Radioiodination of BODIPY and its application to a nuclear and optical dual functional labeling agent for proteins and peptides

In molecular imaging research, the development of multimodal imaging probes has recently attracted much attention. In the present study, we prepared radioiodinated BODIPY and applied it as a nuclear and optical dual functional labeling agent for proteins and peptides. We designed and synthesized [125I]BODIPY with a N-hydroxysuccinimide (NHS) ester, and evaluated its utility as a nuclear and fluorescent dual labeling agent for proteins and peptides. In the radioiodination reaction of BODIPY-NHS with [125I]NaI, [125I]BODIPY-NHS was obtained at a 48% radiochemical yield. When we carried out the conjugation reaction of [125I]BODIPY-NHS with bovine serum albumin (BSA) and RGD (Arg-Gly-Asp) peptide as a model protein and peptide, respectively, [125I]BODIPY-BSA and [125I]BODIPY-RGD peptide were successfully prepared at 98 and 82% radiochemical yields, respectively. Furthermore, we prepared [123I]BODIPY-trastuzumab by this conjugation reaction and successfully applied it to single photon emission computed tomography (SPECT) imaging studies using tumor-bearing mice, suggesting that radioiodinated BODIPY-NHS serves as a dual functional labeling agent for proteins and peptides. Since iodine has various radioisotopes that can be used for SPECT and positron emission tomography (PET) imaging, biological research, and radiotherapy, the radioiodinated BODIPY may be extensively applicable from basic to clinical research.

proteins in the blood does not match that of 18 F. Therefore, to determine the pharmacokinetics of proteins conjugated with [ 18 F]BODIPY, it is necessary to develop new agents that can label the proteins with other radionuclides with a longer half-life than 18 F.
In numerous studies regarding the chemistry of BODIPY 16 , we found that it is possible to introduce iodine into pyrrole rings of the BODIPY scaffold 17,18 . Since iodine has several radionuclides, including 123 I (t 1/2 = 13.2 h, γ), 124 I (t 1/2 = 4.18 d, β + ), 125 I (t 1/2 = 59.4 d, Auger e − ), and 131 I (t 1/2 = 8.02 d, β − ), it can be applied to broad research from basic research to diagnostic imaging and clinical radiotherapy in consideration of their half-life and characteristics of radiation 19 . Therefore, we considered that radioiodinated BODIPY can be used to develop a new method for the dual functional labeling of biologically active peptides and proteins.
In the present study, we synthesized radioiodinated BODIPY ([ 123/125 I]BODIPY) and applied it as a nuclear and optical dual functional labeling agent. We selected bovine serum albumin (BSA), RGD peptide, and trastuzumab as a model protein, peptide, and biologically active protein, respectively, to validate the basic concept for the development of new dual functional probes based on the radioiodinated BODIPY, and evaluated the feasibility of using [ 123/125 I]BODIPY as a dual functional labeling agent for peptides and proteins.

Results and Discussion
Synthesis and characterization of 125 I-labeled BODIPY ([ 125 I]2). We carried out a radioiodination reaction according to the synthetic route shown in Fig. 2. The BODIPY scaffold (1) was radioiodinated with [ 125 I] NaI in the presence of hydrogen peroxide as the oxidant. The radiochemical identity of 125 I-labeled BODIPY ([ 125 I]2) was verified by the HPLC profile of nonradioactive iodo-BODIPY (2), which was synthesized according to a method reported previously (Fig. 3) 17 . We efficiently prepared [ 125 I]2 at a radiochemical yield of 75.1% with a radiochemical purity of >99% after purification by HPLC. This efficiency of radiolabeling was similar to that obtained with the conventional radioiodination including electrophilic reactions with activated aromatic groups, and iodo-demetalation of aryls and alkyenes, using organotin or silicon precursors 19,20 . The new radioiodination reaction of BODIPY may be applicable for a variety of BODIPY derivatives.
Next, in order to determine the in vitro stability of [ 125 I]2, we incubated [ 125 I]2 in murine plasma for 24 h at 37 °C. When we analyzed it with HPLC after 24-h incubation of [ 125 I]2 in murine, the radioactivity peak after 24-h incubation had not changed markedly in comparison with that before incubation (Fig. 4). This result suggests that [ 125 I]2 shows high stability in murine plasma, indicating that the in vivo biodistribution of [ 125 I]2 should be further evaluated.
Then, we evaluated the biodistribution of radioactivity after the intravenous injection of [ 125 I]2 into normal mice (Fig. 5). The radioactivity of [ 125 I]2 after injection into mice displayed a typical biodistribution pattern for low-molecular-weight lipophilic compounds. In other words, as [ 125 I]2 was cleared from the blood, approximately  30%ID/g of [ 125 I]2 accumulated in the liver at 2 min postinjection. High radioactivity accumulation was also observed in the heart (12%ID/g) and lungs (25%ID/g) at 2 min postinjection. Thereafter, the radioactivity observed in the liver, heart, and lungs was gradually excreted into the intestine, and the radioactivity in the intestine reached 20%ID/g at 60 min postinjection. No marked radioactivity accumulation in the thyroid or stomach was observed, suggesting that [ 125 I]2 showed high stability against the deiodination reaction in vivo in addition to  ester as a conjugation site with proteins and peptides. The nonradioactive 4 was synthesized from known compound 3 using N-iodosuccinimide (NIS) at a yield of 54.1% (Fig. 6). Compound 4 had maximum excitation and emission wavelengths of 523 and 541 nm, respectively, and the extinction coefficient (M −1 cm −1 ) and quantum yield (%) of 4 were 90,600 and 11.0, respectively. Furthermore, [ 125 I]4 was synthesized by the radioiodination of compound 3 with [ 125 I]NaI in the presence of N-chlorosuccinimde (NCS) and 10% acetic acid in methanol (Fig. 7). After purification with HPLC by the co-injection of nonradioactive compound 3, [ 125 I]4 was successfully obtained at a radiochemical yield of 47.8% with a radiochemical purity of >99% (Fig. 8). We also attempted radioiodination with a different method using hydrogen peroxide as the oxidant, similarly to the method used in the   Scientific RepoRts | 7: 3337 | DOI:10.1038/s41598-017-03419-z radioiodination of [ 125 I]2. However, we could not obtain [ 125 I]4 at high yields in this reaction, probably because the hydrolysis of the NHS ester of compound 3 occurred due to a reaction with water. Therefore, we changed to the condition of the radioiodination reaction to avoid using water as a solvent. Although we used methanol as a solvent in this reaction, no marked methyl esterification was observed.
Next, we used [ 125 I]4 for a conjugation reaction with proteins and peptides. In the present study, we firstly selected BSA and RGD peptide as a model protein and peptide, respectively, for dual labeling with [ 125 I]4. After the conjugation reaction of [ 125 I]4 with BSA at room temperature for 80 min, we purified [ 125 I]4-BSA with size-exclusion chromatography. The radioactivity peak derived from [ 125 I]4-BSA corresponded to that of absorbance at 280 nm, indicating that the conjugation of [ 125 I]4 with BSA should be successfully achieved (Fig. 9). Furthermore, when we determined the fluorescence at an emission of 520 nm, the fluorescent peak was detected at a similar elution volume to that of absorbance and radioactivity ( Next, we carried out the conjugation reaction of [ 125 I]4 with RGD peptide. Many radiolabeled and fluorescent imaging probes based on RGD peptide have been studied for the in vivo imaging of tumors 21 . Therefore, we selected the RGD peptide as a model peptide in this study to show the basic principle for dual labeling with [ 125 I]4. We synthesized 4-RGD and used it as a nonradioactive standard in the HPLC analysis. The retention time of the radioactivity peak was identical to that of the absorbance of 4-RGD, suggesting that [ 125 I]4 was successfully conjugated to RGD peptide similarly to the conjugation reaction of [ 125 I]2 with BSA (Fig. 11). The results for the conjugation reaction of [ 125 I]4 with BSA and RGD peptide also support the feasibility of nuclear and optical dual functional labeling of various other biologically active proteins and peptides including antibodies, antibody fragments, and other carriers including liposomes, micelles, and polymer conjugates 22,23 .
Then, we applied this conjugation reaction to the labeling of trastuzumab with [ 125 I]4 as one of the examples of biologically active proteins and peptides. Trastuzumab is an antibody against human epidermal growth factor receptor 2 (HER2) that is overexpressed in several cancers, and commonly used as a carrier protein of radioisotopes and fluorescence dyes for the in vivo imaging of cancers 24,25 . According to the similar method used for the conjugation reaction of BSA, we reacted [ 125 I]4 with trastuzumab, and purified the reaction mixture with size-exclusion chromatography. As shown in Fig. 12, the elution volume where the radioactivity peak was detected corresponded to that of UV absorption where intact trastuzumab was eluted, indicating that trastuzumab was labeled with [ 125 I]4. We also analyzed [ 125 I]4-trastuzumab by SDS-PAGE. The radioactivity of [ 125 I]4-trastuzumab was detected in a region where the intact trastuzumab was detected while no marked radioactivity was found in a region where low-molecular-weight compounds were detected (Fig. 13). This suggests that the NHS ester of [ 125 I]4 may bind to the amino groups of trastuzumab via covalent bonds, similarly to [ 125 I]4-BSA.   Next, we prepared [ 123 I]4-trastuzumab and conducted SPECT imaging studies in HER2-positive N87 tumor-bearing mice. As a result, tumors in the mice were clearly detected by SPECT imaging after the injection of [ 123 I]4-trastuzumab (Figs. 14 and S1), suggesting that 123 4 may function as a radiolabeling agent without any loss of biological activity of an antibody. Since no marked radioactivity accumulation was observed in the thyroid (Fig. S1B), [ 123 I]4 shows high stability against in vivo deiodination reactions even after conjugation with an antibody, suggesting that radioiodine may stably bind to the BODIPY scaffold in vivo. In the present study, we did not perform in vivo fluorescence imaging due to the relatively low excitation and emission wavelengths of BODIPY 4 that are inadequate for in vivo fluorescent imaging. However, the successful SPECT imaging with

Conclusions
In this study, for the first time, we revealed that radioiodinated BODIPY can function as a new agent useful for the nuclear and optical dual functional labeling of various biologically active proteins and peptides. Since iodine has several radioisotopes with various features suitable for use in fields from basic research to clinical application, proteins and peptides labeled with radioiodinated BODIPY may be applied extensively for not only clinical diagnosis by SPECT/fluorescence dual imaging with [ 123 I]BODIPY conjugates but also radiotherapy with [ 131 I] BODIPY conjugates.

In vitro stability of [ 125 I]2 in plasma.
[ 125 I]2 (50 µL in a mixed solvent of 90% saline and 10% EtOH, 0.11-0.22 MBq) was diluted with freshly prepared murine plasma (250 µL). The solution was incubated at 37 °C for 24 h. After the addition of acetonitrile (600 µL) and vortexing, the mixture was centrifuged (4 °C, 10,000 g, 5 min). The supernatant was filtered and the filtrate was analyzed by reversed-phase HPLC using a COSMOSIL column (5C 18 AR-II) with an isocratic solvent of CH 3 CN/H 2 O (4/1) at a flow rate of 1.0 mL/min.
In vivo biodistribution in normal mice. Animal experiments were conducted in accordance with our institutional guidelines and approved by Kyoto University. A saline solution (100 μL) containing 0.1% tween 80 and 10% ethanol of [ 125 I]2 (37 kBq) was injected intravenously into the tails of ddY mice (5 weeks old, male, 5 animals per each group). The mice were sacrificed at 2, 10, 30, and 60 min postinjection. The organs of interest were removed and weighed, and the radioactivity was measured with an automatic γ counter (Wizard 1470, PerkinElmer). The results were calculated as the percentage injected dose per gram (%Dose/g) or percentage injected dose (%Dose). Values are expressed as the mean ± SD.