Deuterium- and Fluorine-18-Labeled Glutaminea PET Imaging Agent with Enhanced In Vivo Stability

Hari K Akula; Bao Hu; Jaclyn Brunner; Kaixuan Li; Lemise Saleh; Paul Vaska; Wenchao Qu

doi:10.1021/acsomega.5c03771

. 2025 Jul 3;10(27):29741–29753. doi: 10.1021/acsomega.5c03771

Deuterium- and Fluorine-18-Labeled Glutaminea PET Imaging Agent with Enhanced In Vivo Stability

Hari K Akula ^†,^‡, Bao Hu ^†,^‡, Jaclyn Brunner ^∥,^⊥, Kaixuan Li ^§, Lemise Saleh ^∥,^⊥, Paul Vaska ^‡,^∥,^⊥, Wenchao Qu ^†,^‡,^§,^*

PMCID: PMC12268413 PMID: 40686998

Abstract

Deuterium and fluorine-18 dual-isotope-labeled fluoroglutamine, (2S,4R)-[4-¹⁸F-3,3,4-d ₃]fluoroglutamine (4-[¹⁸F]FGln-d ₃), was designed, synthesized, and biologically evaluated for its potential as a novel glutamine metabolic imaging agent with improved in vivo stability. The tetradeuterated homoserine 11 intermediate was first synthesized via a six-step synthetic pathway, including a chiral HPLC separation process. Next, (2S,4S)-tosylate precursor 14 was prepared following the reference-reported method, and 4-[¹⁸F]FGln-d ₃ was successfully prepared using a semiautomated production process. After that, a head-to-head comparison of 4-[¹⁸F]FGln-d ₃ with its parent compound 4-[¹⁸F]FGln including in vitro cell uptake and in vivo murine animal imaging studies illustrated that the new tracer 4-[¹⁸F]FGln-d ₃ has a similar cell uptake manner and comparable tumor uptake and tumor-to-muscle ratio as 4-[¹⁸F]FGln, but moderately decreased radioactivity bone uptake at 120 min postinjection. Overall, the preliminary data from this research indicate the better in vivo stability of 4-[¹⁸F]FGln-d ₃ compared with its counterpart 4-[¹⁸F]FGln, which warrants further investigation of this radiotracer as the new PET imaging agent with enhanced stability for probing the glutamine metabolism in vivo.

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Introduction

Positron emission tomography (PET) is a clinical and research imaging technique for the noninvasive investigation of biochemical and molecular events in living organisms. , [¹⁸F]Fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) (Figure A) has made a tremendous success in PET imaging for the diagnosis of various diseases, such as cancers, neurological disorders, and cardiovascular diseases, in the past decades. Nowadays, FDG-PET has been proposed as one of the most powerful molecular imaging modalities. , The mechanism of FDG-PET is based upon the upregulation of the PI3K/Akt/mTor pathway, which can boost aerobic glycolysis and support tumor proliferation (Warburg effect). Glutamine is the second most abundant nutrient in the human body, particularly blood and skeletal muscles in high concentration (0.5–1.0 mmol/L). It plays various critical functions such as a substrate for DNA and protein synthesis, a primary source of fuel for cells lining the inside of the small intestine and rapidly dividing immune cells, and a regulator of acid–base balance in the kidney by producing ammonia. , In the brain, the glutamine–glutamate shunt is a critical pathway that controls the inhibitory and excitatory neuronal signals. The ample proofs have shown that upregulated glutaminolysis by overexpression of the oncogene c-Myc (Myc) plays a significant role in tumor growth and metabolism. −

Structures of (A) [¹⁸F]FDG, (B) l-5-[¹¹C]glutamine, (C) l-[5-¹³C-4-d ₂]glutamine, and (D) (2S,4R)-4-[¹⁸F]fluoroglutamine (4-[¹⁸F]FGln).

Based upon this mechanism, a series of radioactive and stable isotope-labeled glutamines, l-5-[¹¹C]glutamine ([¹¹C]Gln, Figure B), l-[5-¹³C-4-d ₂]glutamine (Figure C), and four stereoisomers of 4-[¹⁸F]fluoroglutamine (representative isomer (2S,4R)-4-[¹⁸F]fluoroglutamine (4-[¹⁸F]FGln), as shown in Figure D), have been synthesized and evaluated for tumor imaging purposes. Among them, fluorine-18 (¹⁸F, T _1/2 = 110 min)-labeled glutamine analog 4-[¹⁸F]FGln has been explored broadly in various preclinical and clinical research works, − such as imaging gliomas, neuroblastoma, and brain metastasis; − assessing ASCT2 expression in lung cancer; predicting the response to BRAF^V600E-targeted therapy in preclinical models of colon cancer; detecting glutamine pool size changes in triple-negative breast cancer (TNBC); , imaging 4-[¹⁸F]FGln accumulation in breast cancer; , imaging glutamine metabolism in murine models of hepatocellular carcinoma (HCC) and nonalcoholic steatohepatitis progression; exploiting glutamine consumption in atherosclerotic lesions and addiction in multiple myeloma (MM) cells, , indicating bone marrow metabolism dysfunctions; following the changes in clear cell renal cell carcinoma (ccRCC) metabolism in vivo; finding metabolically targeted osteosarcoma therapy imaging biomarkers; detecting KRAS mutation for noninvasive PDAC diagnosis; imaging inflammatory arthritis and myositis, etc.

There is growing evidence to show that the transport of 4-[¹⁸F]FGln across cell membranes mainly relies on alanine–serine–cysteine, preferring transporter 2 (ASCT2, or SLC1A5), which is frequently found to be highly upregulated in many tumor cells. Once taken up by tumor cells, 4-[¹⁸F]FGln mainly stays unaltered and minimally metabolized to 4-[¹⁸F]fluoroglutamic acid. ,,, This characteristic clearly adds more weight for developing 4-[¹⁸F]FGln as a cancer imaging agent. The other structural characteristic of this molecule, i.e., fluorine-18 attached to the carbon skeleton of the molecule at C-4, which is the α-position to a carbonyl group (C-5), makes the ¹⁸F–C(4) bond vulnerable as compared to a normal sp³ C–F bond. Several reports have shown that 4-[¹⁸F]FGln has propensity toward defluorination in vivo and apparent bone uptake is observed in both animals and humans. ,, This limitation may dampen the interest in using this radiotracer for cancer imaging, especially for metastatic cancers in which the tumor has spread from the original sites to the lymph system and/or bone marrow. To slow down the in vivo defluorination process and lessen the interference from free [¹⁸F]fluoride ions, there is an unmet demand to develop novel ¹⁸F-labeled glutamine analogs with better in vivo stability while maintaining comparable tumor uptake avidity.

In drug discovery and development, the replacement of a hydrogen atom with deuterium has become a valuable approach for improving the pharmacokinetic and/or toxicity properties, which is based on the mechanism of the deuterium kinetic isotope effect (DKIE). − The recent US FDA-approved deuterated drugs such as Austedo and deucravacitinib are such examples (Figure A,B). In the radiopharmaceutical research areas, the concept of DKIE was also employed even back to three decades ago while [¹¹C]l-deprenyl-d ₂ was developed to replace its parent compound [¹¹C]l-deprenyl for achieving better in vivo stability in humans as a MAO B imaging agent (Figure C). − Moreover, this strategy is still actively employed in the development of radiopharmaceuticals for PET imaging. , We hypothesized that the substitution of hydrogen with deuterium in the molecule skeleton of 4-[¹⁸F]FGln might improve its in vivo metabolic stability, while maintaining similar in vivo imaging properties. Therefore, a deuterated 4-[¹⁸F]FGln counterpart molecule, (2S,4R)-[4-¹⁸F-3,3,4-d ₃]fluoroglutamine (4-[¹⁸F]FGln-d ₃, Figure D) was designed with expectation as the new PET tracer for imaging the glutamine metabolism. Herein, we report the first synthesis of 4-[¹⁸F]FGln-d ₃ and its head-to-head biological comparison with 4-[¹⁸F]FGln.

Representative FDA-approved deuterated drugs and PET radiotracers. (A) First FDA-approved deuterated drug deutetrabenazine in 2017, (B) FDA-approved deuterated drug deucravacitinib in 2022, (C) [¹¹C]l-deprenyl-d ₂, and (D) (2S,4R)-[4-¹⁸F-3,3,4-dD ₃]fluoroglutamine (4-[¹⁸F]FGln-d ₃).

Results and Discussion

Chemistry

In the original synthesis of 4-[¹⁸F]FGln reported in 2011, a partially protected homoserine 1 was first synthesized and then converted to aldehyde 2 via a Dess–Martin selective oxidation. Next, the three-component Passerini reaction was employed to assemble a C-4 position-functionalized glutamine derivative, and further transformation provided the stereospecific tosylate 3 as a fluorination precursor. Finally, the desired 4-[¹⁸F]FGln ([¹⁸ F]4) was synthesized via mild fluorination followed by deprotection (Scheme ).

To approach the desired deuterated 4-[¹⁸F]FGln counterpart, 4-[¹⁸F]FGln-d ₃, the deuterated homoserine derivative 11 was assigned as the first synthetic target compound. After the initial attempts to synthesize this compound via the stereospecific or stereoenriched manner, which only presented us with unsatisfying results, a six-step method was designed and implemented to provide us with this deuterated homoserine intermediate 11 (Scheme ). First, an O-benzylating reagent, 2,4,6-tris(benzyloxy)-1,3,5-triazine (TriBOT, 5), was synthesized with in a 40% yield following the report from Yamada et al., and this reagent was next used to provide the benzoxy group-attached tetra-deuterated alkyl bromide intermediate 7. Next, it was reacted with tert-butyl N-(diphenylmethylene)-glycinate 8 using t-BuOK as the base and the following deprotection using 10% citric acid gave a racemic mixture homoserine derivative 9a,b in 51% yield. The chiral preparative HPLC method using a Chiralpak AY-H column successfully separated two isomers, and free amine 9a was obtained in 48% yield. Following Boc N-protection and catalytic debenzylation, two-step reactions provided us with a tetra-deuterated homoserine compound 11 in 96% yield.

With the tetradeuterated homoserine derivative 11 in hand, the synthetic protocol reported for 4-FGln synthesis was next employed to provide the deuterated analogue 4-FGln-d ₃ (16) as the standard compound outlined in Scheme . The synthetic sequence began with a controlled Dess–Martin oxidation of compound 11 to give aldehyde 12 in 72% yield. Intermediate 12 then underwent a Passerini three-component reaction with 2,4,6-trimethoxybenzyl isocyanide (TmobNC) and chloroacetic acid (CAA), followed by dechloroacetylation to afford (2S,4S)-alcohol 13a in 28% overall yield. Subsequent tosylation of intermediate 13a produced (2S,4S)-tosylate 14 in 75% yield. Finally, nucleophilic fluorination of 14 with tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF) and triethylamine trishydrofluoride (Et₃N·3HF) followed by a global deprotection reaction with trifluoroacetic acid/dimethyl sulfide (TFA/DMS) provided 5 mg (20% overall yield) of the desired 4-FGln-d ₃ (16) as the reference standard compound.

Radiochemistry

In the past decade, the synthesis and production of 4-[¹⁸F]FGln have been extensively investigated and the fully automated production process had been reported by several research facilities to replace the initially reported manual synthesis method with the use of different commercial synthesizers. − Based on existing instruments and specific conditions at our radiochemistry facilities, we chose a semiautomated production protocol to synthesize both 4-[¹⁸F]-FGln-d ₃ and 4-[¹⁸F]-FGln tracers. First of all, the intermediate [¹⁸ F]15 was prepared following the reported method with minimal modification using GE TRACERLab FXNPro as the synthesis module. After the radiofluorination, semipreparative HPLC purification, and solid-phase extraction (SPE) using a C18 light cartridge, the [¹⁸ F]15 activity was eluted with ethanol (1 mL) into another reaction vial. Although the next acidic deprotection reaction can also be processed in an automated manner, the volatile and high corrosive properties of TFA potentially could contaminate the synthesis module, shorten the working life of the tubing and valves, and cause the tedious postcleanup work of the synthesizer. To avoid these potential problems, the second step was conducted following the original report with a manual operation process. Once all solvents were removed via an azeotropic evaporation, a series of operations, including acidic full deprotection, purification using a Ag11-A8 resin column, formulation with the addition of sodium acetate (NaOAc) salt, and sterilization via filtration, provided the desired isotonic and sterilized 4-[¹⁸F]FGln-d ₃ ([¹⁸ F]16) product, which is ready for further biological evaluation. The whole production process took around 90–115 min with an overall nondecay-corrected radiochemical yield of 3.6%–12.9% and the radiochemical purity ranged from 81% to 90%. The biological study used 4-[¹⁸F]FGln ([¹⁸ F]4), which was also synthesized in the same aforementioned process, with the radiochemical purity ranging from 88.6% to 92.6%. It is desired that the radiochemical purity of tracers (the isomer of (2S,4R) form) that is used in this biological study is at least more than 80%, and the radiochemical impurity including three radio-isomers and other radiochemical impurities is less than 20%.

In Vitro Cellular Uptake

After 4-[¹⁸F]FGln-d ₃ ([¹⁸ F]16), a rat gliosarcoma cell line, 9L/lacZ, was used to compare the cellular uptake of the radiotracer with 4-[¹⁸F]FGln ([¹⁸ F]4) (Figure ). 9L/lacZ cells were incubated with 4-[¹⁸F]FGln or 4-[¹⁸F]FGln-d ₃ for 5, 30, 60, and 120 min to compare their specific uptake at each time point. In vitro results demonstrated that 4-[¹⁸F]FGln and 4-[¹⁸F]FGln-d ₃ were gradually taken up by 9L/lacZ cells over 120 min. The maximum uptake of 4-[¹⁸F]FGln and 4-[¹⁸F]FGln-d ₃ was 28.4 and 44.7%ID/mg at 120 min, respectively (Figure A).

Cellular uptake of 4-[¹⁸F]FGln and 4-[¹⁸F]FGln-d ₃. (A) Time-dependent uptake of 4-[¹⁸F]FGln and 4-[¹⁸F]FGln-d ₃ in 9L/lacZ cells (n = 3). (B) Uptake of 4-[¹⁸F]FGln and 4-[¹⁸F]FGln-d ₃ by 9L/lacZ cells in the presence of BCH (n = 3), MeAIB (n = 3), l-Ser (n = 3), and l-Gln (n = 3). Data were analyzed using Student’s two-tailed unpaired t test, where **** indicates a p value of <0.0001, *** indicates a p value of <0.001, ** indicates a p value of <0.01, * indicates a p value of <0.05, and ns indicates a p value of >0.05.

Previous reports have confirmed that 4-[¹⁸F]FGln can be transported into 9L/lacZ cells via system L (Na⁺ independent), ASC, and ASC N (Na⁺-dependent) amino acid transporters. ,, Since system A was found to be not responsible for the cellular uptake of 4-[¹⁸F]FGln, 4-[¹⁸F]FGln-d ₃ was not blocked either with 0.5, 1, and 5 mM doses of α-(methylamino)isobutyric acid (MeAIB, system A inhibitor) (Figure B). The uptake of 4-[¹⁸F]FGln-d ₃ was blocked from 16% (0.5 mM) to 38% (5 mM) with 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH, system L inhibitor), from 11% (0.5 mM) to 65% (5 mM) with l-serine (l-Ser, system ASC substrate), and from 71% (0.5 mM) to 80% (5 mM) with l-glutamine (l-Gln, system ASC N substrate), which was comparable to that of 4-[¹⁸F]FGln. As expected, the deuteration at C3 and C4 positions did not impact the cellular uptake of 4-[¹⁸F]FGln-d ₃ as observed for L-[¹⁸F]FAla and L-[¹⁸F]FAla-d ₃. Taken together, 4-[¹⁸F]FGln-d ₃ demonstrated a similar uptake manner to 4-[¹⁸F]FGln. 4-[¹⁸F]FGln-d ₃ was transported into 9L/lacZ cells partially via system L, while predominantly via systems ASC and ASC N.

In Vivo PET ImagingComparison of 4-[¹⁸F]FGln and 4-[¹⁸F]FGln-d ₃ in 9L/lacZ Tumor-Bearing Rats

A 120 min dynamic PET imaging was performed in 9L/lacZ tumor-bearing rats to compare the biodistribution of 4-[¹⁸F]FGln-d ₃ and 4-[¹⁸F]FGln (Figure ). Region of interest (ROI) analysis indicated that the tumor uptake of both radiotracers reached the maximum rapidly and decreased over time (Figure ). 4-[¹⁸F]FGln-d ₃ demonstrated the close amount of tumor uptake (standardized uptake value (SUV) = 0.51 ± 0.14) to 4-[¹⁸F]FGln (SUV = 0.61 ± 0.07, p > 0.05). A close tumor-to-muscle contrast was observed for 4-[¹⁸F]FGln-d ₃ (SUV = 0.92 ± 0.33) and 4-[¹⁸F]FGln (SUV = 1.18 ± 0.24) as well (p > 0.05). The slightly lower tumor uptake and tumor-to-muscle contrast data of 4-[¹⁸F]FGln-d ₃ could be due to several varying factors from the experiments, such as the lower batch radiochemical purity of 4-[¹⁸F]FGln-d ₃ compared with 4-[¹⁸F]FGln (87.0% vs 92.6%) and the change of tumor environment in the different days of imaging (4-[¹⁸F]FGln at the first day and 4-[¹⁸F]FGln-d ₃ at the third day). The blood elimination half-lives of 4-[¹⁸F]FGln-d ₃ (2.08 min; 95% Cl, 1.52–2.97 min) and 4-[¹⁸F]FGln (2.07 min; 95% Cl, 1.56–2.84 min) were comparable to each other as well. The bone uptake of both radiotracers increased gradually, while the bone uptake of 4-[¹⁸F]FGln-d ₃ (2.14 ± 0.28) was significantly lower than that of 4-[¹⁸F]FGln (2.83 ± 0.38) at 120 min postinjection (p < 0.01, Figure ). As expected, the applied deuteration strategy successfully improved the resistance of 4-[¹⁸F]FGln-d ₃ to in vivo defluorination while not influencing its tumor uptake. Bone metastasis is one of the common features observed in various cancers such as breast and prostate cancer. , Early reports have shown the tendency of in vivo defluorination and apparent bone uptake of 4-[¹⁸F]FGln in both animals and humans, , and the later investigation of this tracer in animal models depicted that the defluorination and bone uptake could cause the less-satisfied imaging contrast. Thus, the reduced nonspecific bone uptake of 4-[¹⁸F]FGln-d ₃ could potentially offer better-quality PET images and improve the accuracy of clinical diagnosis, particularly bone metastasis.

PET imaging of 4-[¹⁸F]FGln and 4-[¹⁸F]FGln-d ₃ in 9L/lacZ tumor-bearing rats. Representative dynamic PET images of 4-[¹⁸F]FGln (n = 3) and 4-[¹⁸F]FGln-d ₃ (n = 3) in 9L/lacZ tumor-bearing rats at 30, 60, and 120 min postinjection.

ROI analysis of PET imaging for 4-[¹⁸F]FGln and 4-[¹⁸F]FGln-d ₃ in 9L/lacZ-tumor-bearing rats. Time–activity curves of (A) tumor, (B) bone, (C) muscle, and (D) blood for 4-[¹⁸F]FGln (n = 3) and 4-[¹⁸F]FGln-d ₃ (n = 3). Data were analyzed using Student’s two-tailed paired t test, where ** indicates a p value of <0.01 and ns indicates a p value of >0.05.

Conclusions

In summary, a new synthetic pathway was developed to prepare the deuterium and fluorine-18 dual isotope-labeled glutamine, 4-[¹⁸F]FGln-d ₃ ([¹⁸ F]16). After the successful synthesis of the key intermediate, tetradeuterated homoserine 11 via a six-step preparation process, the next six-step transformation following the previously reported method for 4-FGln synthesis provided us with the (2S,4S) form of tosylate 14 as the labeling precursor as well as the (2S,4R) form of 4-FGln-d ₃ 16 as the reference standard compound. Next, a semiautomated radiosynthesis method was conducted to provide us with the desired new tracer 4-[¹⁸F]FGln-d ₃. Subsequent results from in vitro cellular uptake confirmed that 4-[¹⁸F]FGln-d ₃ was transported and taken up into tumor cells in the same manner as 4-[¹⁸F]FGln. More importantly, the in vivo PET imaging results in murine tumor models clearly demonstrated that 4-[¹⁸F]FGln-d ₃ not only had comparable tumor uptake and tumor-to-muscle ratio as the counterpart tracer 4-[¹⁸F]FGln but also decreased the radioactivity bone uptake at 120 min postinjection. These newly discovered results strongly supported that 4-[¹⁸F]FGln-d ₃ could be utilized as a PET imaging tool to elucidate in vivo glutamine metabolic processes in cancer biology and improve the clinical diagnosis of various chronic diseases.

Materials and Methods

Chemistry

Organic Chemistry

All reagents used were commercial products and were used without further purification unless otherwise indicated. Reactions were carried out under an atmosphere of nitrogen. Analytical thin-layer chromatography was carried out using aluminum-backed plates coated with Merck Kieselgel 60 GF254. Chromatography was performed using silica gel (70-230 mesh, Sigma-Aldrich). ¹H NMR spectra were obtained at either 400, 500, or 700 MHz; ¹³C NMR spectra were recorded either at 125 or 175 MHz; ¹⁹F NMR spectra were recorded at 376 MHz; and ²H NMR spectra were obtained at 61 MHz (Bruker Nanobay 400, Avance III 500, and Avance III 700 spectrometers). Chemical shifts are reported as δ values (parts per million) relative to the remaining protons in the deuterated solvent. Coupling constants are reported in hertz. Multiplicity is defined by s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), br (broad), or m (multiplet). HPLC analyses were performed on Jasco LC-4000 series. LRMS and HRMS were obtained with Agilent 6110 Quadrapole LC/MS and Agilent LC-UV-TOF instruments, respectively. Optical rotation values were measured on an Anton Paar MCP 100 polarimeter.

Tert-butyl(O ⁵-benzyl-3,3,4,4-tetradeutero)homoserinate (9a,b)

Step i

To a solution of 2-bromoethanol-d ₄ 6 (0.73 mL, 10 mmol) in 1,4-dioxane (50 mL) was added 2,4,6-tris(benzyloxy)-1,3,5-triazine (TriBOT 5, 1.6 g, 4 mmol) and 4 Å molecular sieve (MS, 1.25 g) followed by trifluoromethanesulfonic acid (TfOH, 0.18 mL, 2 mmol) and the mixture was stirred at room temperature for 1.5 h under a N₂ atmosphere. At this point, the second portion of TriBOT 5 (1.4 g, 3.5 mmol) was added to the reaction mixture and stirred at room temperature (r.t.) for 16 h under a N₂ atmosphere. The solvent was removed under reduced pressure, and the residue was purified via silica gel column chromatography using 5% ethyl acetate (EtOAc) in hexanes to afford a mixture of (1,1,2,2-d ₄-2-bromoethoxy)benzene (7) and (1,2,2-d ₃-vinyl)benzyl ether (7’) in the ratio 9.4:1 (2.275 g) as a pale-yellow oil.

Step ii

In a flame-dried 250 mL round-bottomed flask, a solution of t-butyl N-(diphenylmethylene) glycinate 8 (1.77 g, 6 mmol) in tetrahydrofuran (THF, 30 mL) was prepared and cooled down to −70 °C under a N₂ atmosphere. To this was added a solution of potassium tert-butyloxide (KOtBu, 0.9 g, 6 mmol) in THF (6 mL) and the mixture was stirred at 0 °C for 30 min. Then a solution of mixture from step 1 (1.137 g) in THF (8 mL) was added to the mixture and the mixture was stirred at r.t. for 16 h. The solvent was removed under reduced pressure and saturated NaHCO₃ (100 mL) was added to the residue and then extracted with CH₂Cl₂ (3 × 75 mL). All organic layers were combined, washed with brine (10 mL), dried with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford a red oil. Another reaction was also conducted on the same scale simultaneously.

Step iii

This residue from the two batches of step ii was dissolved in THF (15 mL). To this solution was added 10% citric acid (15 mL) dropwise and the mixture was stirred overnight at r.t. The mixture was acidified with 0.5 M HCl (20 mL) and then washed with tert-butylmethyl ether (TBME, 50 mL). The organic phase was extracted with water (2 × 50 mL). The combined aqueous layers were washed with TBME (4 × 50 mL) to remove the cleaved benzophenone. The aqueous layer was then basified with solid Na₂CO₃ to adjust the pH to 9 and extracted with CH₂Cl₂ (3 × 50 mL). The combined organic extracts were dried with anhydrous Na₂SO₄ and concentrated in vacuo. The crude product was purified by silica gel chromatography using 1–3% MeOH in CH₂Cl₂ to give the title compounds 9a and 9b (1.39 g, 51%) as a colorless liquid. ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.31 (m, 4H), 7.31–7.27 (m, 1H), 4.51 (s, 2H), 3.49 (s, 1H), 1.44 (s, 9H). HRMS calcd for C₁₅H₂₀D₄NO₃ [M + H]⁺, 270.2002; found, 270.2001.

Separation of (2S)-Tert-butyl(O ⁵-benzyl-3,3,4,4-tetradeutero)homoserinate (9a) and (2R)-Tert-butyl(O ⁵-benzyl-3,3,4,4-tetradeutero)homoserinate (9b) via Chiral Preparative HPLC

The separation of enantiomers 9a and 9b was carried out on a Chiralpak AY-H column (250 × 20 mm, 5 μm) with the mobile phase (EtOH : 0.1% diisopropylethylamine (DIPEA) in hexanes = 4:1) and monitored at 254 nm with a flow rate of 8.0 mL/min (retention time (t _R), t _R-9b: 10.82 min; t _R-9a: 15.39 min). A solution of the racemic mixture 9a,b (16 mg) in the mobile phase (0.8 mL) was used in each injection. A total of 1.765 g (6.552 mmol) of the racemic mixture 9a,b was separated into multiple injections under the aforementioned conditions and both enantiomers 9b and 9a were collected between 10.5–13 min and 14.7–19 min, respectively. All of the fractions corresponding to the enantiomer were combined and concentrated under reduced pressure and then dried under high vacuum for several hours until DIPEA completely evaporated to give 9a (873.6 mg, 49%) as a colorless liquid and 9b (854.3 mg, 48%) as a colorless liquid.

Compound 9a: HPLC purity: >99% (t _R = 9.692 min; column: Amylose-2 (250 × 4.6 mm); UV detector, 254 nm, 20/80/0.1 EtOH/hexanes/DIPEA; flow rate: 1.0 mL/min). ¹H NMR (700 MHz, CDCl₃) δ: 7.36–7.31 (m, 4H), 7.31–7.27 (m, 1H), 4.51 (s, 2H), 3.50 (s, 1H), 1.44 (s, 9H). ²H NMR (61 MHz, CDCl₃) δ 3.58 (s, 2D), 2.06 (s, 1D), 1.75 (s, 1D). ¹³C NMR (175 MHz, CDCl₃) δ: 175.2, 138.5, 128.6, 127.9, 127.8, 81.3, 73.2, 66.4 (m), 52.8, 33.8 (m), 28.2. HRMS calcd for C₁₅H₂₀D₄NO₃ [M + H]⁺, 270.2002; found, 270.2001; ${[α]}_{D}^{25}$ = 5.233° (c = 0.168, EtOH).

Compound 9b: HPLC purity: >99% (t _R = 6.383 min; column: Amylose-2 (250 × 4.6 mm), UV detector, 254 nm, 20/80/0.1 EtOH/hexanes/DIPEA; flow rate: 1.0 mL/min). ¹H NMR (700 MHz, CDCl₃) δ: 7.36–7.31 (m, 4H), 7.31–7.26 (m, 1H), 4.50 (s, 2H), 3.48 (s, 1H), 1.44 (s, 9H). ²H NMR (61 MHz, CDCl₃) δ 3.57 (s, 2D), 2.04 (s, 1D), and 1.74 (s, 1D). ¹³C NMR (175 MHz, CDCl₃) δ 175.3, 138.4, 128.5, 127.8, 127.7, 81.0, 73.1, 66.3 (dt, J = 43.0, 21.5 Hz), 52.6, 33.8 (dt, J = 39.1, 19.4 Hz), 28.1. HRMS calcd for C₁₅H₂₀D₄NO₃ [M + H]⁺, 270.2002; found, 270.2000. ${[α]}_{D}^{25}$ = −15.476° (c = 0.172, EtOH).

(2S)-Tert-butyl(-Butyl (O-benzyl-⁵-benzyl-N-(tert-butoxycarbonyl)-3,3,4,4-tetradeutero)homoserinate (10)

To a solution of compound 9a (852 mg, 3.163 mmol) in 1,4-dioxane (32 mL), (Boc)₂O (828.3 mg, 3.795 mmol) was added and the mixture was stirred at r.t. for 16 h. The mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using 10% EtOAc in hexanes to give compound 10 (1.168 g, quantitative) as a colorless viscous liquid. ¹H NMR (700 MHz, CDCl₃) δ 7.37–7.31 (m, 4H), 7.31–7.26 (m, 1H), 5.39 (d, 1H, J = 7.8 Hz), 4.48 (ABq, Δδ_AB = 0.02, J _AB = 11.9 Hz, 2H), 4.27 (d, 1H, J = 8.0 Hz), 1.44 (s, 9H), 1.43 (s, 9H). ²H NMR (61 MHz, CDCl₃) δ 3.50 (s, 2D), 2.06 (s, 1D), and 1.91 (s, 1D). ¹³C NMR (175 MHz, CDCl₃) δ: 171.8, 155.7, 138.3, 128.6, 128.0, 127.9, 81.9, 79.6, 73.4, 66.2 (m), 52.4, 31.5 (m), 28.6, 28.2. HRMS calcd for C₂₀H₂₈D₄NO₅ [M + H]⁺, 370.2526; found, 370.2523. ${[α]}_{D}^{25}$ = −26.556° (c = 0.9, EtOH).

(2S)-4-Tert-butyl-2-(tert-butoxycarbonylamino)-3,3,4,4-tetradeutero-4-hydroxybutanoate (11)

In a flame-dried, 500 mL two-neck round-bottom flask, a solution of l-homoserinate 10 (1.168 g, 3.161 mmol) in EtOH (234 mL) was prepared to which 10% Pd/C (234 mg) was added and then sealed. The flask was degassed by connecting to a vacuum line. After the solvent in the flask started bubbling, the vacuum was cut off, and the flask was filled with H₂ gas using a balloon filled with hydrogen gas (H₂). This process was repeated three times, and then the reaction was stirred overnight under a H₂ atmosphere. The mixture was filtered through a short plug of silica gel and washed with MeOH (100 mL). The filtrate was concentrated under reduced pressure and dried under high vacuum to give compound 11 (844.1 mg, 96%) as a light yellowish liquid. ¹H NMR (700 MHz, CDCl₃) δ 5.35 (d, 1H, J = 7.2 Hz), 4.32 (d, 1H, J = 7.2 Hz), 3.42 (br s, 1H), 1.45 (s, 9H), 1.43 (s, 9H). ²H NMR (61 MHz, CDCl₃) δ 3.62 (s, 2D), 2.07 (s, 1D), and 1.49 (s, 1D). ¹³C NMR (175 MHz, CDCl₃) δ 172.2, 156.8, 82.5, 80.5, 57.6 (dt, J = 43.3, 21.7 Hz), 50.9, 35.8 (dt, J = 38.6, 19.4 Hz), 28.4, 28.2. HRMS calcd for C₁₃H₂₂D₄NO₅ [M + H]⁺: 280.2057, found: 280.2058. ${[α]}_{D}^{25}$ = −27.453° (c = 1.06, EtOH).

(2S)-4-Tert-butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-oxobutanoate (12)

In a 100 mL round-bottomed flask, a solution of alcohol 11 (465 mg, 1.66 mmol) in CH₂Cl₂ (8.3 mL) was prepared to which solid NaHCO₃ (1.4 g, 16.6 mmol) followed by a solution of Dess–Martin periodinane (DMP, 1.06 g, 2.497 mmol) in CH₂Cl₂ (8.3 mL) was added. The reaction was stirred at r.t. for 1 h. To the reaction mixture, Na₂S₂O₃ (14 mL, 1 M) was added, the resulting biphasic mixture was vigorously stirred for 5 min, and then sat. NaHCO₃ solution (4.6 mL) was added. The mixture was diluted with H₂O (deionized, 20 mL) and extracted with CH₂Cl₂ (3 × 50 mL). The combined organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 5% EtOAc in CH₂Cl₂ followed by 10% EtOAc in CH₂Cl₂ to give aldehyde 12 (351.9 mg, 77% yield). ¹H NMR (400 MHz, CDCl₃) δ 5.35 (d, 1H, J = 6.8 Hz), 4.45 (s, 1H, J = 7.7 Hz), 1.42 (s, 9H), 1.41 (s, 9H). ²H NMR (61 MHz, CDCl₃) δ 9.78 (s, 1D), 2.92 (s, 1D), 2.01 (br s, 1D). ¹³C NMR (175 MHz, CD₃CN) δ 200.5, 171.4, 156.5, 82.6, 80.1, 52.2, 50.2, 28.5, 28.1. HRMS calcd for C₁₃H₂₁D₃NO₅ [M + H]⁺, 277.1837; found, 277.1834. ${[α]}_{D}^{25}$ = −31.5° (c = 1.5, EtOH).

(2S,4S)-Tert-butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-hydroxy-5-oxo-5-(2,4,6-tri methoxybenzylamino)pentanoate (13a) and (2S,4R)-Tert-butyl 2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-hydroxy-5-oxo-5-(2,4,6-trimethoxybenzylamino)pentanoate (13b)

Step i: (2S)-Tert-butyl-2-(tert-butoxycarbonylamino)-4-(2-chloroacetoxy)-3,3,4-trideutero-5-oxo-5-(2,4,6-trimethoxybenzylamino)pentanoate

In a 50 mL round-bottomed flask, a solution of aldehyde 12 (350 mg, 1.267 mmol) in CH₂Cl₂ (9.9 mL) was prepared to which 2,4,6-trimethoxybenzyl isocyanide (288.7 mg, 1.393 mmol) followed by monochloroacetic acid (131.7 mg, 1.393 mmol) was added. The reaction was stirred at r.t. for 24 h. The mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using 20% EtOAc in hexanes followed by 40% EtOAc in hexanes to give the title compound (550.9 mg, 75%) as a white solid. ¹H NMR (700 MHz, CDCl₃) δ 6.84–6.75 (br s, 1H), 6.47 (t, 1H, J = 4.9 Hz), 6.11 (s, 4H), 5.23 (d, 1H, J = 7.6 Hz), 5.03 (d, 1H, J = 9.3 Hz), 4.59 (dd, 1H, J ₁ = 13.9 Hz, J ₂ = 5.8 Hz), 4.54–4.41 (m, 2H), 4.34 (dd, 2H, J ₁ = 13.7 Hz, J ₂ = 4.5 Hz), 4.23 (d, 1H, J = 8.0 Hz), 4.18–4.13 (m, 2H), 4.11–4.06 (m, 2H), 3.81 (s, 18H), 1.45 (s, 18H), 1.42 (s, 9H), 1.39 (s, 9H). ²H NMR (61 MHz, CDCl₃) δ 5.10 (br s, 1D), 2.34 (br s, 1D), 2.08 (br s, 1D). ¹³C NMR (175 MHz, CDCl₃) δ 171.2, 171.1, 168.0, 167.6, 166.6, 166.3, 161.23, 161.18, 159.4, 155.63, 155.61, 106.3, 106.1, 90.7, 82.8, 82.7, 80.3, 80.2, 72.1 (br m), 71.9 (br m), 56.0, 55.57, 55.55, 51.1, 50.8, 41.0, 40.8, 34.1 (br m), 32.6, 32.5, 28.5, 28.16, 28.15. HRMS calcd for C₂₆H₃₇D₃ClN₂O₁₀ [M + H]⁺, 578.2554; found, 578.2551.

Step ii

In a 20 mL round-bottom flask, to a solution of the above 2-chloroacetyl ester (550 mg, 0.951 mmol) in a solvent of EtOH/THF (1/1, v/v, 8.4 mL) were added thiourea (217.3 mg, 2.854 mmol) and NaHCO₃ (239.8 mg, 2.854 mmol), and the mixture was stirred at 50 °C for 1.5 h. The mixture was concentrated under reduced pressure, and the crude product was purified by CombiFlash (24 g silica, Redisep Column, flow rate 3 mL/min, 210 nm) using 30–40% EtOAc in hexanes to give compound 13a (178 mg, 37%) as a white solid and a mixture of 13a and 13b (101.2 mg, 21%) and compound 13b (115 mg, 24%) as a colorless viscous liquid.

Compound 13a: ¹H NMR (700 MHz, CDCl₃) δ 7.24 (br s, 1H), 6.11 (s, 2H), 5.44 (d, J = 7.6 Hz, 1H), 4.88 (s, 1H), 4.52 (dd, J = 13.7, 5.6 Hz, 1H), 4.42 (dd, J = 13.7 Hz, 5.3 Hz, 1H), 4.29 (d, J = 7.7 Hz, 1H), 3.81 (s, 6H), 3.80 (s, 3H), 1.44 (s, 9H), 1.41 (s, 9H). ²H NMR (61 MHz, CDCl₃) δ 3.98 (s, 1D), 1.94 (s, 2D). ¹³C NMR (175 MHz, CDCl₃) δ 171.9, 171.4, 161.0, 159.5, 157.5, 106.7, 90.7, 82.9, 80.9, 68.4–67.6 (m), 56.0, 55.5, 50.8, 39.0–38.1 (m), 31.9, 28.4, 28.1. HRMS calcd for C₂₄H₃₆D₃N₂O₉ [M + H]⁺, 502.2838; found, 502.2833. ${[α]}_{D}^{25}$ = −11.0° (c = 0.1, CH₂Cl₂).

Compound 13b: ¹H NMR (700 MHz, CDCl₃) δ 7.26 (br s, 1H), 6.10 (s, 2H), 5.45 (s, 1H), 4.56 (dd, 1H, J ₁ = 13.2 Hz, J ₂ = 5.2 Hz), 4.37 (dd, 1H, J ₁ = 13.7 Hz, J ₂ = 4.6 Hz), 4.18 (d, 1H, J = 6.8 Hz), 4.07 (br s, 1H), 3.793 (s, 6H), 3.790 (s, 3H), 1.44 (s, 9H), 1.41 (s, 9H). ²H NMR (61 MHz, CDCl₃) δ 4.06 (s, 1D), 2.33 (s, 1D), 1.87 (s, 1D). ¹³C NMR (175 MHz, CDCl₃) δ 172.4, 171.5, 161.0, 159.5, 156.4, 106.7, 90.6, 82.7, 80.5, 69.3 (t, J = 20.8 Hz), 55.9, 55.5, 51.7, 38.6–37.6 (m), 32.3, 28.4, 28.1. HRMS calcd for C₂₄H₃₆D₃N₂O₉ [M + H]⁺, 502.2838; found, 502.2827. ${[α]}_{D}^{25}$ = 9.821° (c = 0.112, CH₂Cl₂).

(2S,4R)-Tert-Butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-5-oxo-4-(tosyloxy)-5-(2,4,6-trimethoxybenzylamino)pentanoate (14)

In an 8 mL vial, to a solution of alcohol 13a (42.5 mg, 84.7 μmol) in CH₂Cl₂ (0.78 mL) at 0 °C, triethylamine (Et₃N, 59 μL, 0.424 mmol), para-toluenesulfonic chloride (TsCl, 32.3 mg, 0.170 mmol), and 4-dimethylaminopyridine (DMAP, 1.0 mg, 8.5 μmol) were added and the mixture was stirred at 0 °C for 15 min. The mixture was brought to r.t. and stirred for 3 h. It was diluted with CH₂Cl₂ (30 mL) and washed with deionized water (3 × 20 mL), brine solution (20 mL), dried with anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography using 30% EtOAc in hexanes, followed by 40% EtOAc in hexanes to give tosylate compound 14 (41.6 mg, 75%) as a white solid. HPLC purity: 95.0% [major peak: t _R = 14.6 min; minor peaks: 10.0 and 19.2 min], column: Cellulose-1 (250 × 4.6 mm), UV detector, 210 nm, 7% 2-propanol in hexanes; flow rate: 1.0 mL/min]. ¹H NMR (400 MHz, CDCl₃) δ 7.74 (d, 2H, J = 7.8 Hz), 7.25 (d, 2H, J = 7.6 Hz), 6.87 (br s, 1H), 6.13 (s, 2H), 5.09 (d, 1H, J = 8.2 Hz), 4.40 (d, 2H, J = 5.0 Hz), 4.00 (d, 1H, J = 8.3 Hz), 3.83 (s, 3H), 3.82 (s, 6H), 2.41 (s, 3H), 1.43 (s, 9H), 1.42 (s, 9H). ²H NMR (61 MHz, CDCl₃) δ 4.88 (s, 1D), 2.19 (s, 2D). ¹³C NMR (175 MHz, CDCl₃) δ 170.7, 167.3, 161.2, 159.4, 155.5, 145.6, 132.8, 130.2, 128.1, 105.9, 90.7, 82.2, 79.9, 77.2, 56.0, 55.6, 51.3, 34.7–33.5 (m), 32.6, 28.5, 28.1, 21.9. HRMS calcd for C₃₁H₄₁D₃N₂O₁₁S [M + H]⁺, 656.2927; found, 656.2925. ${[α]}_{D}^{25}$ = −27.961° (c = 1.03, MeOH).

(2S,4R)-Tert-butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-fluoro-5-oxo-5-(2,4,6-trimethoxy benzylamino)pentanoate (15)

To a stirred solution of tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF, 0.21 g, 0.7625 mmol) in 1:1 THF/CH₂Cl₂ (0.5 mL) was added Et₃N·(HF)₃ (5 μL) dropwise. After that, tosylate 15 (0.100 g, 0.1525 mmol), THF (0.25 mL), and the above “neutralized” TASF solution were added one by one to a 5 mL two-neck flask equipped with a reflux condenser. The mixture was heated to 50 °C by oil bath for 16 h. The reaction mixture was cooled to r.t. and then diluted with EtOAc. The resultant solution was washed with half-saturated NaHCO₃, water, and brine subsequently. The EtOAc phase was collected, dried with Na₂SO₄, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel (60–200 μm, 70–230 mesh) column chromatography using 30% EtOAc in hexanes followed by 40% EtOAc in hexanes to give the title compound 15 (56.3 mg). ¹H NMR (400 MHz, CDCl₃) δ 6.70 (br s, 1H), 6.13 (s, 2H), 4.57 (ddd, 1H, J ₁ = 22.7 Hz, J ₂ = 16.7 Hz, J ₃ = 5.8 Hz), 4.47–4.34 (m, 2H), 3.82 (s, 9H), 1.45 (s, 9H), 1.43 (s, 9H). HRMS calcd for C₂₄H₃₅D₃FN₂O₈ [M + H]⁺: 504.2795; found: 504.2794.

(2S,4R)-2,5-Diamino-3,3,4-trideutero-4-fluoro-5-oxopentanoic acid (16)

To a mixture of 15 (56.3 mg, 0.1118 mmol) with dimethylsulfide (30 μL) cooled with an ice bath (0 °C), trifluoroacetic acid (TFA, 1.5 mL) was added dropwise. After the addition, the reaction was brought to r.t. and stirred for 2.5 h. The solution was evaporated under reduced pressure to remove most TFA. The residue was dissolved in H₂O (5 mL) and washed with CH₂Cl₂ (3 mL × 2). The aqueous part was cooled in an ice bath and neutralized to pH 7 by the slow addition of ice-cold 5% aqueous ammonia. The resulting liquid was lyophilized to give a white solid. The solid was washed several times by trituration with MeOH and 50% EtOH solution to give the final product 4-FGln-d ₃ 16 (5 mg, 20% yield over two steps) as off-white solid. HPLC purity: 95.9% [major peak: t _R = 14.6 min; minor peaks: 11.8 and 18.1 min], column: Chirex 3126 (d)-pencillamine (250 × 4.6 mm), UV detector, 254 nm, 1.0 mM Cu(OAc)₂ solution; flow rate: 1.0 mL/min, column temperature: 10 °C]. ¹H NMR (400 MHz, D₂O) δ 3.97 (s, 1H). ²H NMR (61 MHz, CD₃OD) δ 5.41–5.01 (m, 0.5D), 4.52–4.12 (m, 0.5D), 2.71–2.05 (m, 2D). ¹³C NMR (175 MHz, D₂O + trace CD₃OD) δ: 179.6, 174.8, 174.1, 89.9 (m), 88.9 (m), 52.8, 33.7 (m). ¹⁹F NMR (376 MHz, D₂O) δ −190.33. HRMS calcd for C₅H₇D₃FN₂O₃ [M + H]⁺: 168.0858; found: 168.0853. ${[α]}_{D}^{25}$ = −17.278° (c = 0.2, H₂O).

Radiochemistry

All reagents were purchased from Sigma-Aldrich or Fisher Scientific unless otherwise mentioned. All three cartridges used for radiofluorination chemistry were purchased from Waters Corporation (Sep-Pak Light Accell Plus QMA Carbonate, Sep-Pak Light C18 cartridge, and Sep-Pak Light Alumina N cartridge). Noncarrier-added [¹⁸F]fluoride was produced in-house by bombarding of H₂ ¹⁸O (98 atom %, NUCMEDCOR) with a beam (50 μA) in a 5–25 min range of time using a 16.5 MeV cyclotron (GE, PETtrace 800). Both cartridges (Alumina-N Light and Sep-Pak C18 Light cartridges) were preconditioned with 5.0 mL ethanol and 10 mL water (HPLC grade) just before use. Radioactivity measurements were performed using a Capintec CRC-55t or 55tR PET dose calibrator. Analytical radio-HPLC analysis was performed on a JASCO HPLC system using an Eckert & Ziegler Flow-Count radio-HPLC detection system equipped with a diode detector.

The first step of radiosynthesis was performed in a GE TRACERLab FX2N synthesis module (Scheme ) following the listed sequences:

The [¹⁸F]fluoride in [¹⁸O]water (2.5 mL) was transferred from the cyclotron target to a GE synthesizer, and the radioactivity was trapped on a QMA carbonate cartridge.
The radioactivity trapped in the cartridge was eluted into the reactor with an 18-crown-6/KHCO₃ (8.0 mg/1.44 mg) aqueous MeOH solution (H₂O/MeOH, 0.18 mL/1.0 mL).
The reactor was heated to 70 °C under vacuum with a stream of helium for 5 min and then cooled to 40 °C.
The second portion of solvent for azeotropic drying (MeCN, 1 mL) was added to the reactor, and the reactor was heated again to 70 °C under vacuum with a stream of helium for 5 min. The drying continued for 3 min by raising the heating temperature to 95 °C.
After cooling to 40 °C, a solution of tosylate precursor (compounds 3 or 14, 5.0 mg) in MeCN (anhydrous, 0.60 mL) was added to the reactor and then heated to 70 °C for 15 min.
The reaction mixture was cooled below 40 °C and was diluted with HPLC solvent (2.0 mL) and water (0.6 mL). The diluted solution was passed through a light Al–N cartridge and was injected onto the semiprep HPLC for purification (Agilent XDB-C18 column, 250 × 9.4 mm, 5 μM; mobile phase: 73.5% MeOH/26.5% H₂O (0.1% v/v HCO₂H); 4 mL/min, UV detector set at 210 nm).
The portion of the product-containing radioactive peak (t _R ∼16.1 min) was collected and diluted with water (25 mL). This aqueous solution was next passed through a C18 light cartridge and washed with water (10 mL). The radioactivity was eluted from the cartridge with EtOH (1.0 mL) into a V-shaped microwave reaction tube (0.5–2.0 mL).

The second step of acidic deprotection was operated manually:

The radioactivity collected in V-shaped tube was transferred out from the hot cell, and a gentle nitrogen stream was introduced to evaporate the solvent from the V-shape tubed under the heating (75 °C) condition.
Once all of the solvent was evaporated, the reaction tube was cooled to r.t., TFA/anisole reagent (0.5/0.005 mL) was added, and the reaction mixture was heated at 60 °C for 5 min. The reaction tube was removed from the heater, and a gentle nitrogen flow was introduced to remove volatile liquid.
Ag11-A8 ion retardation and resin purification: once the liquid in the reaction tube became minimal, it was subjected to cooling with ice water bath, and dilution H₂O (1 mL) was added under mild shaking. This radioactive liquid was transferred to a Ag11-A8 resin (2.5 g) column and water (3 × 2 mL) was added to elute radioactivity.
The radioactive fractions (4 mL) were collected and diluted with NaOAc (4 mL, 2.36%) to give isocratic NaOAc (1.18%) solution. This solution was filtered through a filter (Millex-GV, 0.22 μm), and the filtrate was collected into a sterile vial (20 mL) to give the final product 4-[¹⁸F]FGln-d₃ ([¹⁸ F]16) or 4-[¹⁸F]FGln ([¹⁸ F]4).
The radiochemical purities of the final products ([¹⁸ F]16 or [¹⁸ F]4) were analyzed using previously reported conditions with a minor modification (HPLC: JASCO, Column: Chirex 3126 (d-pencillamine), 250 mm × 4.6 mm; final product sample: 20 μL; mobile phase: aqueous Cu(OAc)₂ solution (1 mM); flow rate: 1 mL/min; UV: 254 nm; the column was cooled at 10 °C).

Biology

Culturing of 9L/lacZ Cells

The cell culturing method was followed from a previously published protocol. 9L/lacZ cells (rat nitrosourea-induced gliosarcoma cell line) were purchased from ATCC (cat. #CRL-2200). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) purchased from Gibco (cat. no. 10569-010), which includes 4.5 g/L d-glucose and 110 mg/L sodium pyruvate. The media was supplemented with 10% fetal bovine serum (FBS, cat. #MT35010CV, Thermo Fisher Scientific) and 1% penicillin/streptomycin (10,000 U/mL) (cat. #15140122, Thermo Fisher Scientific) at 37 °C and 5% CO₂. The cell density was maintained between 10⁵ and 10⁶ cells/mL, and the medium was renewed 2–3 times per week.

In Vitro Cellular Uptake of the 4-[¹⁸F]FGln ([¹⁸F]4) and 4-[¹⁸F]FGln-d ₃ ([¹⁸F]16) Time-Dependent Uptake Experiment

9L/lacZ cells were detached from the T-75 flask with trypsin-EDTA solution, centrifuged, washed with Dulbecco’s phosphate buffered saline (DPBS; Ca²⁺ and Mg²⁺ added), and resuspended in DPBS (Ca²⁺ and Mg²⁺ added). Aliquots of 9L/lacZ cells (1 mL each, 1 × 10⁶ cells/mL) were seeded in conical tubes (1.5 mL). Each three replicates of 9L/lacZ cells were incubated with 4-[¹⁸F]FGln ([¹⁸ F]4, 176 kBq) or 4-[¹⁸F]FGln-d ₃ ([¹⁸ F]16, 240 kBq) together with selected inhibitors or substrates at 37 °C for 5, 30, 60, and 120 min. The cells were then centrifuged at 3700 × g for 5 min, after which the supernatant was carefully removed, and the cell pellets were washed with DPBS (1 mL, without Ca²⁺ and Mg²⁺ added) and centrifuged again at 3700 × g for 5 min. The washing step was performed three times. Cell pellets were lysed with cell lysis buffer (60 μL) containing a protease inhibitor and then vortexed for 30 s and seeded on ice for 10 min. The process was repeated three times. The radioactivity of each sample was determined using a gamma counter (WIZARD 2480) and the samples were stored at −80 °C. On the next day, the cell pellets were lysed with Cell Lytic M (catalog no. C2978, Sigma-Aldrich) supplemented with a protease inhibitor (catalog no. A32955, Thermo Fisher Scientific). Lysates were centrifuged at 13,000 × g for 45 min at 4 °C, and the total protein content of the supernatant was quantified using a Pierce Bicinchoninic Acid (BCA) Protein Assay kit (cat. #23225, Thermo Fisher Scientific). The results are expressed as %ID/mg of protein.

Transporter Characterization Experiment

9L/lacZ cells were detached from the T-75 flask with trypsin-EDTA solution, centrifuged, washed with DPBS (Ca²⁺ and Mg²⁺ added), and resuspended in DPBS (Ca²⁺ and Mg²⁺ added). Aliquots of 9L/lacZ cells (1 mL, 1 × 10⁶ cells/mL) were seeded into conical tubes (1.5 mL). 9L/lacZ cells were incubated with 4-[¹⁸F]FGln ([¹⁸ F]4, 176 kBq) or 4-[¹⁸F]FGln-d ₃ ([¹⁸ F]16, 204 kBq) together with selected inhibitors or substrates at 37 °C for 1 h. For inhibiting system L, 9L/lacZ cells were incubated with 2-amino-bicyclo[2.2.1]-heptane-2-carboxylic acid (BCH) at 0.5 mM, 1 mM, and 5 mM. For inhibiting system A, 9L/lacZ cells were incubated with α-(methylamino)isobutyric acid (MeAIB) at 0.5 mM, 1 mM, and 5 mM. For the inhibiting system ASC, 9L/lacZ cells were incubated with l-serine at 0.5 mM, 1 mM, and 5 mM. For inhibiting system ASC N, 9L/lacZ cells were incubated with l-glutamine at 0.5 mM, 1 mM, and 5 mM. The cells were then centrifuged at 3700 × g for 5 min, after which the supernatant was removed, and the cell pellets were washed with DPBS (1 mL, no Ca²⁺ and Mg²⁺ added) and centrifuged again at 3700 × g for 5 min. The washing step was performed three times, after which the radioactivity of each sample was determined using a gamma counter (WIZARD 2480). The results are normalized to the control replicates analyzed by a Student’s two-tailed t-test.

9L/lacZ Tumor-Bearing Rats

All animal work was approved by the Institutional Animal Use and Care Committee of Stony Brook University (IACUC2022-00050). Fisher rats (SAS FISCH, RT1^lv, female) were ordered from Charles River and housed in a maximum isolation room for 1 week in the Division of Laboratory Animal Resources at Stony Brook University. The Fisher rats were then subcutaneously injected with 1 × 10⁶ 9L/lacZ cells (0.1 mL PBS, pH = 7.4) in the right flank. The rats were monitored closely until a palpable tumor was present. The tumor volume was measured two or three times a week and quantified using the formula V = L × W ²/2, where L is the largest dimension of the tumor and W is the perpendicular dimension. After 8–9 weeks, the tumor volume reached around 500 mm³, and the rats were used for imaging experiments.

In Vivo PET/CT Imaging

The 9L/lacZ tumor-bearing Fisher rats were anesthetized by the inhalation of 2.5–3% isoflurane with oxygen as a carrier. PET and CT scans were obtained using a Siemens Inveon multimodality preclinical micro-PET/CT/SPECT scanner (Siemens Medical Solutions USA, Knoxville, TN). Catheters were placed in the tail veins of the rats, after which the animals were positioned on the scanner bed for an 8-min CT scan. Subsequently, formulated tracers 4-[¹⁸F]FGln ([¹⁸ F]4, 0.2–1.0 mL, 6.5–18.7 MBq) were injected through the catheters into the rats followed by saline flush (0.25 mL) while simultaneously initiating the PET scan. Rats were closely monitored during the 120 min dynamic PET scan. Two days later, PET/CT imaging of 4-[¹⁸F]FGln-d ₃ ([¹⁸ F]16, 13.3–14.8 MBq) was performed in the same group of rats by following the above protocol. The PET data were binned into designated time frames, followed by reconstruction using the OSEM 3D method with all quantitative corrections, including CT-based attenuation correction. PET volumes of interest (VOIs) and representative PET/CT images were generated by PMOD version 4.3 (PMOD Technologies Ltd., Zurich, Switzerland), and the standardized uptake value (SUV) of the tissue/organ was calculated. Errors in the average SUV values are reported as the standard deviation. The dynamic PET data from the heart were fit to a one-phase decay model to determine the blood half-life and other pharmacokinetic properties of the radiotracer using GraphPad Prism version 9 (GraphPad Software Inc., San Diego, CA).

Statistical Analysis

Statistical analysis and nonlinear regression were performed by using GraphPad Prism 9.1.0 (GraphPad Software). Quantitative data are expressed as the mean ± standard deviation unless otherwise stated. Mean values were analyzed using the Student’s two-tailed t-test and p values of <0.05 were considered statistically significant.

Supplementary Material

ao5c03771_si_001.pdf^{(1.2MB, pdf)}

Acknowledgments

This work was supported by Dr. Qu’s start-up funds and the pilot grant (2020) from the Department of Psychiatry and Behavioral Health, grants from the 2020 Targeted Research Opportunity Program, Catacosinos Cancer Translational Researcher Award, and the award from NIH NIBIB to Dr. Qu (R01EB031785). In addition, we thank Gino Giacoio from the Stony Brook University PET research core for the cyclotron operation. We also thank Jocelyn A. Marden and Alexa L. Gilberti from the Dr. Peter Tonge group for cell culture, as well as Dr. Peter Tonge for providing assistance with the use of the radiolaboratory and biosafety cabinet. We acknowledge the technical support provided by the Division of Laboratory Animal Resources and Research Histology Core Laboratory.

Glossary

Abbreviation

4-[¹⁸F]FGln-d ₃: (2S,4R)-[4-¹⁸F-3,3,4-d ₃]fluoroglutamine
4-[¹⁸F]FGln: (2S,4R)-4-[¹⁸F]fluoroglutamine
PET: positron emission tomography
[¹⁸F]FDG: [¹⁸F]fluoro-d-glucose
[¹¹C]Gln: l-5-[¹¹C]glutamine
TNBC: triple-negative breast cancer
HCC: hepatocellular carcinoma
MM: multiple myeloma
ccRCC: clear cell renal cell carcinoma
ASCT2: alanine-serine-cysteine–preferring transporter 2
DKIE: deuterium kinetic isotope effect
TriBOT: 2,4,6-tris(benzyloxy)-1,3,5-triazine
SPE: solid-phase extraction
TFA: trifluoroacetic acid
NaOAc: sodium acetate
MeAIB: α-(methylamino)isobutyric acid
BCH: 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid
l-Ser: l-serine
l-Gln: l-glutamine
SUV: standardized uptake value
ROI: region of interest
MS: molecular sieve
TfOH: trifluoromethanesulfonic acid
r.t.: room temperature
EtOAc: ethyl acetate
THF: tetrahydrofuran
KOtBu: potassium tert-butyloxide
TBME: tert-butylmethyl ether
DIPEA: diisopropylethylamine
DMP: Dess–Martin Periodinane
Et₃N: triethylamine
TsCl: para-toluenesulfonic chloride
DMAP: 4-dimethylaminopyridicne
TASF: tris(dimethylamino)sulfonium difluorotrimethyl-silicate
DMEM: Dulbecco’s modified Eagle’s medium
DPBS: Dulbecco’s phosphate-buffered saline
IACUC: Institutional Animal Care and Use Committee
VOI: volume of interest.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c03771.

Synthesis of 2,4,6-tris(benzyloxy)-1,3,5-triazine (TriBOT, 5); analytical HPLC analysis of nonradioactive 9a, 9b, 14, and 16; analytical HPLC analysis of 4-[¹⁸F]FGln-d ₃ ([¹⁸ F]16); compound characterization including ¹H, ²H, ¹³C, NMR spectra (PDF)

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JYAMS PET Research and Development Limited, Nanjing, Jiangsu, 211100, P. R. China

W.Q. contributed to the study’s conception, synthetic chemistry design, and radiosynthesis. H.K.A. performed organic synthesis and radiosynthesis. B.H. conducted the initial exploration of enantio-enriched organic synthesis and established the intermediate chiral HPLC purification method. K.L. performed cell study-related biological evaluation experiments. J.B. and L.S. prepared animal models and performed in vivo micro-PET small animal imaging studies. P.V. codesigned small animal studies with W.Q. and guided micro-PET imaging studies. W.Q., H.A., and K.L. analyzed data, drafted the manuscript, and reviewed and/or revised the manuscript. All authors have approved the final version of the manuscript.

The authors declare no competing financial interest.

References

Fowler J. S., Wolf A. P.. Working against Time: Rapid Radiotracer Synthesis and Imaging the Human Brain. Acc. Chem. Res. 1997;30(4):181–188. doi: 10.1021/ar960068c. [DOI] [Google Scholar]
Miller P. W., Long N. J., Vilar R., Gee A. D.. Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography. Angew. Chem. Int. Ed. 2008;47(47):8998–9033. doi: 10.1002/anie.200800222. [DOI] [PubMed] [Google Scholar]
Weber W. A., Schwaiger M., Avril N.. Quantitative assessment of tumor metabolism using FDG-PET imaging. Nucl. Med. Biol. 2000;27(7):683–687. doi: 10.1016/S0969-8051(00)00141-4. [DOI] [PubMed] [Google Scholar]
Hoh C. K.. Clinical use of FDG PET. Nucl. Med. Biol. 2007;34(7):737–742. doi: 10.1016/j.nucmedbio.2007.07.001. [DOI] [PubMed] [Google Scholar]
Vander Heiden M. G., Cantley L. C., Thompson C. B.. Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science. 2009;324(5930):1029–1033. doi: 10.1126/science.1160809. [DOI] [PMC free article] [PubMed] [Google Scholar]
Cruzat V., Macedo Rogero M., Noel Keane K., Curi R., Newsholme P.. Glutamine: Metabolism and Immune Function, Supplementation and Clinical Translation. Nutrients. 2018;10(11):1564. doi: 10.3390/nu10111564. [DOI] [PMC free article] [PubMed] [Google Scholar]
Newsholme E., Crabtree B., Ardawi M.. The role of high rates of glycolysis and glutamine utilization in rapidly dividing cells. Biosci. Rep. 1985;5:393–400. doi: 10.1007/BF01116556. [DOI] [PubMed] [Google Scholar]
Cooper A. J., Jeitner T. M.. Central Role of Glutamate Metabolism in the Maintenance of Nitrogen Homeostasis in Normal and Hyperammonemic Brain. Biomolecules. 2016;6(2):16. doi: 10.3390/biom6020016. [DOI] [PMC free article] [PubMed] [Google Scholar]
Wise D. R., DeBerardinis R. J., Mancuso A., Sayed N., Zhang X.-Y., Pfeiffer H. K., Nissim I., Daikhin E., Yudkoff M., McMahon S. B., Thompson C. B.. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proc. Natl. Acad. Sci. U. S. A. 2008;105(48):18782–18787. doi: 10.1073/pnas.0810199105. [DOI] [PMC free article] [PubMed] [Google Scholar]
Wise D. R., Thompson C. B.. Glutamine addiction: A new therapeutic target in cancer. Trends Biochem. Sci. 2010;35(8):427–433. doi: 10.1016/j.tibs.2010.05.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
Yang L., Venneti S., Nagrath D.. Glutaminolysis: A Hallmark of Cancer Metabolism. Annu. Rev. Biomed. Eng. 2017;19(1):163–194. doi: 10.1146/annurev-bioeng-071516-044546. [DOI] [PubMed] [Google Scholar]
Qu W., Oya S., Lieberman B. P., Ploessl K., Wang L., Wise D. R., Divgi C. R., Chodosh L. P., Thompson C. B., Kung H. F.. Preparation and characterization of L-[5–11C]-glutamine for metabolic imaging of tumors. J. Nucl. Med. 2012;53(1):98–105. doi: 10.2967/jnumed.111.093831. [DOI] [PubMed] [Google Scholar]
Qu W., Zha Z., Lieberman B. P., Mancuso A., Stetz M., Rizzi R., Ploessl K., Wise D., Thompson C., Kung H. F.. Facile synthesis [5-13C-4-2H2]-L-glutamine for hyperpolarized MRS imaging of cancer cell metabolism. Acad. Radiol. 2011;18(8):932–939. doi: 10.1016/j.acra.2011.05.002. [DOI] [PubMed] [Google Scholar]
Qu W., Zha Z., Ploessl K., Lieberman B. P., Zhu L., Wise D. R., Thompson C. B., Kung H. F.. Synthesis of Optically Pure 4-Fluoro-Glutamines as Potential Metabolic Imaging Agents for Tumors. J. Am. Chem. Soc. 2011;133(4):1122–1133. doi: 10.1021/ja109203d. [DOI] [PubMed] [Google Scholar]
Zhu L., Ploessl K., Zhou R., Mankoff D., Kung H. F.. Metabolic Imaging of Glutamine in Cancer. J. Nucl. Med. 2017;58(4):533–537. doi: 10.2967/jnumed.116.182345. [DOI] [PMC free article] [PubMed] [Google Scholar]
Dunphy M. P. S., Harding J. J., Venneti S., Zhang H., Burnazi E. M., Bromberg J., Omuro A. M., Hsieh J. J., Mellinghoff I. K., Staton K.. et al. In Vivo PET Assay of Tumor Glutamine Flux and Metabolism: In-Human Trial of 18F-(2S,4R)-4-Fluoroglutamine. Radiology. 2018;287(2):667–675. doi: 10.1148/radiol.2017162610. [DOI] [PMC free article] [PubMed] [Google Scholar]
Grkovski M., Goel R., Krebs S., Staton K. D., Harding J. J., Mellinghoff I. K., Humm J. L., Dunphy M. P.. Pharmacokinetic assessment of 18F-(2S, 4R)-4-fluoroglutamine in patients with cancer. J. Nucl. Med. 2020;61(3):357–366. doi: 10.2967/jnumed.119.229740. [DOI] [PMC free article] [PubMed] [Google Scholar]
Xu X., Zhu H., Liu F., Zhang Y., Yang J., Zhang L., Xie Q., Zhu L., Li N., Kung H. F.. et al. Dynamic PET/CT imaging of 18F-(2 S, 4 R) 4-fluoroglutamine in healthy volunteers and oncological patients. Eur. J. Nucl. Med. Mol. Imaging. 2020;47(10):2280–2292. doi: 10.1007/s00259-019-04543-w. [DOI] [PubMed] [Google Scholar]
Thompson C. B., Venneti S., Dunphy M. P., Zhang H., Pitter K. L., Zanzonico P., Campos C., Carlin S. D., La Rocca G., Lyashchenko S.. et al. Glutamine-based PET imaging facilitates enhanced metabolic evaluation of gliomas in vivo. Sci. Transl. Med. 2015;7(274):274ra217. doi: 10.1126/scitranslmed.aaa1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
Lieberman B. P., Ploessl K., Wang L., Qu W., Zha Z., Wise D. R., Chodosh L. A., Belka G., Thompson C. B., Kung H. F.. PET imaging of glutaminolysis in tumors by 18F-(2S,4R)4-fluoroglutamine. J. Nucl. Med. 2011;52(12):1947–1955. doi: 10.2967/jnumed.111.093815. [DOI] [PubMed] [Google Scholar]
Ekici S., Nye J., Neill S., Allen J., Shu H.-K., Fleischer C.. Glutamine imaging: A new avenue for glioma management. Am. J. Neuroradiol. 2022;43(1):11–18. doi: 10.3174/ajnr.A7333. [DOI] [PMC free article] [PubMed] [Google Scholar]
Li C., Huang S., Guo J., Wang C., Huang Z., Huang R., Liu L., Liang S., Wang H.. Metabolic Evaluation of MYCN-Amplified Neuroblastoma by 4-[18F]FGln PET Imaging. Mol. Imaging Biol. 2019;21:1117–1126. doi: 10.1007/s11307-019-01330-9. [DOI] [PubMed] [Google Scholar]
Xu X., Zhu H., Liu F., Zhang Y., Yang J., Zhang L., Zhu L., Li N., Kung H. F., Yang Z.. Imaging Brain Metastasis Patients With 18F-(2S,4R)-4-Fluoroglutamine. Clin Nucl. Med. 2018;43(11):e392–e399. doi: 10.1097/RLU.0000000000002257. [DOI] [PubMed] [Google Scholar]
Miner M. W., Liljenbäck H., Virta J., Merisaari J., Oikonen V., Westermarck J., Li X.-G., Roivainen A.. (2 S, 4 R)-4-[18 F] Fluoroglutamine for In vivo PET Imaging of Glioma Xenografts in Mice: An Evaluation of Multiple Pharmacokinetic Models. Mol. Imaging Biol. 2020;22(22):969–978. doi: 10.1007/s11307-020-01472-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
Hassanein M., Hight M. R., Buck J. R., Tantawy M. N., Nickels M. L., Hoeksema M. D., Harris B. K., Boyd K., Massion P. P., Manning H. C.. Preclinical Evaluation of 4-[18F]Fluoroglutamine PET to Assess ASCT2 Expression in Lung Cancer. Mol. Imaging Biol. 2016;18(1):18–23. doi: 10.1007/s11307-015-0862-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
Schulte M. L., Hight M. R., Ayers G. D., Liu Q., Shyr Y., Washington M. K., Manning H. C.. Non-Invasive Glutamine PET Reflects Pharmacological Inhibition of BRAFV600EIn Vivo. Mol. Imaging Biol. 2017;19(3):421–428. doi: 10.1007/s11307-016-1008-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
Zhou R., Pantel A. R., Li S., Lieberman B. P., Ploessl K., Choi H., Blankemeyer E., Lee H., Kung H. F., Mach R. H., Mankoff D. A.. [18F](2S,4R)4-Fluoroglutamine PET Detects Glutamine Pool Size Changes in Triple-Negative Breast Cancer in Response to Glutaminase Inhibition. Cancer Res. 2017;77(6):1476–1484. doi: 10.1158/0008-5472.CAN-16-1945. [DOI] [PMC free article] [PubMed] [Google Scholar]
Zhou R., Choi H., Cao J., Pantel A., Gupta M., Lee H. S., Mankoff D.. 18F-Fluciclovine PET Imaging of Glutaminase Inhibition in Breast Cancer Models. J. Nucl. Med. 2023;64(1):131–136. doi: 10.2967/jnumed.122.264152. [DOI] [PMC free article] [PubMed] [Google Scholar]
Liu F., Xu X., Zhu H., Zhang Y., Yang J., Zhang L., Li N., Zhu L., Kung H. F., Yang Z.. PET Imaging of 18F-(2 S,4 R)4-Fluoroglutamine Accumulation in Breast Cancer: From Xenografts to Patients. Mol. Pharmaceutics. 2018;15(8):3448–3455. doi: 10.1021/acs.molpharmaceut.8b00430. [DOI] [PubMed] [Google Scholar]
Viswanath V., Zhou R., Lee H., Li S., Cragin A., Doot R. K., Mankoff D. A., Pantel A. R.. Kinetic modeling of 18F-(2S, 4R) 4-fluoroglutamine in mouse models of breast cancer to estimate glutamine pool size as an indicator of tumor glutamine metabolism. J. Nucl. Med. 2021;62(8):1154–1162. doi: 10.2967/jnumed.120.250977. [DOI] [PMC free article] [PubMed] [Google Scholar]
Seo Y., Craig M. C., Murphy S. T., Feng J., Chen X., Yuneva M.. [18F]-(2S, 4R) 4-fluoroglutamine PET imaging of glutamine metabolism in murine models of hepatocellular carcinoma (HCC) Mol. Imaging. 2022;2022:5185951. doi: 10.1155/2022/5185951. [DOI] [PMC free article] [PubMed] [Google Scholar]
Du K., Chitneni S. K., Suzuki A., Wang Y., Henao R., Hyun J., Premont R. T., Naggie S., Moylan C. A., Bashir M. R.. et al. Increased glutaminolysis marks active scarring in nonalcoholic steatohepatitis progression. Cell. Mol. Gastroenterol. Hepatol. 2020;10(1):P1–21. doi: 10.1016/j.jcmgh.2019.12.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
Palani S., Miner M. W. G., Virta J., Liljenbäck H., Eskola O., Örd T., Ravindran A., Kaikkonen M. U., Knuuti J., Li X.-G.. et al. Exploiting Glutamine Consumption in Atherosclerotic Lesions by Positron Emission Tomography Tracer (2S, 4R)-4–18F-Fluoroglutamine. Front. Immunol. 2022;13:821423. doi: 10.3389/fimmu.2022.821423. [DOI] [PMC free article] [PubMed] [Google Scholar]
Valtorta S., Toscani D., Chiu M., Sartori A., Coliva A., Brevi A., Taurino G., Grioni M., Ruffini L., Vacondio F.. et al. [18F](2S,4R)-4-Fluoroglutamine as a New Positron Emission Tomography Tracer in Myeloma. Front. Oncol. 2021;11:760732. doi: 10.3389/fonc.2021.760732. [DOI] [PMC free article] [PubMed] [Google Scholar]
Valtorta S., Toscani D., Chiu M., Sartori A., Coliva A., Brevi A., Taurino G., Grioni M., Ruffini L., Vacondio F.. et al. [18F](2S, 4R)-4-Fluoroglutamine as a New Positron Emission Tomography Tracer in Myeloma. Front. Oncol. 2021;11:760732. doi: 10.3389/fonc.2021.760732. [DOI] [PMC free article] [PubMed] [Google Scholar]
Zhu H., Liu F., Zhang Y., Yang J., Xu X., Guo X., Liu T., Li N., Zhu L., Kung H. F.. et al. (2S, 4R)-4-[18F] Fluoroglutamine as a PET Indicator for Bone Marrow Metabolism Dysfunctional: From Animal Experiments to Clinical Application. Mol. Imaging Biol. 2019;21(5):945–953. doi: 10.1007/s11307-019-01319-4. [DOI] [PubMed] [Google Scholar]
Abu Aboud O., Habib S. L., Trott J., Stewart B., Liang S., Chaudhari A. J., Sutcliffe J., Weiss R. H.. Glutamine Addiction in Kidney Cancer Suppresses Oxidative Stress and Can Be Exploited for Real-Time Imaging. Cancer Res. 2017;77(23):6746–6758. doi: 10.1158/0008-5472.CAN-17-0930. [DOI] [PMC free article] [PubMed] [Google Scholar]
Huang S., Ren L., Beck J. A., Phelps T. E., Olkowski C., Ton A., Roy J., White M. E., Adler S., Wong K.. et al. Exploration of Imaging Biomarkers for Metabolically-Targeted Osteosarcoma Therapy in a Murine Xenograft Model. Cancer Biother. Radiopharm. 2023;38(7):475–485. doi: 10.1089/cbr.2022.0090. [DOI] [PMC free article] [PubMed] [Google Scholar]
Liu S., Liu F., Hou X., Zhang Q., Ren Y. N., Zhu H., Yang Z., Xu X.. KRAS Mutation Detection with (2S,4R)-4-[18F]FGln for Noninvasive PDAC Diagnosis. Mol. Pharmaceutics. 2024;21(4):2034–2042. doi: 10.1021/acs.molpharmaceut.4c00082. [DOI] [PMC free article] [PubMed] [Google Scholar]
Kim M.-J., Akula H. K., Marden J., Li K., Hu B., Vaska P., Qu W.. The Potential Utility of (2S,4R)-4-[18F]fluoroglutamine as a Novel Metabolic Imaging Marker for Inflammation Explored by Rat Models of Arthritis and Paw Edema. Mol. Imaging Biol. 2025;27:10. doi: 10.1007/s11307-024-01967-1. [DOI] [PubMed] [Google Scholar]
Cooper A. J. L., Krasnikov B. F., Pinto J. T., Kung H. F., Li J., Ploessl K.. Comparative enzymology of (2S,4R)4-fluoroglutamine and (2S,4R)4-fluoroglutamate. Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 2012;163(1):108–120. doi: 10.1016/j.cbpb.2012.05.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
Wu Z., Zha Z., Li G., Lieberman B. P., Choi S. R., Ploessl K., Kung H. F.. [18F](2S,4S)-4-(3-Fluoropropyl)glutamine as a Tumor Imaging Agent. Mol. Pharmaceutics. 2014;11(11):3852–3866. doi: 10.1021/mp500236y. [DOI] [PMC free article] [PubMed] [Google Scholar]
Chen J., Li C., Hong H., Liu H., Wang C., Xu M., Han Y., Liu Z.. Side Chain Optimization Remarkably Enhances the in Vivo Stability of 18F-Labeled Glutamine for Tumor Imaging. Mol. Pharmaceutics. 2019;16(12):5035–5041. doi: 10.1021/acs.molpharmaceut.9b00891. [DOI] [PubMed] [Google Scholar]
Klenner M. A., Pascali G., Fraser B. H., Darwish T. A.. Kinetic isotope effects and synthetic strategies for deuterated carbon-11 and fluorine-18 labelled PET radiopharmaceuticals. Nucl. Med. Biol. 2021;96–97:112–147. doi: 10.1016/j.nucmedbio.2021.03.011. [DOI] [PubMed] [Google Scholar]
Di Martino R. M. C., Maxwell B. D., Pirali T.. Deuterium in drug discovery: Progress, opportunities and challenges. Nat. Rev. Drug Discovery. 2023;22(7):562–584. doi: 10.1038/s41573-023-00703-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
Timmins G. S.. Deuterated drugs; updates and obviousness analysis. Expert Opin. Ther. Pat. 2017;27(12):1353–1361. doi: 10.1080/13543776.2017.1378350. [DOI] [PubMed] [Google Scholar]
Fowler J. S., Wang G. J., Logan J., Xie S., Volkow N. D., MacGregor R. R., Schlyer D. J., Pappas N., Alexoff D. L., Patlak C.. Selective reduction of radiotracer trapping by deuterium substitution: Comparison of carbon-11-L-deprenyl and carbon-11-deprenyl-D2 for MAO B mapping. J. Nucl. Med. 1995;36(7):1255–1262. [PubMed] [Google Scholar]
Fowler J. S., Wolf A. P., MacGregor R. R., Dewey S. L., Logan J., Schlyer D. J., Langstrom B.. Mechanistic positron emission tomography studies: Demonstration of a deuterium isotope effect in the monoamine oxidase-catalyzed binding of [11C]L-deprenyl in living baboon brain. J. Neurochem. 1988;51(5):1524–1534. doi: 10.1111/j.1471-4159.1988.tb01121.x. [DOI] [PubMed] [Google Scholar]
Fowler J. S., MacGregor R. R., Wolf A. P., Arnett C. D., Dewey S. L., Schlyer D., Christman D., Logan J., Smith M.. et al. Mapping human brain monoamine oxidase A and B with carbon-11-labeled suicide inactivators and PET. Science. 1987;235(4787):481–485. doi: 10.1126/science.3099392. [DOI] [PubMed] [Google Scholar]
Xiao H., Choi S. R., Zhao R., Ploessl K., Alexoff D., Zhu L., Zha Z., Kung H. F.. A New Highly Deuterated [18F]AV-45, [18F]D15FSP, for Imaging β-Amyloid Plaques in the Brain. ACS Med. Chem. Lett. 2021;12(7):1086–1092. doi: 10.1021/acsmedchemlett.1c00062. [DOI] [PMC free article] [PubMed] [Google Scholar]
Li K., Gilberti A. L., Marden J. A., Akula H. K., Pollard A. C., Guo S., Hu B., Tonge P. J., Qu W.. Synthesis and Biological Evaluation of Fluorine-18 and Deuterium Labeled l-Fluoroalanines as Positron Emission Tomography Imaging Agents for Cancer Detection. J. Med. Chem. 2024;67(12):10293–10305. doi: 10.1021/acs.jmedchem.4c00774. [DOI] [PMC free article] [PubMed] [Google Scholar]
Yamada K., Fujita H., Kunishima M.. A Novel Acid-Catalyzed O-Benzylating Reagent with the Smallest Unit of Imidate Structure. Org. Lett. 2012;14(19):5026–5029. doi: 10.1021/ol302222p. [DOI] [PubMed] [Google Scholar]
Zhang X., Basuli F., Shi Z.-D., Xu B., Blackman B., Choyke P. L., Swenson R. E.. Automated synthesis of [18F](2S,4R)-4-fluoroglutamine on a GE TRACERlab FX-N Pro module. Appl. Radiat. Isot. 2016;112:110–114. doi: 10.1016/j.apradiso.2016.02.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
Li S., Schmitz A., Lee H., Mach R. H.. Automation of the Radiosynthesis of Six Different 18F-labeled radiotracers on the AllinOne. EJNMMI Radiopharm. Chem. 2017;1(1):15. doi: 10.1186/s41181-016-0018-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
Zhang Y., Zhang L., Yang J., Wu Z., Ploessl K., Zha Z., Liu F., Xu X., Zhu H., Yang Z.. et al. Initial experience in synthesis of (2S,4R)-4-[18F]fluoroglutamine for clinical application. J. Labelled Compd. Radiopharm. 2019;62(5):209–214. doi: 10.1002/jlcr.3719. [DOI] [PubMed] [Google Scholar]
Ploessl K., Wang L., Lieberman B. P., Qu W., Kung H. F.. Comparative evaluation of 18F-labeled glutamic acid and glutamine as tumor metabolic imaging agents. J. Nucl. Med. 2012;53(10):1616–1624. doi: 10.2967/jnumed.111.101279. [DOI] [PubMed] [Google Scholar]
Ouvrard E., Kaseb A., Poterszman N., Porot C., Somme F., Imperiale A.. Nuclear medicine imaging for bone metastases assessment: What else besides bone scintigraphy in the era of personalized medicine? Front. Med. 2024;10:1320574. doi: 10.3389/fmed.2023.1320574. [DOI] [PMC free article] [PubMed] [Google Scholar]
Seifert R., Telli T., Opitz M., Barbato F., Berliner C., Nader M., Umutlu L., Stuschke M., Hadaschik B., Herrmann K., Fendler W. P.. Unspecific 18F-PSMA-1007 Bone Uptake Evaluated Through PSMA-11 PET, Bone Scanning, and MRI Triple Validation in Patients with Biochemical Recurrence of Prostate Cancer. J. Nucl. Med. 2023;64(5):738–743. doi: 10.2967/jnumed.118.215434. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

ao5c03771_si_001.pdf^{(1.2MB, pdf)}

[ref1] Fowler J. S., Wolf A. P.. Working against Time: Rapid Radiotracer Synthesis and Imaging the Human Brain. Acc. Chem. Res. 1997;30(4):181–188. doi: 10.1021/ar960068c. [DOI] [Google Scholar]

[ref2] Miller P. W., Long N. J., Vilar R., Gee A. D.. Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography. Angew. Chem. Int. Ed. 2008;47(47):8998–9033. doi: 10.1002/anie.200800222. [DOI] [PubMed] [Google Scholar]

[ref3] Weber W. A., Schwaiger M., Avril N.. Quantitative assessment of tumor metabolism using FDG-PET imaging. Nucl. Med. Biol. 2000;27(7):683–687. doi: 10.1016/S0969-8051(00)00141-4. [DOI] [PubMed] [Google Scholar]

[ref4] Hoh C. K.. Clinical use of FDG PET. Nucl. Med. Biol. 2007;34(7):737–742. doi: 10.1016/j.nucmedbio.2007.07.001. [DOI] [PubMed] [Google Scholar]

[ref5] Vander Heiden M. G., Cantley L. C., Thompson C. B.. Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science. 2009;324(5930):1029–1033. doi: 10.1126/science.1160809. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref6] Cruzat V., Macedo Rogero M., Noel Keane K., Curi R., Newsholme P.. Glutamine: Metabolism and Immune Function, Supplementation and Clinical Translation. Nutrients. 2018;10(11):1564. doi: 10.3390/nu10111564. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref7] Newsholme E., Crabtree B., Ardawi M.. The role of high rates of glycolysis and glutamine utilization in rapidly dividing cells. Biosci. Rep. 1985;5:393–400. doi: 10.1007/BF01116556. [DOI] [PubMed] [Google Scholar]

[ref8] Cooper A. J., Jeitner T. M.. Central Role of Glutamate Metabolism in the Maintenance of Nitrogen Homeostasis in Normal and Hyperammonemic Brain. Biomolecules. 2016;6(2):16. doi: 10.3390/biom6020016. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref9] Wise D. R., DeBerardinis R. J., Mancuso A., Sayed N., Zhang X.-Y., Pfeiffer H. K., Nissim I., Daikhin E., Yudkoff M., McMahon S. B., Thompson C. B.. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proc. Natl. Acad. Sci. U. S. A. 2008;105(48):18782–18787. doi: 10.1073/pnas.0810199105. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref10] Wise D. R., Thompson C. B.. Glutamine addiction: A new therapeutic target in cancer. Trends Biochem. Sci. 2010;35(8):427–433. doi: 10.1016/j.tibs.2010.05.003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref11] Yang L., Venneti S., Nagrath D.. Glutaminolysis: A Hallmark of Cancer Metabolism. Annu. Rev. Biomed. Eng. 2017;19(1):163–194. doi: 10.1146/annurev-bioeng-071516-044546. [DOI] [PubMed] [Google Scholar]

[ref12] Qu W., Oya S., Lieberman B. P., Ploessl K., Wang L., Wise D. R., Divgi C. R., Chodosh L. P., Thompson C. B., Kung H. F.. Preparation and characterization of L-[5–11C]-glutamine for metabolic imaging of tumors. J. Nucl. Med. 2012;53(1):98–105. doi: 10.2967/jnumed.111.093831. [DOI] [PubMed] [Google Scholar]

[ref13] Qu W., Zha Z., Lieberman B. P., Mancuso A., Stetz M., Rizzi R., Ploessl K., Wise D., Thompson C., Kung H. F.. Facile synthesis [5-13C-4-2H2]-L-glutamine for hyperpolarized MRS imaging of cancer cell metabolism. Acad. Radiol. 2011;18(8):932–939. doi: 10.1016/j.acra.2011.05.002. [DOI] [PubMed] [Google Scholar]

[ref14] Qu W., Zha Z., Ploessl K., Lieberman B. P., Zhu L., Wise D. R., Thompson C. B., Kung H. F.. Synthesis of Optically Pure 4-Fluoro-Glutamines as Potential Metabolic Imaging Agents for Tumors. J. Am. Chem. Soc. 2011;133(4):1122–1133. doi: 10.1021/ja109203d. [DOI] [PubMed] [Google Scholar]

[ref15] Zhu L., Ploessl K., Zhou R., Mankoff D., Kung H. F.. Metabolic Imaging of Glutamine in Cancer. J. Nucl. Med. 2017;58(4):533–537. doi: 10.2967/jnumed.116.182345. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref16] Dunphy M. P. S., Harding J. J., Venneti S., Zhang H., Burnazi E. M., Bromberg J., Omuro A. M., Hsieh J. J., Mellinghoff I. K., Staton K.. et al. In Vivo PET Assay of Tumor Glutamine Flux and Metabolism: In-Human Trial of 18F-(2S,4R)-4-Fluoroglutamine. Radiology. 2018;287(2):667–675. doi: 10.1148/radiol.2017162610. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref17] Grkovski M., Goel R., Krebs S., Staton K. D., Harding J. J., Mellinghoff I. K., Humm J. L., Dunphy M. P.. Pharmacokinetic assessment of 18F-(2S, 4R)-4-fluoroglutamine in patients with cancer. J. Nucl. Med. 2020;61(3):357–366. doi: 10.2967/jnumed.119.229740. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref18] Xu X., Zhu H., Liu F., Zhang Y., Yang J., Zhang L., Xie Q., Zhu L., Li N., Kung H. F.. et al. Dynamic PET/CT imaging of 18F-(2 S, 4 R) 4-fluoroglutamine in healthy volunteers and oncological patients. Eur. J. Nucl. Med. Mol. Imaging. 2020;47(10):2280–2292. doi: 10.1007/s00259-019-04543-w. [DOI] [PubMed] [Google Scholar]

[ref19] Thompson C. B., Venneti S., Dunphy M. P., Zhang H., Pitter K. L., Zanzonico P., Campos C., Carlin S. D., La Rocca G., Lyashchenko S.. et al. Glutamine-based PET imaging facilitates enhanced metabolic evaluation of gliomas in vivo. Sci. Transl. Med. 2015;7(274):274ra217. doi: 10.1126/scitranslmed.aaa1009. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref20] Lieberman B. P., Ploessl K., Wang L., Qu W., Zha Z., Wise D. R., Chodosh L. A., Belka G., Thompson C. B., Kung H. F.. PET imaging of glutaminolysis in tumors by 18F-(2S,4R)4-fluoroglutamine. J. Nucl. Med. 2011;52(12):1947–1955. doi: 10.2967/jnumed.111.093815. [DOI] [PubMed] [Google Scholar]

[ref21] Ekici S., Nye J., Neill S., Allen J., Shu H.-K., Fleischer C.. Glutamine imaging: A new avenue for glioma management. Am. J. Neuroradiol. 2022;43(1):11–18. doi: 10.3174/ajnr.A7333. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref22] Li C., Huang S., Guo J., Wang C., Huang Z., Huang R., Liu L., Liang S., Wang H.. Metabolic Evaluation of MYCN-Amplified Neuroblastoma by 4-[18F]FGln PET Imaging. Mol. Imaging Biol. 2019;21:1117–1126. doi: 10.1007/s11307-019-01330-9. [DOI] [PubMed] [Google Scholar]

[ref23] Xu X., Zhu H., Liu F., Zhang Y., Yang J., Zhang L., Zhu L., Li N., Kung H. F., Yang Z.. Imaging Brain Metastasis Patients With 18F-(2S,4R)-4-Fluoroglutamine. Clin Nucl. Med. 2018;43(11):e392–e399. doi: 10.1097/RLU.0000000000002257. [DOI] [PubMed] [Google Scholar]

[ref24] Miner M. W., Liljenbäck H., Virta J., Merisaari J., Oikonen V., Westermarck J., Li X.-G., Roivainen A.. (2 S, 4 R)-4-[18 F] Fluoroglutamine for In vivo PET Imaging of Glioma Xenografts in Mice: An Evaluation of Multiple Pharmacokinetic Models. Mol. Imaging Biol. 2020;22(22):969–978. doi: 10.1007/s11307-020-01472-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref25] Hassanein M., Hight M. R., Buck J. R., Tantawy M. N., Nickels M. L., Hoeksema M. D., Harris B. K., Boyd K., Massion P. P., Manning H. C.. Preclinical Evaluation of 4-[18F]Fluoroglutamine PET to Assess ASCT2 Expression in Lung Cancer. Mol. Imaging Biol. 2016;18(1):18–23. doi: 10.1007/s11307-015-0862-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref26] Schulte M. L., Hight M. R., Ayers G. D., Liu Q., Shyr Y., Washington M. K., Manning H. C.. Non-Invasive Glutamine PET Reflects Pharmacological Inhibition of BRAFV600EIn Vivo. Mol. Imaging Biol. 2017;19(3):421–428. doi: 10.1007/s11307-016-1008-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref27] Zhou R., Pantel A. R., Li S., Lieberman B. P., Ploessl K., Choi H., Blankemeyer E., Lee H., Kung H. F., Mach R. H., Mankoff D. A.. [18F](2S,4R)4-Fluoroglutamine PET Detects Glutamine Pool Size Changes in Triple-Negative Breast Cancer in Response to Glutaminase Inhibition. Cancer Res. 2017;77(6):1476–1484. doi: 10.1158/0008-5472.CAN-16-1945. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref28] Zhou R., Choi H., Cao J., Pantel A., Gupta M., Lee H. S., Mankoff D.. 18F-Fluciclovine PET Imaging of Glutaminase Inhibition in Breast Cancer Models. J. Nucl. Med. 2023;64(1):131–136. doi: 10.2967/jnumed.122.264152. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref29] Liu F., Xu X., Zhu H., Zhang Y., Yang J., Zhang L., Li N., Zhu L., Kung H. F., Yang Z.. PET Imaging of 18F-(2 S,4 R)4-Fluoroglutamine Accumulation in Breast Cancer: From Xenografts to Patients. Mol. Pharmaceutics. 2018;15(8):3448–3455. doi: 10.1021/acs.molpharmaceut.8b00430. [DOI] [PubMed] [Google Scholar]

[ref30] Viswanath V., Zhou R., Lee H., Li S., Cragin A., Doot R. K., Mankoff D. A., Pantel A. R.. Kinetic modeling of 18F-(2S, 4R) 4-fluoroglutamine in mouse models of breast cancer to estimate glutamine pool size as an indicator of tumor glutamine metabolism. J. Nucl. Med. 2021;62(8):1154–1162. doi: 10.2967/jnumed.120.250977. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref31] Seo Y., Craig M. C., Murphy S. T., Feng J., Chen X., Yuneva M.. [18F]-(2S, 4R) 4-fluoroglutamine PET imaging of glutamine metabolism in murine models of hepatocellular carcinoma (HCC) Mol. Imaging. 2022;2022:5185951. doi: 10.1155/2022/5185951. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref32] Du K., Chitneni S. K., Suzuki A., Wang Y., Henao R., Hyun J., Premont R. T., Naggie S., Moylan C. A., Bashir M. R.. et al. Increased glutaminolysis marks active scarring in nonalcoholic steatohepatitis progression. Cell. Mol. Gastroenterol. Hepatol. 2020;10(1):P1–21. doi: 10.1016/j.jcmgh.2019.12.006. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref33] Palani S., Miner M. W. G., Virta J., Liljenbäck H., Eskola O., Örd T., Ravindran A., Kaikkonen M. U., Knuuti J., Li X.-G.. et al. Exploiting Glutamine Consumption in Atherosclerotic Lesions by Positron Emission Tomography Tracer (2S, 4R)-4–18F-Fluoroglutamine. Front. Immunol. 2022;13:821423. doi: 10.3389/fimmu.2022.821423. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref34] Valtorta S., Toscani D., Chiu M., Sartori A., Coliva A., Brevi A., Taurino G., Grioni M., Ruffini L., Vacondio F.. et al. [18F](2S,4R)-4-Fluoroglutamine as a New Positron Emission Tomography Tracer in Myeloma. Front. Oncol. 2021;11:760732. doi: 10.3389/fonc.2021.760732. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref35] Valtorta S., Toscani D., Chiu M., Sartori A., Coliva A., Brevi A., Taurino G., Grioni M., Ruffini L., Vacondio F.. et al. [18F](2S, 4R)-4-Fluoroglutamine as a New Positron Emission Tomography Tracer in Myeloma. Front. Oncol. 2021;11:760732. doi: 10.3389/fonc.2021.760732. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref36] Zhu H., Liu F., Zhang Y., Yang J., Xu X., Guo X., Liu T., Li N., Zhu L., Kung H. F.. et al. (2S, 4R)-4-[18F] Fluoroglutamine as a PET Indicator for Bone Marrow Metabolism Dysfunctional: From Animal Experiments to Clinical Application. Mol. Imaging Biol. 2019;21(5):945–953. doi: 10.1007/s11307-019-01319-4. [DOI] [PubMed] [Google Scholar]

[ref37] Abu Aboud O., Habib S. L., Trott J., Stewart B., Liang S., Chaudhari A. J., Sutcliffe J., Weiss R. H.. Glutamine Addiction in Kidney Cancer Suppresses Oxidative Stress and Can Be Exploited for Real-Time Imaging. Cancer Res. 2017;77(23):6746–6758. doi: 10.1158/0008-5472.CAN-17-0930. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref38] Huang S., Ren L., Beck J. A., Phelps T. E., Olkowski C., Ton A., Roy J., White M. E., Adler S., Wong K.. et al. Exploration of Imaging Biomarkers for Metabolically-Targeted Osteosarcoma Therapy in a Murine Xenograft Model. Cancer Biother. Radiopharm. 2023;38(7):475–485. doi: 10.1089/cbr.2022.0090. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref39] Liu S., Liu F., Hou X., Zhang Q., Ren Y. N., Zhu H., Yang Z., Xu X.. KRAS Mutation Detection with (2S,4R)-4-[18F]FGln for Noninvasive PDAC Diagnosis. Mol. Pharmaceutics. 2024;21(4):2034–2042. doi: 10.1021/acs.molpharmaceut.4c00082. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref40] Kim M.-J., Akula H. K., Marden J., Li K., Hu B., Vaska P., Qu W.. The Potential Utility of (2S,4R)-4-[18F]fluoroglutamine as a Novel Metabolic Imaging Marker for Inflammation Explored by Rat Models of Arthritis and Paw Edema. Mol. Imaging Biol. 2025;27:10. doi: 10.1007/s11307-024-01967-1. [DOI] [PubMed] [Google Scholar]

[ref41] Cooper A. J. L., Krasnikov B. F., Pinto J. T., Kung H. F., Li J., Ploessl K.. Comparative enzymology of (2S,4R)4-fluoroglutamine and (2S,4R)4-fluoroglutamate. Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 2012;163(1):108–120. doi: 10.1016/j.cbpb.2012.05.010. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref42] Wu Z., Zha Z., Li G., Lieberman B. P., Choi S. R., Ploessl K., Kung H. F.. [18F](2S,4S)-4-(3-Fluoropropyl)glutamine as a Tumor Imaging Agent. Mol. Pharmaceutics. 2014;11(11):3852–3866. doi: 10.1021/mp500236y. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref43] Chen J., Li C., Hong H., Liu H., Wang C., Xu M., Han Y., Liu Z.. Side Chain Optimization Remarkably Enhances the in Vivo Stability of 18F-Labeled Glutamine for Tumor Imaging. Mol. Pharmaceutics. 2019;16(12):5035–5041. doi: 10.1021/acs.molpharmaceut.9b00891. [DOI] [PubMed] [Google Scholar]

[ref44] Klenner M. A., Pascali G., Fraser B. H., Darwish T. A.. Kinetic isotope effects and synthetic strategies for deuterated carbon-11 and fluorine-18 labelled PET radiopharmaceuticals. Nucl. Med. Biol. 2021;96–97:112–147. doi: 10.1016/j.nucmedbio.2021.03.011. [DOI] [PubMed] [Google Scholar]

[ref45] Di Martino R. M. C., Maxwell B. D., Pirali T.. Deuterium in drug discovery: Progress, opportunities and challenges. Nat. Rev. Drug Discovery. 2023;22(7):562–584. doi: 10.1038/s41573-023-00703-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref46] Timmins G. S.. Deuterated drugs; updates and obviousness analysis. Expert Opin. Ther. Pat. 2017;27(12):1353–1361. doi: 10.1080/13543776.2017.1378350. [DOI] [PubMed] [Google Scholar]

[ref47] Fowler J. S., Wang G. J., Logan J., Xie S., Volkow N. D., MacGregor R. R., Schlyer D. J., Pappas N., Alexoff D. L., Patlak C.. Selective reduction of radiotracer trapping by deuterium substitution: Comparison of carbon-11-L-deprenyl and carbon-11-deprenyl-D2 for MAO B mapping. J. Nucl. Med. 1995;36(7):1255–1262. [PubMed] [Google Scholar]

[ref48] Fowler J. S., Wolf A. P., MacGregor R. R., Dewey S. L., Logan J., Schlyer D. J., Langstrom B.. Mechanistic positron emission tomography studies: Demonstration of a deuterium isotope effect in the monoamine oxidase-catalyzed binding of [11C]L-deprenyl in living baboon brain. J. Neurochem. 1988;51(5):1524–1534. doi: 10.1111/j.1471-4159.1988.tb01121.x. [DOI] [PubMed] [Google Scholar]

[ref49] Fowler J. S., MacGregor R. R., Wolf A. P., Arnett C. D., Dewey S. L., Schlyer D., Christman D., Logan J., Smith M.. et al. Mapping human brain monoamine oxidase A and B with carbon-11-labeled suicide inactivators and PET. Science. 1987;235(4787):481–485. doi: 10.1126/science.3099392. [DOI] [PubMed] [Google Scholar]

[ref50] Xiao H., Choi S. R., Zhao R., Ploessl K., Alexoff D., Zhu L., Zha Z., Kung H. F.. A New Highly Deuterated [18F]AV-45, [18F]D15FSP, for Imaging β-Amyloid Plaques in the Brain. ACS Med. Chem. Lett. 2021;12(7):1086–1092. doi: 10.1021/acsmedchemlett.1c00062. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref51] Li K., Gilberti A. L., Marden J. A., Akula H. K., Pollard A. C., Guo S., Hu B., Tonge P. J., Qu W.. Synthesis and Biological Evaluation of Fluorine-18 and Deuterium Labeled l-Fluoroalanines as Positron Emission Tomography Imaging Agents for Cancer Detection. J. Med. Chem. 2024;67(12):10293–10305. doi: 10.1021/acs.jmedchem.4c00774. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref52] Yamada K., Fujita H., Kunishima M.. A Novel Acid-Catalyzed O-Benzylating Reagent with the Smallest Unit of Imidate Structure. Org. Lett. 2012;14(19):5026–5029. doi: 10.1021/ol302222p. [DOI] [PubMed] [Google Scholar]

[ref53] Zhang X., Basuli F., Shi Z.-D., Xu B., Blackman B., Choyke P. L., Swenson R. E.. Automated synthesis of [18F](2S,4R)-4-fluoroglutamine on a GE TRACERlab FX-N Pro module. Appl. Radiat. Isot. 2016;112:110–114. doi: 10.1016/j.apradiso.2016.02.016. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref54] Li S., Schmitz A., Lee H., Mach R. H.. Automation of the Radiosynthesis of Six Different 18F-labeled radiotracers on the AllinOne. EJNMMI Radiopharm. Chem. 2017;1(1):15. doi: 10.1186/s41181-016-0018-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref55] Zhang Y., Zhang L., Yang J., Wu Z., Ploessl K., Zha Z., Liu F., Xu X., Zhu H., Yang Z.. et al. Initial experience in synthesis of (2S,4R)-4-[18F]fluoroglutamine for clinical application. J. Labelled Compd. Radiopharm. 2019;62(5):209–214. doi: 10.1002/jlcr.3719. [DOI] [PubMed] [Google Scholar]

[ref56] Ploessl K., Wang L., Lieberman B. P., Qu W., Kung H. F.. Comparative evaluation of 18F-labeled glutamic acid and glutamine as tumor metabolic imaging agents. J. Nucl. Med. 2012;53(10):1616–1624. doi: 10.2967/jnumed.111.101279. [DOI] [PubMed] [Google Scholar]

[ref57] Ouvrard E., Kaseb A., Poterszman N., Porot C., Somme F., Imperiale A.. Nuclear medicine imaging for bone metastases assessment: What else besides bone scintigraphy in the era of personalized medicine? Front. Med. 2024;10:1320574. doi: 10.3389/fmed.2023.1320574. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref58] Seifert R., Telli T., Opitz M., Barbato F., Berliner C., Nader M., Umutlu L., Stuschke M., Hadaschik B., Herrmann K., Fendler W. P.. Unspecific 18F-PSMA-1007 Bone Uptake Evaluated Through PSMA-11 PET, Bone Scanning, and MRI Triple Validation in Patients with Biochemical Recurrence of Prostate Cancer. J. Nucl. Med. 2023;64(5):738–743. doi: 10.2967/jnumed.118.215434. [DOI] [PubMed] [Google Scholar]

PERMALINK

Deuterium- and Fluorine-18-Labeled Glutaminea PET Imaging Agent with Enhanced In Vivo Stability

Hari K Akula

Bao Hu

Jaclyn Brunner

Kaixuan Li

Lemise Saleh

Paul Vaska

Wenchao Qu

Abstract

Introduction

1.

2.

Results and Discussion

Chemistry

1. Original Reported Method for the Synthesis of 4-[18F]­FGln ([ 18 F]­4).

2. Synthesis of Deuterated Homoserine Intermediate 11 .

3. Synthesis of Deuterated and Fluorinated (2S,4R)-4-Fluoroglutamines 4-[18F]­FGln-d 3 ([ 18 F]­16) and 4-FGln-d 3 (16).

Radiochemistry

In Vitro Cellular Uptake

3.

In Vivo PET ImagingComparison of 4-[18F]­FGln and 4-[18F]­FGln-d 3 in 9L/lacZ Tumor-Bearing Rats

4.

5.

Conclusions

Materials and Methods

Chemistry

Organic Chemistry

Tert-butyl­(O 5-benzyl-3,3,4,4-tetradeutero)­homoserinate (9a,b)

Step i

Step ii

Step iii

Separation of (2S)-Tert-butyl­(O 5-benzyl-3,3,4,4-tetradeutero)­homoserinate (9a) and (2R)-Tert-butyl­(O 5-benzyl-3,3,4,4-tetradeutero)­homoserinate (9b) via Chiral Preparative HPLC

(2S)-Tert-butyl­(-Butyl (O-benzyl-5-benzyl-N-(tert-butoxycarbonyl)-3,3,4,4-tetradeutero)­homoserinate (10)

(2S)-4-Tert-butyl-2-(tert-butoxycarbonylamino)-3,3,4,4-tetradeutero-4-hydroxybutanoate (11)

(2S)-4-Tert-butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-oxobutanoate (12)

(2S,4S)-Tert-butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-hydroxy-5-oxo-5-(2,4,6-tri methoxybenzylamino)­pentanoate (13a) and (2S,4R)-Tert-butyl 2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-hydroxy-5-oxo-5-(2,4,6-trimethoxybenzylamino)­pentanoate (13b)

Step i: (2S)-Tert-butyl-2-(tert-butoxycarbonylamino)-4-(2-chloroacetoxy)-3,3,4-trideutero-5-oxo-5-(2,4,6-trimethoxybenzylamino)­pentanoate

Step ii

(2S,4R)-Tert-Butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-5-oxo-4-(tosyloxy)-5-(2,4,6-trimethoxybenzylamino)­pentanoate (14)

(2S,4R)-Tert-butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-fluoro-5-oxo-5-(2,4,6-trimethoxy benzylamino)­pentanoate (15)

(2S,4R)-2,5-Diamino-3,3,4-trideutero-4-fluoro-5-oxopentanoic acid (16)

Radiochemistry

Biology

Culturing of 9L/lacZ Cells

In Vitro Cellular Uptake of the 4-[18F]­FGln ([18F]­4) and 4-[18F]­FGln-d 3 ([18F]­16) Time-Dependent Uptake Experiment

Transporter Characterization Experiment

9L/lacZ Tumor-Bearing Rats

In Vivo PET/CT Imaging

Statistical Analysis

Supplementary Material

Acknowledgments

Glossary

Abbreviation

#.

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

1. Original Reported Method for the Synthesis of 4-[¹⁸F]FGln ([¹⁸ F]4).

3. Synthesis of Deuterated and Fluorinated (2S,4R)-4-Fluoroglutamines 4-[¹⁸F]FGln-d ₃ ([¹⁸ F]16) and 4-FGln-d ₃ (16).

In Vivo PET ImagingComparison of 4-[¹⁸F]FGln and 4-[¹⁸F]FGln-d ₃ in 9L/lacZ Tumor-Bearing Rats

Tert-butyl(O ⁵-benzyl-3,3,4,4-tetradeutero)homoserinate (9a,b)

Separation of (2S)-Tert-butyl(O ⁵-benzyl-3,3,4,4-tetradeutero)homoserinate (9a) and (2R)-Tert-butyl(O ⁵-benzyl-3,3,4,4-tetradeutero)homoserinate (9b) via Chiral Preparative HPLC

(2S)-Tert-butyl(-Butyl (O-benzyl-⁵-benzyl-N-(tert-butoxycarbonyl)-3,3,4,4-tetradeutero)homoserinate (10)

(2S,4S)-Tert-butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-hydroxy-5-oxo-5-(2,4,6-tri methoxybenzylamino)pentanoate (13a) and (2S,4R)-Tert-butyl 2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-hydroxy-5-oxo-5-(2,4,6-trimethoxybenzylamino)pentanoate (13b)

Step i: (2S)-Tert-butyl-2-(tert-butoxycarbonylamino)-4-(2-chloroacetoxy)-3,3,4-trideutero-5-oxo-5-(2,4,6-trimethoxybenzylamino)pentanoate

(2S,4R)-Tert-Butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-5-oxo-4-(tosyloxy)-5-(2,4,6-trimethoxybenzylamino)pentanoate (14)

(2S,4R)-Tert-butyl-2-(tert-butoxycarbonylamino)-3,3,4-trideutero-4-fluoro-5-oxo-5-(2,4,6-trimethoxy benzylamino)pentanoate (15)

In Vitro Cellular Uptake of the 4-[¹⁸F]FGln ([¹⁸F]4) and 4-[¹⁸F]FGln-d ₃ ([¹⁸F]16) Time-Dependent Uptake Experiment