Investigation of the role of the base in the synthesis of [18F]FLT
Introduction
Recently, 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) has been developed as a radiopharmaceutical to image cellular proliferation in vivo with positron emission tomography (PET) (Vesselle et al., 2002; Been et al., 2004; Barthel et al., 2005). Intra-cellularly, [18F]FLT is phosphorylated by the enzyme thymidine kinase-1, which is expressed during the DNA synthesis phase of the cell cycle (Sherley and Kelly, 1988; Munch-Petersen et al., 1991). Subsequently, 18F-labeled thymidine monophosphate accumulates and is trapped within the cell. Unlike 11C-thymidine, [18F]FLT is not incorporated into DNA (Been et al., 2004). However, its accumulation into proliferating cells shows a high correlation with DNA replication. It has been shown that the uptake of [18F]FLT reflects the cell proliferation rate in tumors better than that of the widely utilized tumor imaging agent [18F]FDG (Chen et al., 2005; Reske and Deisenhofer, 2006). Furthermore, [18F]FLT images were demonstrated to differentiate tumor from inflammation (Buck et al., 2002; Van Waarde et al., 2004; Chen et al., 2005; Reske and Deisenhofer, 2006). The compound has also been evaluated in detecting tumor response to chemo- and radiotherapies (Sugiyama et al., 2004; Leyton et al., 2005; Dorow et al., 2006; Pio et al., 2006).
Synthesis of [18F]FLT via nucleophilic displacement of the nosyl functionality at the 3′ position with [18F]fluoride has been reported by Grierson et al. (1997), Grierson and Shields (2000), Martin et al. (2002), Yun et al. (2003) and Moon et al. (2006). Among them, the in-depth investigation by Yun et al. gives important information regarding optimization of the [18F]FLT preparation procedure. They report that their decay-corrected radiochemical yields reached 40–42% using 30–40 mg of the two precursors, 1-[5-O-(4,4′-dimethoxytrityl)-3-O-nosyl-2-deoxy-β-d-lyxofuranosyl]thymine and its 3-N-Boc analog. We initiated our [18F]FLT radiosyntheses based on their published method. However, we could not reproduce some of their results: (a) after heating the reaction mixture containing 10 mg of the N-Boc protected precursor, [18F]fluoride and Kryptofix 222/K2CO3 in acetonitrile at 100 °C for 5 min, 60–70% of 18F was found incorporated into the molecule whereas they report only 7% incorporation and (b) under the same reaction conditions, we discovered that 80–90% of the precursor was lost in 5 min. Furthermore, if the reaction was heated at 120 °C, the loss was greater than 99% while they report that an increase in temperature up to 120 °C resulted in higher 18F-incorporation. These problems prompted us to suspect that there might be some other critical factors determining the rate of the substitution reaction and ultimately the yield of [18F]FLT synthesis. We therefore undertook to further investigate the nucleophilic substitution reaction that produces [18F]FLT. Here we report our findings.
Section snippets
Chemicals
The precursors, 3-N-Boc-1-[5-O-(4,4′-dimethoxytrityl)-3-O-nosyl-2-deoxy-β-d-lyxofuranosyl]thymine (Precursor A) and 1-[5-O-(4,4′-dimethoxytrityl)-3-O-nosyl-2-deoxy-β-d-lyxofuranosyl]thymine (Precursor B) were purchased from ABX Advanced Biomedical Compounds (Radeberg, Germany). Kryptofix 222 (K222) and potassium carbonate were purchased from Sigma-Aldrich (Milwaukee, WI, USA) and 2 N NaOH and 1 N HCl from Fisher Scientific. Anhydrous acetonitrile was obtained from Sigma-Aldrich and Pierce
Synthesis of [18F]FLT using Precursor A
The nucleophilic substitution reaction with [18F]fluoride in acetonitrile (Fig. 1, top) proceeded efficiently at 100 °C with 6–13 mg of the precursor (average: 8.8±2.2 mg, 11 μmol, n=11) in the presence of 5–6 mg of K222 (5.3±0.5 mg, 14 μmol) and 1.4 mg of K2CO3 (1.35±0.11 mg, 10 μmol). 18F-incorporation into the intermediate (Compound B, Fig. 1) was approximately 60–70% (average 64.4±10.5%) in 5 min. Two radioactive peaks were observed, one corresponding to the [18F]FLT intermediate (Compound B) and the
Discussion
This investigation reveals that the 18F-incorporation into the [18F]FLT intermediate (Compound B) is dramatically decreased with increase in the amount of the K222/K2CO3 complex (Fig. 3 and Table 1) suggesting that the basicity of the reaction plays a critical role in determining the rate of the nucleophilic substitution reaction with [18F]fluoride in [18F]FLT production. It is well known that base-induced elimination competes with nucleophilic substitution when the leaving group, such as a
Conclusion
The basicity of the reaction medium appears to play a critical role in determining the rate of nucleophilic substitution of the nosyl leaving group of Precursor A with [18F]fluoride. In the presence of an excess amount of the base vs that of the precursor, the elimination reaction appears to dominate, and the precursor is consumed rapidly before the nucleophilic substitution is complete. Under optimal conditions, where the precursor-to-base ratio is approximately 1.2–1.5, [18F]FLT can be
Acknowledgments
We thank Mr. Howard Sheh and Mr. Calvin Lom of Sloan-Kettering Institute for their careful management of the cyclotron facility and in particular for 18F production essential for this work. We also thank Dr. Alan A. Wilson, Chief Radiochemist of Vivian M Rokoff PET Imaging Center, University of Toronto, for kindly taking the time to discuss this work.
References (26)
- et al.
Base-mediated decomposition of a mannose triflate during the synthesis of 2-deoxy-2-18F-fluoro-d-glucose
Appl. Radiat. Isot.
(1995) - et al.
Automated synthesis of [18F]FDG using tetrabutylammonium bocarbonate
Appl. Radiat. Isot.
(1995) - et al.
Synthesis of deoxyfluoro sugars from carbohydrate precursors
Carbohydr. Res.
(2000) - et al.
Radiosynthesis of 3′-deoxy-3′-[18F]fluorothymidine: [18F]FLT for imaging of cellular proliferation in vivo
Nucl. Med. Biol.
(2000) - et al.
Selective action of 2′,3′-didehydro-2′,3′-dideoxythymidine triphosphate on human immunodeficiency virus reverse transcriptase and human DNA polymerases
J. Biol. Chem.
(1992) - et al.
A new precursor for the radiosynthesis of [18F]FLT
Nucl. Med. Biol.
(2002) - et al.
Diverging substrate specificity of pure human thymidine kinase 1 and 2 against antiviral dideoxynucleosides
J. Biol. Chem.
(1991) - et al.
Regulation of human thymidine kinase during the cell cycle
J. Biol. Chem.
(1988) - et al.
High radiochemical yield synthesis of 3′deoxy-3′-[18F]fluorothymidine using (5′-O-dimethoxytrityl-2′-deoxy-3′-O-nosyl-β-d-threopentofuranosyl)thymine and its 3-N-Boc-protected analogue as a labeling precursor
Nucl. Med. Biol.
(2003) - et al.
The uptake of 3′-deoxy-3′-[18F]fluorothymidine into L5178Y tumors in vivo is dependent on thymidine kinase 1 protein levels
Eur. J. Nucl. Med. Mol. Imaging
(2005)
[18F]FLT-PET in oncology: current status and opportunities
Eur. J. Nucl. Med. Mol. Imaging
3-Deoxy-3-[18F]fluorothymidine-positron emission tomography for noninvasive assessment of proliferation in pulmonary nodules
Cancer Res.
Imaging proliferation in brain tumors with 18F-FLT PET: comparison with 18F-FDG
J. Nucl. Med.
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