F-18 Labeling Protocol of Peptides Based on Chemically Orthogonal

Article pubs.acs.org/bc

F-18 Labeling Protocol of Peptides Based on Chemically Orthogonal Strain-Promoted Cycloaddition under Physiologically Friendly Reaction Conditions Kalme Sachin,† Vinod H. Jadhav,† Eun-Mi Kim,† Hye Lan Kim, Sang Bong Lee, Hwan-Jeong Jeong, Seok Tae Lim, Myung-Hee Sohn, and Dong Wook Kim* Department of Nuclear Medicine, Cyclotron Research Center, Research Institute of Clinical Medicine, Chonbuk National University Medical School, Jeonju, Jeonbuk 561-712, Korea S Supporting Information *

ABSTRACT: We introduce the high-throughput synthesis of various 18F-labeled peptide tracers by a straightforward 18F-labeling protocol based on a chemo-orthogonal strain-promoted alkyne azide cycloaddition (SPAAC) using aza-dibenzocyclootyne-substituted peptides as precursors with 18F-azide synthon to develop peptide based positron emission tomography (PET) molecular imaging probes. The SPAAC reaction and subsequent chemo-orthogonal purification reaction with azide resin proceeded quickly and selectively under physiologically friendly reaction conditions (i.e., toxic chemical reagents-free, aqueous medium, room temperature, and pH ≈7), and provided four 18F-labeled tumor targetable bioactive peptides such as cyclic Arg-Gly-Asp (cRGD) peptide, bombesin (BBN), c-Met binding peptide (cMBP), and apoptosis targeting peptide (ApoPep) in high radiochemical yields as direct injectable solutions without any HPLC purification and/or formulation processes. In vitro binding assay and in vivo PET molecular imaging study using the 18F-labeled cRGD peptide also demonstrated a successful application of our 18F-labeling protocol.

1. INTRODUCTION Positron emission tomography (PET) molecular imaging in living subjects provides exciting opportunities to understand fundamental biological processes, disease pathologies and pharmaceutical development.1,2 Specific molecular imaging probes labeled with positron-emitting radioisotopes must be prepared to measure these processes with PET.3,4 Among various radioisotopes, fluorine-18 is considered as an ideal positron-emitter due to its excellent chemical, physiological, and nuclear characteristics.3−7 In this regard, the efficient application of rapid, simple, and reliable 18F-labeling strategies is an extremely important topic to synthesize and obtain new radiopharmaceuticals for PET molecular imaging studies.8,9 The implementation of phase display and combinatorial chemistry technologies has allowed the development of novel and diverse peptide-based probes, which can specifically address molecular targets in vivo and be rapidly evolving radiolabeled bioactive peptides.10 In particular, 18F-labeled peptides have received much attention in the field of PET molecular imaging and diagnosis.11 In general, the direct 18F-labeling method by the radiofluorination with no-carrier-added (nca) for introducing a fluorine-18 into the bioactive peptide at specific sites requires harsh strong basic reaction conditions in organic media, which may not be suitable for these bioactive peptide molecules containing various sensitive functional groups.12,13 As a result, diverse 18F-labeled building blocks referred to as prosthetic groups as well as the conjugation methods between these prosthetic groups and peptides such as acylation, alkylation, amidation, and hydrazone formation with amino© 2012 American Chemical Society

or thiol- reactive groups, have been developed for the preparation of 18F-labeled peptide based probes.12,13 However, these established labeling protocols can frequently show synthetic limitations due to the short half-life of fluorine-18 (t1/2 = 109.8 min), the denaturation of peptides by reactive reagents or organic media, the complicated synthesis procedure, or the time-consuming purification process, thereby providing relatively low radiochemical yields or/and low purities including low radiospecific activities.12−14 Additionally, when performing the labeling reactions with 18F-labeled synthon or [18F]fluorine atom, which are usually produced in trace quantities with high radiospecific activity, the use of excess amounts of precursor should be required for a reasonable reaction time and radiochemical yield (RCY), and the resulting mixtures should be purified by high performance liquid chromatography (HPLC) to separate trace quantities of radioprobes from both nonlabeled compounds and chemical reagents for increasing PET imaging quality and reducing the toxicity or the side-effects of these radioprobes.3,4 Therefore, an ideal 18F-labeled method should provide (1) a short reaction time and high RCY with a high purity including a high radiospecific activity; (2) proceed under mild reaction conditions (i.e., room temperature, pH 7, and aqueous medium) to prevent the denaturation of the biomolecules; and (3) make the procedure simple for easy handling and automatic production. Received: May 4, 2012 Revised: June 20, 2012 Published: July 9, 2012 1680

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2.2. Representative Procedure for the Preparation of [18F]2. [18F]fluoride was produced in a cyclotron by the 18O(p, n)18F reaction. A volume of 100−200 μL of [18F]fluoride (1665 MBq) in water was added to a vacutainer containing n-Bu4NHCO3 (40% aq, 3.7 μL, 7.68 μmol). The azeotropic distillations were conducted with 200 μL aliquots of CH3CN at 75 °C under a stream of nitrogen. A [18F]fluoride displacement reaction of 14 (2.5 mg, 7.32 μmol) with n-Bu4N[18F]F in tertamyl alcohol (500 μL) was carried out in a reaction vial at 100 °C for 20 min. After cooling to room temperature, the solvent was removed with a gentle stream of nitrogen. The crude compound was injected onto reverse-phase HPLC with acetonitrile (1 mL) and purified. The desired compound [18F]2 was collected from HPLC (tR = 11.1 min; C18 silica gel, 10 μm, 4.6 × 250 mm; 0.1% TFA in H2O/acetonitrile = 30:70 (v/v); 214 nm; 2 mL/min). Then 790 MBq of [18F]2 was obtained after evaporation under a stream of nitrogen in 63% decay-corrected radiochemical yield with high radiochemical purity (>99%). The total synthesis time was 45 min. For the identification of the radioactive-product, the collected HPLC fraction was coinjected with the nonradioactive 2. 2.3. ADIBO Scavenger Azide-Resin 4. Pentaethylene glycolic azide 13 (3.3 g, 12.5 mmol) was added to a suspension of NaH (60% in mineral oil, 500 mg, 12.5 mmol) in dried THF at 0 °C, and stirred for 30 min at 0 °C. Merrifield resin (1.0 g, 2.5 mmol, 1% DVB, 2.5 mmol Cl/g) was then added to the reaction mixture, which was followed by the addition of tetrabutylammonium iodide (920 mg, 2.5 mmol). The reaction mixture was then stirred over 3 days at 25 °C. After filtration, the resin was washed repeatedly with THF, water, acetone, water, methanol, and finally with dichloromethane. After drying under high vacuum, 1.1 g of resin 4 was obtained and identified by solid state NMR and elemental analysis: 13C NMR (solid state) δ 41 (aliphatic polystyrene skeleton), 51 (terminal azido methylene carbon), 71−74 (ether carbons), 120−150 (aromatic polystyrene skeleton). Anal. N, 4.7 (1.05 mmol of azide portion per gram). 2.4. Typical Procedure for the Preparation of cRGDADIBOT-18F. To a solution of cRGD-ADIBO (1.0 mg, 0.77 μmol) in 0.1 mL of EtOH/water (1/1) was added a solution of [18F]2 (500 MBq) in 0.1 mL of EtOH/water (1/1). The reaction mixture was stirred at room temperature for 15 min. To remove nonreacted cRGD-ADIBO precursor, 50 mg of ADIBOprecursor−scavenger-resin 4 (1.05 mmol of azide portion/g) was added to the reaction mixture, and this heterogeneous solution was then stirred for approximately 20 min at room temperature. After the filtration and washing with PBS solution (2 × 0.5 mL), 368 MBq of cRGD-ADIBOT-18F was obtained in 92% decay-corrected radiochemical yield (35 min total reaction time) as a direct injectable solution for animal PET image study. Both isomers of the triazole were collected and treated as one compound. 2.5. In Vitro Binding Assays. In vitro integrin αvβ3 binding affinity and specificity of cRGD-ADIBOT-F was assessed via a displacement cell-binding assay using 125I-echistatin (μCi/well) as a radioligand. Experiments were performed on U87MG cells by a previously published method. For the integrin αvβ3 binding affinity assay, U87MG cells were seeded onto 96-well plates at 3 × 104 cells per well and incubated overnight at 37 °C. Serial dilutions of cRGD-ADIBOT-F and cRGDyk (corresponding to concentrations of 0.45 nM to 1.4 μM) were added to 96-well plates. The plates were then incubated for 0.5 h at 37 °C, washed, and dried, and 0.1 mL of 2 N NaOH solution was added to each

Recently, the copper(I)-catalyzed azide−alkyne [3 + 2] cycloaddition (CuAAC), which is the so-called “click reaction”, has become an emerging method to ligate biomolecules with radioprosthetic groups for the synthesis of radiopharmaceuticals.15−19 However, the possibility that the Cu(I) catalyst can bind to biomolecules, may lead to various problems, such as blocking or reducing the biological activity, as well as the rate of the CuAAC reaction, and the cytotoxicity by residual copper in products, in the synthetic process of radiolabeled biomolecules using the CuAAC conjugation method.20−22 In a recent significant advance, an interesting copper-free “click chemistry” based on strain-promoted alkyne−azide cycloaddition (SPAAC) has been developed as a fast and bioorthogonal conjugation protocol for the biological application in live-cell imaging, radioisotope labeling, and surface modification.23−31 In particular, dibenzocyclooctyne (DIBO) or aza-dibenzocyclooctyne (ADIBO) derivatives have shown good performance in this SPAAC reaction for these purposes (Figure 1).32−35 In this article, we introduce a

Figure 1. Strain-promoted compounds and their corresponding products by copper-free click reactions. DIBOs = dibenzocyclooctynes; ADIBO = aza-dibenzocyclooctynes; ADIBOT = aza-dibenzocycloocta-triazoles.

facile 18F-labeling protocol based on a copper-free SPAAC reaction using the ADIBO peptide system with [18F]fluoroazide synthon to prepare 18F-labeled peptide tracers, and this protocol exactly meets the above requirement.

2. EXPERIMENTAL SECTION 2.1. General. Unless otherwise noted, all reagents and solvents were commercially available. cRGDyK, C-Bombesin, and cMBP were purchased from PEPTRON Co. (South Korea), and ApoPep was purchased from FutureChem Co. (South Korea). Reaction progress was followed by TLC on 0.25 mm silica gel glass plates containing F-254 indicator. Visualization on TLC was monitored by UV light or a radio-TLC scanner. Flash chromatography was performed using 230−400 mesh silica gel (Merck KGaA). 1H spectra were recorded on a 600 MHz spectrometer. Chemical shifts were reported in δ units (ppm) relative to tetramethylsilane, and coupling constants were reported in hertz. 13C NMR spectra were acquired at 100 or 150 MHz. Low- and high-resolution electron impact (EI) or fast atom bombardment (FAB) mass spectra were obtained. [18F]Fluoride ion was produced from a cyclotron (KIRAMS 13 MeV, South Korea) using the 18O(p,n)18F nuclear reaction with 13 MeV proton irradiation of an enriched [18O]H2O target. High performance liquid chromatography (HPLC) was performed with a spectra system (Thermo Scientific, Waltham, MA) using a semipreparative column (C18 silica gel, 10 μm, 10 × 250 mm) and an analytic column (C18 silica gel, 5 μm, 4.6 × 250 mm). The eluant was simultaneously monitored by a UV detector (215 and 254 nm) and a NaI(Tl) radioactivity detector. Radioactivity was measured in a dose calibrator. 1681

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obtained from Orient-Bio (Seoul, Korea). Mice were anesthetized by subcutaneous injection with a mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg). After sedation, glioblastoma (U87MG) tumors were established by the subcutaneous injection in the right flank of 5 × 106 cells mixed with matrigel. The mice underwent PET/CT studies when the tumor volume reached 150−250 mm3 (eq = length × W2 × 0.5 4−5 wk after inoculation). PET and CT scans and image analyses were performed using a microPET/CT scanner (GE Healthcare). About 1.8−2.6 MBq of cRGD-ADIBOT-18F was intravenously injected into each mouse under isoflurane anesthesia (1.5%). PET images were acquired at 10, 30, and 60 min, 2 and 3 h after postinjection. For each microPET scan, regions of interest (ROIs) were drawn over the tumor and muscle on decay-corrected whole-body images that were gained by 3D-OSEM iterative image reconstruction (Amira 3.1, GE Healthcare). For receptor-blocking experiments, nude mice bearing U87MG tumors were scanned after coinjection with cRGD-ADIBOT-18F (1.8−2.6 MBq) and cRGDyk (10 mg/kg). The radioactivity concentration within the tumor or muscle was obtained from the mean value, and then these values were compared. 2.7. Biodistribution Study. Female BALB/c mice (n = 4, aged 4 weeks with 15 g) were obtained from Orient Bio, Inc. (Seoul, Korea). All animal experiments were performed according to the Institutional Animal Care and Use Committee for animal treatment of Chonbuk National University. U87MG tumor xenografted mice were subjected to the biodistribution study when the tumor reached in 0.7−1.0 cm. All mice received intravenous (i.v.) injections through the tail vein with approximately 1.11 MBq of cRGD-ADIBOT-18F. At 120 min after the radiotracer injection, the animals were anesthetized and

Figure 2. Copper-free strain-promoted alkyne azide cycloaddition (SPAAC) reaction of the model ADIBO compound 1 with [18F]fluorohexaethylene glycolic azide ([18F]2) based on levels of 1.

well to facilitate cell lysis. The lysates were collected and counted in a gamma counter (Perkin-Elmer, USA). Binding affinities (IC50) for both receptors were calculated by nonlinear regression analysis (sigmoidal dose response equation) using the GraphPad Prism 4.0 computer-fitting program (Graph-Pad Software, San Diego, CA, USA). 2.6. In Vivo MicroPET Imaging. All animal experiments were performed in compliance with the policies and procedures of Institutional Animal Care and Use Committee for animal treatment. Female athymic nude mice (4-wk, nu/nu) were

Figure 3. (A) Schematic procedure for the preparation of cRGD-ADIBOT-18F with [18F]2 and subsequent chemo-orthogonal purification using a polystyrene-supported azide resin 4 as a ADIBO-precursor−scavenger. (B) HPLC chromatograms of the cRGD-ADIBOT-18F reaction mixture before and after treatment with the ADIBO-precursor−scavenger-resin 4. (C) Schematic procedure for the preparation of [18F]fluoropentaethylene glycolic azide ([18F]2) and ADIBO scavenger azide-resin 4. 1682

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conjugation reaction using different quantities (0.1−1.0 μmol) of a simple model ADIBO precursor compound 1 with a 481 MBq (specific radioactivity: 42 GBq/μmol) of [18F]fluoropentaethylene glycolic azide ([18F]2, see Figure 3C) as a 18 F-labeled synthon at room temperature in aqueous solution (ethanol/water = 3/2) as shown in Figure 2. On a 1.0−0.5 μmol reaction scale of the ADIBO compound 1, the SPAAC reactions were completed within a reasonable reaction time (15−30 min), affording the 18F-labeled aza-dibenzocyclooctatriazole (ADIBOT) product ([18F]3) in nearly quantitative RCYs. However, the use of 98% of radiochemical purity as a direct injectable solution for an animal PET image study without any HPLC purification and formulation process by this F-18 labeling protocol. 3.3. In Vitro and in Vivo Evaluation of cRGD-ADIBOT-18F. To verify that the incorporation of the 18F-labeled ADIBOT prosthetic group into the peptide did not affect its biological behavior in vitro, the affinities of the nonradioactive standard cRGD-ADIBOT-F compound and the nonmodified corresponding peptide (cRGDyK) were determined for binding to integrin αvβ3 expressing U87MG cells in a competitive binding assay against 125I-echistatin. Figure 4A showed that the modified cRGD-ADIBOT-F had a similar binding affinity with the nonmodified cRGDyK, suggesting that the attachment of the 18F-labeled ADIBOT prosthetic group into the peptide had no effect on its specific binding. We also conducted an in vivo evaluation of cRGD-ADIBOT-18F using the micro-PET-CT system. A transaxial image of a mouse bearing a U87MG tumor on the right front leg at 120 min postinjection of 1.8 MBq of cRGD-ADIBOT-18F is shown in Figure 4B (left). The tumor was clearly visible with a high contrast to contralateral background. As shown in Figure 4B (right), uptake of a tumor coinjected with 10 mg/kg nonradioactive cRGD-ADIBOT-F was significantly lower than that in a nonblocked a mouse

Figure 4. (A) Competitive binding assay of cRGD-ADIBOT-F in comparison with the nonmodified cRGDyK with 125I-echistatin using U87MG cells. (B) In vivo evaluation of cRGD-ADIBOT-18F; transaxial microPET-CT images of U87MG tumor bearing mice at 120 min postinjection of 1.8 MBq of cRGD-ADIBOT-18F without and with (denoted as “blocking”) a coinjection of nonradioactive cRGDADIBOT-F (10 mg/kg). Tumors are indicated by white arrows. (C) Biodistribution of cRGD- ADIBOT-18F at 2 h. Data are expressed as normalized accumulation of activity in % ID/g ± SD (n = 4).

necropsied. Tissues were weighed and counted on a gamma counter. Uptake in each tissue was expressed as the percentage injected dose per gram of tissue (% ID/g). All experiments was repeated many times, and the results were presented as the mean ± standard deviation. A p-value of less than 0.05 was considered statistically significant.

3. RESULTS AND DISCUSSION 3.1. Model SPAAC Reactions Using a Simple ADIBO Precursor with 18F-Labeled Synthon. Considering the highly bio- and chemo-orthogonal features of the SPAAC reaction, we attempted to find the minimal amount of ADIBO peptide precursor enabling a reasonable reaction time and a high RCY. For this purpose, we examined the SPAAC based 1683

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Figure 5. (A) Schematic high-throughput synthesis of BBN-ADIBOT-18F, cMBP-ADIBOT-18F, and ApoPep-ADIBOT-18F. (B, C, and D) HPLC chromatograms of the BBN-ADIBOT-18F (B), cMBP-ADIBOT-18F (C), and ApoPep-ADIBOT-18F (D) reaction mixtures before and after treatment with the ADIBO scavenger-resin 4.

BOT-18F), cMBP (cMBP-ADIBOT-18F), and ApoPep (ApoPep-ADIBOT-18F) were obtained readily and simultaneously in excellent dcRCYs (90−92%) and radiochemical purities (>98%) within only a 35 min total reaction time by this copper-free SPAAC reaction and subsequent treatment of the azide-resin 4. Figure 5B−D demonstrated the successful removal of all three unreacted ADIBO precursors from the corresponding reaction mixture solutions by the scavenger resin 4. In particular, the ApoPep-ADIBOT-18F tracer showed almost the same retention time as its precursor on the HPLC chromatogram (Figure 5D), which can make the separation of this tracer using a conventional HPLC system difficult or impossible, whereas the chemo-orthogonal scavenger method can provide a highly efficient and easy purification process even though the tracer has the same HPLC retention time as its precursor.

model. These in vivo studies demonstrated a successful specific visualization of integrin αvβ3 expression, indicating a high potential for our sequential F-18 labeling protocol in future PET imaging studies. Figure 4C showed the result of the biodistribution experiment. The tissue distribution studies showed that there was a significantly high uptake of cRGD-ADIBOT-18F radiotracer in tumor−principal target tissues for integrin αvβ3 compared to nontarget tissues such as muscle. 3.4. High-Throughput Synthesis of 18F-Labeled Peptides. To demonstrate that our strategy is generally applicable to other more complicated peptides, we carried out the SPAAC reactions with three ADIBO substituted peptide precursors, such as BBN-ADIBO, cMBP-ADIBO, and ApoPep-ADIBO, under the same reaction conditions as that described in Figure 3A for F-18 labeling. Figure 5A showed that, with [18F]2 from its one time production, three 18F-labeled BBN (BBN-ADI1684

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(7) Kim, D. W., Jeong, H.-J., Lim, S. T., and Sohn, M.-H. (2010) Recent trends in the nucleophilic [18F]-radiolabeling method with nocarrier-added [18F]fluoride. Nucl. Med. Mol. Imaging 44, 25−32. (8) Kim, D. W., Ahn, D.-S., Oh, Y.-H., Lee, S., Kil, H. S., Oh, S. J., Lee, S. J., Kim, J. S., Ryu, J. S., Moon, D. H., and Chi, D. Y. (2006) A new class of SN2 reaction catalyzed by protic solvents: facile fluorination for isotopic labeling of diagnostic molecules. J. Am. Chem. Soc. 128, 16394−16397. (9) Miller, P. W., Long, N. J., Vilar, R., and Gee, A. D. (2008) Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography. Angew. Chem., Int. Ed. 47, 8998−9033. (10) Deutscher, S. L. (2010) Phage display in molecular imaging and diagnosis of cancer. Chem. Rev. 110, 3196−3211. (11) Chen, K., and Conti, P. S. (2010) Target-specific delivery of peptide-based probes for PET imaging. Adv. Drug Delivery Rev. 62, 1005−1022. (12) Okarvi, S. M. (2001) Recent progress in fluorine-18 labelled peptide radiopharmaceuticals. Eur. J. Nucl. Med. 28, 929−938. (13) Wuest, F. (2005) Aspects of positron emission tomography radiochemistry as relevant for food chemistry. Amino Acids 29, 323− 339. (14) Lee, S., Xie, J., and Chen, X. (2010) Peptides and peptide hormones for molecular imaging and disease diagnosis. Chem. Rev. 110, 3087−3111. (15) Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001) Click chemistry: diverse chemical function from a few good reactions. Angew. Chem., Int. Ed. 40, 2004−2021. (16) Mamat, C., Ramenda, T., and Wuest, F. R. (2009) Application of click chemistry for the synthesis of radiotracers for molecular imaging. Mini-Rev. Org. Chem. 6, 21−34. (17) Mindt, T. L., Struthers, H., Brans, L., Anguelov, T., Schweinsberg, C., Maes, V., Tourwé, D., and Schibli, R. (2006) ″Click to chelate″: synthesis and installation of metal chelates into biomolecules in a single step. J. Am. Chem. Soc. 128, 15096−15097. (18) Thonon, D., Kech, C., Paris, J., Lemaire, C., and Luxen, A. (2009) New strategy for the preparation of clickable peptides and labeling with 1-(azidomethyl)-4-[18F]-fluorobenzene for PET. Bioconjugate Chem. 20, 817−823. (19) Maschauer, S., Einsiedel, J., Haubner, R., Hocke, C., Ocker, M., Hübner, H., Kuwert, T., Gmeiner, P., and Prante, O. (2010) Labeling and glycosylation of peptides using click chemistry: a general approach to 18F-glycopeptides as effective imaging probes for positron emission tomography. Angew. Chem., Int. Ed. 49, 976−979. (20) Baskin, J. M., Prescher, J. A., Laughlin, S. T., Agard, N. J., Chang, P. V., Miller, I. A., Lo, A., Codelli, J. A., and Bertozzi, C. R. (2007) Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl. Acad. Sci. U.S.A. 104, 16793−16797. (21) Lallana, E., Fernandez-Megia, E., and Riguera, R. (2009) Surpassing the use of copper in the click functionalization of polymeric nanostructures: a strain-promoted approach. J. Am. Chem. Soc. 131, 5748−5750. (22) Kennedy, D. C., McKay, C. S., Legault, M. C. B., Danielson, D. C., Blake, J. A., Pegoraro, A. F., Stolow, A., Mester, Z., and Pezacki, J. P. (2011) Cellular consequences of copper complexes used to catalyze bioorthogonal click reactions. J. Am. Chem. Soc. 133, 17993−18001. (23) Sletten, E. M., and Bertozzi, C. R. (2011) From mechanism to mouse: a tale of two bioorthogonal reactions. Acc. Chem. Res. 44, 666− 676. (24) Debets, M. F., van Berkel, S. S., Dommerholt, J., Dirks, A. J., Rutjes, F. P. J. T., and van Delft, F. L. (2011) Acc. Chem. Res. 44, 805− 815. (25) Jewett, J. C., and Bertozzi, C. R. (2010) Bioconjugation with strained alkenes and alkynes. Chem. Soc. Rev. 39, 1272−1279. (26) Sletten, E. M., and Bertozzi, C. R. (2009) Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem., Int. Ed. 48, 6974−6998. (27) Laughlin, S. T., Baskin, J. M., Amacher, S. L., and Bertozzi, C. R. (2008) In vivo imaging of membrane-associated glycans in developing zebrafish. Science 320, 664−667.

4. CONCLUSIONS In summary, we have described a tremendously efficient and straightforward 18F-labeling protocol platform based on the highly chemo-orthogonal copper-free SPAAC reaction using the ADIBO peptide precursors with the 18F-azide synthon for the high-throughput synthesis of 18F-labeled peptide tracers. In this method, the SPAAC conjugation reaction and subsequent chemo-orthogonal treatment of the azide-resin proceeded quickly and selectively under physiologically friendly reaction conditions (i.e., toxic chemical reagents-free, aqueous medium, room temperature, and pH ≈7), and provided 18F-labeled peptide tracers in high RCY as direct injectable solutions without any HPLC purification and formulation processes for the in vivo molecular imaging study. The successful application of our F-18 labeling protocol was also demonstrated by in vitro and in vivo studies on cRGD-ADIBOT-18F. We believe that this is a very versatile protocol for numerous applications in the field of radiopharmaceutical science and molecular imaging. Further studies on the development of a more efficient protocol for automated production and application of in vivo PET molecular imaging studies using 18F-labeled peptides prepared by this protocol are currently underway.

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ASSOCIATED CONTENT

S Supporting Information *

Full experimental procedures and characterization data of all compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-63-250-2396. Fax: +82-63-255-1172. E-mail: [email protected]. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Nuclear Research & Development Program of the National Research Foundation of Korea (NRF) grant, funded by the Korean government (MEST) (grant code: 2011-0006322 and 2011-0030952), and the Conversing Research Center Program through the MEST (grant code: 2011K000705).

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REFERENCES

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Bioconjugate Chemistry

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dx.doi.org/10.1021/bc3002425 | Bioconjugate Chem. 2012, 23, 1680−1686