Development of [¹⁸F]F-5-OMe-Tryptophans through Photoredox Radiofluorination: A New Method to Access Trp-based PET Agents

Abstract

Although various radiolabeled tryptophan analogs have been developed to monitor tryptophan metabolism using positron emission tomography (PET) for various human diseases including melanoma and other cancers, their application can be limited due to the complicated synthesis process. In this study, we demonstrated that photoredox radiofluorination represents a simple method to access novel tryptophan-based PET agents. In brief, 4-F-5-OMe-tryptophans (L/D-T13) and 6-F-5-OMe-tryptophans (L/D-T18) were easily synthesized. The ¹⁸F-labeled analogs were produced by photoredox radiofluorination with radiochemical yields (RCYs) ranging from 2.6±0.5% to 32.4±4.1% (3 ≤ n ≤ 5, enantiomeric excess (ee) ≥ 99.0%) and over 98.0% radiochemical purity (RCP). Small animal imaging showed that L-[¹⁸F]T13 achieved 9.58±0.26 %ID/g tumor uptake and good contrast in B16F10 tumor-bearing mice (n=3). Clearly, L-[¹⁸F]T13 exhibited prominent tumor uptake, warranting future evaluations of its potential usage in precise immunotherapy monitoring.

Graphical Abstract

INTRODUCTION

L-Tryptophan (Trp) is an essential amino acid in mammals for protein synthesis and anabolic processes. However, most (99%) of ingested Trp is a biochemical precursor of metabolites involved in crucial physiologic roles regulating gut homeostasis, immunity, and neuronal function.¹ While the gut microbiota metabolizes a significant proportion of ingested Trp, the remaining absorbed fraction is metabolized primarily via the kynurenine pathway by the action of rate-limiting enzymes tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenases (IDO1/IDO2). The remainder of the absorbed Trp is metabolized via the serotonin pathway by the action of the rate-limiting enzymes tryptophan hydroxylase 1 and 2 (TPH1/TPH2; Figure 1). Imbalances in the synthesis and availability of Trp metabolites are associated with various psychiatric (depression, schizophrenia), mental (anxiety), and inflammatory diseases (cancer, diabetes, obesity, cardiovascular diseases).² Therefore, it is unsurprising that Trp metabolism has been an ideal pharmacologic target for the treatment of various diseases.³ The use of such drugs ranges from sleep supplements (e.g., melatonin) to FDA-approved drugs for depression/anxiety (selective serotonin reuptake inhibitors) and from carcinoid syndrome (telotristat ethyl)⁴ to investigational drugs for pulmonary hypertension, idiopathy pulmonary fibrosis (rodatristat ethyl),⁵ and cancer (various TDO/IDO1 inhibitors).⁶ Therefore, the ability to assess global and/or pathway-specific Trp metabolism may assist clinicians in better prognosticating and/or predicting responses to specific therapies. For example, ¹¹C-labeled alpha methyl tryptophan ([¹¹C]AMT), a Trp-like analog, has been used across various clinical trials for positron emission tomography (PET) imaging of various benign and malignant conditions, such as the epileptogenic foci associated with tuberous sclerosis complex, primary and metastatic brain tumors, and metastatic breast cancer.^{7, 8} In our recent prospective clinical trial of patients with metastatic PD1 inhibitor-naïve melanoma, we used [¹¹C]AMT as a PET tracer to image intratumoral Trp metabolism. We found baseline [¹¹C]AMT appeared to be a better predictor of poor antitumor response to pembrolizumab than fluorodeoxyglucose (FDG) PET results.⁹ Thus, the ability to predict response to this growing class of cancer immunotherapies that render at best 20–40% antitumor responses across various solid tumors, is appealing because it could limit financial cost and decrease unnecessary toxicity to a potentially toxic drug.

FIGURE 1.

Trp metabolisms¹⁷ and radiotracers based on Trp analogs¹⁴

Although [¹¹C]AMT PET imaging has been an excellent tool compound to support prospective clinical trials for more than two decades, its production and use have severe limitations. First, the moisture-sensitive reaction conditions and short half-life of ¹¹C (20.36 minutes) have limited its use to a single academic institution with the capacity for such complexed manufacturing.¹⁰ Moreover, [¹¹C]AMT is a universal substrate for most of the Trp enzymes, which would be unsuitable for conditions that only affect a specific pathway due to the lack of specificity.⁸ Therefore, as a measure of global, non-pathway-specific Trp metabolism, [¹¹C]AMT is difficult to be used for human conditions that predominantly affect the serotonin as opposed to the kynurenine pathways (and vice versa). To address the challenges posted by the use of [¹¹C]AMT, several ¹⁸F-labeled tryptophan derivatives have been developed, including [¹⁸F]FETrp,^{11, 12} 4-[¹⁸F]F-Trp, 5-[¹⁸F]F-Trp, 6-[¹⁸F]F-Trp,¹³ which were all reported to be related to IDO1 enzyme. While 7-[¹⁸F]F-Trp¹³ exhibited good in-vivo stability and selective accumulation in serotonergic and melatonin-producing areas in the brain. The intensive studies on these Trp-based radiotracers show the Trp backbone have been attractive owing to its great potential as a radiotracer for broad applications. However, challenges remain in terms of metabolism pathways,¹⁴ synthesis feasibility, and in vivo stability.^{12, 14, 15} Recently, our lab demonstrated that the PET imaging outcomes for B16F10 tumor could be improved by substituting fluorine for a hydrogen atom at the 5-position of AMT, resulting 5-[¹⁸F]F-AMT.¹⁶ However, as a multi-step synthesis starting with L-Trp, the production of the precursor was complicated. In addition, the radiolabeling conditions were harsh, as the ¹⁸F-labeling involves (copper) metal catalysis followed by strong acid and base deprotection, resulting in 10.9–14.9% decay-corrected radiochemical yield (RCY).¹⁶

To establish a complete profile of the [¹⁸F]fluorinated AMT, which might lead to better Trp-based ¹⁸F-PET agents, we synthesized and evaluated several [¹⁸F]AMT analogs. One attempt was to radiofluorinate the α-methyl group of the AMT molecule (Figure 2). The protected precursor 6 could be synthesized over five steps in a good total yield of 31%.^{16, 18} Although the radio chemical yield is less than 2%, [¹⁸F]7 could be obtained by heating the [¹⁸F]TBAF and precursor 6 in DMSO. However, the deprotection reaction mainly led to the decomposed product(s) instead of [¹⁸F]T8.¹⁹

FIGURE 2. Synthesis of an α-[¹⁸F]F-methyl tryptophan radiotracer: designs and attempts.

Reaction conditions: i: SO₂Cl₂, MeOH; ii: ClCOOMe, Et₃N, DCM; iii: H₃PO4; iv: PhSO₂Cl, pyridine; v: LDA, CH₂I₂, THF, −45 °C; vi: [¹⁸F]TBAF, DMSO, 120 °C; vii: Mes-Acr-Ph⁺ClO₄⁻, DIPEA, 390 nm irradiation.²⁰ viii: TFA, Δ.

In addition to introducing the fluoro group to position 5 of the tryptophan backbone, other functional groups can be installed at the 5-position to minimize the TPH metabolic pathway. We focused on the methoxy group due to its good stability and previous report of 5-methoxy-D,L-tryptophan (5-MTP) as an IDO²¹ or IDO1²² substrate. Indeed, 5-MTP is synthesized in vivo from intracellular tryptophan via two enzymatic steps: TPH converts L-tryptophan (L-Trp) to 5-hydroxytryptophan (5-HTP), and hydroxy indole O-methyltransferase (HIOMT) converts 5-HTP to 5-MTP.²³ Theoretically, 5-MTP-based-Trp analogs should not be metabolized by TPH. In light of previous reports, here we report the synthesis of 5-MTP-based ¹⁸F-radiotracers (L-[¹⁸F]T13, D-[¹⁸F]T13, and L-[¹⁸F]T18, D-[¹⁸F]T18) using our recently reported photoredox radiolabeling method.²⁴

RESULTS AND DISCUSSIONS

Chemistry

Unlike some previously reported Trp-based precursors, the preparation of precursors 12 and 17 was straightforward and included only three-step synthetic routes (Figures 3 and 4). In brief, 3-Iodo-4-F-5-OMe-indole (10) was obtained from 9 by direct iodization, which afforded the indole compound 11 in 70% isolation yield after 1-N-Boc protection. The chiral α-amino acid precursors L-12 and D-12 were constructed through nickel-catalyzed reductive cross-coupling from 11 and (R)-iodo-N-Boc-serine methyl ester or (S)-iodo-N-Boc-serine methyl ester respectively. L-17 and D-17 could be prepared using similar methods. Another advantage of this approach is that the authentic product standards (T13 and T18) could be obtained in high yield after simple deprotection from the precursors. Finally, we would like to point out that the method could be easily expanded to the preparation of Trp analogs bearing other functional groups.

FIGURE 3.

Synthesis of novel 4-[¹⁸F]F-5-OMe-Tryptophan radiotracer by photoredox radiofluorination

FIGURE 4.

Synthesis of novel 6-[¹⁸F]F-5-OMe-Tryptophan radiotracer by photoredox radiofluorination

Radiochemistry

With the fluorine-containing precursor on hand, we then explored the feasibility of converting them into PET agents through the newly developed photoredox labeling strategy. Using 1-N-Boc-4-F-5-OMe-indole (1-N-Boc-9) as a model compound, photoredox radiofluorination was successfully performed with a good RCY (49.5%, decay corrected, See Table S1). The molar activity (A_m) of the 1-N-Boc-[¹⁸F]9 was 0.39GBq/μmol (See Table S2). With the encouraging results, we then tested the photoredox-radiofluorination using enantiopure precursor L-12. In a multi-solvent system consisting of t-BuOH, DCE, and MeCN. 30 min blue laser (450 nm) irradiation led to L-[¹⁸F]12 in 45.6±3.2% RCY (n=5, decay corrected), which was then converted to L-[¹⁸F]T13 after deprotection with hydrochloric acid (71.0±5.1%, n=5, decay corrected). The radiochemical purity (RCP) of L-[¹⁸F]T13 was determined to be ≥ 98.0% and the enantiomeric excess (ee) was ≥ 99.0%. Similarly, D-[¹⁸F]T13 was synthesized in 26.1±5.1% overall RCY (n=4, d.c, RCP ≥ 98.0%, ee ≥ 99.0%), whose purity and identity were validated by HPLC analysis. In addition to position 4, we also introduced F to position 6 of the indole ring. Starting from 6-F-5OMe-indole (14), precursors L-17 and D-17 were synthesized using the same chemistry (Figure 4). L-[¹⁸F]T18 and D-[¹⁸F]T18 were successfully synthesized and purified with a 2.9±1.5% RCY for L-[¹⁸F]T18 (n=3, decay corrected, RCP ≥ 98.0%, ee ≥ 99.0%), and a 2.6±0.5% RCY for D-[¹⁸F]T18, (n=3, decay corrected, RCP ≥ 98.0%, ee ≥ 99.0%). Overall, these yields were much lower than those of L-[¹⁸F]T13 and D-[¹⁸F]T13. Chiral HPLC was used to confirm the enantiopurity of the four PET agents.

We observed more radioactive byproducts when L-17 and D-17 were labeled via a photoredox-mediated direct ¹⁸F-¹⁹F exchange, compared to those of L-12 and D-12. The increased radio byproducts may be due to a reduced reactivity at the 6-position compared with the 4-position. For compound 17, one potential competitive fluorination site might be the 2- or 3-position of the 5-membered indole ring, which may be explored further in the future. We also must point out that the PET agents obtained from direct ¹⁸F-¹⁹F exchange had relatively lower A_m. Like ¹⁸F-FDOPA, this low A_m does not affect Trp PET imaging results including brain uptake as they are substrate-based probes. However, additional experiment is needed to confirm it. Moreover, PET agents with high A_m may be prepared using photoredox-mediated Cl/¹⁸F exchange (reported A_m = 71.25 GBq/μmol)²⁴ or C-O S_NAr radiofluorination (reported A_m = 74.7 ± 14.1 GBq/μmol)²⁵, which can be explored in the future if there is a need. We used an ice-bath condition to avoid overheating under laser irradiation, but if an LED light was used for the photoredox-mediated radiolabeling, the ice bath could be removed. Currently, we are exploring and developing more applicable labeling apparatus with LED irradiation and simplified procedures. For T18, we observed a significant amount of decomposition during the deprotection process. We tried lowering the temperature or acid volume, but this reduced the RCY due to partial hydrolysis. The deprotection condition could be optimized further to increase the RCY.

In vitro assay and analysis

We performed IDO and TPH in vitro enzymatic assays to investigate whether the T13 and T18 enantiomers are metabolized via the kynurenine or the serotonin pathway, respectively. L-Trp was used as the positive control for both pathways. The negative control or baseline was the reading when no amino acid substrate was added to the incubated reaction mixture. TPH enzyme assays were carried out using the LC-MS technique, which showed that none of the four agents (L-T13, D-T13, and L-T18, D-T18) appears to be metabolized via the serotonin pathway, while L-Trp gave 5-hydroxyl-Trp as the product of the TPH enzyme. We then performed IDO1 in vitro assay, which was carried out using UV-Vis spectroscopy to monitor the UV absorbance at 321 nm, the maximum absorption peak of the principle kynurenine metabolite, formylkynurenine. IDO1 enzyme assay results showed that L-T13, L-T18, and L-Trp are IDO1 substrates according to the observed UV absorbance enhancement at 321 nm versus the negative control (Figure 5b). We also noticed a high UV peak at 334 nm for L-T18 (L-6-F-5-OMe-Trp) (Figure S15). This 334 nm peak was very close to the peak of formylkynurenine, indicating that a similar metabolic product was produced in abundance from L-T18 with the IDO1 enzyme. However, additional experiments are needed before a conclusion can be drawn about the metabolic pathways of these novel Trp analogs. We would also like to point out that there are reports suggesting AMT and 6-F-AMT may not be IDO substrate.^{26, 27} Studies comparing the reported agents and AMT/6-F-AMT would be interesting to pursue in the future. In fact, there is also debate on whether 5-MTP is IDO1 or IDO2 substrate.²⁸ Our method used UV absorbance to measure IDO activity, which should be further confirmed using HPLC method²⁷ in the future. It is also not clear whether the agents are substrates of TDO and whether they can act as an inhibitor of TPH. The current report focuses on the new method to develop Trp based PET agent and initial biological exploration. A follow up study should be devoted to evaluating the substrate specificity towards all Trp enzymes.

FIGURE 5.

IDO and TPH assay.

a.) TPH assay by LC-MS: Only L-Trp (positive control) is the substrate for the TPH enzyme.

b.) IDO1 assay: L-T13, L-T18, and L-Trp are substrates for the IDO1 enzyme. (﹡﹡﹡﹡ p < 0.0001)

Small-Animal PET Imaging

After demonstrating their in vitro selectivity towards the IDO pathway, we tested four ¹⁸F-labeled Trp analogs (T13 and T18 enantiomers) in B16F10 tumor-bearing animal models. Both L- and D-[¹⁸F]T13 showed substantial tumor uptake and high contrast. However, the absolute uptake value of L-[¹⁸F]T13 (9.58±0.26 and 2.10±0.31 %ID/g at 0.5 and 3 h post-injection, respectively) was much higher than that of D-[¹⁸F]T13 (2.17±0.10% and 0.34±0.04% at 0.5 and 3 h p.i.). For D-[¹⁸F]T13, the tumor/muscle ratio increased from 4.98±1.69 at 0.5h to 10.33±5.21 at 3.0h (Figure 6f). Compared with previously reported 5-[¹⁸F]F-AMT, the tumor uptake value of L-[¹⁸F]T13 remained at comparable range. However, to select the best candidate for further evaluation, side-by-side comparisons are needed to assess tumor retention/washout of each agent. D-[¹⁸F]T18 showed prominent tumor uptake and high contrast as well. The initial tumor uptake of D-[¹⁸F]T18 reached 5.78±0.68 %ID/g at 0.5h. It remained at 2.15±0.59 %ID/g at 1.5h. While kidney uptake decreased from 4.95±0.34 %ID/g at 0.5h to 0.98±0.32 %ID/g at 1.5h. Although L-T18 showed good IDO activity in enzyme assay, its contrast between the tumor and background was poor with defluorination and apparent bone uptake in vivo. Therefore, it is not sufficient that a suitable substrate-type PET agent is an efficient and selective substrate of the corresponding enzyme. Furthermore, a good cellular retention of the PET-tracer by itself and/or its radiometabolites to prevent washout of the radioactive signal from the target tissue is also required. Because L-T18 is stable in PBS for 3h, the degradation is likely be caused by enzymes in vivo. The byproduct identity still needs to be studied in the future. Of note, L-T13 showed reduced IDO activity compared with L-T18. The observed higher tumor uptake of L-T13 could be a combination of IDO reactivity, LAT transporters and stability.

FIGURE 6.

PET imaging with L-[¹⁸F]T13 and D-[¹⁸F]T13 in B16F10 tumor-bearing mice.

a.) ROI analysis of tumor and major organs, b.) Representative PET images, and c.) Tumor/organ uptake ratios of L-[¹⁸F]T13 at 0.5, 1.5, and 3.0 h post-injection (n=3); d.) ROI analysis of tumor and major organs, e.) Representative PET images, and f.) Tumor/organ uptake ratios of D-[¹⁸F]T13 at 0.5, 1.5, and 3.0 h post-injection (n=3).

The kidney was the organ with the highest initial uptake for our newly developed Trp agents. For example, kidney uptake of L-[¹⁸F]T13 was 11.30±0.09 %ID/g at 0.5h and quickly decreased by 83% at the 3.0-hour time point. Due to LAT1 expression in pancreatic beta cells,²⁹ we observed substantial pancreas uptake of all four tracers. In addition to the B16F10 model, we also performed a preliminary pilot study of [¹⁸F]T13 using MCF-7, H358, LnCap, and UPPL tumor-bearing animal models. PET scan was performed at 1.5 and 3.0h post-injection. In all four tumor models, the tumor uptake of L-[¹⁸F]T13 was higher than that of D-[¹⁸F]T13 (Figure S10). This is similar to the results obtained from the B16F10 tumor model. However, the IDO expression of these tumors still needs to be studied further. Since the radiochemical yield of [¹⁸F]T18 was much lower than that of [¹⁸F]T13, we did not perform further imaging studies with the [¹⁸F]T18 agent.

Biodistribution Studies

Radioactivity biodistribution studies were performed after imaging data collection at 3 h post-injection. Tumors and other tissues were collected, and radioactive uptake in tumors and organs was countered. As shown in Figure 7 and Figure S11, radioactivities in the collected tissues were consistent with the imaging-derived ROI analysis (Figure 6 and Figure S7). Among the four agents, D-[¹⁸F]T18 had the best tumor-to-background contrast (Figure 7d). D-[¹⁸F]T13 also demonstrated a high tumor-muscle ratio (20.09±5.96) (Figure 7c). L-/D-[¹⁸F]T13 showed good tumor retention (Figures 7a and 7c). Defluorination of L-[¹⁸F]T18 led to high bone uptake, similar to the PET result (Figure 7b).

FIGURE 7. Biodistribution analysis of four agents in B16F10 tumor-bearing mice.

B16F10 tumor-bearing mice (n = 3) were injected with (a.)L-[¹⁸F]T13, (c.)D-[¹⁸F]T13, (b.)L-[¹⁸F]T18, and (d.)D-[¹⁸F]T18 via tail veins and various tissues were collected at 3.5 h post-injection. Radioactivities in the tissues were determined using a gamma counter.

CONCLUSION

In conclusion, we have established a facile photoredox radiofluorination method to produce Trp-based radiotracers, by which enantiopure precursors and standard radiolabeled PET reagents can be directly synthesized without racemization. Using our method, L-[¹⁸F]T13/T18 and D-[¹⁸F]T13/T18 were synthesized with acceptable to good RCY. The L-[¹⁸F]T13 exhibited higher tumor uptake than the D enantiomer in B16F10 tumor-bearing mice. Good tumor to major organ/tissue ratios, especially tumor/muscle ratio, were obtained for both enantiomers. The initial evaluation suggested L-[¹⁸F]T13 had prominent tumor uptake in the B16F10 tumor-bearing mice, which warrants future evaluations for potential usage in immunotherapy monitoring and further investigation on the metabolism of these agents. Due to its simplicity, our photoredox radiofluorination method can also be used to develop other novel tryptophan-based PET agents.

Experimental Section

General.

Commercially available chemicals reagents and solvents were purchased from Sigma-Aldrich, Alfa Aesar, TCI, Acros, Combi-Blocks, Matrix Scientific, Oakwood Chemical, Chem Impex International, and Fisher Scientific, etc. Anhydrous acetonitrile (MeCN), dichloromethane (DCM), tetrahydrofuran (THF), and dimethylformamide (DMF) were dried by an Inert solvent purification system (PS-MD-5). Nuclear magnetic resonance spectra were obtained using a Varian 400 MR spectrometer and a Varian 500 MHz spectrometer. High Resolution Mass Spectra (HRMS) were analyzed on the ThermoFisher Q-Exactive HF (QE) Quadrupole-Obitrap mass spectrometer (ThermoFisher, Bremen, Germany) mass spectrometer with positive mode heated electrospray ionization (HESI) in the University of North Carolina’s Department of Chemistry Mass Spectrometry Core Laboratory. The photocatalyst Mes-Acr-Ph⁺ClO₄⁻ was provided by David Nicewicz research group (Department of Chemistry, UNC at Chapel Hill), and prepared according to reported procedures. The characterization of data was well matched with that reported by Chen et al.³⁰. Tetrabutylammonium bicarbonate (TBAHCO₃) solution (20%, w/w) and TBAHCO₃ solution in MeCN (~60 mg/ml) were prepared according to our reported procedures²⁴. HPLC was carried out with a Shimadzu LC-20AT Prominence equipped with a UV detector. The purity of all final compounds was ≥95%, as determined by HPLC analysis.

Chemistry

Trp-based precursors 12 and 17 were obtained through a facile three-step synthetic route (Figures 3 and 4).

4-fluoro-3-iodo-5-methoxy-1H-indole (10).

3-Iodination was carried out according to reference.³¹ To a 50 mL round-bottom flask in which 4-fluoro-5-methoxy-1H-indole (1.0 g) dissolved in 10 mL DMSO, N-iodosuccinimide (NIS, 1.05 equiv) was added, and the reaction mixture was stirred for 18 h. After completion of the reaction, as monitored by TLC, the reaction mixture was diluted with 30 mL of ethyl acetate, and washed with a saturated solution of Na₂S₂O₃ (30 mL × 2) and 30 mL of brine. The organic phase was subsequently dried over anhydrous Na₂SO_4, and the solvent was removed in vacuo. The resultant crude product compound was subjected to silica gel column chromatography (Hexanes/EA: 10/1 to 3/1) to isolate the desired white-to-gray product (1.65g, 93.0% yield). ¹H NMR (500 MHz, Chloroform-d) δ 8.33 (s, 1H), 7.21 (d, J = 2.5 Hz, 1H), 7.08 (dd, J = 8.8, 1.0 Hz, 1H), 6.98 (t, J = 4.5 Hz, 1H), 3.93 (s, 3H). ¹⁹F NMR (376 MHz, Chloroform-d) δ −148.54 (d, J = 6.7 Hz). ¹³C NMR (101 MHz, Chloroform-d) δ 146.25 (d, J = 249.7 Hz), 140.91 (d, J = 9.4 Hz), 133.63 (d, J = 8.1 Hz), 130.60, 125.31, 113.88, 106.46 (d, J = 24.8 Hz), 98.77, 59.23 (d, J = 14.1 Hz).

tert-butyl 4-fluoro-3-iodo-5-methoxy-1H-indole-1-carboxylate (11)

The obtained solid (1.6 g, 5.5 mmol) was suspended with 5 mL dichloromethane in a 50mL round bottom flask. DMAP (4-Dimethylaminopyridine) (67 mg, 0.55 mmol, 10 mol %) and di-tert-butyl dicarbonate (Boc anhydride) (1.8 g, 8.25 mmol, 1.50 equiv.) were dissolved in 10 mL dichloromethane in a 20mL vial before they were added into the flask at room temperature. Then the mixture was stirred for 3 hours at room temperature until the completion of the reaction as monitored by TLC. Then the reaction mixture was washed with 35 mL 0.1N HCl, and the aqueous phase was extracted with dichloromethane (3 × 35 mL). The combined organic layers were dried with anhydrous Na₂SO₄, the solvents were removed under reduced pressure, and the residue was purified by silica chromatography to harvest 1.5 g (70% yield) off-white solid as the desired product. ¹H NMR (500 MHz, Chloroform-d) δ 7.87 (d, J = 9.0 Hz, 1H), 7.63 (s, 1H), 7.04 (t, J = 8.5 Hz, 1H), 3.94 (s, 3H), 1.65 (s, 9H). ¹⁹F NMR (376 MHz, Chloroform-d) δ −148.84 (d, J = 7.5 Hz, 1F). ¹³C NMR (101 MHz, Chlroform-d) δ 148.53, 145.46 (d, J = 250.7 Hz), 143.13 (d, J = 8.9 Hz), 132.21, 131.59 (d, J = 6.6 Hz), 121.08, 113.58, 110.45 (d, J = 5.0 Hz), 84.72, 58.17 (d, J = 1.0 Hz), 55.41 (d, J = 1.3 Hz), 28.26.

Cross coupling

procedures were modified from the reference.³² In a glove box, NiCl₂ (6.5 mg, 0.05 mmol, 10 mol%), 4,7-diphenyl-1,10-phenanthroline (16.6 mg, 0.05 mmol, 10 mol%), Mn powder (83 mg, 1.5 mmol, 3.0 equiv.), and anhydrous Dimethylacetamide (DMA, 2 mL) were added to a 10 mL glass vial before it was sealed with an aluminum-rubber crimp cap. The reaction mixture was brought out to the fume hood and then stirred at 80 °C for 30 min. The reaction mixture was cooled to room temperature, and the pre-mixed iodo-1H-indole-1-carboxylate (195.5 mg, 0.5 mmol, 1.0 equiv.) and derived chiral serine iodides (0.7 mmol, 1.4 equiv.) in anhydrous 1-Methyl-2-pyrrolidinone (NMP, 1 mL) were added by a syringe under an argon balloon pressure. The reaction mixture was stirred at 25 °C for 20 h. To remove the NMP and DMA, the reaction mixture was poured into 50 mL of ice water, and the resulting mixture was extracted with ethyl acetate (30 mL × 4). The combined organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The product was then isolated by silica flash chromatography (Hexanes / EA = 20/1 to 3/1) as a colorless solid film after drying over a vacuum pump. The products and yields are listed below.

4F-5OMe-L-Trp precursor (L-12, white powder, 81 mg, 34.8% yield), tert-butyl (S)-3-(2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-4-fluoro-5-methoxy-1H-indole-1-carboxylate ¹H NMR (500 MHz, Chloroform-d) δ 7.81 (s, 1H), 7.32 (s, 1H), 6.98 (t, J = 8.5 Hz, 1H), 5.14 – 4.74 (m, 1H), 4.65 – 4.44 (m, 1H), 3.92 (s, 3H), 3.74 (s, 3H), 3.35 (dd, J = 14.8, 5.3 Hz, 1H), 3.18 – 2.86 (m, 1H), 1.64 (s, 9H), 1.38 (s, 9H). ¹⁹F NMR (376 MHz, Chloroform-d) δ −146.65 (d, J = 78.8 Hz, 1F). ¹³C NMR (101 MHz, CDCl₃) δ 172.64, 155.21, 149.45, 146.04 (d, J = 246.1 Hz), 142.71 (d, J = 9.7 Hz), 131.98, 125.47, 120.18 (d, J = 14.4 Hz), 113.46 (d, J = 2.2 Hz), 112.75, 110.77, 84.02, 79.95, 58.08, 54.06, 52.45, 29.29, 28.38, 28.32. C23H31FN2O7, [M+H]+: 467.2194, found: 467.2187.

4F-5OMe-D-Trp precursor (D-12, white powder, 156 mg, 67.0% yield), tert-butyl (R)-3-(2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-4-fluoro-5-methoxy-1H-indole-1-carboxylate ¹H NMR (400 MHz, Chloroform-d) δ 7.81 (d, J = 7.9 Hz, 1H), 7.32 (s, 1H), 6.98 (t, J = 8.5 Hz, 1H), 5.10 (d, J = 8.5 Hz, 1H), 4.61 (d, J = 7.1 Hz, 1H), 3.92 (d, J = 1.3 Hz, 3H), 3.73 (s, 3H), 3.34 (dd, J = 14.7, 5.3 Hz, 1H), 3.14 (dd, J = 14.7, 7.5 Hz, 1H), 1.64 (d, J = 1.1 Hz, 8H), 1.38 (s, 9H). ¹⁹F NMR (376 MHz, Chloroform-d) δ −146.66 (d, J = 86.5 Hz, 1F). ¹³C NMR (101 MHz, Chloroform-d) δ 172.64, 155.21, 149.45, 146.02 (d, J = 246.1 Hz), 142.70 (d, J = 9.6 Hz), 131.93, 125.46, 120.18 (d, J = 14.6 Hz), 113.45 (d, J = 2.2 Hz), 112.71, 110.74 (d, J = 3.9 Hz), 84.00, 79.93, 58.05, 54.03, 52.44, 29.27, 28.36, 28.30. C23H31FN2O7, [M+H]+: 467.2194, found: 467.2186.

1-N-Boc-4-F-5-OMe-indole (1-N-Boc-9) was prepared with the same procedure as the preparation of 11 directly from 83 mg (0.5 mmol) of the indole 9 with a yield of (95.0%). ¹H NMR (400 MHz, Chloroform-d) δ 7.82 (d, J = 8.9 Hz, 1H), 7.53 (d, J = 3.8 Hz, 1H), 7.00 (t, J = 8.5 Hz, 1H), 6.63 (dd, J = 3.8, 0.8 Hz, 1H), 3.94 (s, 3H), 1.66 (s, 9H). ¹⁹F NMR (376 MHz, Chloroform-d) δ −144.44 (d, J = 8.3 Hz, 1F). ¹³C NMR (101 MHz, CDCl₃) δ 149.74, 145.14 (d, J = 246.9 Hz), 142.45 (d, J = 9.0 Hz), 131.62 (d, J = 9.5 Hz), 126.92, 120.79 (d, J = 18.8 Hz), 112.47 (d, J = 1.0 Hz), 110.65 (d, J = 4.5 Hz), 102.80, 84.06, 58.02, 28.32.

The precursors L-17 and D-17 were synthesized with the same procedures as described above by starting with 6-fluoro-5-methoxy-1H-indole (14, 1.0 g).

6-fluoro-3-iodo-5-methoxy-1H-indole (15).

Gray-to-brown solid isolated (1.7g, 96.0%). ¹H NMR (400 MHz, Chloroform-d) δ 8.27 (s, 1H), 7.24 (d, J = 2.5 Hz, 1H), 7.14 (d, J = 11.0 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 3.97 (s, 3H). ¹⁹F NMR (376 MHz, Chloroform-d) δ −135.94 (t, J = 10.1 Hz, 1F). ¹³C NMR (101 MHz, Chloroform-d) δ 151.57 (d, J = 242.3 Hz), 144.42 (d, J = 13.1 Hz), 128.77 (d, J = 3.8 Hz), 128.63 (d, J = 11.0 Hz), 125.67 (d, J = 1.2 Hz), 104.14 (d, J = 2.5 Hz), 98.89 (d, J = 23.8 Hz), 57.13 (d, J = 1.5 Hz), 56.85.

tert-butyl 6-fluoro-3-iodo-5-methoxy-1H-indole-1-carboxylate (16).

Gray-to-brown solid isolated (1.82g, 85.0%). ¹H NMR (500 MHz, Chloroform-d) δ 7.89 (d, J = 12.0 Hz, 1H), 7.66 (s, 1H), 6.88 (d, J = 8.1 Hz, 1H), 3.97 (s, 3H), 1.66 (s, 9H). ¹⁹F NMR (376 MHz, Chloroform-d) δ −135.93 (t, J = 10.2 Hz, 1F). ¹³C NMR (126 MHz, Chloroform-d) δ 151.94 (d, J = 243.7 Hz, C-F), 148.53, 145.64 (d, J = 12.7 Hz), 130.30 (d, J = 2.4 Hz), 127.96, 127.82, 104.69 (d, J = 2.6 Hz), 103.52 (d, J = 26.0 Hz), 84.76, 64.69 (d, J = 1.8 Hz, C-I), 56.69, 28.25.

6F-5OMe-L-Trp precursor (L-17, white powder, 113 mg, 48.5% yield), tert-butyl (S)-3-(2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-6-fluoro-5-methoxy-1H-indole-1-carboxylate. ¹H NMR (400 MHz, Chloroform-d) δ 7.84 (s, 1H), 7.34 (s, 1H), 7.02 (d, J = 8.1 Hz, 1H), 5.15 (d, J = 8.2 Hz, 1H), 4.63 (q, J = 6.4 Hz, 1H), 3.93 (s, 3H), 3.68 (s, 3H), 3.17 (dt, J = 13.4, 6.3 Hz, 2H), 1.64 (s, 8H), 1.44 (s, 8H). ¹⁹F NMR (376 MHz, Chloroform-d) δ – 76.03(s, TFA), −136.80 (d, J = 83.9 Hz, 1F). ¹³C NMR (101 MHz, Chloroform-d) δ 172.42, 155.20, 151.38 (d, J = 242.0 Hz), 149.36, 144.88 (d, J = 12.7 Hz), 128.71, 126.13, 124.25, 114.96, 103.75 (d, J = 25.6 Hz), 102.62, 84.07, 80.19, 56.79, 53.71, 52.51, 29.37, 28.41, 28.27. C23H31FN2O7, [M+H]+: 467.2194, found: 467.2186.

6F-5OMe-D-Trp precursor (D-17, white powder, 65 mg, 28.0% yield), tert-butyl (R)-3-(2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-6-fluoro-5-methoxy-1H-indole-1-carboxylate ¹H NMR (400 MHz, Chloroform-d) δ 7.85 (s, 1H), 7.34 (s, 1H), 7.03 (d, J = 8.1 Hz, 1H), 5.13 (d, J = 8.0 Hz, 1H), 4.64 (d, J = 7.3 Hz, 1H), 3.94 (s, 3H), 3.69 (s, 3H), 3.23 – 3.08 (m, 2H), 1.65 (s, 9H), 1.42 (s, 8H). ¹⁹F NMR (376 MHz, CDCl₃) δ −136.79 (d, J = 89.8 Hz, 1F). ¹³C NMR (101 MHz, Chloroform-d) δ 172.45, 155.23, 151.43 (d, J = 242.2 Hz), 149.40, 144.93 (d, J = 12.8 Hz), 128.94, 126.16, 124.29, 114.98, 103.79 (d, J = 25.8 Hz), 102.67, 84.12, 80.24, 56.84, 53.74, 52.54, 29.83, 28.44, 28.31. C23H31FN2O7, [M+H]+: 467.2194, found: 467.2188.

The authentic product standards T13 and T18 were also prepared directly by removing tert-butyloxycarbonyl (Boc) groups in 12 or 17 using trifluoroacetic acid (TFA), followed by hydrolysis under mild aqueous basic condition.

Boc deprotection.

The prepared ¹⁹F tryptophan precursor (10.0 mg) was dissolved in 300 uL TFA in a 20 mL glass vial, and stirred for 30–60 min at room temperature. Then the TFA was removed by gently blowing N₂ into the open reaction vial for 30 min in the fume hood. Both TLC and ¹HNMR were utilized to monitor the deprotection of the Boc groups in the cases of D-4F-5-OMe-Trp precursor and L-4F-5-OMe-Trp precursor. The disappearance of the Boc groups and retention of the methyl ester group were observed on the ¹HNMR spectrum (See Figures S4 and S5 in the SI).

Hydrolysis.

The ¹⁹F tryptophan methyl ester product mixture obtained from the last step was dissolved in 300 uL MeOH without further purification. 150 uL 1.5N NaOH aq solution was added to the reaction vial, followed by stirring overnight (16–24 hours) to afford the hydrolyzed ¹⁹F tryptophan authentic standard product. After neutralizing the product mixture with 225 uL 1N HCl aq solution, the product mixture was then loaded into a C18 cartridge (Waters, Sep-Pak^® Plus C18, preconditioned with 10 mL EtOH and 10mL H₂O) and eluted with pure water (4 mL) followed by 10% (volume) MeCN aqueous acidic solution (0.1% TFA, 10 mL). The pure desired product was obtained in the 10% MeCN eluents. The eluent contents were monitored by HPLC and LC-MS. Generally, 3.0–4.9 mg products could be obtained with 2-step-deprotection yields from 56.0% to 90.7% for 4 different ¹⁹F tryptophan standard compounds.

(S)-2-amino-3-(4-fluoro-5-methoxy-1H-indol-3-yl) propanoic acid-2,2,2-trifluoroacetic acid (L-T13), white solid powder, ¹H NMR (400 MHz, D₂O) δ 7.26 (t, J = 4.4 Hz, 2H), 7.13 (t, J = 8.4 Hz, 1H), 4.04 (dd, J = 9.0, 4.9 Hz, 1H), 3.96 (s, 3H), 3.56 (dd, J = 15.1, 4.9 Hz, 1H), 3.22 (dd, J = 15.2, 9.1 Hz, 1H). ¹⁹F NMR (376 MHz, D₂O) δ −75.63 (s), −147.95 (d, J = 8.1 Hz, 1F). ¹³C NMR (101 MHz, D₂O) δ 174.47, 145.78 (d, J = 241.9 Hz), 138.59 (d, J = 9.6 Hz), 134.34 (d, J = 10.5 Hz), 126.48, 116.10 (d, J = 17.1 Hz), 112.12, 107.43 (d, J = 4.2 Hz), 105.97 (d, J = 2.2 Hz), 58.75, 55.94 (d, J = 2.3 Hz), 27.63. C12H14FN2O3, [M+H]+: 253.0988, found: 253.0981.

(R)-2-amino-3-(4-fluoro-5-methoxy-1H-indol-3-yl) propanoic acid-2,2,2-trifluoroacetic acid (D-T13), white solid powder, ¹H NMR (400 MHz, D₂O) δ 7.22 (t, J = 4.4 Hz, 2H), 7.09 (t, J = 8.5 Hz, 1H), 4.03 (dd, J = 9.0, 4.8 Hz, 1H), 3.93 (s, 3H), 3.54 (dd, J = 15.3, 4.9 Hz, 1H), 3.20 (dd, J = 15.1, 9.1 Hz, 1H). ¹⁹F NMR (376 MHz, D₂O) δ −75.66 (s), −148.07 (d, J = 8.0 Hz, 1F). ¹³C NMR (101 MHz, D₂O) δ 174.21 (s), 145.72 (d, J = 241.6 Hz), 138.56 (d, J = 9.7 Hz), 134.32 (d, J = 10.6 Hz), 126.47 (s), 116.06 (d, J = 17.1 Hz), 112.05 (s), 107.41 (d, J = 4.2 Hz), 105.85 (d, J = 2.3 Hz), 58.70 (s), 55.88 (d, J = 2.1 Hz), 27.52 (s). C12H14FN2O3, [M+H]+: 253.0988, found: 253.0983.

(S)-2-amino-3-(6-fluoro-5-methoxy-1H-indol-3-yl) propanoic acid-2,2,2-trifluoroacetic acid (L-T18), white solid powder, ¹H NMR (400 MHz, D₂O) δ 7.38 – 7.32 (m, 1H), 7.30 – 7.22 (m, 2H), 4.02 (dd, J = 8.0, 4.8 Hz, 1H), 3.94 (d, J = 1.7 Hz, 3H), 3.41 (dd, J = 15.9, 5.4 Hz, 1H), 3.25 (dd, J = 15.3, 8.1 Hz, 1H). ¹⁹F NMR (376 MHz, D₂O) δ −75.67 (s), −141.43 (dd, J = 11.8, 8.4 Hz, 1F). ¹³C NMR (101 MHz, D₂O) δ 174.36 (s), 150.00 (d, J = 236.7 Hz), 141.75 (d, J = 12.3 Hz), 129.76 (d, J = 11.8 Hz), 125.14 (d, J = 3.7 Hz), 122.09 (s), 107.47 (s), 102.02 (s), 98.91 (d, J = 23.3 Hz), 56.64 (s), 54.76 (s), 26.33 (s). C12H14FN2O3, [M+H]+: 253.0988, found: 253.0982.

(R)-2-amino-3-(6-fluoro-5-methoxy-1H-indol-3-yl) propanoic acid-2,2,2-trifluoroacetic acid (D-T18), white solid powder, ¹H NMR (400 MHz, D₂O) δ 7.22 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 11.9 Hz, 1H), 7.11 (s, 1H), 3.89 (dd, J = 8.0, 4.8 Hz, 1H), 3.81 (s, 3H), 3.28 (dd, J = 15.3, 4.7 Hz, 1H), 3.12 (dd, J = 15.3, 8.1 Hz, 1H). ¹⁹F NMR (376 MHz, D₂O) δ −75.60 (s), −141.39 (dd, J = 12.0, 8.3 Hz, 1F). ¹³C NMR (101 MHz, D₂O) δ 174.35 (s, COO), 150.02 (d, J = 236.7 Hz, C6-F), 141.77 (d, J = 13.0 Hz), 129.78 (d, J = 11.8 Hz), 125.16 (d, J = 3.6 Hz), 122.09 (s), 107.48 (s), 102.05 (d, J = 2.1 Hz), 98.94 (d, J = 23.2 Hz, C7), 56.67 (s), 54.77 (s), 26.33 (s). C12H14FN2O3, [M+H]+: 253.0988, found: 253.0982.

Radiochemistry

[¹⁸F]F-5-methoxy(5OMe)-tryptophan was synthesized using a simple photoredox radiofluorination method. In brief, ¹⁸F-tetrabutylammonium fluoride ([¹⁸F]TBAF, 20–50 μL in MeCN, 370 MBq to 3700 MBq), precursor 12 (7.0 mg, 0.015 mmol) or 17 (9.3 mg, 0.02 mmol), acridinium catalyst (Mes-Acr-Ph⁺ClO₄⁻, 1.5 mg) and TBAHCO₃ (20%, 25 μL in MeCN) were dissolved in the solution of dichloroethane (DCE)/t-butanol (tBuOH)/acetonitrile (MeCN) (300 μL/400 μL/100 μL) in a 5 ml V-vial. The resulting solution (~850 μL) was illuminated top-down using a 450 nm laser (450 nm, 3.5 W after fiber coupling) with an N₂ balloon sparge in an ice bath for 30 min (See Figure S6 for the radiolabeling set-up in a hot cell). The resulting reaction solution was diluted with 0.5 ml MeCN and passed through an aluminum cartridge (preconditioned with 5 ml DI water) to remove the unconverted ¹⁸F-fluoride. Then the reaction vial and cartridge were rinsed with another 0.3 mL MeCN. Part of the elution was then purified by high-performance liquid chromatography (HPLC) (See SI for detailed HPLC condition) to give the product [¹⁸F]12 or [¹⁸F]17. After removing the solvent under 95 °C with an argon stream, 250 μL HCl (36 wt.% in H₂O) was then added to the V-vial, and the mixture was heated at 120 °C for 10 min. After that, water (200 μL) and saturated NaHCO₃ solution (500 μL) were slowly added to the cooled V-vial. The resulting solution was purified on HPLC (See SI for detailed HPLC condition) to yield the final product, [¹⁸F]F-5OMe-tryptophan [¹⁸F]T13 (15.0 MBq to 37.0 MBq) or [¹⁸F]T18 (3.7 MBq to 10.0 MBq) in good to acceptable RCY with high radiochemical purity (RCP, 98.0%). Quality controls were conducted using HPLC equipped with a chiral column (Astec CHIROBIOTICR T Chiral Column, 5μm, 250 mm × 4.6 mm, SUPELCO).

Indoleamine 2,3-dioxygenase 1 activity in vitro assay

IDO enzyme assay was conducted in a 100 μL reaction system as described previously.³³ The reaction buffer contained 50 mM potassium phosphate (pH 6.5), 20 mM ascorbate (neutralized to pH 7.0), 50 μM methylene blue, 100 μg/mL catalase from bovine liver, 300 ng of recombinant human IDO1 (R&D Systems), and 400 μM L/D-T13, L/D-T18 or L-Trp. L-Trp was used as the positive control, and a tryptophan-free sample mixture was used as the negative control. After one hour of incubation at 37 °C, the absorbance of the reaction mixture was measured at 321 nm with a Thermo Scientific Evolution 201 UV-Visible Spectrophotometer.

Tryptophan hydroxylase 1 activity assay

TPH activity assay was conducted in a 100 μL reaction system using the reported protocol with modifications.³⁴ In brief, the reaction buffer contained 50 mM MOPS (pH 7.0), 100 mM ammonium sulfate, 100 μM ferrous ammonium sulfate, 500 μM TCEP, 300 μM 6-methyl tetrahydropterin, 50 μg/mL catalase from bovine liver, 1 mM DTT, 100 ng recombinant human TPH1 enzyme (Abcam), and 60 μM of L/D-T13, L/D-T18 or L-Trp. L-Trp was used as the positive control. The product mixture was analyzed using a Finnigan LTQ – Ion Trap LC-MS system.

Cell culture

Human and mouse melanoma cell lines (SK-MEL-2, B16F10) were obtained from American Type Culture Collection (Manassas, Virginia, USA). SK-MEL-2 cells were maintained in MEM medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU ml⁻¹ penicillin, and 100 μg ml⁻¹ streptomycin (GIBCO, Gaithersburg, MD, USA) at 37°C with 5% CO₂ in the air. B16F10 cells were cultured in RPMI medium containing 10% FBS, 2 mM L-glutamine, 100 IU ml⁻¹ penicillin, and 100 μg ml⁻¹ streptomycin.

Western Blotting analysis

Western blotting analysis was performed as described previously³⁵. Twenty micrograms of cell lysates were denatured and separated by SDS-PAGE. The separated proteins were then transferred to a nitrocellulose membrane (BioRad). The membranes were blocked with 2% BSA in PBS for 1h and incubated with IDO antibody overnight. After incubation with IgG horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA), the immunoblots were developed using the enhanced chemiluminescence (ECL) reagent (Cell signaling) and visualized using an Imaging processor (Biorad).

Tumor-Bearing Animal Models

Male C57BL/6 mice (purchased from Jackson Laboratory) were used in our study. 2×10⁶ of human B16F10 melanoma cells suspended in a 1:1 mixture of PBS and Matrigel were subcutaneously injected into the right shoulder area of the mice. When tumors reached 100 mm³ in size, mice were used for imaging studies. In addition, other tumor models were also tested, including MCF7 (breast cancer), H358 (Non-Small Cell Lung Cancer), LnCap (prostate carcinoma), and UPPL (bladder cancer) tumor-bearing mouse models. These tumor xenografts were established in nu/nu mice using the same methods described above. All cancer cell lines were purchased from the Tissue Culture Facility, UNC Lineberger Comprehensive Cancer Center. Animal procedures were approved by the University of North Carolina Institutional Animal Care and Use Committee.

Small-Animal PET Imaging

We injected approximately 3.7 MBq (~ 0.12 MBq/g) of L-[¹⁸F]T13, D-[¹⁸F]T13, L-[¹⁸F]T18, and D-[¹⁸F]T18 into the tail veins of the mouse. There are 3 animals in each group. We acquired 15 min static PET and CT images at 0.5, 1.5, and 3 h post-injection with animals under isoflurane anesthesia. The animals were awake during the intervals of the scans. List-mode data were chosen in the PET scan and reconstructed with random attenuation correction. CT scans were acquired with a resolution of 768*486 pixels, a current of 300uA, a voltage of 70kV, and an exposure time of 39 milliseconds. The images were reconstructed using 2D-OSEM. The regions of interest (ROIs) were drawn in Amide software (http://amide.sourceforge.net).³⁶ Organ uptakes were converted to mean ± standard deviation percentage injected dose per gram (%ID/g).

Biodistribution Studies

B16F10 mice were euthanized under isoflurane at 3.5h after the administration of [¹⁸F]-tracers. Major organs were harvested and weighed. The radioactivity of each organ was measured using a Gamma Counter (Wizard). The data was converted to %ID/g of each organ and corrected for radioactivity decay and background noise.

Statistical Analysis

Organ uptakes were expressed as mean ± standard deviation %ID/g with each group containing three animals. Enzyme assays were also conducted in triplicates, and the results were expressed as relative abundance (of the desired mass spectroscopy peak) in the TPH assay and absolute read number (UV absorbance) in the IDO assay. Student t-test was performed between the experiment groups (L-T13, L-T18, and L-Trp) and negative control. A P-value less than 0.05 was considered statistically significant.

ABBREVIATIONS

AMT

alpha methyl tryptophan

Boc

tert-butyloxycarbonyl

ee

enantiomeric excess

IDO1

Indoleamine 2, 3-dioxygenase 1

5-MTP

5-methoxy-D,L-tryptophan

PET

positron emission tomography

RCP

radiochemical purity

RCY

radiochemical yield

TBAF

tetrabutylammonium fluoride

TFA

trifluoroacetic acid

TPH

tryptophan hydroxylase

Trp

Tryptophan