 mL/min). The desired fraction was collected, neutralized with NaHCO 3 , and sterile filtered. Quality Control Radiochemical identity and purity were confirmed by analytical HPLC (Fig. 7 ) with co-injection of a non-radioactive reference standard. Enantiomeric excess was determined using chiral HPLC. Stability was monitored by HPLC over 4 hours post-synthesis at room temperature. Results The final precursor was obtained in high purity (> 95%) as determined by 1H NMR. Enantiomeric excess of the intermediates was 92% and 97%, respectively. Automated radiosynthesis consistently yielded 2-[18F]BPA in 3–5% non-decay-corrected radiochemical yield (NDCRY, n = 8) with a total synthesis time of 60–70 minutes. Radiochemical purity exceeded 98% in all batches, and molar activity ranged from 85–120 GBq/µmol at end of synthesis. The final formulation was stable for at least 4 hours post-synthesis at room temperature with residual solvents below ICH (International Council for Harmonisation) limits. The results of the radiolabeling tests are shown in Table 2 . Table 2 Summary of radiochemistry production runs, including the non-decay corrected radiochemical yield (NDCRY) Experiment Initial Activity (mCi) Product (mCi) NDCRY % 001 270 10 3.7 002 120 0.6 0.5 003 450 7 1.6 004 600 32.7 5.5 005 700 25 3.6 006 850 29 3.4 007 650 25 3.8 008 510 26 5.1 Discussion This work establishes a practical, automated radiosynthetic pathway for 2-[¹⁸F]BPA. Compared to electrophilic fluorination approaches (Mairinger et al., 2015; Ishiwata, 2019), our method delivers superior radiochemical purity and acceptable molar activity, while avoiding the need for specialized electrophilic fluorine production. 2-[¹⁸F]BPA is a promising agent for boron neutron capture therapy (BNCT); however, its application has been limited by the complexity of its multi-step radiosynthesis. Previous methods relied on laborious, multi-step and multi-pot reactions involving the use of ¹⁸F₂ gas, which yielded low radiochemical product yields. These approaches pose significant challenges for routine clinical implementation, as most automated synthesis modules are designed with only a single reactor. More recently, Chang et al. demonstrated that 2-[¹⁸F]BPA could be produced with high radiochemical yield, but the process was performed manually and required multiple reaction vessels, complicating its clinical translation.⁵ In this study, we developed a robust, two-step, single-pot automated synthesis protocol for multi-dose production of 2-[¹⁸F]BPA using the GE TRACERlab™ FXFN radio synthesizer (Figs. 4 and 5 ), which is cGMP-enabled and suitable for future clinical applications. Although the NDCRY remains modest (3–5%), this is within the range reported for electrophilic syntheses, and sufficient for preclinical imaging when starting activity exceeds 500 mCi. Future optimization could target improved yields through fine-tuning precursor loading, copper complex stoichiometry, or solvent ratios. Another important consideration is the complexity of the analytical radio-HPLC profile, which demonstrates the presence of multiple radiolabeled byproducts and closely eluting impurities. This complexity makes purification challenging, as baseline separation from the desired 2-[¹⁸F]BPA peak is required to ensure radiochemical purity and identity. Optimization of the semi-preparative HPLC conditions was therefore critical to achieving consistent recovery of the target fraction. We found that careful control of mobile phase composition and isocratic conditions was essential to reproducibly isolate 2-[¹⁸F]BPA without significant product loss. These parameters will be especially important for future cGMP adaptation of the method, where batch-to-batch reproducibility is mandatory. Importantly, the entire workflow is compatible with a commercial synthesis module, supporting potential cGMP implementation. The robustness of the procedure, demonstrated across multiple runs, indicates suitability for routine production. In the context of BNCT, 2-[¹⁸F]BPA PET has the potential to refine patient selection and dosimetry, ultimately improving therapeutic outcomes. Our results represent a critical step toward enabling such clinical applications. Tracerlab vs. Fastlab and rationale to optimize the single pot reaction Compared to cassette-based systems such as the GE Fastlab, the GE Tracerlab FX series offers greater flexibility and control over the radiosynthetic process. The Tracerlab allows for direct modification of reaction parameters, tubing configuration, and reagent delivery sequences, enabling rapid optimization of labeling conditions and adaptation to novel tracers or non-standard chemistries. This open architecture is particularly advantageous during early-stage development, when synthetic steps may require iterative fine-tuning or non-routine manipulations. In contrast, cassette-based modules are designed primarily for standardized, routine production under GMP conditions. While they provide improved reproducibility, reduced risk of operator error, and simplified regulatory compliance, their fixed design limits the user’s ability to modify the process or incorporate custom reagents. Thus, the Tracerlab system offers a more versatile platform for research and development, whereas cassette-based modules such as the Fastlab are better suited for high-throughput clinical manufacturing. Conclusions We report a robust, automated radiosynthetic method for 2-[ 18 F]BPA using copper-mediated nucleophilic fluorination of a rationally designed precursor. The method circumvents the challenges of electrophilic fluorination, delivers high radiochemical purity, acceptable molar activity, and formulation stability, and is compatible with commercial synthesis platforms. These features position the tracer for preclinical evaluation and pave the way for cGMP-compliant production in support of BNCT clinical workflows. Declarations Acknowledgements Not applicable Author contributions VP, RTT, DKG and CDP designed, performed and analyzed the experiments. HCM evaluated the results. All authors read and approved the final manuscript. Funding This work was supported by : The Cancer Prevention Research Institute of Texas (CPRIT) RR200046; H. C. Manning is a CPRIT Scholar of Cancer Research. TAE Life Sciences Availability of data All data generated or analyzed during this study are included in this manuscript Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Nedunchezhian K, Aswath N, Thiruppathy M, Thirugnanamurthy S. Boron Neutron Capture Therapy - A Literature Review. J Clin Diagn Res. 2016 Dec;10(12):ZE01-ZE04. doi: 10.7860/JCDR/2016/19890.9024. Epub 2016 Dec 1. PMID: 28209015; PMCID: PMC5296588. Mishima Y, Ichihashi M, Hatta S, Honda C, Yamamura K, Nakagawa T, Obara H, Shirakawa J, Hiratsuka J, Taniyama K, et al. First human clinical trial of melanoma neutron capture. Diagnosis and therapy. Strahlenther Onkol. 1989 Feb-Mar;165(2-3):251-4. PMID: 2494743. Ishiwata K. 4-Borono-2- 18 F-fluoro-L-phenylalanine PET for boron neutron capture therapy-oriented diagnosis: overview of a quarter century of research. Ann Nucl Med. 2019 Apr;33(4):223-236. doi: 10.1007/s12149-019-01347-8. Epub 2019 Feb 28. PMID: 30820862; PMCID: PMC6450856. Mairinger, S., Stanek, J., Wanek, T., Langer, O., Kuntner, C., 2015. Automated electrophilic radiosynthesis of [18F]FBPA using a modified nucleophilic GE TRACERlab FXFDG. Applied Radiation and Isotopes 104, 124-127. Chang TY, et al. Comparison of the synthesis and biological properties of no-carrier-added and carrier-added 4-borono-2-[18F]fluorophenylalanine ([18F]FBPA). Nucl Med Biol. 2023 Jan-Feb;116-117:108313. doi: 10.1016/j.nucmedbio.2022.108313. Epub 2022 Dec 30. PMID: 36621257. Naka, S., et al., 2012. Optimization of 4-borono-2-[18F]fluoro-L-phenylalanine (FBPA) synthesis: PET tracer for boron neutron capture therapy (BNCT) for cancer treatment. Journal of Nuclear Medicine, 53 (supplement 1) 1689 He, et al., 2021. Nucleophilic radiosynthesis of boron neutron capture therapy-oriented PET probe [18F]FBPA using aryldiboron precursors. Chem. Commun. 57, 8953. Ishiwata, K., 2019. 4-Borono-2-18F-fluoro-L-phenylalanine PET for boron neutron capture therapy-oriented diagnosis: overview of a quarter century of research. Ann. Nucl. Med. 33, 223–236. Schemes Scheme 1 and 2 are available in the Supplementary Files section. Supplementary Files Schemes.docx Cite Share Download PDF Status: Published Journal Publication published 05 Mar, 2026 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted Editorial decision: Major revision 07 Jan, 2026 Reviewers agreed at journal 17 Dec, 2025 Reviewers invited by journal 17 Dec, 2025 Editor assigned by journal 10 Dec, 2025 First submitted to journal 09 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-8289343\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":561764213,\"identity\":\"fc013cc3-b1d1-4111-8190-12cbf2861de8\",\"order_by\":0,\"name\":\"Vincenzo Paolillo\",\"email\":\"data:image/png;base64,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\",\"orcid\":\"https://orcid.org/0000-0003-0691-691X\",\"institution\":\"The University of Texas MD Anderson Cancer Center\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Vincenzo\",\"middleName\":\"\",\"lastName\":\"Paolillo\",\"suffix\":\"\"},{\"id\":561764214,\"identity\":\"90991f81-6d26-4b6a-98cd-0ebe942ea0b4\",\"order_by\":1,\"name\":\"Cong-Dat Pham\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Indivumed GmbH\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Cong-Dat\",\"middleName\":\"\",\"lastName\":\"Pham\",\"suffix\":\"\"},{\"id\":561764215,\"identity\":\"207e3c77-1d12-4d09-845a-613a06db8523\",\"order_by\":2,\"name\":\"Robert Ta\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"The University of Texas MD Anderson Cancer Center\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Robert\",\"middleName\":\"\",\"lastName\":\"Ta\",\"suffix\":\"\"},{\"id\":561764216,\"identity\":\"6c41e530-70cf-452c-ad85-560afaf61241\",\"order_by\":3,\"name\":\"Dimitra K. 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The numbers denoted in RED represent the designation of item and position of reagents and consumables indicated in Table 1.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8289343/v1/2ceb5123be4c40591ecfbd3f.png\"},{\"id\":98763881,\"identity\":\"59cb4a4e-616b-4817-8f2b-18b1fd82b1cc\",\"added_by\":\"auto\",\"created_at\":\"2025-12-22 10:06:36\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":203499,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSemi-preparative HPLC radiochromatogram showing isolation of 2-[\\u003csup\\u003e18\\u003c/sup\\u003eF]BPA. The top portion shows the gamma signal, the bottom shows the correspondent UV signal.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8289343/v1/18d3a3d7f4c9830b3790cfb7.png\"},{\"id\":98778769,\"identity\":\"09d3c614-02b6-4ac8-bd76-8cdc5570cb4e\",\"added_by\":\"auto\",\"created_at\":\"2025-12-22 12:29:38\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":25106,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAnalytical HPLC chromatogram of the final product \\u003csup\\u003e18\\u003c/sup\\u003eF-BPA (quality control).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8289343/v1/6e89919db3c9234be69e5570.png\"},{\"id\":104251504,\"identity\":\"e738cc4e-a691-4b94-b005-2e909845486e\",\"added_by\":\"auto\",\"created_at\":\"2026-03-09 16:13:36\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2525634,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8289343/v1/94db7fd4-5f6b-4bac-acf4-4e94001021e1.pdf\"},{\"id\":98779351,\"identity\":\"7a5c9f76-f0df-4894-a574-1de086009288\",\"added_by\":\"auto\",\"created_at\":\"2025-12-22 12:30:16\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":142676,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Schemes.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8289343/v1/c785162af50cb5f83b8bebaf.docx\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Automated Radiosynthesis of 2-[18F]BPA for PET-based Planning of Boron Neutron Capture Therapy (BNCT): Rational Precursor Design, Radiofluorination, and Characterization of Methodology\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003eBoron neutron capture therapy (BNCT) is a binary therapeutic strategy that relies on the selective accumulation of boron-containing compounds in tumor cells, followed by targeted irradiation with low-energy neutrons\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. Among boron delivery agents, p-boronophenylalanine (BPA) has demonstrated clinical relevance due to its preferential uptake in malignant cells\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. However, the success of BNCT depends critically on the ability to quantify BPA biodistribution in patients prior to treatment.\\u003c/p\\u003e \\u003cp\\u003ePositron emission tomography (PET) with a fluorine-18 labeled analog, 2-[18F]BPA, offers a powerful tool to support patient selection, treatment planning, and therapy monitoring\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e. Despite this potential, existing radiosynthetic methods for 2-[18F]BPA are limited by poor yields, low molar activity, and workflows that are often incompatible with clinical good manufacturing practice (cGMP). Several reported approaches rely on electrophilic fluorination\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e, which is low-yielding and operationally challenging.\\u003c/p\\u003e \\u003cp\\u003eAnother study\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e described automated nucleophilic fluorination using a two-pot strategy, which limits its reproducibility on the more common single-reactor automated synthesis modules.\\u003c/p\\u003e \\u003cp\\u003eTo address these limitations, we sought to: (i) design and synthesize a rationally protected precursor for nucleophilic fluorination, (ii) develop a fully automated radiosynthetic method using copper-mediated fluorination on a commercial synthesis platform, and (iii) characterize the resulting radiotracer for radiochemical purity, molar activity, and stability to support preclinical applications and potential cGMP translation.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eGeneral\\u003c/h2\\u003e \\u003cp\\u003eThe automated radiosynthesis of 2-[\\u003csup\\u003e18\\u003c/sup\\u003eF]BPA using GE TRACERlab\\u0026trade; FXFN was performed inside the lead-shielded COMECER hot cell. The Cryptand-222 (Kryptofix\\u0026reg; [2.2.2]) (Prod. # 800) and the preconditioned QMA light cartridges (Prod. # K-920) were purchased from ABX GmbH (Radeberg, Germany). Deionized water was obtained through a Milli-Q water (18 MΩ\\u0026bull;cm) taken from a Millipore Milli-Q Integral 5 water purification system. All the other reagents were purchased from MilliporeSigma (St. Louis, MO) The ethanol 200 PROOF was obtained from Pharmco. Nitrogen and argon gas used primarily in drying and transferring of solutions were provided through Matheson Tri-gas. The automation synthesis on the TRACERlab\\u0026trade; FXFN module was controlled by the TRACERLab FX software. The Alumina N Plus Light Cartridge (Part # WAT023561) and tC18 Plus Short Cartridge (Part # WAT036810) were acquired through Waters (Milford, MA). Purification was performed using a Phenomenex Luna C18 column (250 mm \\u0026Aring;~ 4.6 mm, 5 um). The Kryptofix stock solution was prepared, in-house, with ratio of Cryptand-222 (10 mg/ml) and potassium carbonate (6 mg/ml) in methanol. Both BPA precursor (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) and FBPA reference standard are produced in-house, as shown below.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003ePrecursor Synthesis Overview\\u003c/h3\\u003e\\n\\u003cp\\u003eA multi-step synthetic route was designed to generate a BPA precursor stabilized by tert-butyloxycarbonyl (Boc) protection of the amino group and a pinacol ester to stabilize the boronic acid moiety. This precursor was tailored for copper-mediated [\\u003csup\\u003e18\\u003c/sup\\u003eF]fluorination.\\u003c/p\\u003e \\u003cp\\u003eThe precursor was synthesized through a multi-step route (Scheme \\u003cspan refid=\\\"Sch1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) involving Boc protection of the amine group and pinacol ester formation of the boronic acid moiety to enhance stability during coupling and fluorination. Key intermediates were purified by column chromatography, and their identity confirmed by NMR and MS. Enantiomeric purity was assessed by chiral HPLC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eProcedure of Preparation of Intermediate 3\\u003c/h3\\u003e\\n\\u003cp\\u003eTo a solution of N-(diphenylmethylene)glycerine tert-butyl ester 1 (4 g, 13.54 mmol), 2,4-dibromo-1-(bromomethyl)benzene 2 (4.45 g, 13.54 mmol) and tetrabutylammonium bromide (TBAB) (43.66 mg, 0.135 mmol) in 30 mL toluene, KOH (10 g, 0.178 mmol) in 8 mL H2O was added. Then the mixture was stirred at room temperature for 12 h and was monitored by thin layer chromatography (TLC) (hexanes:EtOAc\\u0026thinsp;=\\u0026thinsp;20:1). After completion of the reaction, the mixture was diluted with 20 mL EtOAc and extracted with EtOAc (20 mL x 2). The combined organic layers were washed with brine (30 mL x 2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/EtOAc\\u0026thinsp;=\\u0026thinsp;100:1 to 20:1). Yield: 3.64 g (91%) 1H NMR (300 MHz, CDCl3): δ\\u0026thinsp;=\\u0026thinsp;7.62 (3H, m), 7.35 (7H, m), 7.11 (1H, d, J\\u0026thinsp;=\\u0026thinsp;7.82 Hz), 6.70 (2H, J\\u0026thinsp;=\\u0026thinsp;6.75 Hz), 4.34 (1H, dd, J1\\u0026thinsp;=\\u0026thinsp;4.07 Hz, J2\\u0026thinsp;=\\u0026thinsp;9.53 Hz), 3.42 (1H, dd, J1\\u0026thinsp;=\\u0026thinsp;4.06 Hz, J2\\u0026thinsp;=\\u0026thinsp;13.40 Hz), 3.21 (1H, dd, J1\\u0026thinsp;=\\u0026thinsp;9.59 Hz, J2\\u0026thinsp;=\\u0026thinsp;13.59 Hz), 1.49 (9H, s). MS (ESI+): m/z\\u0026thinsp;=\\u0026thinsp;544.2 [M\\u0026thinsp;+\\u0026thinsp;H]+.\\u003c/p\\u003e\\n\\u003ch3\\u003eProcedure of preparation of intermediate 4\\u003c/h3\\u003e\\n\\u003cp\\u003eTo a solution of compound 3 (1.2 g, 2.21 mmol) in 2.8 mL THF citric acid (1.27 g, 6.63 mmol) in 4.8 mL H2O was added and stirred for 12 h. Then Na2CO3 (1.17 g, 11.04 mmol) in 6 mL H2O and Boc2O (530.26 mg, 2.43 mmol) was added to the mixture and stirred for 4 h and is monitored by TLC (hexane:EtOAc\\u0026thinsp;=\\u0026thinsp;10:1) until compound 3 was consumed completely. The reaction mixture was extracted with EtOAc (8 mL x 2). The combined organic layers were washed with (8 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/EtOAc\\u0026thinsp;=\\u0026thinsp;100:1 to 10:1). Yield: 787.9 mg (74.4%). 1H NMR (300 MHz, CDCl3): δ\\u0026thinsp;=\\u0026thinsp;7.75 (1H, d, J\\u0026thinsp;=\\u0026thinsp;1.82 Hz), 7.37 (1H, dd, J1\\u0026thinsp;=\\u0026thinsp;1.81 Hz, J2\\u0026thinsp;=\\u0026thinsp;8.15 Hz), 7.14 (1H, d, J\\u0026thinsp;=\\u0026thinsp;8.15 Hz), 5.06 (1H, d, J\\u0026thinsp;=\\u0026thinsp;7.80 Hz), 4.51 (1H, m), 3.24 (1H, dd, J1\\u0026thinsp;=\\u0026thinsp;5.87 Hz, J2\\u0026thinsp;=\\u0026thinsp;13.84 Hz), 3.01 (1H, m), 1.43 (9H, s), 1.38 (9H, s). MS (ESI+): m/z\\u0026thinsp;=\\u0026thinsp;480.1 [M\\u0026thinsp;+\\u0026thinsp;H]+.\\u003c/p\\u003e\\n\\u003ch3\\u003eProcedure of Preparation of Intermediate 4a and 4b\\u003c/h3\\u003e\\n\\u003cp\\u003eThe compound 4 (787.9 mg, 0.747 mmol) was separated by multiple rounds of chiral chromatography (loading concentration 20\\u0026ndash;30 mg/ml) to obtain the enantiomers 4a and 4b. An isocratic gradient of 62% MeCN\\u0026thinsp;+\\u0026thinsp;0.1% TFA (phase A) and 38% H2O\\u0026thinsp;+\\u0026thinsp;0.1% TFA (phase B) was used as the mobile phase (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Compound 4a was eluted at -18.5-20 min and 4b at 21\\u0026ndash;22 min (flowrate 10 ml/min). Compound 4a: Yield: (48.1%). 1H NMR (300 MHz, CDCl3): δ\\u0026thinsp;=\\u0026thinsp;7.74 (1H, d, J\\u0026thinsp;=\\u0026thinsp;1.81 Hz), 7.38 (1H, dd, J1\\u0026thinsp;=\\u0026thinsp;1.81 Hz, J2\\u0026thinsp;=\\u0026thinsp;8.15 Hz), 7.13 (1H, d, J\\u0026thinsp;=\\u0026thinsp;8.15 Hz), 5.07 (1H, d, J\\u0026thinsp;=\\u0026thinsp;7.80 Hz), 4.53 (1H, m), 3.25 (1H, dd, J1\\u0026thinsp;=\\u0026thinsp;5.87 Hz, J2\\u0026thinsp;=\\u0026thinsp;13.84 Hz), 3.02 (1H, m), 1.44 (9H, s), 1.39 (9H, s). MS (ESI+): m/z\\u0026thinsp;=\\u0026thinsp;480.1 [M\\u0026thinsp;+\\u0026thinsp;H]+. Compound 4b: Yield: (45.8%). 1H NMR (300 MHz, CDCl3): δ\\u0026thinsp;=\\u0026thinsp;7.73 (1H, d, J\\u0026thinsp;=\\u0026thinsp;1.81 Hz), 7.39 (1H, dd, J1\\u0026thinsp;=\\u0026thinsp;1.81 Hz, J2\\u0026thinsp;=\\u0026thinsp;8.15 Hz), 7.14 (1H, d, J\\u0026thinsp;=\\u0026thinsp;8.15 Hz), 5.06 (1H, d, J\\u0026thinsp;=\\u0026thinsp;7.80 Hz), 4.53 (1H, m), 3.24 (1H, dd, J1\\u0026thinsp;=\\u0026thinsp;5.87 Hz, J2\\u0026thinsp;=\\u0026thinsp;13.84 Hz), 3.01 (1H, m), 1.44 (9H, s), 1.38 (9H, s). MS (ESI+): m/z\\u0026thinsp;=\\u0026thinsp;480.1 [M\\u0026thinsp;+\\u0026thinsp;H]+. Enantiomeric excess: Compound 4a\\u0026thinsp;~\\u0026thinsp;92%, compound 4b\\u0026thinsp;~\\u0026thinsp;97%.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eProcedure of Preparation of Precursors P1a and P1b.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eA mixture of compound 4a (124 mg, 0.259 mmol), B\\u003csub\\u003e2\\u003c/sub\\u003ePin\\u003csub\\u003e2\\u003c/sub\\u003e (329 mg, 1.3 mmol), AcOK (101.74 mg, 1.04 mmol), Pd(dppf)Cl\\u003csub\\u003e2\\u003c/sub\\u003e (18.91 mg, 0.026 \\u0026micro;mol) in 1.5 mL dioxane was degassed and purged with Ar gas for 3 times. Then the mixture was stirred at 90\\u0026deg;C for 1 h under argon atmosphere. The crude reaction mixture was filtered and diluted with 14 mL H\\u003csub\\u003e2\\u003c/sub\\u003eO and extracted with EtOAc (7 mL x 3). The organic phase was washed with brine, dried over anhydrous Na\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e, filtered and concentrated. The combined crude product was purified by preparative RP C18 HPLC with water and MeCN mixture as mobile phase. The precursor was also prepared using the same procedure from compound P1a: compound 4b (117 mg, 0.244 mmol), B\\u003csub\\u003e2\\u003c/sub\\u003ePin\\u003csub\\u003e2\\u003c/sub\\u003e (310.27 mg, 1.22 mmol), AcOK (95.93 mg, 0.977 mmol), Pd(dppf)Cl\\u003csub\\u003e2\\u003c/sub\\u003e (17.83 mg, 0.024 \\u0026micro;mol). P1a yield: 106.9 mg (71%); P1b yield: 91.20 mg (67%). (\\u003csup\\u003e1\\u003c/sup\\u003eH NMR (300 MHz, CDCl\\u003csub\\u003e3\\u003c/sub\\u003e): δ\\u0026thinsp;=\\u0026thinsp;8.16 (1H, s), 7.76 (1H, dd, \\u003cem\\u003eJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;1.39 Hz, \\u003cem\\u003eJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;7.71 Hz), 7.22 (1H, dd, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.71 Hz), 5.82 (1H, d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.28 Hz), 4.13 (1H, m), 3.13 (2H, m), 1.39 (9H, s), 1.30 (12H, s), 1.25 (12H, s), 1.19 (9H, s). MS (ESI\\u003csup\\u003e+\\u003c/sup\\u003e): m/z\\u0026thinsp;=\\u0026thinsp;596.6 [M\\u0026thinsp;+\\u0026thinsp;Na]\\u003csup\\u003e+\\u003c/sup\\u003e. P1b: \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (300 MHz, CDCl\\u003csub\\u003e3\\u003c/sub\\u003e): δ\\u0026thinsp;=\\u0026thinsp;8.15 (1H, s), 7.75 (1H, dd, \\u003cem\\u003eJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;1.39 Hz, \\u003cem\\u003eJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;7.71 Hz), 7.21 (1H, dd, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.71 Hz), 5.81 (1H, d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;8.28 Hz), 4.15 (1H, m), 3.13 (2H, m), 1.39 (9H, s), 1.31 (12H, s), 1.26 (12H, s), 1.20 (9H, s). MS (ESI\\u003csup\\u003e+\\u003c/sup\\u003e): m/z\\u0026thinsp;=\\u0026thinsp;596.6 [M\\u0026thinsp;+\\u0026thinsp;Na]\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eProcedure of Preparation of the Precursors P2a and P2b\\u003c/h2\\u003e \\u003cp\\u003eA mixture of compound P1a (106.9 mg, 0.186 mmol), DMAP (24.83 mg, 0.203 mmol) and Boc\\u003csub\\u003e2\\u003c/sub\\u003eO (134.29 mg, 0.615 mmol) were dissolved in 30 mL anhydrous MeCN in a round-bottom flask stirred at room temperature for 24 h and monitored by TLC (hexane:EtOAc\\u0026thinsp;=\\u0026thinsp;4:1) until the consumption of compound P1a was complete. The solvent of mixture was distilled under reduced pressure and purified with RP C18 HPLC (MeCN\\u0026thinsp;+\\u0026thinsp;0.1% TFA 90\\u0026ndash;100%, H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u0026thinsp;+\\u0026thinsp;TFA 0.1%, 10\\u0026thinsp;\\u0026minus;\\u0026thinsp;0%, eluted at 16.7 min, flow rate 10 ml/min). The fraction of compound P2a and P2b were collected, neutralized with saturated aqueous Na\\u003csub\\u003e2\\u003c/sub\\u003eCO\\u003csub\\u003e3\\u003c/sub\\u003e, extracted with 2x 50 ml dichloromethane, evaporated under reduced pressure, and repurified with silica gel chromatography (hexane:EtOAc\\u0026thinsp;=\\u0026thinsp;4:1) to afford P2a 45 mg (36%). The compound P2b was also prepared using the same procedure from compound P1b: P1b (91.20 mg, 0.132 mmol), Boc\\u003csub\\u003e2\\u003c/sub\\u003eO (95.27 mg, 0.436 mmol), DMAP (17.61 mg, 0.144 mmol). Yield: 37 mg (31%). P2a: \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (300 MHz, CDCl\\u003csub\\u003e3\\u003c/sub\\u003e): δ\\u0026thinsp;=\\u0026thinsp;8.13 (1H, s), 7.67 (1H, dd, \\u003cem\\u003eJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;1.21 Hz, \\u003cem\\u003eJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;7.51 Hz), 6.96 (1H, d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.59 Hz), 5.15 (1H, d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;3.82 Hz), 3.89 (1H, d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;3.88 Hz), 3.06 (2H, dd, \\u003cem\\u003eJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;13.30 Hz, \\u003cem\\u003eJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;11.36 Hz), 1.42 (9H, s), 1.27 (42H, s). MS (ESI\\u003csup\\u003e+\\u003c/sup\\u003e): m/z\\u0026thinsp;=\\u0026thinsp;696.7 [M\\u0026thinsp;+\\u0026thinsp;Na]\\u003csup\\u003e+\\u003c/sup\\u003e. P2b: \\u003csup\\u003e1\\u003c/sup\\u003eH NMR (300 MHz, CDCl\\u003csub\\u003e3\\u003c/sub\\u003e): δ\\u0026thinsp;=\\u0026thinsp;8.13 (1H, s), 7.67 (1H, dd, \\u003cem\\u003eJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;1.21 Hz, \\u003cem\\u003eJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;7.51 Hz), 6.97 (1H, d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;7.59 Hz), 5.13 (1H, d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;3.82 Hz), 3.89 (1H, d, \\u003cem\\u003eJ\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;3.88 Hz), 3.07 (2H, dd, \\u003cem\\u003eJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e1\\u003c/em\\u003e\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;13.31 Hz, \\u003cem\\u003eJ\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;11.36 Hz), 1.41 (9H, s), 1.26 (42H, s). MS (ESI\\u003csup\\u003e+\\u003c/sup\\u003e): m/z\\u0026thinsp;=\\u0026thinsp;696.7 [M\\u0026thinsp;+\\u0026thinsp;Na]\\u003csup\\u003e+\\u003c/sup\\u003e. P2b as the precursor with the naturally occurring (\\u003cem\\u003eS\\u003c/em\\u003e)-configuration was used in the next step for radiofluorination. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e represents the chiral HPLC chromatograms showing enantiomeric excess of intermediates.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eAutomated Radiosynthesis of 2-[F]BPA\\u003c/h3\\u003e\\n\\u003cp\\u003eThe automatic radiosynthesis of 2-[\\u003csup\\u003e18\\u003c/sup\\u003eF]BPA is produced by copper-mediated nucleophilic substitution of aryl boronic ester precursor on a GE Tracerlab FX FN module, Scheme \\u003cspan refid=\\\"Sch2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. The automatic radiosynthesis, including the nucleophilic radiolabeling of the precursor and the acidic hydrolysis of the radiolabeled intermediate, was performed with a single-pot reactor.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe list of the reagents and their corresponding vials in the Tracerlab are shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eMaterial and reagent list used in the radiosynthesis of 2-[\\u003csup\\u003e18\\u003c/sup\\u003eF]BPA via TRACERlab\\u0026trade; FXFN module.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"2\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eItem #\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eReagents or consumables\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e1\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003ePotassium Carbonate/Kryptofix QMA Elution Solution,1.0 mL\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e2\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eMethanol, 0.6 mL\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e3\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBPA precursor (10-15mg), Cu(OTf)\\u003csub\\u003e2\\u003c/sub\\u003ePy\\u003csub\\u003e4\\u003c/sub\\u003e (20 mg) dissolved in DMA/nBuOH/pyridine (800,100,100 \\u0026micro;l), 1.0 mL\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e4\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e6M HCl, 0.6 mL\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e5\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eHPLC MP, 1.5 mL\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e6\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003ePre-conditioned QMA light Sep-Pak cartridge, 1 cartridge\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e7\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eIon exchange cartridge/Glass membrane filter assembly, 1 set\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e8\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eFinal Product Vial, 1 vial\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe [\\u003csup\\u003e18\\u003c/sup\\u003eF]Fluoride was produced by irradiating 2.5 mL of enriched [\\u003csup\\u003e18\\u003c/sup\\u003eO]H2O with 60 \\u0026micro;Ah beam current from the 16.5 MeV GE PETrace cyclotron. The [\\u003csup\\u003e18\\u003c/sup\\u003eF]Fluoride was separated from the [\\u003csup\\u003e18\\u003c/sup\\u003eO]H2O and capture on the preconditioned QMA light cartridge then eluted with 1 ml of the Kryptofix solution. After drying the Kryptofix/[\\u003csup\\u003e18\\u003c/sup\\u003eF]KF solution by heating at 100 \\u0026ordm;C under vacuum and nitrogen gas flow, the precursor (15 mg) and Cu(OTf)\\u003csub\\u003e2\\u003c/sub\\u003ePy\\u003csub\\u003e4\\u003c/sub\\u003e (20 mg) dissolved in DMA/nBuOH/pyridine (800,100,100 \\u0026micro;l) were added to the reactor and heated at 120 \\u0026ordm;C for 25 minutes. After the completion of the labeling step, the reactor was cooled to 40 \\u0026ordm;C and 0.6 mL of 6 M HCl was added to the reaction mixture. Hydrolysis occurs as the reactor is heated at 130 \\u0026ordm;C for 15 minutes. Purification was achieved using semi-preparative HPLC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e) with isocratic elution (0.1% acetic acid/1% methanol, 4 mL/min). The desired fraction was collected, neutralized with NaHCO\\u003csub\\u003e3\\u003c/sub\\u003e, and sterile filtered.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eQuality Control\\u003c/h3\\u003e\\n\\u003cp\\u003eRadiochemical identity and purity were confirmed by analytical HPLC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e) with co-injection of a non-radioactive reference standard. Enantiomeric excess was determined using chiral HPLC. Stability was monitored by HPLC over 4 hours post-synthesis at room temperature.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003eThe final precursor was obtained in high purity (\\u0026gt;\\u0026thinsp;95%) as determined by 1H NMR. Enantiomeric excess of the intermediates was 92% and 97%, respectively. Automated radiosynthesis consistently yielded 2-[18F]BPA in 3\\u0026ndash;5% non-decay-corrected radiochemical yield (NDCRY, n\\u0026thinsp;=\\u0026thinsp;8) with a total synthesis time of 60\\u0026ndash;70 minutes. Radiochemical purity exceeded 98% in all batches, and molar activity ranged from 85\\u0026ndash;120 GBq/\\u0026micro;mol at end of synthesis. The final formulation was stable for at least 4 hours post-synthesis at room temperature with residual solvents below ICH (International Council for Harmonisation) limits.\\u003c/p\\u003e \\u003cp\\u003eThe results of the radiolabeling tests are shown in Table \\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eSummary of radiochemistry production runs, including the non-decay corrected radiochemical yield (NDCRY)\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"4\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eExperiment\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eInitial Activity (mCi)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eProduct (mCi)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eNDCRY %\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e001\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e270\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e10\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.7\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e002\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e120\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.6\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e003\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e450\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e7\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.6\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e004\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e600\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e32.7\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e5.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e005\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e700\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e25\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.6\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e006\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e850\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e29\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.4\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e007\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e650\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e25\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.8\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e008\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e510\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e26\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e5.1\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThis work establishes a practical, automated radiosynthetic pathway for 2-[\\u0026sup1;⁸F]BPA. Compared to electrophilic fluorination approaches (Mairinger et al., 2015; Ishiwata, 2019), our method delivers superior radiochemical purity and acceptable molar activity, while avoiding the need for specialized electrophilic fluorine production.\\u003c/p\\u003e \\u003cp\\u003e2-[\\u0026sup1;⁸F]BPA is a promising agent for boron neutron capture therapy (BNCT); however, its application has been limited by the complexity of its multi-step radiosynthesis. Previous methods relied on laborious, multi-step and multi-pot reactions involving the use of \\u0026sup1;⁸F₂ gas, which yielded low radiochemical product yields. These approaches pose significant challenges for routine clinical implementation, as most automated synthesis modules are designed with only a single reactor. More recently, Chang et al. demonstrated that 2-[\\u0026sup1;⁸F]BPA could be produced with high radiochemical yield, but the process was performed manually and required multiple reaction vessels, complicating its clinical translation.⁵ In this study, we developed a robust, two-step, single-pot automated synthesis protocol for multi-dose production of 2-[\\u0026sup1;⁸F]BPA using the GE TRACERlab\\u0026trade; FXFN radio synthesizer (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e and \\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e), which is cGMP-enabled and suitable for future clinical applications. Although the NDCRY remains modest (3\\u0026ndash;5%), this is within the range reported for electrophilic syntheses, and sufficient for preclinical imaging when starting activity exceeds 500 mCi. Future optimization could target improved yields through fine-tuning precursor loading, copper complex stoichiometry, or solvent ratios.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eAnother important consideration is the complexity of the analytical radio-HPLC profile, which demonstrates the presence of multiple radiolabeled byproducts and closely eluting impurities. This complexity makes purification challenging, as baseline separation from the desired 2-[\\u0026sup1;⁸F]BPA peak is required to ensure radiochemical purity and identity. Optimization of the semi-preparative HPLC conditions was therefore critical to achieving consistent recovery of the target fraction. We found that careful control of mobile phase composition and isocratic conditions was essential to reproducibly isolate 2-[\\u0026sup1;⁸F]BPA without significant product loss. These parameters will be especially important for future cGMP adaptation of the method, where batch-to-batch reproducibility is mandatory.\\u003c/p\\u003e \\u003cp\\u003eImportantly, the entire workflow is compatible with a commercial synthesis module, supporting potential cGMP implementation. The robustness of the procedure, demonstrated across multiple runs, indicates suitability for routine production.\\u003c/p\\u003e \\u003cp\\u003eIn the context of BNCT, 2-[\\u0026sup1;⁸F]BPA PET has the potential to refine patient selection and dosimetry, ultimately improving therapeutic outcomes. Our results represent a critical step toward enabling such clinical applications.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTracerlab vs. Fastlab and rationale to optimize the single pot reaction\\u003c/h2\\u003e \\u003cp\\u003eCompared to cassette-based systems such as the GE Fastlab, the GE Tracerlab FX series offers greater flexibility and control over the radiosynthetic process. The Tracerlab allows for direct modification of reaction parameters, tubing configuration, and reagent delivery sequences, enabling rapid optimization of labeling conditions and adaptation to novel tracers or non-standard chemistries. This open architecture is particularly advantageous during early-stage development, when synthetic steps may require iterative fine-tuning or non-routine manipulations. In contrast, cassette-based modules are designed primarily for standardized, routine production under GMP conditions. While they provide improved reproducibility, reduced risk of operator error, and simplified regulatory compliance, their fixed design limits the user\\u0026rsquo;s ability to modify the process or incorporate custom reagents. Thus, the Tracerlab system offers a more versatile platform for research and development, whereas cassette-based modules such as the Fastlab are better suited for high-throughput clinical manufacturing.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eWe report a robust, automated radiosynthetic method for 2-[\\u003csup\\u003e18\\u003c/sup\\u003eF]BPA using copper-mediated nucleophilic fluorination of a rationally designed precursor. The method circumvents the challenges of electrophilic fluorination, delivers high radiochemical purity, acceptable molar activity, and formulation stability, and is compatible with commercial synthesis platforms. These features position the tracer for preclinical evaluation and pave the way for cGMP-compliant production in support of BNCT clinical workflows.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003eAuthor contributions\\u003c/p\\u003e\\n\\u003cp\\u003eVP, RTT, DKG and CDP designed, performed and analyzed the experiments. HCM evaluated the results. All authors read and approved the final manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003eFunding\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by :\\u003c/p\\u003e\\n\\u003cul\\u003e\\n \\u003cli\\u003eThe Cancer Prevention Research Institute of Texas (CPRIT) RR200046; H. C. Manning is a CPRIT Scholar of Cancer Research.\\u0026nbsp;\\u003c/li\\u003e\\n \\u003cli\\u003eTAE Life Sciences\\u003c/li\\u003e\\n\\u003c/ul\\u003e\\n\\u003cp\\u003eAvailability of data\\u003c/p\\u003e\\n\\u003cp\\u003eAll data generated or analyzed during this study are included in this manuscript\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eNedunchezhian K, Aswath N, Thiruppathy M, Thirugnanamurthy S. Boron Neutron Capture Therapy - A Literature Review. J Clin Diagn Res. 2016 Dec;10(12):ZE01-ZE04. doi: 10.7860/JCDR/2016/19890.9024. Epub 2016 Dec 1. PMID: 28209015; PMCID: PMC5296588.\\u003c/li\\u003e\\n \\u003cli\\u003eMishima Y, Ichihashi M, Hatta S, Honda C, Yamamura K, Nakagawa T, Obara H, Shirakawa J, Hiratsuka J, Taniyama K, et al. First human clinical trial of melanoma neutron capture. Diagnosis and therapy. Strahlenther Onkol. 1989 Feb-Mar;165(2-3):251-4. PMID: 2494743.\\u003c/li\\u003e\\n \\u003cli\\u003eIshiwata K. 4-Borono-2-\\u003csup\\u003e18\\u003c/sup\\u003eF-fluoro-L-phenylalanine PET for boron neutron capture therapy-oriented diagnosis: overview of a quarter century of research. Ann Nucl Med. 2019 Apr;33(4):223-236. doi: 10.1007/s12149-019-01347-8. Epub 2019 Feb 28. PMID: 30820862; PMCID: PMC6450856.\\u003c/li\\u003e\\n \\u003cli\\u003eMairinger, S., Stanek, J., Wanek, T., Langer, O., Kuntner, C., 2015. Automated electrophilic radiosynthesis of [18F]FBPA using a modified nucleophilic GE TRACERlab FXFDG. Applied Radiation and Isotopes 104, 124-127.\\u003c/li\\u003e\\n \\u003cli\\u003eChang TY, et al. Comparison of the synthesis and biological properties of no-carrier-added and carrier-added 4-borono-2-[18F]fluorophenylalanine ([18F]FBPA). Nucl Med Biol. 2023 Jan-Feb;116-117:108313. doi: 10.1016/j.nucmedbio.2022.108313. Epub 2022 Dec 30. PMID: 36621257.\\u003c/li\\u003e\\n \\u003cli\\u003eNaka, S., et al., 2012. Optimization of 4-borono-2-[18F]fluoro-L-phenylalanine (FBPA) synthesis: PET tracer for boron neutron capture therapy (BNCT) for cancer treatment. Journal of Nuclear Medicine,\\u0026nbsp;53\\u0026nbsp;(supplement 1)\\u0026nbsp;1689\\u003c/li\\u003e\\n \\u003cli\\u003eHe, et al., 2021. Nucleophilic radiosynthesis of boron neutron capture therapy-oriented PET probe [18F]FBPA using aryldiboron precursors. Chem. Commun. 57, 8953.\\u003c/li\\u003e\\n \\u003cli\\u003eIshiwata, K., 2019. 4-Borono-2-18F-fluoro-L-phenylalanine PET for boron neutron capture therapy-oriented diagnosis: overview of a quarter century of research. Ann. Nucl. Med. 33, 223\\u0026ndash;236.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"Schemes\",\"content\":\"\\u003cp\\u003eScheme 1 and 2 are available in the Supplementary Files section.\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"ejnmmi-radiopharmacy-and-chemistry\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"erpc\",\"sideBox\":\"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/erpc/default.aspx\",\"title\":\"EJNMMI Radiopharmacy and Chemistry\",\"twitterHandle\":\"@officialEANM\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"2-[18F]BPA, Radiosynthesis, Boron Neutron Capture Therapy, Copper-mediated fluorination, PET tracer\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8289343/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8289343/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground:\\u003c/h2\\u003e \\u003cp\\u003eBoron neutron capture therapy relies on the selective accumulation of boron-containing compounds in tumor tissue, making accurate quantification of boron distribution essential for effective treatment planning. The amino acid analog boronophenylalanine is widely used as a boron delivery agent, yet direct assessment of its biodistribution remains challenging. A fluorine-18 labeled analog, 2-fluoro-boronophenylalanine, offers the potential to visualize and quantify uptake through positron emission tomography. However, reported radiosynthetic methods often suffer from low radiochemical yield, complex workflows, and limited compatibility with automated production platforms. The aim of this study was to design a stable precursor suitable for nucleophilic fluorination, develop a fully automated single-reactor radiosynthesis, and characterize the resulting tracer to support both preclinical use and future clinical translation.\\u003c/p\\u003e\\u003ch2\\u003eResults:\\u003c/h2\\u003e \\u003cp\\u003eA rationally protected precursor incorporating tert-butyloxycarbonyl and pinacol ester groups was synthesized and isolated with high chemical and enantiomeric purity. Using this precursor, an automated single-pot radiosynthesis was implemented on a commercial synthesis module employing copper-mediated nucleophilic fluorination followed by acidic hydrolysis. Across eight production runs, the method yielded 2-fluoro-boronophenylalanine with non-decay-corrected radiochemical yields of 3\\u0026ndash;5 percent and a total synthesis time of approximately 60\\u0026ndash;70 minutes. Radiochemical purity consistently exceeded 98 percent, and the molar activity at the end of synthesis ranged from 85 to 120 gigabecquerels per micromole. The final formulation remained chemically and radiochemically stable for at least four hours at room temperature, and residual solvent levels were within accepted safety limits. Analytical and chiral chromatographic assessments confirmed product identity, purity, and retention of stereochemical configuration.\\u003c/p\\u003e\\u003ch2\\u003eConclusions:\\u003c/h2\\u003e \\u003cp\\u003eThis study establishes a practical and fully automated radiosynthetic approach for producing 2-fluoro-boronophenylalanine using a single-reactor nucleophilic fluorination strategy. The\\u003c/p\\u003e \\u003cp\\u003emethod overcomes key limitations of electrophilic fluorination and multi-pot workflows, provides high radiochemical purity and suitable molar activity, and is compatible with commercially available synthesis equipment. These features support routine preclinical application and position the method for future current good manufacturing practice adaptation to enable clinical use in boron neutron capture therapy planning.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Automated Radiosynthesis of 2-[18F]BPA for PET-based Planning of Boron Neutron Capture Therapy (BNCT): Rational Precursor Design, Radiofluorination, and Characterization of Methodology\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-12-22 10:06:26\",\"doi\":\"10.21203/rs.3.rs-8289343/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Major revision\",\"date\":\"2026-01-07T07:55:41+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2025-12-17T13:27:55+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-12-17T12:08:57+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-12-10T08:09:09+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"EJNMMI Radiopharmacy and Chemistry\",\"date\":\"2025-12-09T11:26:22+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"ejnmmi-radiopharmacy-and-chemistry\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"erpc\",\"sideBox\":\"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/erpc/default.aspx\",\"title\":\"EJNMMI Radiopharmacy and Chemistry\",\"twitterHandle\":\"@officialEANM\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"6e570bb3-e315-4cb7-a1df-3acadb3cb1a7\",\"owner\":[],\"postedDate\":\"December 22nd, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-03-09T16:09:35+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-8289343\",\"link\":\"https://doi.org/10.1186/s41181-026-00436-0\",\"journal\":{\"identity\":\"ejnmmi-radiopharmacy-and-chemistry\",\"isVorOnly\":false,\"title\":\"EJNMMI Radiopharmacy and Chemistry\"},\"publishedOn\":\"2026-03-05 15:58:40\",\"publishedOnDateReadable\":\"March 5th, 2026\"},\"versionCreatedAt\":\"2025-12-22 10:06:26\",\"video\":\"\",\"vorDoi\":\"10.1186/s41181-026-00436-0\",\"vorDoiUrl\":\"https://doi.org/10.1186/s41181-026-00436-0\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8289343\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8289343\",\"identity\":\"rs-8289343\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}