Un protocolo optimizado para el radiomarcaje eficaz de nanopartículas de oro mediante el uso de una<sup> 125</sup> Azida grupo prostético marcado con I
Summary
Se describe un procedimiento detallado para la síntesis de una azida marcado con I125 y el radiomarcado de dibenzocyclooctyne (DBCO) -grupo-conjugados, las nanopartículas de oro de 13 nm de tamaño utilizando una reacción clic libre de cobre.
Abstract
Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 ± 10%, n = 8) and excellent radiochemical purity (>99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.
Introduction
The strain-promoted copper-free click reaction between azides and cyclooctynes has been extensively applied to the efficient bioorthogonal labeling of a wide range of biomolecules, nanomaterials, and living subjects1-7. Due to the excellent site-specificity and rapid reaction rate of this conjugation reaction, it has also been used to synthesize radiolabeled tracers. A few 18F-labeled azide or DBCO prosthetic groups have been prepared for in vitro labeling of various cancers targeting peptides and antibodies, as well as for in vivo pre-targeted imaging of tumors8-13. In addition to these examples, the same conjugation reaction was applied to the metal-radioisotope-labeling of nanomaterials for positron emission tomography (PET) imaging studies14-16.
For several decades, radioactive iodines have been used for biomedical research and clinical trials through PET imaging (124I), single-photon emission computed tomography (SPECT) imaging (123I, 125I), and thyroid cancer treatment (131I)17-21. Therefore, an efficient method for radioactive iodine labeling is fundamentally important for various investigations, including molecular imaging studies, analysis of organ distribution of biomolecules, biomarker identification, and drug development. A copper-free click reaction strategy could be used in radioactive iodine labeling. However, this application has not been investigated as extensively as 18F-labeled biomolecules22-23. Here, we will provide a step-by-step protocol for the synthesis of an 125I-labeled azide for radiolabeling of DBCO-group-derived molecules. The procedures in the present report will include radioiodination of the stannylated precursor, purification steps with HPLC, and solid phase extraction. We also demonstrate efficient radiolabeling of DBCO-group-modified 13-nm-sized gold nanoparticles using the 125I-labeled azide. The detailed protocol in this report will help synthetic chemists understand a new radiolabeling methodology for the synthesis of radiolabeled products.
Protocol
Representative Results
Discussion
En general, el rendimiento radioquímico observado de la purificada 125 azida marcado con I (1) fue 75 ± 10% (n = 8). El radiomarcaje se realizó con 50-150 MBq de radiactividad, y los resultados radioquímica son bastante consistentes. Si [125 I] NaI (t 1/2 = 59,4 d) que se sometió a la desintegración radiactiva durante más de un mes se utilizó en la reacción de radioyodado, se observó el rendimiento radioquímico de 1 a ser disminuido l…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the National Research Foundation of Korea, funded by the government of the Republic of Korea, (Grant nos. 2012M2B2B1055245 and 2012M2A2A6011335) and by the RI-Biomics Center of Korea Atomic Energy Research Institute.
Materials
| Chloramine T trihydrate | Sigma | 402869 | |
| [125I]NaI in aq. NaOH | Perkin-Elmer | NEZ033A010MC | |
| Sodium metabisulfite | Sigma | S9000 | |
| Formic acid | Sigma | 251364 | |
| Sep-Pak tC18 plus cartridge | Waters | WAT036800 | |
| Dimethyl sulfoxide | Sigma | D2650 | |
| Acetone | Sigma | 650501 | |
| Ethanol | Sigma | 459844 | |
| Gold(III) chloride trihydrate | Sigma | 520918 | |
| Tween 20 | Sigma | P1379 | |
| DBCO PEG SH (MW 5000) | NANOCS | PG2-DBTH-5k | |
| TLC silica gel 60 F254 | Merck | ||
| Analytical HPLC | Agilent | 1290 Infinity | Model number |
| Preparative HPLC | Agilent | 1260 Infinity | Model number |
| Analytical C18 reverse-phase column | Agilent | Zorbax Eclipse XDB-C18 | |
| Preparative C18 reverse-phase column | Agilent | PrepHT XDB-C18 | |
| Radio TLC scanner | Bioscan | AR-2000 | Model number |
| Radioisotope dose calibrator | Capintec, Inc | CRC -25R dose calibrator | Model number |
References
- Jewett, J. C., Bertozzi, C. R. Cu-free Click Cycloaddition Reactions in Chemical Biology. Chem. Soc. Rev. 39, 1272-1279 (2010).
- Debets, M. F., et al. Bioconjugation with Strained Alkenes and Alkyne. Acc. Chem. Res. 44, 805-815 (2011).
- Sletten, E. M., Bertozzi, C. R. From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions. Acc. Chem. Res. 44, 666-676 (2011).
- Koo, H., et al. Bioorthogonal Cu-Free Click Chemistry in vivo for Tumor-Targeted Delivery of Nanoparticles. Angew. Chem. Int. Ed. 51, 11836-11840 (2012).
- Chang, P. V., et al. Copper-Free Click Chemistry in Living Animals. Proc. Natl. Acad. Sci. U. S. A. 107, 1821-1826 (2010).
- Bostic, H. E., Smith, M. D., Poloukhtine, A. A., Popik, V. V., Best, M. D. Membrane Labeling and Immobilization via copper-free Click Chemistry. Chem. Commun. 48, 1431-1433 (2012).
- Someya, T., Ando, A., Kimoto, M., Hirao, I. Site-Specific Labeling of RNA by Combining Genetic Alphabet Expansion Transcription and Copper-Free Click Chemistry. Nucl. Acids Res. 43, 6665-6676 (2015).
- Lee, S. B., et al. Mesoporous Silica Nanoparticle Pretargeting for PET Imaging Based on a Rapid Bioorthogonal Reaction in a Living Body. Angew. Chem. Int. Ed. 52, 10549-10552 (2013).
- Sachin, K., et al. F-18 Labeling Protocol of Peptides Based on Chemically Orthogonal Strain-Promoted Cycloaddition under Physiologically Friendly Reaction Conditions. Bioconjugate Chem. 23, 1680-1686 (2012).
- Evans, H. L., et al. Copper-Free Click – A Promising Tool for Pre-targeted PET Imaging. Chem. Commun. 48, 991-993 (2012).
- Campbell-Verduyn, L. S., et al. Strain-Promoted Copper-Free "Click" Chemistry for 18F Radiolabeling of Bombesin. Angew. Chem. Int. Ed. 50, 11117-11120 (2011).
- Arumugam, S., Chin, J., Schirrmacher, R., Popik, V. V., Kostikov, A. P. 18F]Azadibenzocyclooctyne ([18F]ADIBO): A Biocompatible Radioactive Labeling Synthon for Peptides using Catalyst Free [3+2] Cycloaddition. Bioorg. Med. Chem. Lett. 21, 6987-6991 (2011).
- Bouvet, V., Wuest, M., Wuest, F. Copper-Free Click Chemistry with the Short-Lived Positron Emitter Fluorine-18. Org. Biomol. Chem. 9, 7393-7399 (2011).
- Satpati, D., Bauer, N., Hausner, S. H., Sutcliffe, J. L. Synthesis of [64Cu]DOTA-ADIBON3-Ala-PEG28-A20FMDV2 via Copper-Free Click Chemistry for PET Imaging of Integrin αvβ6. J. Radioanal. Nucl. Chem. 302, 765-771 (2014).
- Lee, D. E., et al. Facile Method To Radiolabel Glycol Chitosan Nanoparticles with 64Cu via Copper-Free Click Chemistry for MicroPET Imaging. Mol. Pharmaceutics. 10, 2190-2198 (2013).
- Zeng, D. 64Cu Core-Labeled Nanoparticles with High Specific Activity via Metal-Free Click Chemistry. ACS Nano. 6, 5209-5219 (2012).
- Jeon, J., et al. Radiosynthesis and in vivo Evaluation of [125I]2-4(iodophenethyl)-2-Methylmalonic Acid as a Potential Radiotracer for Detection of Apoptosis. J. Radioanal. Nucl. Chem. 308, 23-29 (2016).
- Adam, M. J., Wilbur, D. S. Radiohalogens for Imaging and Therapy. Chem. Soc. Rev. 34, 153-163 (2005).
- Jeon, J., et al. Radiosynthesis of 123I-Labeld Hesperetin for Biodistribution Study of Orally Administered Hesperetin. J. Radioanal. Nucl. Chem. 306, 437-443 (2015).
- Kil, K. E., et al. Development of [123I]IPEB and [123I]IMPEB as SPECT Radioligands for Metabotropic Glutamate Receptor Subtype. ACS Med. Chem. Lett. 5, 652-656 (2014).
- Chen, M. K., et al. The Utility of I-123 Pretherapy Scan in I-131 Radioiodine Therapy for Thyroid Cancer. Thyroid. 22, 304-309 (2012).
- Jeon, J., et al. Efficient Method for Iodine Radioisotope Labeling of Cyclooctyne-Containing Molecules using Strain-Promoted Copper-Free Click Reaction. Bioorg. Med. Chem. 23, 3303-3308 (2015).
- Choi, M. H., et al. Synthesis and Evaluation of an 125I-Labeled Azide Prosthetic Group for Efficient and Bioorthogonal Radiolabeling of Cyclooctyne-Group Containing Molecules using Copper-Free Click Reaction. Bioorg. Med. Chem. Lett. 26, 875-878 (2016).
- Kim, Y. H., et al. Tumor Targeting and Imaging Using Cyclic RGD-PEGylated Gold Nanoparticle Probes with Directly Conjugated Iodine-125. Small. 7, 2052-2060 (2011).
