“Click Labeling” with 2-[18F]Fluoroethylazide for Positron Emission

Apr 13, 2007 - As an effort in the development of more flexible 18F-labeling chemistry, we report ...... Xinghai Ning , Wonewoo Seo , Seungjun Lee , K...
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Bioconjugate Chem. 2007, 18, 989−993

989

“Click Labeling” with 2-[18F]Fluoroethylazide for Positron Emission Tomography Matthias Glaser and Erik A° rstad* Hammersmith Imanet Ltd, Cyclotron Building, Du Cane Road, London W12 ONN, U.K. Received September 29, 2006; Revised Manuscript Received March 1, 2007

As an effort in the development of more flexible 18F-labeling chemistry, we report herein on the use of the Cu(I)-catalyzed Huisgen cycloaddition, also known as the “click reaction”, to form 18F-labeled 1,2,3-triazoles. Nucleophilic fluorination of 2-azidoethyl-4-toluenesulfonate followed by distillation provided 2-[18F]fluoroethylazide in 55% radiochemical yield (decay-corrected). 2-[18F]fluoroethylazide was reacted with a small library of terminal alkynes in the presence of excess Cu2+/ascorbate or copper powder. The most reactive alkyne, N-benzylpropynamide provided nearly quantitative incorporation of 2-[18F]fluoroethylazide after 15 min at ambient temperature, whereas the majority of the alkyne substrates provided excellent yields of the corresponding 18F-labeled 1,2,3-triazoles following heating to 80 °C. Using the method described, a model peptide was obtained in 92.3 ( 0.3% (n ) 3) radiochemical yield (decay-corrected) after purification by semipreparative HPLC.

INTRODUCTION Positron emission tomography (PET) allows high-resolution quantitative imaging of biochemical processes in vivo by detecting the distribution pattern of labeled biomarkers over time. PET is increasingly used for diagnostic imaging, medical research, and drug development (1-3). 18F is particularly attractive for PET, as it can be produced in high yields and has favorable decay characteristics (t1/2 ) 110 min) (4-6). As formation of electrophilic 18F-labeling agents invariably leads to isotopic dilution (2), the use of nucleophilic [18F]fluoride, which can be produced with excellent specific activity, is usually preferred for labeling. However, due to the low reactivity and high proton affinity of [18F]fluoride, 18F-labeled prosthetic groups are necessary for labeling complex biomolecules such as peptides and small molecules with H-acidic functions (7). Currently, peptide labeling is mostly confined to conjugation of free amino groups, either by direct acylation using 18Ffluorinated activated esters (7) or indirectly by functionalization, e.g., to an aminooxy group, which subsequently can undergo condensation with 18F-fluorinated aldehydes (8). The lack of alternative labeling positions and the high lipophilicity of the coupling groups that are formed make optimization of peptidebased biomarkers difficult. An additional limitation is that 18Flabeling agents often are complicated to produce, which restricts their use to specialized centers. The recent discovery that Cu(I) catalyzes the Huisgen 1,3dipolar cycloaddition of terminal alkynes and organic azides to form 1,2,3-triazoles (9, 10) often referred to as “click chemistry” (11) has proven particularly valuable in combinatorial chemistry (12-16). The reaction provides a versatile tool for coupling druglike fragments in high yields and under mild conditions, and the 1,2,3-triazole formed is biologically stable, with a polarity and size similar to amide bonds (17). The reaction can be carried out in aqueous media, and as both alkynes and organic azides are relatively inert, there is no need for protection groups. In addition, there is a wide selection of commercially available alkynes, providing building blocks for library synthesis. * Erik A° rstad, E-mail: [email protected], Tel. +44 208 383 3714, Fax +44 208 383 8573.

As an effort in the development of more flexible 18F-labeling chemistry, we report herein on the use of Cu(I)-catalyzed Huisgen cycloaddition to form 18F-labeled 1,2,3-triazoles (18).

EXPERIMENTAL PROCEDURES General. Reagents and solvents were purchased from SigmaAldrich Co. Ltd. (Gillingham, United Kingdom) and used without further purification. Ethyl-2-acetylamino-4-pentynoate was purchased from Bachem (Merseyside, U.K.). The model peptide 11 was provided by the Proteomics and Peptide Synthesis Facility, MRC Clinical Science Center/Imperial College, Hammersmith Hospital London. Mass spectra were recorded on a Finnigan LCQ system using electrospray ionization (ESI). MALDI-TOF mass spectra were measured on a Finnigan Lasermat 2000 instrument. Accurate mass spectra (HRMS) were measured on a Micromass Q-Tof Ultima machine coupled with a nanoLC system using Mass Lynx 3.5 software. NMR spectra were obtained from a JEOL Eclipse instrument (500 MHz), and the chemical shifts are reported in parts per million (ppm) relative to residual solvent peaks. The melting points were measured using an SMP-1 apparatus (Fisher Scientific UK). GC-MS data were acquired under electron ionization using an Agilent 6890N system. FT-IR spectra were recorded with a SpectrumBX2 (Perkin-Elmer). Analytical HPLC was carried out using a Beckman Gold instrument with Karat32 software. The radio HPLC system was a Beckman System Gold instrument equipped with a γ detector (Bioscan Flow-count). A Phenomenex Luna C18(2) column (50 × 4.6 mm, 3 µm; flow rate 1 mL/min) was used for analytical HPLC. A semipreparative column (Phenomenex Onyx Monolithic C18, 100 × 10 mm, with a flow rate of 4 mL/min) was used for the final purification of the peptide [18F]12. The following mobile phase systems were used both for analytical and preparative HPLC: solvent A, water/TFA (0.1%); solvent B, acetonitrile/TFA (0.1%); linear gradient of 5-80% solvent B over 15 min (gradient I), 5-60% solvent B over 15 min (gradient II), or 30-80% solvent B over 15 min (gradient III). Non-radioactive compounds were purified using an automated flash machine (Companion Presearch Ltd., 4 g RediSep silica cartridges, flow rate 18 mL/min), with the exception of peptide 12, which was purified using a preparative HPLC (Agilent1200, column Phenomenex Luna C18(2), 75 ×

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990 Bioconjugate Chem., Vol. 18, No. 3, 2007

30 mm, 5 µm, gradient I, flow rate 15 mL/min). [18F]Fluoride was produced by a cyclotron (Scanditronix MC40) using the 18O(p,n)18F nuclear reaction with 19 MeV proton irradiation of an enriched [18O]H2O target. 2-Fluoroethylazide (2). To a solution of 2-fluoroethyl-4toluenesulfonate (1) (19) (128 mg, 0.586 mmol) in DMF (anhydr., 10 mL) was added sodium azide (114 mg, 1.76 mmol), and the resulting mixture was stirred at ambient temperature for 48 h. The reaction mixture was filtered, and the filtrate containing the title compound was used without isolation for subsequent reactions. WARNING: Attempts to isolate neat 2-fluoroethylazide may result in an explosion. 1-(2-Fluoroethyl)-4-phenyl-1H-[1,2,3]triazole (3). To a stirred solution of copper(II) sulfate pentahydrate (12 mg, 0.0489 mmol) and L-ascorbic acid (16 mg, 0.0977 mmol) in water (0.3 mL) under nitrogen was added a solution of phenylacetylene (105 µL, 0.977 mmol) in DMF (1 mL). After addition of 2-fluoroethylazide (1.172 mmol) in DMF (5 mL), stirring was continued at room temperature for 21 h. The reaction mixture was diluted with water (5 mL), and the crude product was extracted with dichloromethane (3 × 5 mL) and washed with sodium bicarbonate solution (10%, 3 × 10 mL) and brine (1 × 5 mL). After drying over sodium sulfate, the solvent was removed under reduced pressure, and the crude material purified by means of flash chromatography (25% to 100% ethyl acetate in hexane for 10 min, tR ) 3.0 min) to provide the title compound (32 mg, 17% yield) as a white crystalline material. mp 83-85 °C. 1H NMR (CDCl3): δ 4.70 (m, 1H, CH2), 4.76 (m, 1H, CH2), 4.80 (m, 1H, CH2), 4.89 (m, 1H, CH2), 7.35 (tt, J4,2 ) 1.0 Hz, J4,3 ) 7.5 Hz, 1H, HAr), 7.44 (m, 2 H, HAr), 7.84 (m, 2H, HAr), 7.89 (d, J ) 1.0 Hz, 1H, CH-triazole). GCMS: m/z ) 191 [M]+. TOF-ES-MS: found m/z ) 192.0935 [MH]+, calcd for C10H10N3F m/z ) 192.0932 [MH]+. 1-(2-Fluoroethyl)-1H-[1,2,3]triazol-4-yl]carboxylic Acid (4). To a stirred solution of copper(II) sulfate pentahydrate (12 mg, 0.0489 mmol) and L-ascorbic acid (34 mg, 0.135 mmol) in water (0.4 mL) under nitrogen was added a solution of propargylic acid (60 µL, 0.977 mmol) in DMF (0.5 mL). After addition of 2-fluoroethylazide (1.172 mmol) in DMF (2.5 mL), stirring was continued at ambient temperature for 4 h. The reaction mixture was quenched with HCl (20 mL, 1 M), and the crude product was extracted with ethyl acetate (3 × 20 mL). After washing with brine (5 mL) and drying over sodium sulfate, the solvent was removed under reduced pressure. The residue was purified by recrystallization from ethyl acetate/hexane to give the title compound (16 mg, 10% yield) as a white crystalline material. mp 160-165 °C. 1H NMR (DMSO-d6): δ 4.74 (m, 1H, CH2), 4.80 (m, 2H, CH2), 4.90 (m, 1H, CH2), 8.71 (s, 1H, CH-triazole). TOF-ES-MS: found m/z ) 160.0518 [MH]+, calcd for C5H6N3O2F m/z ) 160.0517 [MH]+. 4-[1-(2-Fluoroethyl)-1H-[1,2,3]triazol-4-yl]benzoic Acid (5). To a stirred solution of copper(II) sulfate pentahydrate (3.7 mg, 0.0149 mmol) and L-ascorbic acid (10.5 mg, 0.0595 mmol) in water (0.2 mL) under nitrogen was added a solution of sodium 4-ethynylbenzoate (50 mg, 0.297 mmol) in DMF (1.5 mL). After addition of 2-fluoroethylazide (0.356 mmol) in DMF (0.76 mL), stirring was continued at ambient temperature for 12 h. The reaction mixture was diluted with HCl (20 mL, 1 M), the crude product was extracted with ethyl acetate (3 × 10 mL), and the organic phase washed with brine (10 mL). After drying over sodium sulfate, the solvent was removed under reduced pressure, and the crude material recrystallized from ethyl acetate/hexane to give the title compound (37 mg, 52% yield) as a white crystalline material. mp 236-240 °C (decomp.). 1H NMR (DMSO-d6): δ 4.74 (m, 1H, CH2), 4.80 (m, 2H, CH2), 4.90 (m, 1H, CH2), 7.99 (m, 4H, HAr), 8.70 (s, 1H, CH-triazole).

Glaser and A° rstad

TOF-ES-MS: found m/z ) 236.0838 [MH]+, calcd for C11H10N3O2F m/z ) 236.0830 [MH]+. 4-[1-(2-Fluoroethyl)-1H-[1,2,3]triazol-4-yl]aniline (6). To a stirred solution of copper(II) sulfate pentahydrate (129 mg, 0.516 mmol) and L-ascorbic acid (182 mg, 1.032 mmol) in water (1.2 mL) under nitrogen was added a solution of 4-ethynylaniline (40 mg, 0.344 mmol) in DMF (0.7 mL). After addition of 2-fluoroethylazide (0.287 mmol) in DMF (2.45 mL), stirring was continued at ambient temperature for 4 h. The reaction mixture was quenched with sodium hydroxide solution (1 M, 5 mL). The product was extracted with ethyl acetate (3 × 5 mL), and the organic phase was washed with water (5 mL) and brine (2 mL). After drying over sodium sulfate, the solvent was removed under reduced pressure and the crude material purified by means of flash chromatography (40% to 90% ethyl acetate in hexane for 10 min, tR ) 8.0 min) to give the title compound (15 mg, 25% yield) as a tan crystalline material. mp 79-82 °C. 1H NMR (CDCl3): δ 4.70 (m, 1H, CH2), 4.72 (m, 1H, CH2), 4.77 (m, 1H, CH2), 4.88 (m, 1H, CH2), 6.74 (d, J ) 8.7 Hz, 2H, HAr), 7.64 (d, J ) 8.7 Hz, 2H, HAr), 7.74 (d, J ) 0.1 Hz, 1H, CH-triazole). TOF-ES-MS: found m/z ) 207.1030 [MH]+, calcd for C10H11N4F m/z ) 207.1040 [MH]+. N-Benzyl-[1-(2-Fluoroethyl)-1H-[1,2,3]triazol-4-yl]formamide (7). A solution of N-benzylpropynamide (20) (50 mg, 0.314 mmol) in DMF (1 mL) was added to a stirred solution of copper(II) sulfate pentahydrate (3.9 mg, 0.0157 mmol) and L-ascorbic acid (11 mg, 0.0628 mmol) in water (0.4 mL) under nitrogen. After addition of 2-fluoroethylazide (0.377 mmol) in DMF (3.2 mL), stirring was continued at room temperature for 48 h. The reaction mixture was diluted with sodium bicarbonate (10%, 5 mL), the crude product extracted with dichloromethane (3 × 5 mL), and the organic phase washed with brine (5 mL). After drying over sodium sulfate, the solvent was removed under reduced pressure and the crude material purified by recrystallization from ethyl acetate/diethyl ether to give the title compound (8 mg, 10% yield) as a white crystalline material. mp 165-167 °C. 1H NMR (CDCl3): δ 4.70 (m, 6H, CH2), 7.34 (m, 5H, HAr), 7.46 (m, 1H, NH), 8.20 (s, 1H, CH-triazole). TOF-ES-MS: found m/z ) 249.1143 [MH]+, calcd for C12H13N4OF m/z ) 249.1146 [MH]+. N-Benzyl-3-[1-(2-fluoroethyl)-1H-[1,2,3]triazol-4-yl]propionamide (8). N-Benzyl-4-pentynamide was prepared from 4-pentynoic acid according to the method of Coppola and Damon (20). The spectroscopic data were consistent with reported values (21). To a stirred suspension of copper(I) iodide (255 mg, 1.335 mmol) in methanol (0.8 mL) under nitrogen was added a solution of N-benzyl-4-pentynamide (50 mg, 0.267 mmol) in methanol (0.5 mL), 2-fluoroethylazide (0.320 mmol) in DMF (2.62 mL), and diisopropylamine (0.233 mL, 1.335 mmol). Stirring was continued at ambient temperature for 2 h. The reaction mixture was diluted with a solution of sodium hydrogen phosphate (1 g) in water (10 mL) and filtered through Celite. The crude product was extracted with ethyl acetate (3 × 20 mL), and the organic phase was washed with brine (20 mL). After drying over sodium sulfate, the solvent was removed under reduced pressure and the crude material purified by column chromatography (silica, conditioned with ethyl acetate/hexane 1:1, elution with ethyl acetate/hexane 4:1) to afford the title compound (19 mg, 26% yield) as a white crystalline material. mp 127-133 °C. 1H NMR (CDCl3): δ 2.66 (t, J ) 7.0 Hz, 2H, CH2), 3.09 (t, J ) 7.0 Hz, 2H, CH2), 4.40 (d, J ) 5.7 Hz, 2H, CH2-benzyl), 4.56 (m, 1H, CH2), 4.61 (m, 1H, CH2), 4.70 (m, 1H, CH2), 4.80 (m, 1H, CH2), 6.0 (s, 1H, NH), 7.25 (m, 5H, HAr), 7.44 (s, 1H, CH-triazole). TOFES-MS: found m/z ) 277.1474 [MH]+, calcd for C14H17N4OF m/z ) 277.1459 [MH]+.

Technical Notes

Ethyl-(2-acetylamino-3-[1-(2-fluoroethyl)-1H-[1,2,3]triazol4-yl])propionate (9). A solution of ethyl-2-acetylamino-4pentynoate (146 mg, 0.799 mmol) in DMF (1 mL) was added to copper powder (200 mg, 40 mesh) under nitrogen, followed by a solution of 2-fluoroethylazide (0.799 mmol) in DMF (2.16 mL). The mixture was stirred and heated at 80 °C for 3 h. Purification by means of reversed-phase flash chromatography (4.3 g RediSep C18-RP cartridge, 5% to 95% acetonitrile in water for 10 min, tR ) 3.5 min) provided the title compound as a white solid (196 mg, 90% yield). mp 55-60 °C. 1H NMR (DMSO-d6): δ 1.13 (t, J ) 7.1 Hz, 3H, CH2CH3), 1.82 (s, 3H, CH3), 3.02 (m, 2H, CH2-propionate), 4.05 (m, 2H, OCH2CH3), 4.47 (m, 1H, CH), 4.46 (m, 1H, CH2), 4.64 (m, 1H, CH2), 4.70 (m, 1H, CH2), 4.81 (m, 1H, CH2), 7.89 (s, 1H, CH-triazole), 8.31 (d, 1H, NH). TOF-ES-MS: found m/z ) 273.1372 [MH]+, calcd for C11H17N4O3F m/z ) 273.1357 [MH]+. N-[1-(2-Fluoroethyl)-1H-[1,2,3]triazol-4-ylmethylene]benzamide (10). A solution of N-propargylbenzamide (22) (100 mg, 0.628 mmol) in DMF (1 mL) was added to copper powder (200 mg, 200 mesh) under nitrogen followed by a solution of 2-fluoroethylazide (0.628 mmol) in DMF (1.75 mL). The mixture was stirred for 30 min at ambient temperature and subsequently heated to 80 °C for 1 h. After evaporation of the solvent under high vacuum, water (5 mL) and saturated sodium bicarbonate (1 mL) were added. The crude product was extracted with ethyl acetate (3 × 10 mL), and the organic phase was washed with brine (5 mL), filtered, and dried over sodium sulfate. The triazole 10 was obtained by recrystallization from ethyl acetate/hexane as a white solid (68 mg, 44% yield). mp 100-105 °C. 1H NMR (CDCl3): δ 4.63 (t, J ) 17.0 Hz, 1H, CH2), 4.68 (t, J ) 17.0 Hz, 1H, CH2), 4.75 (t, J ) 17.0 Hz, 1H, CH2), 4.84 (t, J ) 17.0 Hz, 1H, CH2), (d, J ) 23 Hz, 2H, CH2-amide), 6.87 (s, 1H, NH), 7.43 (m, 3H, HAr), 7.73 (s, 1H, CH-triazole), 7.85 (m, 2H, HAr). TOF-ES-MS: found m/z ) 249.1133 [MH]+, calcd for C12H13N4OF m/z ) 249.1146 [MH]+. (S)-6-Amino-2-(2-{(S)-2-[2-((S)-6-amino-2-{[4-(2-fluoroethyl)-1H[1,2,3]triazol-4-yl-1-carbonyl]amino}hexanoylamino)acetyl-amino]-3-phenylpropionylamino}acetylamino)hexanoic Acid (12). To a stirred mixture of 11 (20.7 mg, 35.2 µmol), copper powder (200 mg, 40 mesh), and sodium phosphate buffer (0.2 mL, pH 6.0, 0.25 M) under nitrogen was added a solution of 2 (36.8 µmol) in DMF (97 µL). The mixture was heated to 80 °C for 2 h, the heating bath was removed, and stirring was continued for 48 h. The filtered solution was concentrated under vacuum and purified by means of preparative HPLC to provide the title compound 12 as a colorless oil (12 mg, 50% yield). MALDI-MS: found m/z ) 676.18 [M]+, calcd for C30H45N10O7F m/z ) 676.75 [M]+. Radiochemistry. 2-[18F]Fluoroethylazide ([18F]2). A mixture of Kryptofix 222 (5 mg, 13.3 µmol), potassium carbonate (1 mg, 7.2 µmol, dissolved in 50 µL water), and acetonitrile (1 mL) was added to [18F]fluoride (74-370 MBq) in water (1 mL). The solvent was removed by heating at 80 °C under a stream of nitrogen (100 mL/min). Afterward, acetonitrile (0.5 mL) was added, and the distillation was continued. This procedure was repeated twice. After cooling to room temperature, a solution of 2-azidoethyl-4-toluenesulfonate (1) (1.5 µL, 7.5 µmol) in anhydrous acetonitrile (0.2 mL) was added. The reaction mixture was stirred for 15 min at 80 °C. After addition of acetonitrile (0.3 mL), [18F]2 was distilled at 130 °C under a flow of nitrogen (15 mL/min) into a trapping vial containing acetonitrile (0.1 mL). Compound [18F]2 was collected with a radiochemical yield of 54% (decay-corrected) and 63% distillation efficiency. Preparation of [18F]3-10 (See Table 1 for reaction times, temperatures, and yields). Method A. To a solution of copper(II) sulfate (50 µL, 0.45 M), sodium L-ascorbate (50 µL, 1.5

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M), and sodium phosphate buffer (100 µL, pH 6.0, 50 mM) under nitrogen was added a solution of the alkyne (0.015 mmol) in DMF (0.1 mL) followed by [18F]2 in acetonitrile (0.2 mL, 18.5-37 MBq). Method B: To a mixture of sodium phosphate buffer (200 µL, pH 6.0, 50 mM) and copper powder (200 mg, 70 µm) was added a solution of the alkyne (0.015 mmol) in DMF (0.1 mL) followed by [18F]2 in acetonitrile (0.2 mL, 18.5-37 MBq). [18F](S)-6-Amino-2-(2-{(S)-2-[2-((S)-6-amino-2-{[4-(2-fluoroethyl)-1H[1,2,3]triazol-4-yl-1-carbonyl]amino}hexanoylamino)acetylamino]-3-phenylpropionylamino}acetylamino)hexanoic Acid ([18F]12). To a solution of copper(II) sulfate pentahydrate (4.3 mg, 17 µmol) in water (50 µL) was added sodium ascorbate (3.4 mg, 17 µmol) in water (50 µL) under nitrogen. After 1 min at room temperature, a solution of alkyne peptide precursor 11 was added (2 mg, 3.4 µmol, in sodium phosphate buffer, pH 6.0, 0.5 M, 50 µL), followed by a solution of [18F]2 (24-32 MBq) in acetonitrile (50 µL). The mixture was left at room temperature for 15 min and diluted with HPLC mobile phase A (0.3 mL). The labeled peptide [18F]12 was isolated using semipreparative HPLC (gradient II, UV at 216 nm, tR ) 6.4 min).

RESULTS AND DISCUSSION 2-[18F]-Fluoroethylazide ([18F]2) was designed as the labeling agent, since alkynes in general are more readily available and less hazardous than organic azides. Nucleophilic fluorination of 2-azidoethyl-4-toluenesulfonate (1) (23) with anhydrous nocarrier-added Kryptofix 222 K+[18F]F- (acetonitrile, 80 °C, 15 min) provided the azide [18F]2 in 95% analytical yield, of which 55% yield (decay-corrected) was isolated by distillation (Scheme 1). While the “click reaction” has been extensively studied, the conditions reported so far either involve too long reaction times for 18F-labeling or the use of microwave heating, which is difficult to implement in automated radiotracer production. Our main concern was therefore to investigate the scope and efficiency of the reaction with conventional heating within a time frame suitable for PET. Reportedly, the two most robust catalytic systems, Cu2+/ascorbate (10) (method A) and copper metal (24) (method B), were used to evaluate the reaction of 2-[18F]fluoroethylazide ([18F]2) with a small library of terminal alkynes (Scheme 1, Table 1). The reactions were carried out in a buffered solution (pH 6.0, water/acetonitrile/DMF) under an inert atmosphere, using excess copper relative to the alkynes. In order to evaluate the effect of the catalytic systems and the functional groups on the kinetics, radiochemical yields were measured after 15 min reaction time at ambient temperature and following subsequent heating to 80 °C for an additional 15 min. At ambient temperature, the degree of incorporation of [18F]2 varied considerably depending on the alkyne substrate and the catalytic system. N-Benzylpropynamide (entry 5, method A) performed particularly well, providing 98% yield as measured by radio-HPLC. Following heating to 80 °C for 15 min, excellent conversions of 2-[18F]fluoroethylazide ([18F]2) to the corresponding 1,2,3-triazoles were observed for the majority of the substrates, with the exception of propargylic acid, which provided 61% radiochemical yield (entry 2, method A). Thus, the click reaction is indeed compatible with the half-life of 18F and has a high tolerance for functionalities R to terminal alkynes, including N- and C-terminal amides, which are attractive for labeling of peptides. With a suitable match of catalytic system and alkyne substrate, excellent yields can be obtained within a short time under very mild conditions, i.e., ambient temperature and neutral pH, which opens up the possibility of labeling fragile biomolecules that otherwise cannot be labeled with 18F. To further investigate the scope of the method, propargylic acid was coupled to a model peptide to provide the N-

Glaser and A° rstad

992 Bioconjugate Chem., Vol. 18, No. 3, 2007 Scheme 1. Radiosynthesis of 2-[18F]fluoroethylazide ([18F]2) and 1,3-Dipolar Cycloaddition with Terminal Alkynes

Table 1. Alkyne Substrates and Radiolabeling Yields with 2-[18F]fluoroethylazide ([18F]2)a

Figure 1. Preparative HPLC trace of a reaction mixture containing [18F]12. (a) Radioactivity channel, [ 18F]12 at 6:24 min. (b) UV channel at 216 nm. Scheme 2. Labeling of Model Peptide 11 with 2-[18F]fluoroethylazide ([18F]2) Using Cu2+/Ascorbate Catalysis (method A)

a Yields measured by analytical radio-HPLC after 15 min reaction time at ambient temperature (in brackets) and after subsequent heating to 80 °C for 15 min. Method A: To a solution of copper(II) sulfate (50 µL, 0.45 M), sodium L-ascorbate (50 µL, 1.5 M), and sodium phosphate buffer (100 µL, pH 6.0, 50 mM) under nitrogen was added a solution of the alkyne (0.015 mmol) in DMF (0.1 mL) followed by [18F]2 in acetonitrile (0.2 mL). Method B: To a mixture of sodium phosphate buffer (200 µL, pH 6.0, 50 mM) and copper powder (200 mg, 70 µm) was added a solution of the alkyne (0.015 mmol) in DMF (0.1 mL) followed by [18F]2 in acetonitrile (0.2 mL).

propargylamide precursor 11. In accordance with previous reports (17), “click labeling” (method A, 15 min room temperature, Scheme 2) was unaffected by the functional groups present and provided nearly quantitative incorporation of 2-[18F]fluoroethylazide ([18F]2) to form the corresponding triazole [18F]12. Purification by means of semipreparative HPLC provided [18F]12 in 92.3 ( 0.6% (n ) 3) radiochemical yield (decaycorrected) with a radiochemical purity of 99% (Figure 1). While the click reaction proceeds well in a wide range of pH values (10), it should be noted that 2-[18F]fluoroethylazide ([18F]2) rapidly decomposes in the presence of copper and trifluoroacetic acid (25). Interestingly, the labeling agent [18F]2 appears otherwise to be stable in acidic media and also in the presence of copper at neutral or basic pH values. As peptides often contain residual trifluoroacetic acid, the addition of a buffer to the reaction mixture is therefore recommended to achieve optimal results for click labeling with [18F]2-fluoroethylazide ([18F]2).

CONCLUSION The Cu-catalyzed Huisgen 1,3-dipolar cycloaddition provides a simple, flexible and highly efficient method for 18F-labeling of small molecules and peptides. The labeling agent 2-[18F]fluoroethylazide ([18F]2) can be produced in high yield in a onestep procedure, and subsequent Cu-catalyzed coupling with terminal alkynes to form the corresponding 2-[18F]-fluoroethyl1,2,3-triazoles proceeds in excellent yields within a short time under mild conditions.

ACKNOWLEDGMENT We thank Geeta Patel (Proteomics and Peptide Synthesis Facility, MRC Clinical Science Center/Imperial College, Ham-

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Technical Notes

mersmith Hospital London) for the preparation of the model peptide and Emmanuel Samuel, School of Pharmacy London, for carrying out the mass spectrometry analysis. Finally, we thank Colin Steel and the cyclotron operators of Hammersmith Imanet Ltd. for providing us with 18F. Supporting Information Available: HPLC chromatograms of reference compounds and 18F-labeled derivatives. This material is available free of charge via the Internet at http://pubs.acs.org.

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(14) Whiting, M., Muldoo, J., Lin, Y.-C., Silverman, S. M., Lindstrom, W., Olson, A. J., Kolb, H. C., Finn, M. G., Sharpless, K. B., Elder, J. H., and Fokin, V. V. (2006) Inhibitors of HIV-1 protease by using in situ click chemistry. Angew. Chem., Int. Ed. 45, 1435-1439. (15) Krasinski, A., Radic, Z., Manetsch, R., Raushel, J., Taylor, P., Sharpless, K. B., and Kolb, H. C. (2005) In situ selection of lead compounds by click chemistry: target-guided optimization of acetylcholinesterase inhibitors. J. Am. Chem. Soc. 127, 66866692. (16) Agard, N. J., Prescher, J. A., and Bertozzi, C. R. (2004) A strainpromoted [3+2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126, 1504615047. (17) Bock, V. D., Hiemstra, H., and van Maarseveen, J. H. (2006) CuI-Catalyzed alkyne-azide “Click” cycloadditions form a mechanistic and synthetic perspective. Eur. J. Org. Chem. 51-68. (18) While this manuscript was in preparation, a similar account on the use of click chemistry for 18F-labeling of peptides was published (Marik, J., and Sutcliffe, J. L. (2006) Click for PET: Rapid preparation of [18F]fluoropeptides using CuI catalyzed 1,3-dipolar cycloaddition. Tetrahedron Lett. 47, 6681-6684). (19) van Velzen, E. U. T., Engbersen, J. F. J., and Reinhoudt, D. T. (1995) Synthesis of self-assembling resorcin[4]arene tetrasulfide adsorbates. Synthesis 989-997. (20) Coppola, G. M., and Damon, R. E. (1993) Acetylenic amides. i: synthesis of N-substituted-2-propynamide. Synth. Commun. 23, 2003-2010. (21) Jacobi, P. A., Brielmann, H. L., and Hauck, S. I. (1996) Toward the synthesis of biologically important chlorines, isobacteriochlorins, and corrins. Cyclic enamides from acetylenic amides. J. Org. Chem. 61, 5013-5023. (22) Hashmi, A. S. K., Weyrauch, J. P., Frey, W., and Bats, J. W. (2004) Gold catalysis: mild conditions for the synthesis of oxazoles from N-propargylcarboxamides and mechanistic aspects. Org. Lett. 6, 4391-4394. (23) Demko, Z. P., and Sharpless, K. B. (2001) An intramolecular [2+3] cycloaddition route to fused 5-heterosubstituted tetrazoles. Org. Lett. 3, 4091-4094. (24) Himo, F., Lovell, T., Hilgraf, R., Rostovtsev, V. V., Noodleman, L., Sharpless, K. B., and Fokin, V. V. (2004) Copper(I)-catalyzed synthesis of azoles. DFT study predicts unprecedented reactivity and intermediates. J. Am. Chem. Soc. 127, 210-216. (25) A solution of [18F]2 (13 MBq) in acetonitrile (50 µL) was added to a mixture of trifluoroacetic acid in water (5%, 50 µL) and copper powder (200 mg, 200 mesh). The resulting mixture was left for 15 min at room temperature. Subsequent HPLC analysis revealed a new peak (45%) that was assigned to [18F]fluoride. No decomposition was detected after incubation of [18F]2 in 5% TFA solution only.

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