A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.
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.
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.
Caution: The oxidized form of radioactive iodine is quite volatile and must be handled with adequate lead shields and lead vials. All radiochemical steps should be carried out in a well-ventilated charcoal-filtered hood, and the experimental procedures need to be monitored by radioactivity detection devices.
1. Preparation of Chemicals and the Reverse Phase Cartridge for the Synthesis of the 125I-labeled Azide
2. Radiosynthesis of the 125I-labeled Azide Prosthetic Group
3. Synthesis of DBCO-group-conjugated Gold Nanoparticles
4. Radiolabeling of DBCO-group-modified Gold Nanoparticles via the Copper-free Click Reaction
The radioiodination reaction of the stannylated precursor (2) was carried out using 150 MBq of [125I]NaI, acetic acid, and chloramine T at room temperature for 15 min to provide the radiolabeled product (1). After preparative HPLC purification of the crude mixture, the desired product was obtained with 75 ± 10% (n = 8) of radiochemical yield. Analytical HPLC revealed that the radiochemical purity of the 125I-labeled product was more than 99% (Figure 2), and the observed specific radioactivity of product 1 is 40.7 MBq/µmol. Solid phase extraction of the fraction containing the purified product by using the cartridge provided an acetone solution of 1. Using a stream of nitrogen or argon gas, the organic solvent can be evaporated, and then the residue can be dissolved again in DMSO or absolute ethanol for the next step.
For 125I-labeling of polyethylene-glycol-modified gold nanoparticles, the DBCO-group-modified gold nanoparticles were prepared by the procedure shown in Figure 1. An excess amount of polyethylene glycol thiol (MW 5,000) with DBCO groups was reacted with the citrated, stabilized 13-nm gold nanoparticles. After the modification step, the product was purified by successive centrifugation to give the DBCO-functionalized gold nanoparticles (3). In the radiolabeling step, 3.7 MBq of 1 was added to 2 µM of 3 (~400 µM of the DBCO groups), and the labeling reaction was carried out at 40 °C for 1 hr. Radio-TLC analysis showed that more than 95% of 1 was reacted with the DBCO-group-functionalized gold nanoparticles (3) within 60 min. The reaction was carried out for 60 min, and then the crude product was purified by centrifugation. 125I-labeled gold nanoparticles (4) were obtained with >99% (n = 4) radiochemical yield as determined by radio TLC (Figure 3).
Figure 1. Radiosynthesis of the 125I-labeled azide (1) and 125I-labeled gold nanoparticles (4). Reagents and conditions: (a) [125I]NaI, acetic acid, chloramine T, RT, 15 min, 75 ± 10% (n = 8) radiochemical yield; (b) DBCO-PEG-SH (MW 5,000), H2O, RT, 2 hr; ~40 °C, 60 min, >99% radiochemical yield. Please click here to view a larger version of this figure.
Figure 2. Analytical HPLC chromatogram of the 125I-labeled azide (1). (a) Radiochromatogram of the crude product. (b) Radiochromatogram of the purified product. (c) UV chromatogram (254 nm) of the purified product. Please click here to view a larger version of this figure.
Figure 3. Radio-TLC results of the 125I-labeled gold nanoparticles (4) (Rf of 4 = 0.05, Rf of 1 = 0.45, eluent: ethyl acetate) (a) after a 60 min reaction and (b) after purification. Please click here to view a larger version of this figure.
In general, the observed radiochemical yield of the purified 125I-labeled azide (1) was 75 ± 10% (n = 8). The radiolabeling was accomplished with 50-150 MBq of radioactivity, and the radiochemical results are quite consistent. If [125I]NaI (t1/2 = 59.4 d) that underwent radioactive decay for more than a month was used in the radioiodination reaction, the radiochemical yield of 1 was observed to be slightly decreased (53-65%). Therefore, it is recommended that [125I]NaI be utilized as soon as it is produced or is delivered to the lab in order to obtain optimized radiochemical yield. In addition, a freshly prepared chloramine T solution should also be used in the reaction to obtain the desired radiochemical yield.
Because the precursor (2) was quite hydrophobic, 150 µl absolute ethanol should be added to dissolve 1 mg of 2 in the radioiodination reaction. Otherwise, the whole reaction mixture could become turbid after adding an aqueous solution of the oxidant and [125I]NaI. Decreased solubility of the precursor often results in low radiochemical yield of 1. DMSO can also be used for dissolving 2 in the radiolabeling step. In addition, acetic acid should be added to the precursor solution to obtain high radiochemical yield in the radioiodination step.
Before using preparative HPLC for purification of the crude product containing 1, the reverse phase HPLC column needs to be washed with solvents A and B (flow rate: 10 ml/min; eluent gradient: 100% solvent B for 0-10 min, 100-0% solvent B for 10-25 min, and 0% solvent B for 25-30 min) to remove trace amount of impurities from the system. Next, the reverse phase HPLC column is equilibrated with 20% solvent B in 80% solvent A for at least 20 min to obtain the consistent retention time of 1.
The fraction containing purified 1 should be diluted with more than 4 times the volume of H2O in the solid phase extraction procedure. Otherwise, some of the purified product cannot be trapped in the tC18 cartridge. When acetone is used to elute purified 1 from the cartridge, the final volume can be reduced by evaporation of acetone with a stream of nitrogen or argon gas at ambient temperature.
Among several radioactive iodines, 125I was selected and used in the current research. Different kinds of iodine radioisotopes need to be tested using the present method in other biological and medical studies (e.g.,124I for PET imaging, 131I for therapeutic purposes).
As far as we understand, the present radiolabeling protocol is the first report describing detailed synthetic steps for a radioiodine-labeled azide group. Recently, we published another azide prosthetic group, which has a different structure23. However, the radiolabeled azide (1) in the current method provided slightly better radiochemical results than the other in terms of radiolabeling efficiency with DBCO-group-containing molecules. Existing prosthetic groups (i.e., N-hydroxysuccinimide and maleimide) for the labeling of radioactive iodine could not provide site-specificity. However, the present method demonstrates straightforward radiolabeling efficiency along with excellent bioorthogonality. Since the azide functional group is known to be highly stable in physiological conditions and in vivo environments, the radiolabeled product (1) can be utilized in pre-targeted in vivo imaging studies. We anticipate that this method will be efficiently applied to both in vitro and in vivo iodine radioisotope labeling of biomolecules and nanomaterials that contain a strained cyclooctyne structure.
Based on the specific radioactivity of 1, the calculated molar ratio of 125I and gold nanoparticles is ~1:1. 125I-labeled gold nanoparticles (4) can be used in molecular imaging and biodistribution studies of nanomaterials. The current method can also be applied to radioactive iodine labeling of different sizes and shapes of gold nanomaterials.
The authors have nothing to disclose.
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.
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 |