A Novel Prosthetic Group for Site-Selective ... - ACS Publications

Efficient methodologies for the radiolabeling of peptides with [18F]fluoride are a prerequisite to enabling commercialization of peptide-containing ra...
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Bioconjugate Chem. 2008, 19, 1301–1308

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A Novel Prosthetic Group for Site-Selective Labeling of Peptides for Positron Emission Tomography Dag Erlend Olberg,*,† Ole Kristian Hjelstuen,†,‡ Magne Solbakken,‡ Joseph Arukwe,‡ Hege Karlsen,‡ and Alan Cuthbertson‡ University of Tromsø, Institute of Pharmacy, Department of Pharmaceutics and Biopharmaceutics, N- 9037 Tromsø, Norway, and GE Healthcare, Medical Diagnostics Discovery Research, P.O. Box 4220, 0401 Oslo, Norway. Received January 7, 2008; Revised Manuscript Received April 4, 2008

Efficient methodologies for the radiolabeling of peptides with [18F]fluoride are a prerequisite to enabling commercialization of peptide-containing radiotracers for positron emission tomography (PET) imaging. It was the purpose of this study to investigate a novel chemoselective ligation reaction comprising conjugation of an [18F]-N-methylaminooxy-containing prosthetic group to a functionalized peptide. Twelve derivatives of general formula R1-CO-NH-Lys-Gly-Phe-Gly-Lys-OH were synthesized where R1 was selected from a short list of moieties anticipated to be reactive toward the N-methylaminooxy group. Conjugation reactions were initially carried out with nonradioactive precursors to assess, in a qualitative manner, their general suitability for PET chemistry with only the most promising pairings progressing to full radiochemical assessment. Best results were obtained for the ligation of O-[2-(2-[18F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine 18 to the maleimidopropionyl-Lys-GlyPhe-Gly-Lys-OH precursor 10 in acetate buffer (pH 5) after 1 h at 70 °C. The non-decay-corrected isolated yield was calculated to be 8.5%. The most encouraging result was observed with the combination 18 and 4-(2-nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH, 9, where the conjugation reaction proceeded rapidly to completion at 30 °C after only 5 min. The corresponding non-decay-corrected radiochemical yield for the isolated 18 F-labeled product 27 was 12%. The preliminary results from this study demonstrate the considerable potential of this novel strategy for the radiolabeling of peptides.

INTRODUCTION In recent years, 18F-labeled peptides have gained increasing interest as an important class of radiotracer well-suited to functional imaging with PET (1–3). The intrinsic pharmacokinetic properties, high affinity, and versatility of solid-phase chemistry make small proteins and peptides attractive candidates for diagnostic imaging. The short-lived positron emitter fluorine-18 is currently the radionuclide of choice from the perspective of radiotracer manufacturability and distribution. It has excellent physical characteristics including a relatively low positron energy of 635 keV and a half-life of 109.7 min. This allows for extended radiochemical synthesis times and prolonged PET imaging protocols. There are, however, still considerable challenges to finding faster, more efficient methods for radiolabeling of peptides with this radionuclide. Although direct labeling routes have been described (4), the most common method utilizes a two-step process whereby the fluorine-18 is first introduced into a prosthetic group followed by conjugation to the peptide. This strategy separates the harsh reaction conditions required for [18F]fluoride incorporation from the mild conditions preferred for conjugation, ameliorating the risk of significant degradation of the peptide precursor. A variety of 18F-labeled prosthetic groups have been developed for incorporation into peptides, exploiting many different chemical reactions. These include acylation, alkylation, oxime formation, hydrazone formation, and 1,3-dipolar cycloaddition (4–7). Although alkylation and Michael-type reactions are often used in conjugation chemistry, * Dag Erlend Olberg. E-mail: [email protected]. Tel. +47 23185213. Fax +47 23186014. † University of Tromsø. ‡ GE Healthcare.

there are limited cases reported for peptide labeling with 18Fcontaining prosthetic groups. Examples include the [18F]thiol conjugation to R-haloacetylated or maleimide-modified peptides and [18F]maleimide conjugation to peptides bearing a free thiol group (7, 8). Thiols however, have the potential to oxidize to disulfides and readily take part in exchange reactions under a variety of conditions leading to complex mixtures (9). 4-[18F]Fluorobenzaldehyde has been demonstrated to be a useful prosthetic group in the labeling of peptides bearing an aminooxy function (10–13). Oxime-bond formation proceeds rapidly and in good yield but often requires heating during the conjugation step, leading in some cases to the accelerated degradation of the peptide. In addition, the high reactivity of the aminooxy group complicates the routine handling and storage of the precursors and demands high quality standards for the reagents and solvents used in the process. In contrast to aminooxy, the N-methylaminooxy group is unreactive toward aldehydes and ketones but reacts with Michael acceptors and a wide range of alkyl halides (14). The Nmethylaminooxy nitrogen can also be selectively reacted with active esters at slightly acidic pH in the presence of competing nucleophiles such as the thiol group of cysteine or amino group of lysine (15). Peptides containing N-methylaminooxy amino acids have been chemoselectively alkylated with allylic, benzylic, and R-haloacetyl, N-ethylmaleimide, and hexyl acrylate groups in mildly acidic aqueous/organic solutions (14, 16). It was therefore postulated that an 18F-labeled N-methylaminooxy prosthetic group could provide a useful addition to the toolbox of PET labeling strategies. This study reports on the radiosynthesis of the novel prosthetic O-[2-(2-[18F]fluoroethoxy)ethyl]N-methyl-N-hydroxylamine 18 and demonstrates its suitablility for radiolabeling in a model peptide system. Conjugation proceeded rapidly to completion in acetate buffer at pH 5 with

10.1021/bc800007h CCC: $40.75  2008 American Chemical Society Published on Web 05/29/2008

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4-(2-nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH, 9, at 30 °C after only 5 min. The desired conjugate 27 was isolated after purification with a non-decay-corrected radiochemical yield of 12%.

MATERIAL AND METHODS General. 9-Fluorenylmethoxycarbonyl (Fmoc) amino acids, Fmoc-Lys(Boc)-Sasrin resin, and peptide coupling reagents were purchased from Novabiochem, Bachem, and Applied Biosystems, respectively. All other reagents and solvents were purchased from Acros, Fluka, and Sigma-Aldrich. Mass spectra were recorded on a LCQ DECA XP MAX instrument using electrospray ionization (ESI) operated in positive mode at 4.5 kV and scan rate 5500 Da/s coupled to Finnigan Surveyor PDA chromatography system. Accurate mass spectra were recorded on a QToF-micro orthogonal acceleration time-of-flight mass spectrometer (Micromass UK Ltd., Manchester, UK) coupled to an Agilent 1100 chromatography system (Agilent Technology, Stockport, UK) using Leucine Enkephalin (Sigma Aldrich, St Louis, MO, USA) as reference compound. Data acquisition and processing were performed using Masslynx 4.0 data system. All NMR-spectra were recorded at 25 °C in 5 mm tubes on a Varian Unity Inova 500 NMR spectrometer using a 1 H{broadband} indirect detection pfg probe. Thin layer chromatography (TLC) was run on precoated plates of silica gel 60F254 (Merck). Analytical and preparative reversedphase HPLC runs were performed on Finnigan, Shimadzu, and Beckman chromatography systems. Purification of nonradioactive compounds, with the exception of the peptide derivatives, was performed using standard manual flash chromatography or an automated flash chromatography instrument (Combiflash companion, Teledyne ISCO). Details on HPLC and LC-MS conditions and analytical results are deposited as Supporting Information. Preparation of Peptide Precursors. Assembly of the resinbound amino acid sequence Lys(Boc)-Gly-Phe-Gly-Lys(Boc)OH was done using fully automated synthesis (ABI 433A synthesis machine) on Fmoc-Lys(Boc)-Sasrin resin (0.25 mmol) with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU) activation. Further derivatization of the peptide to yield compounds 1-12 was performed on the solid phase using a manual nitrogen bubbler apparatus (0.05-0.1 mmol peptide loading). Anhydride formation was mediated by N,N′-dicyclohexylcarbodiimide in dichloromethane. Coupling reactions were monitored by the Kaiser test (17). Simultaneous removal of the peptides from the resin and deprotection of sidechain protecting groups was carried out in trifluoroacetic acid containing triisopropylsilane and water (95:2.5:2.5 v/v/v). After filtration, the solution was concentrated under reduced pressure and the residue was washed with diethyl ether. Crude products were purified by reversed-phase preparative HPLC and isolated by lyophilization. Chloroacetyl-Lys-Gly-Phe-Gly-Lys-OH (1). Freshly prepared chloroacetic anhydride (0.4 mmol) and diisopropylethylamine (DIPEA) (0.4 mmol) were added to a suspension of the resin-bound peptide (0.05 mmol) in dimethylformamide (DMF) (5 mL). The reaction was run until complete conversion was achieved (1 h). After cleavage, the crude material was purified yielding 13 mg (42%). Bromoacetyl-Lys-Gly-Phe-Gly-Lys-OH (2). The compound was synthesized as described for the corresponding chloro compound 1 yielding 17 mg (52%) after purification. Acryloyl-Lys-Gly-Phe-Gly-Lys-OH (3). Acrylic acid (0.25 mmol) was preactivated for 3 min using (7-azabenzotriazol-1yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) (0.25 mmol) and DIPEA (0.4 mmol) in DMF (3 mL). The mixture was added to the resin-bound peptide (0.06 mmol) and

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left to react until complete conversion (2 h). Yield after cleavage and purification was 18 mg (51%). 2-Fluoroacryloyl-Lys-Gly-Phe-Gly-Lys-OH (4). A solution of freshly prepared 2-fluoroacrylic anhydride (0.2 mmol) in DMF (5 mL) was added to the resin-bound peptide (0.05 mmol) and left to react for 2 h. The procedure was repeated until complete conversion was achieved. After cleavage, the crude material was purified affording 24 mg (79%). 2-(Trifluoromethyl)acryloyl-Lys-Gly-Phe-Gly-Lys-OH (5). 2(Trifluoromethyl)acryloyl chloride was prepared from 2-(trifluoromethyl)acrylic acid following the procedure by Yamazaki et al. (18). Coupling of the acid chloride (0.3 mmol) to the resinbound peptide (0.1 mmol) in dry dichloromethane containing sym-collidine (0.1 mmol) was performed as described by Volonterio et al. (19) at 0 °C for 1 h and was repeated until complete conversion was achieved. After cleavage, the crude product was purified affording 13 mg (23%). 4-Bromocrotonoyl-Lys-Gly-Phe-Gly-Lys-OH (6). Freshly prepared 4-bromocrotonic anhydride (0.3 mmol) was added to a suspension of the resin-bound peptide (0.1 mmol) in DMF (3 mL) and left to react until complete conversion was achieved (2 h). After cleavage, the crude material was purified affording 50 mg (70%). 4-(Bromomethyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH (7). 4(Bromomethyl)benzoic acid (0.15 mmol) was preactivated for 3 min using a solution of PyAOP (0.15 mmol) and DIPEA (0.3 mmol) in DMF (3 mL). The mixture was added to the resinbound peptide (0.05 mmol) and left to react until complete conversion was achieved (2 h) followed by cleavage. The product was identified but decomposed during purification. 4-(Bromoacetyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH (8). Freshly prepared 4-(bromoacetyl)benzoic acid anhydride (0.3 mmol) was added to the resin-bound peptide (0.1 mmol) and left to react until complete conversion was achieved (2 h). After cleavage, the crude product was purified affording 17 mg (22%). 4-(2-Nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH (9). 4[(1E)-2-Nitrovinyl]benzoic acid (0.3 mmol), prepared according to a method of Park and Pei (20), was preactivated for 3 min in a solution of PyAOP (0.3 mmol) and DIPEA (0.6 mmol) in DMF (5 mL). The mixture was added to the resin-bound peptide (0.1 mmol) and left to react until complete conversion was achieved (1 h). After cleavage, the crude material was purified affording 50 mg (70%). 3-(Maleimido)propionyl-Lys-Gly-Phe-Gly-Lys-OH (10). To a suspension of the resin-bound peptide (0.1 mmol) in DMF (3 mL) were added 3-(maleimido)propionic acid N-hydroxysuccinimide ester (0.3 mmol) and DIPEA (0.35 mmol). The mixture was left to react until a complete conversion was achieved (4 h). After cleavage, the crude product was purified affording 60 mg (87%). Vinylsulfonyl-Lys-Gly-Phe-Gly-Lys-OH (11). The vinylsulfonyl derivative was prepared following a procedure by Li et al. (21). To a stirred suspension of the resin-bound peptide (0.05 mmol) in dichloromethane (3 mL) cooled to 0 °C were added 2-chloroethanesulfonyl chloride (0.375 mmol) and triethylamine (0.375 mmol). The mixture was stirred for 1 h at room temperature. Triethylamine (3.75 mmol) was added and the mixture was stirred for 1 h to effect elimination and formation of the vinylsulfone. The product was cleaved off the resin and purified to give 24 mg (76%). 2-Methyloxirane-2-carbonyl-Lys-Gly-Phe-Gly-Lys-OH (12). 2-Methyloxirane-2-carboxylic acid was prepared by hydrolysis of methyl 2-methyloxirane-2-carboxylate following a procedure by Roush et al. (22). Freshly prepared 2-methyloxirane-2-caboxylic acid anhydride (0.4 mmol) was added to the resin-bound peptide (0.1 mmol) in DMF and left to react until

Novel [18F]Fluorinated Prosthetic Group

a complete conversion was achieved (1 h). After cleavage, the crude product was purified affording 28 mg (45%). Methyl[4-[[(4-methylphenyl)sulfonyl]oxy]butoxy]carbamic Acid t-Butyl Ester (13). N-Boc-N-methylhydroxylamine was synthesized from N-methylhydroxylamine after a procedure by Tamura et al. (23). To a solution of 4-bromo-1-butanol (2.80 g, 18.0 mmol) and DIPEA (10 mL) in dichloromethane (10 mL) was added diphenyl-t-butylsilyl chloride (5.0 mL, 18 mmol) under argon atmosphere. The solution was stirred at room temperature for 2 h, concentrated in Vacuo, and purified by flash chromatography (hexane/ethyl acetate, 10:1) to afford (4-bromobutoxy)(t-butyl)diphenylsilane as a colorless oil (4.39 g, 62%). 1H NMR (CDCl3, 500 MHz) δ 1.05 (9H, s, (CH3)3), 1.69 (2H, m, CH2), 1.98 (2H, m, CH2), 3.42 (2H, t, J ) 6.8 Hz, CH2Br), 3.69 (2H, t, J ) 6.1 Hz, CH2O), 7.41 (3H, m, 3 × ArH), 7.66 (2H, m, 2 × ArH). A solution of N-Boc-N-methylhydroxylamine (0.74 g, 5.0 mmol) in DMF (10 mL) was treated with sodium hydride (200 mg, 60% dispersion in mineral oil, 4.80 mmol) and stirred for 1 h under argon. To the mixture was added a solution of (4bromobutoxy)(t-butyl)diphenylsilane (1.56 g, 4.00 mmol) in DMF (10 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 3 h and then concentrated. The residue was taken up in ethyl acetate (150 mL) and washed with 0.1 M NaOH (5 × 50 mL), water (50 mL), 0.1 M KHSO4, and brine (50 mL), and dried (MgSO4). After concentration, the residue was purified by flash chromatography (hexane/ethyl acetate, 10:1) affording [4-[[(t-butyl)diphenylsilyl]oxy]butoxy]methylcarbamic acid tbutyl ester as a colorless oil (0.59 g, 24%). 1H NMR (CDCl3, 500 MHz) δ 1.04 (9H, s, (CH3)3), 1.48 (9H, s, (CH3)3), 1.69 (4H, m, 2 × CH2), 3.07 (3H, s, CH3O), 3.68 (2H, t, J ) 6.2 Hz, 2 × CH2O), 3.83 (2H, t, J ) 6.2 Hz, 2 × CH2O), 7.39 (6H, m, 6 × ArH), 7.66 (4H, m, 4 × ArH). ESI-MS: found m/z ) 457.8 (MH)+, calcd m/z ) 458.3. Tetrabutylammonium fluoride (TBAF) (1 M solution in THF, 1.6 mL, 1.6 mmol) was added to a solution of [4-[[(tbutyl)diphenylsilyl]oxy]butoxy]methylcarbamic acid t-butyl ester (0.59 g, 1.2 mmol) in dry THF (20 mL). The reaction was stirred overnight under argon. Saturated ammonium chloride solution (40 mL) was added to the mixture and THF was evaporated off. The product was extracted into dichloromethane and dried (Na2SO4). The organic phase was removed under reduced pressure and the residue was purified by flash chromatography (hexane/ethyl acetate 1:1) affording (4-hydroxybutoxy)methylcarbamic acid t-butyl ester as a colorless oil (0.17 g, 63%). 1H NMR (CDCl3, 500 MHz) δ 1.49 (9H, s, (CH3)3), 1.71 (4H, m, 2 × CH2), 1.78 (1H, t, J ) 5.3 Hz, OH), 3.10 (3H, s, CH3O), 3.69 (2H, m, CH2OH), 3.89 (2H, m, CH2O). ESI-MS: found m/z ) 241.9 (M+Na)+, calcd m/z ) 242.1. To a stirred solution of (4-hydroxybutoxy)methylcarbamic acid t-butyl ester (170 mg, 0.800 mmol) and triethylamine (161 µL, 1.16 mmol) in dry dichloromethane (10 mL) was added tosyl chloride (191 mg, 1.00 mmol) in dry dichloromethane (5 mL). The reaction mixture was stirred overnight under argon. The organic phase was washed with 10% NaHCO3 (3 × 10 mL) and dried (MgSO4). After concentration, the residue was purified by flash chromatography (hexane/ethyl acetate 8:2) to give 13 as a colorless oil (64 mg, 22%). 1H NMR (CDCl3, 500 MHz) δ 1.47 (9H, s, (CH3)3), 1.64 (2H, m, CH2), 1.79 (2H, m, CH2), 2.45 (3H, s, ArCH3), 3.05 (3H, s, CH3N), 3.79 (2H, t, J ) 6.1 Hz, CH2O), 4.07 (2H, t, J ) 6.3 Hz, CH2O), 7.34 (2H, m, 2 × ArH), 7.79 (2H, m, 2 × ArH). ESI-MS: found m/z ) 396.1 (M+Na)+, calcd m/z ) 396.2. (4-Fluorobutoxy)methylcarbamic Acid t-Butyl Ester (14). A mixture of potassium fluoride (4.64 mg, 0.0800 mmol) and 4,7,13,16,21,24-hexaoxa-1,10-diazabicylo[8.8.8]hexacosane (Kryptofix 222) (30.1 mg, 80.0 µmol) in dry acetonitrile (0.75

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mL) was stirred for 5 min. A solution of 13 (15 mg, 40 µmol) in dry acetonitrile (0.250 mL) was added and the mixture was heated at 60 °C for 1 h. After concentration, the residue was purified by flash chromatography (hexane/ethyl acetate, 1:1) affording 14 as a colorless oil (4.5 mg, 51%). 1H NMR (CDCl3, 500 MHz) δ 1.49 (9H, s, (CH3)3), 1.73 (2H, m, CH2), 1.82 (2H, m, CH2), 3.10 (3H, s, CH3N), 3.87 (2H, t, J ) 6.2 Hz, CH2O), 4.49 (2H, d of t, JHF ) 47.2 Hz, J ) 6.0 Hz, CH2F). ESI-MS: found m/z ) 221.7 (M+H)+, calcd m/z ) 222.2. Methyl[2-[2-[[(4-methylphenyl)sulfonyl]oxy]ethoxy]ethoxy]carbamic Acid t-Butyl Ester (16). To a stirred solution of diethylene glycol (22.0 g, 208 mmol) and triethylamine (10.5 g, 104 mmol) in dichloromethane (100 mL) was added tosyl chloride (9.9 g, 52 mmol). The solution was stirred at room temperature for 1 h. The reaction mixture was washed with aqueous 0.1 M KHSO4 and 5% NaHCO3 and dried (Na2SO4). After concentration, the residue was purified by flash chromatography (hexane/ethyl acetate, 1:1) to give 2-(2-hydroxyethoxy)ethyl tosylate as a colorless oil (7.62 g, 56%). 1H NMR (CDCl3, 500 MHz) δ 1.86 (1H, s, OH), 2.45 (3H, s, ArCH3), 3.54 (2H, m, CH2O), 3.67 (2H, m, CH2O), 3.70 (2H, m, CH2O), 4.20 (2H, m, CH2O), 7.35 (2H, m, 2 × ArH), 7.81 (2H, m, 2 × ArH). ESI-MS: found m/z ) 261.3 (M+H)+, calcd m/z ) 261.1. To a solution of 2-(2-hydroxyethoxy)ethyl tosylate (7.62 g, 29.3 mmol) in dichloromethane (30 mL) containing DIPEA (6.0 mL, 35 mmol) were added diphenyl-t-butylsilyl chloride (9.0 mL, 35 mmol) and a catalytic amount of 4-dimethylaminopyridine under argon atmosphere. The solution was stirred at room temperature for 48 h. The reaction mixture was diluted with dichloromethane (100 mL) and washed with water (2 × 100 mL) and brine (2 × 50 mL) and dried (MgSO4). After concentration, the residue was purified by automated flash chromatography (RediSep 120 g column, flow 85 mL/min) using a gradient of 0-50% ethyl acetate in hexane over 30 min affording 2-[2-[[(t-butyl)diphenylsilyl]oxy]ethoxy]ethyl tosylate as a colorless oil (9.2 g, 63%). 1H NMR (CDCl3, 500 MHz) δ 1.03 (9H, s, (CH3)3), 2.41 (3H, s, ArCH3), 3.52 (2H, m, CH2O), 3.67 (2H, m, CH2O), 3.73 (2H, m, CH2O), 4.17 (2H, m, CH2O), 7.28 (2H, m, 2 × ArH), 7.37 (4H, m, 4 × ArH), 7.42 (2H, m, 2 × ArH), 7.66 (4H, m, 4 × ArH), 7.78 (2H, m, 2 × ArH). ESI-MS: found m/z ) 499.2 (M+H)+, calcd m/z ) 499.2. A solution of N-Boc-N-methylhydroxylamine (1.53, 1.04 mmol) in THF (5 mL) was added slowly to a stirred suspension of sodium hydride (60% in mineral oil, 0.48 g, 12 mmol) in THF (5 mL) under argon. The reaction mixture was stirred for 30 min until no more gas evolution was observed and then cooled to 0 °C. 2-[2-[[(t-Butyl)diphenylsilyl]oxy]ethoxy]ethyl tosylate (4.0 g, 8.0 mmol) was added and the solution was stirred at 0 °C for 30 min and then at room temperature overnight. After concentration, the residue was taken up in ethyl acetate (200 mL) and washed consecutively with 0.1 M NaOH (5 × 50 mL), water (50 mL), 0.1 M KHSO4 (50 mL), and brine (50 mL); dried (MgSO4), and concentrated. The product was purified by flash chromatography (RediSep 40 g column, flow 40 mL/ min) using a gradient 0-20% methanol in dichloromethane over 20 min affording [2-[2-[[(t-butyl)diphenylsilyl]oxy]ethoxy]ethoxy]methylcarbamic acid t-butyl ester as a colorless oil (3 g, 78%). 1H NMR (CDCl3, 500 MHz) δ 1.05 (9H, s, (CH3)3), 1.48 (9H, s, (CH3)3), 3.09 (3H, s, CH3N), 3.61 (2H, m, CH2O), 3.68 (2H, m, CH2O), 3.81 (2H, m, CH2O), 3.98 (2H, m, CH2O), 7.37 (4H, m, 4 × ArH), 7.42 (2H, m, 2 × ArH), 7.68 (4H, m, 4 × ArH). ESI-MS: found m/z ) 496.1 (M+Na]+, calcd m/z ) 496.3. TBAF (1 M solution in THF, 6.8 mL, 6.8 mmol) was added to a stirred solution of [2-[2-[[(t-butyl)diphenylsilyl]oxy]ethoxy]ethoxy]methylcarbamic acid t-butyl ester (3.0 g, 6.0 mmol) in dry THF (15 mL). The reaction was stirred overnight at room

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temperature. After concentration, the residue was taken up in dichloromethane and washed consecutively with saturated ammonium chloride solution (40 mL), water (2 × 50 mL), and brine (2 × 50 mL); dried (MgSO4), and concentrated. The product was purified by automated flash chromatography (RediSep 40 g column, flow 30 mL/min) using gradient 0-5% methanol in dichloromethane over 20 min affording [2-(2hydroxyethoxy)ethoxy]methylcarbamic acid t-butyl ester as a colorless oil (1.36 g, 91%). 1H NMR (CD2Cl2, 500 MHz) δ 1.46 (9H, s, (CH3)3), 2.41 (1H, t, J ) 6.2 Hz, OH), 3.08 (3H, s, CH3N), 3.57 (2H, m, CH2O), 3.65 (2H, m, CH2O), 3.67 (2H, m, CH2O), 3.97 (2H, m, CH2O). ESI-MS: found m/z ) 258.2 (M+Na)+, calcd m/z ) 258.1. A solution of tosyl chloride (1.6 mg, 8.5 mmol), [2-(2hydroxyethoxy)ethoxy]methylcarbamic acid t-butyl ester (1.0 g, 4.3 mmol), and triethylamine (1.25 mL, 9.00 mmol) in dry dichloromethane (30 mL) was stirred under argon overnight. Dichloromethane (50 mL) was added and the reaction mixture was washed consecutively with 5% NaHCO3 (100 mL), brine (100 mL), and water (2 × 100 mL); dried (MgSO4) and concentrated. The product was purified by automated flash chromatography (RediSep 40 g column, flow 40 mL/min) using gradient 10-65% ethyl acetate in hexane over 20 min to give 16 as a colorless oil (1.23 g, 74%). 1H NMR (CDCl3, 500 MHz) δ 1.48 (9H, s, (CH3)3), 2.45 (3H, s, ArCH3), 3.07 (3H, s, CH3N), 3.62 (2H, m, CH2O), 3.70 (2H, m, CH2O), 3.95 (2H, m, CH2O), 4.17 (2H, m, CH2O), 7.34 (2H, m, 2 × ArH), 7.80 (2H, m, 2 × ArH). ESI-MS: found m/z ) 412.0 (M+Na)+, calcd m/z ) 412.2. [2-(2-Fluoroethoxy)ethoxy]methylcarbamic Acid t-Butyl Ester (17). A mixture of potassium fluoride (58 mg, 1.0 mmol) and Kryptofix 222 (376 mg, 1.00 mmol) in dry acetonitrile (3 mL) was stirred for 5 min. A solution of 16 (230 mg, 0.600 mmol) in dry acetonitrile (2 mL) was added and the mixture was heated at 80 °C for 1 h. After concentration, the residue was purified by flash chromatography (hexane/ethyl acetate, 1:1) affording 17 as a colorless oil (83 mg, 58%). 1H NMR (CDCl3, 500 MHz) δ 1.49 (9H, s, (CH3)3), 3.12 (3H, s, CH3N), 3.73 (2H, m, CH2O), 3.76 (2H, d of m, JHF ) 29.5 Hz, FCH2CH2O), 4.03 (2H, m, CH2O), 4.57 (2H, d of m, JHF ) 47.6 Hz, FCH2CH2O). ESI-MS: found m/z ) 260.0 (M+Na)+, calcd m/z ) 260.1. Compounds 10-31. General Conjugation Procedure. To a conical vial charged with 14 or 17 (2 mg, 8 µmol) was added 2 M HCl in diethylether (0.3 mL), and the reaction mixture was left at room temperature for 5 min removing the Boc-groups quantitatively to give 15 and 18, respectively. The solution was concentrated to dryness under reduced pressure and a solution of peptide derivative (3 mg, 2.8-5.1 µmol) in 0.4 M sodium acetate buffer pH 5 (0.8 mL) was added. Progress of the reaction was monitored by LC-MS analysis. If formation of conjugate could not be detected after reaction at room temperature for 30 min, temperature was increased to 70 °C and aliquots were analyzed at 45, 60, 90, and 120 min reaction times. Analytical results are deposited as Supporting Information. Radiochemistry. All anhydrous chemicals were purchased from Sigma-Aldrich (Norway). HPLC solvents were obtained from Merck KGaA (VWR). Radiochemistry and semipreparative HPLC purifications were performed using the TracerLab FxFN (GE Medical Systems) with manual interventions when required. Thin layer chromatography (TLC) was run on precoated plates of silica gel 60F254 (Merck) developed in hexane/ethyl acetate (1:1). Instant Imager (Packard BioScience) was used to measure the Radio-TLC scan. Analytical HPLC was performed on an Agilent system (1100 series) with UV detection equipped in series with a γ-detector (Bioscan flow-count). Preparative purification was run on a TracerLab HPLC system. [18F]Fluoride

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was produced by a cyclotron (GE PETtrace 6) using 18O(p,n)18F nuclear reaction with a 16.5 MeV proton irradiation of an enriched [18O]H2O target. Preparation of the K[18F]F-K222 Complex. Aqueous 18 [ F]fluoride (1 mL, 150-250 MBq) was passed through an anion-exchange resin (Sep-Pak light Waters Accell Pluss QMA Cartridge, preconditioned with K2CO3). The [18F]fluoride was then eluted from the resin into the TracerLab reaction vessel using a solution of Kryptofix 222 (56 mg) and K2CO3 (10 mg) in a mixture of water (215 µL) water and acetonitrile (785 µL). The solution was then concentrated to dryness by heating at 100 °C under reduced pressure and a flow of nitrogen for 2 min. Acetonitrile (0.8 mL) was added and evaporated off as above. This step was repeated twice. (4-[18F]Fluorobutoxy)methylcarbamic Acid t-Butyl Ester 18 ([ F]14). To the dried K[18F] F-K222 complex was added a solution of 13 (3 mg, 8 µmol) in acetonitrile (1 mL). The sealed reaction vessel was heated at 70 °C for 20 min and then analyzed by TLC and HPLC. The reaction mixture was passed through an alumina column (Sep-Pak, Waters) removing free [18F]F-. Finally, the reaction vessel was rinsed with acetonitrile (1 mL) and this solution was passed through the same alumina column to isolate as much of the labeled material as possible. [2-(2-[18F]Fluoroethoxy)ethoxy]methylcarbamic Acid tButyl Ester ([18F]17). To the dried K[18F] F-K222 complex was added a solution of 16 (3.0 mg, 7.7 µmol or 5.0 mg, 12.5µmol) acetonitrile (1 mL). The sealed reaction vessel was heated to 90 °C for 10 min. and then analyzed by TLC and HPLC. The reaction mixture was diluted with water (9 mL) and loaded onto an Oasis HLB Sep-Pak column (Waters). The column was rinsed with 25% methanol in water solution (50 mL) and then dried with a flow of nitrogen. The labeled product was eluted off the column using acetonitrile (1.5 mL). [18F]23. Conjugation of Prosthetic Group [18F]15 to Peptide 10. The solution of [18F]14 in acetonitrile (2 mL) from the labeling procedure was evaporated to dryness at 70 °C under vacuum. To the residue was added 2 M HCl in diethyl ether (1 mL) and the reaction mixture was stirred for 5 min. The mixture was concentrated at 65 °C under reduced pressure with a flow of nitrogen. To the residue was added a solution of peptide 10 (5.0 mg, 7.3 µmol) in 0.4 M sodium acetate buffer pH 5 (0.8 mL). The reaction mixture was heated to 70 °C and aliquots were removed after 20, 40, and 60 min reaction time. HPLC analysis showed reappearance of free [18F]fluoride, unreacted [18F]15, and the desired peptide conjugate [18F]23. [18F]27. Conjugation of Prosthetic Group [18F]18 to Peptide 9. To the solution of [18F]17 in acetonitrile (1.5 mL) from the labeling procedure was added 2 M HCl in diethyl ether (0.2 mL) and the reaction mixture was stirred for 5 min. The mixture was concentrated at 65 °C under reduced pressure with a flow of nitrogen for 3 min. Acetonitrile (0.8 mL) was added and the concentration procedure was repeated. To the residue was added a solution of peptide 9 (5 mg, 7 µmol) in 0.4 M sodium acetate buffer pH 5 (0.8 mL). The reaction mixture was heated at 30 °C for 5 min, diluted with water (3 mL), and analyzed by HPLC showing unreacted [18F]18 and desired conjugate [18F]27. The labeled peptide was purified on a preparative HPLC and shown to coelute with reference standard 27. [18F]28. Conjugation of Prosthetic Group [18F]18 to Peptide 10. The deprotection of Boc-protected [18F]17 and removal of the organic solvents was carried out in the same manner as that described in the section above. The dry residue was reconstituted in 0.8 mL of 0.4 M sodium acetate buffer containing 5 mg (7.3 µmol) of peptide derivative 10. The reaction mixture was left for 60 min at 70 °C. After diluting the reaction mixture with 3 mL of water, an aliquot was analyzed

Novel [18F]Fluorinated Prosthetic Group Table 1. Structures of the Peptide Derivatives Studied

a

indicates product degradation on purification.

by HPLC showing [18F]18 and [18F]28. The reaction mixture was subjected to preparative HPLC and the isolated fraction was shown to colelute with reference standard 28.

RESULTS AND DISCUSSION The aim of this study was to investigate the utility of two novel 18F-containing prosthetic groups bearing the N-methylaminooxy functionality as a new methodology for the radiolabeling of peptides. Because of the alpha effect, N-methylaminooxy compounds are much more powerful nucleophiles than the ε-amino group of lysine, allowing site-specific conjugation under mildly acidic conditions (24). Furthermore, although structurally related, the N-methylaminooxy group has a quite different set of reactivities compared to aminooxy being unreactive toward aldehydes and ketones (14). Carrasco et al. reported site-specific ligations of N-methylaminooxy groups in peptide systems to Michael-type acceptors and halides through nucleophilic substitution (16). One potential drawback however in applying this chemistry to the labeling of peptides for PET imaging was the relatively long reaction times reported for the conjugation step. We therefore adopted a strategy designed to rapidly screen a range of reactive groups linked to the N-terminal end of the model peptide KGFGK. Our approach was to assess, in a qualitative manner, the suitability and general applicability to PET chemistry. The conjugation reactions were first carried out using nonradioactive compounds with promising chemistries transferred to full radiochemical evaluation with [18F]fluoride. Table 1 shows a list of peptide derivatives synthesized for the initial conjugation studies. The reactivity of peptide precursors 1-12 were then assessed using the two prosthetic groups 15 and 18 whose structures and synthetic routes are illustrated in Scheme 1 below. Starting from 4-bromo-1-butanol, 13 was readily prepared in 4 steps by protection of the hydroxyl group as the t-butyl diphenyl silyl ether followed by reaction with the anion of

Bioconjugate Chem., Vol. 19, No. 6, 2008 1305

N-Boc-N-methylhydroxylamine generated by sodium hydride, removal of the silyl ether with TBAF and reaction with tosyl chloride. The Boc-protected product 14 was obtained by direct fluorination of 13 with the KF-Kryptofix complex. Subsequent removal of the Boc-group with 2 M HCl in ether produced 15 in quantitative yield. In a similar manner, 16 was prepared from diethylene glycol by monotosylation of the diol followed by protection as the t-butyl diphenyl silyl ether. Once again, reaction with the anion of N-Boc-N-methylhydroxylamine generated by sodium hydride, TBAF mediated removal of the silyl ether, and subsequent reaction with tosyl chloride gave the desired product in acceptable yield. The fluorinated compound 18 was prepared by reaction of 16 with KF-Kryptofix 222 followed by removal of the Boc-group with 2 M HCl in ether. Nonradioactive conjugations were performed with prosthetic groups 15 and 18 and peptide derivatives 1-12 dissolved in 0.4 M acetate buffer at pH 5. The reactions were initially stirred for 30 min at room temperature and an aliquot analyzed by LCMS. In cases where no detectable product had formed, the temperature of the solution was elevated to 70 °C and further aliquots removed for LC-MS analysis at 45, 60, 90, and 120 min. The reactions were performed on a milligram scale using 2 equiv of the prosthetic group and conjugation yields and product identity were confirmed by HPLC and mass spectrometry respectively. Table 2 summarizes the test reactions carried out with peptide derivatives 1, 3, 5, 6, and 10 in combination with prosthetic group 15. Precursors 1 and 3 reacted sluggishly with 15, forming only low yields of the conjugates 19 and 20 and were not selected for further evaluation. Although the 2-trifluormethyl derivative 5 gave product 21 in a 17% yield after 45 min at 70 °C, significant degradation of the starting material was observed by HPLC, complicating the analysis of the product mixture. A similar observation was made for peptide derivative 6. Indeed, the complexity of the product mixtures led us to exclude these derivatives, and no attempt at radiochemical assessment was made. In contrast, the maleimide derivative 10 gave a 28% yield of 23 after 90 min at 70 °C, and the conjugate could be readily isolated from the starting material by HPLC. In a similar set of experiments, the prosthetic group 18 was used to prepare conjugates with the peptide derivatives 2, 4, 8, 9, 10, 11, and 12, and the yields are shown in Table 3 below. Interestingly, the conjugation yields were in general higher using 18 compared to 15. The maleimide derivative 10 was converted in 50% yield to product 28 with heating to 70 °C for 45 min. Most significantly, the 4-(2-nitrovinyl)benzoyl derivative 9 was converted to conjugate 27 in greater than 90% yield at 30 °C. Indeed, the reaction time could be reduced from 30 min to only 5 min with no significant impact on yield. The vinylsulfonamide 11 gave a clean reaction product, but the reaction was slower than for both maleimide and 4-(2-nitrovinyl)benzoyl-functionalized peptides. Once again, significant degradation of all other derivatives was observed, and from the initial round of screening, derivatives 9 and 10 were selected for full radiochemical assessment. Radiochemical Assessment. Incorporation of [18F]fluoride into precursor 13 using [18F]KF-Kryptofix/ carbonate in acetonitrile at 70 °C for 20 min typically gave ∼60% yield of [18F]14 as analyzed by radio-TLC. Removal of free fluoride was performed on an alumina column (Sep-Pak, Waters) before cleavage of the Boc group with 2 N HCl in diethyl ether to quantitatively liberate [18F]15. The organic phase was evaporated in Vacuo and the product reconstituted by addition of sodium acetate buffer pH 5. On subsequent reaction with the maleimide derivative 10 at 70 °C, the presence of a new

1306 Bioconjugate Chem., Vol. 19, No. 6, 2008

Olberg et al.

Scheme 1. Preparation of Prosthetic Groups 15 and 18 from Corresponding Tosylates 13 and 16a

a Reagents and conditions: (i) 1. t-BDPSiCl, i-PrNEt, 24 h; 2. N-tert-butoxycarbonyl-N-methylhydroxylamine, NaH, 4 h; 3. TBAF, 24 h; 4. TosCl, i-PrNEt, 12 h; (ii) KF, Kryptofix, 70 °C, 60 min; (iii) 2 M HCl in diethylether, 5 min; (iv) 1. TosCl, i-PrNEt, 2 h; 2. t-BDPSiCl, i-PrNEt, 48 h; 3. N-tert-butoxycarbonyl-N-methylhydroxylamine, NaH, 12 h; 4. TBAF, 24 h; 5. TosCl, i-PrNEt, 24 h; (v) KF, Kryptofix, 70 °C, 60 min; (vi) 2 M HCl in diethyl ether.

Table 2. Reaction Yields of Prosthetic Group 15 with Peptide 1, 3, 5, 6, and 10

a indicates conjugation yield determined by HPLC integration (UV detection at 254 nm) of all peptide products relative to starting peptide. b indicates significant degradation of starting peptide.

Table 3. Reaction Yields of Prosthetic Group 18 with Peptide 2, 4, 8, 9, 10, 11, and 12

Figure 1. Radioactive HPLC analysis of [18F]15 in the presence of peptide 10 after heating to 70 °C for (top) 20 min and (bottom) 60 min in 0.4 M sodium acetate buffer at pH 5. Peak A ) [18F]fluoride, B ) [18F]15, and C ) [18F]23. Scheme 2. Proposed Route of Degradation of [18F]15 at 80 °C in 0.4 M Sodium Acetate Buffer at pH 5

a indicates conjugation yield determined by HPLC integration (UV detection at 254 nm) of all peptide products relative to starting peptide. b indicates significant degradation of starting peptide.

radioactive peak corresponding to the desired product 23 was observed (peak C, Figure 1). However, a second, more prominent peak was also present eluting in the void volume (peak A, Figure 1). This byproduct could be explained by

liberation of [18F]fluoride through cyclization of the prosthetic group to give the six-membered ring 31 as shown in Scheme 2. Figure 1 shows the radiochromatograms of [18F]15 (peak B) on heating for 20 and 60 min at 70 °C in the presence of peptide derivative 10. The presence of product 31 in the reaction mixture was confirmed by mass spectrometry. This was surprising, as direct nucleophilic substitution on alkyl fluorides is relatively uncommon; one example found in the literature relates to their reactivity with hydrazines (25). As a result of these findings, no further work was carried out on [18F]15. Radiolabeling of precursor 16 gave [18F]17 in typical radiochemical yields ranging 60-80% (n ) 9) starting with 5 mg (12.9 µmol) of precursor at 90 °C for 5 min in 1 mL acetonitrile. Increasing the reaction time to either 10 or 15 min had no detectable impact on yield, as the tosylate 16 appeared to rapidly

Novel [18F]Fluorinated Prosthetic Group

Bioconjugate Chem., Vol. 19, No. 6, 2008 1307 Table 4. Radiochemical Yields (not corrected for decay) for [18F]27 and [18F]28 peptide reaction conjugation conjugate temp (°C) time (min) 27 28

30 70

5 60

% isolated yielda 12.0 (n ) 3) 8.5 (n ) 3)

% total synthesis timeb (min) RCPc 80 140

>99 >99

a Yields of isolated [18F]fluoropeptide, based on starting [18F]fluoride (not corrected for decay). b Time of synthesis measured from incoming [18F]fluoride to end of purification. c Radiochemical purity analyzed by radio-HPLC.

Figure 2. Analysis of an aliquot taken from the crude reaction mixture after 5 min at 30 °C. (a) Radioactivity channel, (b) UV channel at 254 nm. [18F]27, retention time ) 10.8 min.

hydrolyze to the alcohol. This was not observed for 13 and may reflect a neighboring group effect of the ether oxygen. Purification was carried out by trapping the products of the reaction mixture on an Oasis HLB Sep-Pak cartridge, first eluting the excess alcohol with five aliquots of 10 mL 25% aqueous MeOH. Product [18F]17 was then eluted in 1.5 mL acetonitrile with a radiochemical purity of greater than 95% and a preparation time of 35 min. This compares well with the 50% radiochemical yield and synthesis time of 30-55 min reported for [18F]FB-CHO by Phoetko et al. (10). The amount of alcohol carried over to the conjugation stage was found to have an impact on the final conjugation yield. Reducing the amount of precursor 16 to 3 mg (8 µmol) gave an isolated yield of [18F]17 after Oasis SepPak purification (decay-corrected) of 43 ( 4% (n ) 13). Removal of the Boc group was performed by addition of 0.2 mL 2 M HCl in diethyl ether as previously described, liberating [18F]18. Excess solvent was removed in Vacuo at 65 °C under a stream of nitrogen for 3 min. To the residue was added a further 0.8 mL aliquot of acetonitrile and the drying process repeated before progressing to the next step. Conjugation was then initiated by dissolving [18F]18 in 0.4 M acetate buffer pH 5 (0.8 mL), followed by addition of either 9 or 10. In contrast to the [18F]15, [18F]18 was found to be stable with no observable cyclization in solution following Boc deprotection. Efficient conjugation with the maleimide precursor 10 was achieved by heating for 1 h at 70 °C giving [18F]28 in yields ranging 64-78% (n ) 3) as analyzed by radio-HPLC. In contrast, peptide 9, which in the nonradioactive experiments had proven to be the most reactive compound, demonstrated excellent reactivity at 30 °C forming [18F]27 in yields ranging 80-89% (n ) 5) after only 5 min (Figure 2a, 10.8 min). Indeed, the clean conjugation of the prosthetic group to the precursor was considered noteworthy, as closely eluting radioactive byproducts can be difficult to remove even by HPLC. These results generally compare favorably with other methodologies currently being employed in PET chemistry such as the [18F]GalactoRGD radiosynthesis using the prosthetic group [18F]fluoropropionate, which has a reported RCY for the acylation step of ∼85% (26). The thiol-based prosthetic groups gave comparable conjugation yields but required heating to 70-80 °C for 30 min (7). Likewise, for an aminooxy-modified somatostatin analogue, conjugation with [18F]FB-CHO gave a 60% RCY after 15 min at 60 °C, and for an RGD peptide, 60-80% RCY after 10-15 min at 60 °C has been reported (10).

The overall efficiency of the reaction sequence was calculated from starting activity of [18F]fluoride following isolation of final product by reversed-phase chromatography. Isolated yields of 12.0% and 8.5% were achieved for [18F]27 and [18F]28, respectively, as summarized in Table 4. The major significance of these preliminary findings were the rapid kinetics demonstrated by the N-methylaminooxy prosthetic group [18F]18 in combination with peptide precursor 9 at 30 °C. By avoiding high-temperature conjugation reactions, we can improve the chemical purity of the final conjugation mixture and reduce the complexity of the purification process. Peptides are known to have an inherent instability at elevated temperatures, and strategies which minimize the formation of byproduct are advantageous. We therefore anticipate that the methodology described in this study will provide a useful alternative for radiolabeling sensitive peptides for PET imaging.

CONCLUSION 18

F-Containing N-methylaminooxy compounds are useful prosthetic groups suitable for labeling of peptides. The 4-(2nitrovinyl)benzoyl moiety covalently linked to a model peptide was found to be highly reactive and selective toward the prosthetic group, enabling efficient labeling at 30 °C after 5 min in sodium acetate buffer at pH 5. This protocol therefore represents a novel methodology for the radiolabeling of peptides.

ACKNOWLEDGMENT We would like to thank David Grace and Tone Hauk Fritzell (GE Healthcare) for kindly supplying the NMR spectra. We finally thank Roger Smeets and the staff at Kjeller for technical assistance. Supporting Information Available: Additional information as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) Okarvi, S. M. (2004) Peptide-based radiopharmaceuticals: Future tools for diagnostic imaging of cancers and other diseases. Med. Res. ReV. 24, 357–397. (2) Massoud, T. F., and Gambhir, S. S. (2003) Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes DeV. 17, 545–580. (3) Lundqvist, H., and Tolmachev, V. (2002) Targeting peptides and positron emission tomography. Biopolymers (Peptide Science) 66, 381–392. (4) Okarvi, S. M. (2001) Recent progress in fluorine-18 labelled peptide radiopharmaceuticals. Eur. J. Nucl. Med. 28, 929–938. (5) de Bruin, B., Kuhnast, B., Hinnen, F., Yaouancq, L., Amessou, M., Johannes, L., Samson, A., Boisgard, R., Tavitian, B., and Dolle´, F. (2005) 1-[3-(2-[18F]Fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione: Design, synthesis, and radiosynthesis of a new [18F]Fluoropyridine-based maleimide reagent for the labeling of peptides and proteins. Bioconjugate Chem. 16, 406–420. (6) 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.

1308 Bioconjugate Chem., Vol. 19, No. 6, 2008 (7) Glaser, M., Karlsen, H., Solbakken, M., Arukwe, J., Brady, F., Luthra, S. K., and Cuthbertson, A. (2004) 18F-Fluorothiols: A new approach to label peptides chemoselectively as potential tracers for positron emission tomography. Bioconjugate Chem. 15, 1447–1453. (8) Cai, W., Zhang, X., Wu, Y., and Chen, X. (2006) A thiolreactive 18F-labeling agent, N-[2-(4-[18F)fluorobenzamido)ethyl]maleimide, and synthesis of RGD peptide-based tracer for PET imaging of Rvβ3 integrin expression. J. Nucl. Med. 47, 1172– 1180. (9) Moroder, L., Musiol, H. J., Schaschke, L., Chen, B., Hargittal, B., and Barany (2004) Thiol group. Synthesis of peptides and peptidomimetics (G. Goodman, M., Felix, A., Moroder, L. and Toniolo, C., Eds) pp 384-423, Chapter 2, Houben-Weyl, New York. (10) Poethko, T., Schottelius, M., Thumshirn, G., Hersel, U., Herz, M., Henriken, G., Kessler, H., Schwaiger, M., and Wester, H. J. (2004) Two-step methodology for high-yield routine radiohalogenation of peptides: 18F-labeled RGD and octreotide analogs. J. Nucl. Med. 45, 892–902. (11) Cuthbertson, A., Solbakken, M., Arukwe, J., Karlsen, H., and Glaser, M. WO Patent 2004/080492. (12) Schottelius, M., Poethko, T., Herz, M., Reubi, J-C., Kessler, H., Schwaiger, M., and Wester, H-J. (2004) First 18F-labeled tracer suitable for routine clinical imaging of SST receptorexpressing tumors using positron emission tomography. Clin. Cancer. Res. 10, 3593–3606. (13) Glaser, M., Morrison, M., Solbakken, M., Arukwe, J., Karlsen, H., Wiggen, U., Champion, S., Kinberg, G. M., Cuthbertson, A. ( 2008) Radiosynthesis and biodistribution of cyclic RGD peptides conjugated with novel [18F]fluorinated aldehydecontaining prosthetic groups Bioconjugate Chem. 19, 951–957. (14) Carrasco, M. ( 2005) N-Alkylaminooxy amino acids as versatile derivatives for“post translation”modifications of synthetic peptides, In Understanding Biology Using Peptides (Blondelle, S. E., Ed) pp 300-302, Springer, New York. (15) Bark, S. J., Schmid, S., and Hahn, K. M. (2000) A highly efficient method for site-specific modification of unprotected peptides after chemical synthesis. J. Am. Chem. Soc. 122, 3567– 3573.

Olberg et al. (16) Carrasco, M., Silva, O., Rawls, K. A., Sweeney, M. S., and Lombardo, A. A. (2006) Chemoselective alkylation of Nalkylaminooxy-containing peptides. Org. Lett. 8, 3529–3532. (17) Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595– 598. (18) Yamazaki, T., Ichige, T., and Kitazume, T. (2002) Convenient preparation of 2-phenylethyl 3,3-difluoro-2-methylpropionate. Collect. Czech. Chem. Commun. 67, 1479–1485. (19) Volonterio, A., Chiva, G., Fustero, S., Piera, J., Rosello, M. S., Sani, M., and Zanda, M. (2003) Stereocontrolled solid-phase synthesis of fluorinated partially-modified retropeptides via tandem aza-Michael/enolate-protonation. Tetrahedron Lett. 44, 7019–7022. (20) Park, J., and Pei, D. (2004) trans-β-Nitrostyrene derivatives as slow inhibitors of protein tyrosine phosphatases. Biochemistry 43, 15014–15021. (21) Li, M., Wu, R. S., Tsai, J. S. C., and Salamone, S. J. (2003) Evaluation of vinylsulfamides as sulfhydryl selective alkylation reagents in protein modification. Bioorg. Med. Chem. Lett. 13, 383–386. (22) Roush, W. R., Hernandez, A. A., and Zepeda, G. (1999) A new synthesis of peptidyl epoxysuccinates for probing cysteine protease-inhibitor P3/S3 binding interactions. Synthesis 1500– 1504. (23) Tamura, Y., Ikeda, H., Morita, I., Tsubouchi, H., and Ikeda, M. (1982) O-Arenesulfonyl-N-alkylhydroxylamines as aminating reagents. Chem. Pharm. Bull. 30, 1221–1224. (24) Langenhan, J. M., and Thorson, J. S. (2005) Recent carbohydrate-based chemoselective ligation applications. Curr. Org. Synth. 2, 59–81. (25) Thomesen, B., Bols, E., and Bols, M. (1997) Synthetic studies of fluorinated analogues of 1-azafagomine: Remarkable nucleophilic substitution of fluorine by hydrazine. Tetrahedron 53, 9357–9364. (26) Haubner, R., Kuhnast, B., Mang, C., Weber, W. A., Kessler, H., Wester, H., and Schwaiger, M. (2004) [18F]Galacto-RDG: Synthesis, radiolabeling, metabolic stability, and radiation dose estimates. Bioconjugate Chem. 15, 61–69. BC800007H