New Strategy for the Preparation of Clickable Peptides and Labeling

Mar 26, 2009 - Development and Evaluation of Endothelin-A Receptor (Radio)Ligands for Positron Emission Tomography. Kristin Michel , Katrin Büther ...
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Bioconjugate Chem. 2009, 20, 817–823

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New Strategy for the Preparation of Clickable Peptides and Labeling with 1-(Azidomethyl)-4-[18F]-fluorobenzene for PET David Thonon,* Ce´cile Kech, Je´roˆme Paris, Christian Lemaire, and Andre´ Luxen Cyclotron Research Center, Liege University, Sart-Tilman B.30, B-4000 Liege, Belgium. Received December 16, 2008; Revised Manuscript Received February 16, 2009

The alkyne-azide Cu(I)-catalyzed Huisgen cycloaddition, a click type reaction was used to label a peptide with fluorine-18. A novel solid phase synthesis approach for the preparation of clickable peptides has been developed and has also permitted the straightforward preparation of reference compounds. A complementary azide labeling agent (1-(azidomethyl)-4-[18F]-fluorobenzene) has been produced in a four step procedure in 75 min with a 34% radiochemical yield (decay corrected). Conjugation of [18F]fluoroazide with a model alkyne-neuropeptide produced the desired 18F-radiolabeled peptide in less than 15 min with a yield of 90% and excellent radiochemical purity.

INTRODUCTION Molecular imaging allows in vivo imaging of gene expression, optimizing drug therapy and imaging drug effect at the molecular and cellular level and may also be useful in detecting pathology at a predisease state (1). Thanks to its high sensitivity (10-9-10-12 M) and quantitative nature, positron emission tomography (PET1) is ideally suited to image molecular processes. Among a number of positron-emitting nuclides, fluorine-18 is particularly attractive for labeling biomolecules due to its favorable physical and nuclear properties. The challenge for the radiochemist is to introduce a short half-life radioisotope (fluorine-18, t1/2 109.7 min) onto biomolecules that are specific to the biological process studied. As 18F fluoride introduction onto organic compounds requires pretty harsh reaction conditions and is not compatible with H-acidic functions (2), biocompounds (e.g., peptides, proteins, antibodies, and oligonucleotides) are usually labeled with 18F by means of prosthetic groups (3). A large number of 18F labeled prosthetic groups have been developed (4), but none has emerged as a standard method because each has its own advantages and limitations (5). The main limitation of most advanced methods is that they are not site-specific as the labeling is accomplished through an amino group (N-terminus or lysine side chain), which is frequently contained in multiple copies in peptides. Therefore, the search for a rapid, selective, and generic method for radiolabeling biomolecules continues. Reactions defined as click reactions respond well to these criteria (6, 7). Moreover, these reactions are particularly well adapted to the preparation of radiopharmaceuticals as they require only benign reaction conditions and simple workup and purification procedures. Among these reactions, the Cu(I) catalyzed formation of 1,2,3triazole using Huisgen 1,3-dipolar cycloaddition of alkynes with * Corresponding author. Phone: +32 4 3662310. Fax: +32 4 3662946. E-mail: [email protected]. 1 Abbreviations: DIEA, N,N-diisopropylethylamine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; Enk, enkephalin (H-Tyr-GlyGly-Phe-Xxx-OH); [Leu5]-Enk (XxxdLeu); Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; Kryptofix 222, 4,7,13,16,21,24-hexaoxa-1,10diazabicyclo[8.8.8]-hexacosane; PET, positron emission tomography; pEPA and Phe (t), p-ethynyl-L-phenylalanine; QMA, quaternary methyl ammonium; SPPS, solid-phase peptide synthesis; TBAF, tetrabutylammonium fluoride; TFA, trifluoracetic acid; THF, tetrahydrofuran; TMS, tetramethylsilane; TMS-t, trimethylsilylacetylene.

azides is the most explored reaction. In principle, this cycloaddition can be performed in the presence of water and oxygen and is orthogonal to any functional group and can thus be performed without the protection of other functional groups (6, 7). For these reasons, our group and several other laboratories around the world have begun to evaluate this reaction to bind 18 F tagged molecules to biocompounds. In 2006, Marik and Sutcliffe reported the labeling of peptides bearing N-(3azidopropionyl)-groups with volatile ω-[18F]fluoroalkynes in 10 min with yields of 54-99% (coupling step) (8). Two other authors have recently reported the preparation of 18F labeled synthons bearing both an alkyne function and a short polyethylene glycol linker to reduce volatility and obtain water solubility (9, 10). Various azido-containing molecules have been labeled with these bifunctional tracers. In particular, Li has labeled azido-RGD peptides in 20 min with a yield of 69% (coupling step). A more indirect approach has been followed by Ramenda and Wuest who have prepared a click building block by derivatizing the well-known N-succinimidyl-4[18F]fluorobenzoate [18F]SFB with propargyl amine. This synthon has been used to label azido-neurotensin in 20 min with a yield of 66% (decay corrected, coupling step). Although a lot of effort has been applied to the preparation of an alkyne synthon, only one publication by Glaser and Årstad has described an azide synthon (11). After purification by distillation, 2-[18F]fluoroethylazide was linked to a model peptide derivatized with propargylic acid in 15 min with a yield of 92% (decay corrected, coupling step). In this technical note, we describe the radiosynthesis of a new azide synthon: 1-(azidomethyl)-4-[18F]-fluorobenzene [18F]5 bearing an aromatic bound fluorine, which is known to be less susceptible to in ViVo radiodefluorination (12). An original method to functionalize peptides with alkyne is also depicted. Finally, this new synthon has been used to label a model alkyne amino acid and an alkynylated neuropeptide by click chemistry (Figure 1).

EXPERIMENTAL PROCEDURES General. All solvents and chemicals were of analytical grade and used without further purification. The [18O]-enriched water was purchased from Cambridge Isotope Laboratories. Mass spectra were recorded with a Finnigan TSQ7000 mass spectrometer (ThermoElectronCorp.) operating in full-scan MS mode with an ESI+ source and with a Bruker Daltonics micrOTOF

10.1021/bc800544p CCC: $40.75  2009 American Chemical Society Published on Web 03/26/2009

818 Bioconjugate Chem., Vol. 20, No. 4, 2009

Figure 1. Synthetic strategies for the preparation of 18F labeled peptides and 19F references.

spectrometer (TOF-ES-MS). 1H and 13C NMR spectra were recorded on a Bruker AM 400 (1H at 400 MHz and 13C at 100 MHz). 1H and 13C spectra were referenced to TMS using the 13 C or residual proton signals of the deuterated solvents as internal standards. HPLC analyses were run on a Waters system (616 pump, a manual Rheodyne injector, 996 PDA detector,and NaI(Tl) scintillation detector from Eberline) controlled by the Empower program. Analyses were performed on an XTerra C18 column from Waters (150 × 4.6 mm, 3.5 µM) with CH3CN and H2O containing 0.1% TFA mixture (proportions given in parentheses) at 1 mL/min. Preparative HPLC was performed on a SymmetryPrep C18 column from Waters (300 × 7.8 mm, 7 µM). A Gilson model 233 XL reactarray automatic injection system was remotely controlled to achieve the radiosynthesis of [18F]5. A Bioscan TLC scanner model AR2000 was used for analysis of the 18F labeled compounds. Radiochemical yields are given decay corrected and take into account the losses due to transfer. p-Iodophenylalanine has been synthetized according to Ousmer (13), and Fmoc protection was carried out according to the standard procedure (14). 4-Ethynyl-L-phenylalanine (6) has been prepared according Zimmer (15). Peptide synthesis were carried out on an automated solid phase peptide synthesizer PS3 (Protein Technologies) by a Fmoc strategy employing HBTU as the coupling reagent. Enantiomerically pure L-amino acids were obtained from IRIS Biotech. Chemistry. (S)-2-Amino-3-(4-(1-(4-fluorobenzyl)-1H-1,2,3triazol-4-yl)phenyl)propanoic Acid (8). Twenty milligrams of 4-ethynyl-L-phenylalanine (6) (0.106 mmol), 40 mg of copper (I) iodide (2 equiv., 0.210 mmol), 55 mg of DIEA (4 equiv., 0.424mmol), and 16 mg of 1-(azidomethyl)-4-fluorobenzene (5) (0.106 mmol) were mixed in 3 mL of DMF. This solution was stirred for 4 h. The solvent was eliminated under vacuum. The residue obtained was suspended in acetonitrile and pre-purified by dry flash chromatography on silicagel (the column was washed by EtOAc and MeOH, and compound 8 was eluted by MeOH/NH3 25% 3:1). The solid obtained was finally purified by HPLC (4 mL/min, CH3CN/H2O 25:75, 0.1% TFA) to furnish 13 mg (0.038 mmol, 36%) of the desired compound. TOF-ESMS: m/z 341.1413 for [MH]+ (C18H18FN4O2, calculated [MW] 341.1408). 1H NMR (CD3OD), δ: 8.36 (s, 1H, CH triazole), 7.84 (m, 2H, HAr, C6H4), 7.40 (m, 4H), 7.13 (m, 2H, HAr, C6H4F), 5.64 (s, 2H, CH2C6H4F), 4.29 (m, 1H, CR,PheH), 3.33-3.17 (m, 2H, Cβ,PheH2) ppm.13C NMR (CD3OD) δ 178.6 (CdO), 166.75 (d, J ) 246 Hz, C-F), 134.4, 131.4, 130.0, 129.9, 129.8, 129.7, 125.9, 121.0, 115.3 (d, 3J ) 22 Hz, Cortho-F), 52.9 (CH2C6H4F), 47.6 (CR,Phe), 35.7 (Cβ,Phe) ppm. [Ac-Tyr(tBu)1, Phe (t)4, Leu5)]-Enk bound to Wang Resin 11. Four hundred milligrams of peptide 10 (e0.250 mmol) still

Technical Notes

bound to the resin was suspended in 4.5 mL of a degassed mixture of DMF/NEt3 (2:1). Then 179 mg of trimethylsilylacetylene (1.82 mmol) was added to the reaction mixture followed by 15 min of degassing. Then 25 mg of CuI (0.13 mmol) and 50 mg of Pd(PPh3)2Cl2 (0.071 mmol) were added, and the suspension was stirred overnight. The reaction mixture was filtered through a Bu¨chner funnel, and the resin was successively rinsed with DMF, H2O, EtOH, and CH2Cl2 and dried under reduced pressure. Two hundred milligrams (e0.125 mmol) of the obtained solid was suspended in 20 mL of THF at -78 °C. To this suspension was added 1600 µL of a solution 1 M of tetrabutylammonium fluoride in THF. This mixture was first stirred for 15 min at -78 °C then 30 min at RT. The reaction mixture was filtered through a Bu¨chner funnel, and the resin was successively rinsed with THF, H2O, EtOH, and CH2Cl2 and dried under reduced pressure. [Ac-Tyr1, Phe (1-(4-fluorobenzyl))-1H-1,2,3-triazol-4-yl)4, Leu5]-Enk 13. Eighty milligrams of peptide 11 bound to the Wang resin (e0.05 mmmol), 18 mg of copper (I) bromide (2 equiv., 0.126 mmol), 47 mg of DIEA (0.360 mmol), and 9 mg of 4-fluorobenzyle azide 5 (0.060 mmol) were mixed in 3 mL of DMF. The reaction medium was stirred under an inert atmosphere at RT overnight. The suspension was filtered through a Bu¨chner funnel and the resin was successively rinsed with DMF, H2O, EtOH, and CH2Cl2. Final deprotection and cleavage from the resin was performed with a TFA mixture (TFA/H2O/anisole ) 90:5:5, 5.5 mL, 3 h). The suspension was filtered and the resin washed once again with TFA. The filtrate was evaporated to dryness. The obtained oily residue was dissolved in a minimum of TFA, and ether was added to precipitate the peptide. This suspension was centrifuged (2500 rpm, 10 min). The supernatant was eliminated, and the obtained solid was washed with ether. The compound was purified by preparative HPLC (CH3CN/H2O 40:60 containing 0.1% TFA). Elimination of the solvent provided 10 mg of the targeted compound (0.013 mmol; yield starting from the commercial Wang resin, 26%). TOF-ESMS: m/z 773.3397 for [MH]+ (C39H46FN8O8, calculated [MW] 773.3417).1H NMR (CD3OD), δ: 8.29 (s, 1H, CH triazole), 7.73 (d, 2H, HAr), 7.40 (m, 4H, HAr), 7.10 (m, 4H, HAr), 6.70 (d, 2H, HAr), 5.63 (s, 2H, CH2C6H4F), 4,70 (m, 1H) and 4,40 (m, 2H) CR,Tyr, Leu, ethynylPheH, 3.68-3.86 (m, 4H, CR,GlyH), 2.87-3.33 (m, 4H, Cβ,Tyr, EthynylPheH2), 1.95 (s, 3H, CH3, acetyl), 1.68 (m, 1H, CH(CH3)2), 1.65 (m, 2H, Cβ,Leu), 0.93 (m, 6H, CH(CH3)2).13C NMR (CD3OD) 178.2, 173.8, 172.4, 170.8, 170.8, 170.0 (CdO), 169.7 (d, J ) 240 Hz, C-F), 156.0 (C-OHtyr), 137.4,131.8 129.8, 129.7, 129.6, 128.6, 128.1,127.4, 125.3, 120.7 (CH triazole), 115.5 (d, 3J ) 21 Hz, Cortho-F), 114.8 (Cortho C-OH Tyr), 56.9 (CH2C6H4F), 55.7, 54.3, 52.8 (CR,Tyr, Leu, ethynylPhe), 42.6, 41.9 (CR,Gly), 40.1, 37.0, 36.34 (Cβ,Tyr, Leu, ethynylPhe), 24.5, 21.9, 21.1, 20.4 (CH3,Leu, Acetyl, CHLeu) δ ppm. [Ac-Tyr(tBu)1, Phe (t)4, Leu5]-Enk 14. Eighty milligrams of peptide 11 bound to the resin (e0.05 mmmol) was suspended in 3 mL of (CH2Cl)2. Forty-five milligrams of trimethyltin hydroxide (0.16 mmol) was added to this suspension at RT under nitrogen. The reaction mixture was refluxed (83 °C) overnight under an inert atmosphere. The suspension was filtered through a Bu¨chner funnel and washed with (CH2Cl)2, CH2Cl2, MeOH, and EtOAc. The filtrate was brought to dryness under reduced pressure, and the resulting residue was dissolved in EtOAc, washed with an aqueous solution of HCl (0.05 N), and extracted with a 5% NaHCO3 solution. The pH of the combined aqueous solution was lowered to 3-4 with 2.5 N H2SO4 and extracted with EtOAc. The combined organic solution was dried (Na2SO4) and evaporated to dryness to yield 8 mg of a colorless solid (0.012 mmol; yield starting from the commercial Wang resin, 24%). TOF-ESMS: m/z 678.3489 for [MH]+ (C36H48N5O8, calculated [MW]

Technical Notes

678.3497).1H NMR (CD3OD), δ: 7.48 (d, 2H, HAr,EthynylPhe), 7.37 (d, 2H, HAr,EthynylPhe), 7.25 (d, 2H, HAr,tBuTyr), 7.02 (d, 2H, HAr,tBuTyr), 4.78 (dd, 1H), 4.60 (dd, 1H), 4.51 (dd, 1H), CR,Tyr, Leu, EthynylPheH, 3.77-3.97 (m, 4H, CR,GlyH), 3.42 (s, 1H, tCH), 3.00-3.30 (m, 4H, Cβ,Tyr, EthynylPheH2), 1.95 (s, 3H, CH3 acetyl), 1.68 (m, 1H, CH(CH3)2), 1.65 (m, 2H, Cβ,Leu), 1.29 (s, 9H, CH3, t-Bu), 0.91 (m, 6H, CH(CH3)2) ppm.13C NMR (CD3OD) 174.6, 174.0, 173.1, 172.5, 171.4, 170.6 (CdO), 154.7 (CAr, tBuTyr), 138.8, 132.8, 132.4, 130.1, 130.0, 124.6, 121.4 (CAr, tBuTyr, EthynylPhe), 83.8 (CtC), 79.2 (C(CH3)3, 77.7 (CHtC), 56.3, 54.9, 51.5 (CR,Tyr, Leu, EthynylPhe), 43.3, 42.7 (CR,Gly), 40.9, 37.9, 37.3 (Cβ,Tyr, Leu, EthynylPhe), 30.0 (CH3,t-Bu), 30.0, 29.9, 28.5, 25.3 (CH3Leu, Acetyl, CHLeu) δ ppm.

[Ac-Tyr(tBu)1, Phe (1-(4-fluorobenzyl))-1H-1,2,3-triazol-4yl)4, Leu5]-Enk 15. Protected peptide 15 was obtained by replacing the TFA treatment applied to 12 by a cleavage with trimethyltin hydroxide analogous to that applied to obtain peptide 14. Compound 15 was isolated by preparative HPLC purification (CH3CN/H2O 40:60 containing 0.1% TFA) to give 5 mg of a colorless solid (0.006 mmol, 12% starting from the commercial Wang resin). TOF-ES-MS: m/z 829.4012 for [MH]+ (C43H54FN8O8, calculated [MW] 829.4043).1H NMR (CD3OD), δ: 8.28 (s, 1H, CH triazole), 7.71 (d, 2H, HAr), 7.35 (m, 4H, HAr), 7.13 (m, 4H, HAr), 6.90 (d, 2H, HAr), 5.62 (s, 2H, CH2C6H4F), 4,72 (m, 1H), 4,48 (m, 2H) CR,Tyr, Leu, EthynylPheH, 3.70-3.87 (m, 4H, CR,GlyH), 2.82-3.28 (m, 4H, Cβ,Tyr, EthynylPheH2), 1.93 (s, 3H, CH3, acetyl), 1.68 (m, 1H, CH(CH3)2), 1.65 (m, 2H, Cβ,Leu), 1.35 (s, 9H, (CH3, t-Bu), 0.94 (m, 6H, CH(CH3)2).13C NMR

(CD3OD) 178.2, 173.8, 172.4, 170.8, 170.8, 170.0 (CdO), 169.7 (d, J ) 240 Hz, C-F), 156.0 (C-OH,tyr), 137.4,131.0 129.4, 129.7, 129.6, 128.6, 128.1,127.4, 123.3, 120.7 (CH triazole), 115.54 (d, 3J ) 21 Hz, Cortho-F), 114.8 (Cortho C-OH Tyr), 79.4 (C(CH3)3, 56.9 (CH2C6H4F), 55.7, 54.3, 52.8 (CR,Tyr, Leu, EthynylPhe), 42.6, 41.9 (CR,Gly), 40.1, 37.0, 36.34 (Cβ,Tyr, Leu, EthynylPhe), 30.4 (CH3 t-Bu), 24.5, 21.9, 21.1, 20.4 (CH3Leu, Acetyl, CHLeu) δ ppm.

Radiochemistry. 4-[18F]-Fluorobenzaldehyde [18F]2. Nocarrier-added [18F] fluoride was obtained by proton bombardment of an [18O]-enriched water target via the 18O(p,n)18F reaction. The activity was trapped by passing the target water through a Sep-Pak light QMA cartridge (Waters) previously conditioned under carbonate form. Then, 500 µL of a 50:50 CH3CN/H2O solution of K2CO3 (6 mg) and Kryptofix 222 (20 mg) were used to elute the fluoride from the cartridge into a heated conical glass vial (120 °C). This eluate (110-740 MBq) was brought to dryness by azeotropic distillation after the addition of acetonitrile (3 × 250 µL) under a gentle stream of nitrogen gas. 4-N,N,N-Trimethylammonium benzaldehyde triflate (0.064 mmol) in DMSO (1 mL) was added to the dried residue, and the mixture was heated at 130 °C for 5 min. The reaction medium was then diluted with water (15 mL). Labeling efficiency was checked by radio-TLC (silica gel, CH2Cl2; Rf values: [18F]fluoride ) 0; [18F]2 ) 0.6). HPLC analysis: tR ) 3.9 min (CH3CN/H2O 50/50 0.1% TFA). Radiochemical yield (decay corrected) ) 65%. 1-(Bromomethyl)-4-[18F]-fluorobenzene [18F]4. The crude labeled compound [18F]2 (250 MBq) diluted in H2O was adsorbed onto a C18 environmental Sep-Pak cartridge. NaBH4 (125 mg dissolved in 4 mL of H2O) was passed with a flow rate of 3 mL/min through the cartridge then washed with water (5 mL). The 4-[18F]-fluorobenzyl alcohol formed was not isolated but directly converted into the corresponding bromide derivative by heating the Sep-Pak (50 °C) with a stream of hot air while passing 48% aqueous HBr (vol ) 2.8 mL; flow rate ) 3 mL/min) through the cartridge. After a delay of 4 min, an additional portion of concentrated HBr (1.4 mL) was passed in the same manner. After 4 min, heating was stopped, and 6 mL of an ammonium formate solution (pH 4.0) was added to the Sep-Pak. The bromide compound [18F]4 was finally eluted with

Bioconjugate Chem., Vol. 20, No. 4, 2009 819

DMF (3.5 mL) and was checked by radio-TLC (silica gel, CH2Cl2; Rf values: [18F]4 ) 1.0; in n-hexane, Rf [18F]4 ) 0.3). HPLC analysis: tR ) 9.0 min (CH3CN/H2O 40/60 0.1% TFA). Radiochemical yield (decay corrected) from 2 was 75%. 1-(Azidomethyl)-4-[18F]-fluorobenzene [18F]5. Compound [18F]4 (150 MBq) in DMF (3.5 mL) was transferred into a reaction vial containing 500 mg of azido resin (1.9 mmol). This suspension was agitated for 15 min by gentle N2 bubbling and then filtered. The resin was washed with 1 mL of DMF, then filtered again. The filtrates were combined and analyzed by radio-TLC (silica gel, CH2Cl2; Rf values: [18F]5 ) 1.0; in n-hexane, Rf [18F]5 ) 0.1). HPLC analysis: tR ) 7.8 min (CH3CN/H2O 40/60 0.1% TFA). Radiochemical yield (decay corrected) from [18F]4 70%. (S)-2-Amino-3-(4-(1-(4-[18F]-fluorobenzyl)-1H-1,2,3-triazol4-yl)phenyl)propanoic Acid [18F]8. A solution of 1-(azidomethyl)-4-[18F]-fluorobenzene [18F]5 (20MBq) in DMF (1 mL) was added to (S)-2-amino-3-(4-ethynylphenyl) propanoic acid (6) (pEPA, 0.6 mg, 0.0032 mmol), CuI (3.2 mg, 0.017 mmol), sodium ascorbate (25 mg, 0.13mmol), and DIEA (50 µL, 0.28 mmol). The mixture was stirred at room temperature for 15 min. The reaction mixture was then diluted with water (15 mL) and passed through a Sep-Pak Plus Short tC18 cartridge. The cartridge was washed with water (9 mL), and activity was eluted with CH3CN (1.5 mL). Analysis by radio-TLC (silica gel, EtOAc; Rf values: [18F]8 ) 0; C18, CH3CN/H2O 50/50, Rf values, [18F]8 ) 0.5). HPLC analysis: tR ) 3.1 min (CH3CN/H2O 40/60 0.1% TFA). Radiochemical yield (decay corrected) from [18F]5 90%. [Ac-Tyr1, Phe (1-(4-[18F] fluorobenzyl))-1H-1,2,3-triazol-4yl)4, Leu5] Enk [18F]13. A solution of [18F] fluoroazide [18F]5 in DMF (1 mL) was added to peptide 14 (2 mg, 0.0032 mmol), CuI (3.2 mg, 0.017 mmol), sodium ascorbate (25 mg, 0.13 mmol), and DIEA (50 µL, 0.28 mmol). The mixture was stirred at room temperature for 15 min. Analysis by radio-TLC (silica gel, CH2Cl2; Rf values: [18F]15 ) 0; C18, CH3CN/H2O 60/40, Rf values, [18F]15 ) 0.6). HPLC analysis: tR ) 6.3 min (CH3CN/H2O 50/50 0.1% TFA). The reaction mixture was then diluted with water (15 mL) and passed through a Sep-Pak Plus Short tC18 cartridge. The column was washed with water (9 mL), and activity was directly eluted with TFA (1.5 mL) in the deprotection vial. Deprotection was finished under stirring in less than 3 min. Analysis by radio-TLC (silica gel, CH2Cl2; Rf values: [18F]13 ) 0; C18, CH3CN/H2O 50/50, Rf values, [18F]13 ) 0.30). HPLC analysis: tR ) 5.6 min (CH3CN/H2O 40/60 0.1% TFA). Radiochemical yield (decay corrected) from [18F]5 90%. Radiochemical purity >95%.

RESULTS AND DISCUSSION Synthesis. The strategy we have followed to functionalize peptides with alkynes was based on the Sonogashira reaction. This cross-coupling reaction between iodoaryl and alkyne is catalyzed by Pd0 complexes in mild conditions and is insensitive to all side groups. Furthermore, functionalization through Sonogashira coupling generates regioselectively and quantitatively a very stable C-C bond (16). Therefore, we planned to introduce the residue p-iodophenylalanine, a non-natural amino acid, into the peptide chain and to convert it to p-ethynylphenylalanine through the Sonogashira coupling reaction of ethynyltrimethylsilane (Scheme 1). The direct introduction of an ethynyl group is also possible but is not compatible with trifluoroacetic acid (17), which is predominantly used in peptidic synthesis. Moreover, the synthesis of p-ethynylphenylalanine is more demanding than the simple preparation of p-iodophenylalanine (13, 18). A similar approach has been described by Hoffmans who has used the Sonogashira coupling to label

820 Bioconjugate Chem., Vol. 20, No. 4, 2009

Technical Notes

Scheme 1. Synthesis of References (13, Deprotected, and 15, Protected) and Clickable Alkyne Peptide 14

[Leu5]Enkephalin (a neuropeptide that is a natural ligand of the opiate receptors) with ethynylferrocene (19). Thus, we have also chosen to use [Leu5]Enkephalin as our model peptide because it is a small peptide, easy-to-handle to set up its labeling with fluorine-18 by click chemistry with 4-[18F]fluorobenzyl azide. In the context of this work, no effort was made to preserve or improve the targeting ability or the in vivo stability of the labeled peptide. Readers interested in the imaging of the opiate receptors should read the following publications (20-22). Unlike Hoffmans, we decided to keep the [Leu5]Enk peptide bound to the solid support (Wang resin) for the next steps to facilitate purification and isolation of the peptides 11-15. The Sonogashira coupling between iodophenyl-derivatized peptide and trimethylsilylacetylene was carried out in conditions close to those described by Hoffmans. Excess reagents and catalysts were simply eliminated by filtration and washing. Completion of the reaction was checked by ES-MS after cleavage of small aliquots of the peptide by TFA. Deprotection of the alkyne group was achieved in the presence of TBAF in THF to furnish alkyne-peptide 11 still bound to the Wang resin. Reference Compound Preparation. Conjugation of 4-ethynyl-L-phenylalanine (6) with 1-(azidomethyl)-4-fluorobenzene was conducted in DMF or acetonitrile, in the presence of DIEA

and excess copper iodide (2 equiv.). When catalytic amounts of copper were used, the reaction did not start, possibly because of copper ion complexation by the amino acid (23). For cold chemistry, click conjugation of the peptide was carried out with the alkyne-peptide still bound to the resin support (Scheme 1). In the conditions used with 4-ethynyl-L-phenylalanine, compound 13 was obtained but iodo- and hydroxytriazole were also formed as secondary products. This problem was circumvented by replacing copper iodide with copper bromide and using an inert atmosphere. The reference peptide 13 was deprotected and removed from the resin by treatment with TFA. The protected reference peptide 15 was isolated by replacing the TFA treatment by a cleavage with trimethyltin hydroxide as described by Furlan (24). This solid phase method permits a rapid and easy preparation of reference peptides, which could be very valuable in a combinatorial approach. Radiolabeling. The synthesis of 1-(azidomethyl)-4-[18F]fluorobenzene [18F]5, a labeling agent for the subsequent click reaction, was carried out in four steps (Scheme 2) starting from the well-known nucleophilic fluorination of 4-formylN,N,N-trimethylanilinium triflate (1) (25-28). Radiofluorination was accomplished at 130 °C in DMSO for 5 min with an overall yield of 65%. This crude medium was diluted in

Technical Notes Scheme 2. Radiosynthesis of 1-(Azidomethyl)-4-[18F]-fluorobenzene

water, then passed on a Waters C18 environmental Sep-Pak cartdrige to trap compound 2[18F]. This compound was reduced to 4-[18F]-fluorobenzyl alcohol [18F]3 by slowly passing a solution of NaBH4 in water through the C18 cartridge following a procedure previously described (29, 30). The alcohol formed was not eluted from the cartridge but directly converted to the bromide [18F]4 by passing 48% aqueous HBr by small portions through the C18 cartdridge. At RT, less than 25% of the alcohol was converted into the bromide derivative. Fortunately, by heating the cartridge at 50 °C via a stream of hot air, the conversion was nearly quantitative. After neutralization of the medium by passing an ammonium formate buffer, the bromide [18F]4 was eluted by DMF (CH3CN, toluene, and CH2Cl2 were also used). The bromide was obtained with a radiochemical yield of around 75% (decay corrected). Previously, this bromination was conducted in solution using PBr3 or Ph3PBr2 (29, 31), SOBr2 (32, 33), HBr 48% (34), or gaseous HBr (35). None of these methods is well suited for an easily automated routine production. Our method avoids using gas reagents, and excess reagents are readily and easily eliminated. This methodology may be extended beyond the present application as the fluorobenzyl bromide synthon is a common structure in some radiopharmaceuticals used for PET imaging (30, 31, 34). Finally, 1-(azidomethyl)-4-[18F]-fluorobenzene [18F]5 was formed using a commercially available azide exchange resin (azide on Amberlite IRA-400) as described by Hassner (36). In DMF at RT, the reaction was completed in 15 min using 500 mg of the resin. In a less polar solvent such as acetonitrile, the reaction was slower (75% of conversion after 25 min). Tests conducted in the laboratory have shown that this reaction is insensitive to the presence of water and carbonate or formate salts. Nevertheless, acidic media inhibit the reaction. Resin is simply eliminated by filtration and the targeted azide collected in solution in DMF, ready to use for click reactions. Hence, starting from 700 MBq [18F]F-, a total amount of 238 MBq (34%, decay corrected) of [18F]5 could be synthesized within 75 min. Click Reactions with 1-(Azidomethyl)-4-[18F]-fluorobenzene [18F]5. Several catalytic systems have been described for Huisgen 1,3-dipolar cycloaddition (6, 7). In experiments to link [18F]fluoroalkyne groups onto peptides, Marik and Sutcliffe have obtained better results using CuI than with Cu(I) generated in situ from copper sulfate and sodium ascorbate (8). Paradoxically, other teams have preferred in situ generation to click [18F]fluoroprosthetic groups onto biomolecules. As we have obtained good results with copper (I) salts to prepare the cold references, we have chosen to follow the Marik and Sutcliffe approach. First, experiments with various conditions have been conducted on p-EPA (Table 1 and Scheme 3). The solvent greatly influences the reaction rate, which is much higher in DMF than in CH3CN. This could be due at least partially to the better solubility of the alkyne in DMF. The reduction of the alkyne concentration also has a great impact on the reaction rate, which becomes insufficient at 0.003 M in the initial conditions (entry 4). Acceptable reaction rates were recovered by adding sodium ascorbate in the reaction medium (entry 5). Moreover, copper

Bioconjugate Chem., Vol. 20, No. 4, 2009 821 Table 1. Influence of Reagent Concentration on the Radiochemical Yield for the Preparation of [18F] 8 with 1-(Azidomethyl)-4-[18F]-fluorobenzene [18F]5 (RT, DMF) alkyne (M)

CuI (M)

DIEA (M)

ascorbate (M)

solvent

reaction time (min)

yield (TLC)

0.050 0.050 0.05 0.003 0.003

0.105 0.105 0.105 0.105 0.017

0.600 0.600 0.600 0.600 0.280

0 0 0 0 0.130

AN AN:DMF 1:1 DMF DMF DMF

50 30 7 15 15

67% 85% 90% 40% 95%

Scheme 3. 1,3-Dipolar Cycloaddition of 1-(Azidomethyl)-4-[18F]-fluorobenzene with Alkyne Peptides or Amino Acid

ions and base concentrations can also be diminished when ascorbate is used since the reaction still completes in less than 15 min at RT. Thus, similar conditions have been applied to conjugate alkyne-peptide 14 with 1-(azidomethyl)-4-[18F]fluorobenzene [18F]5. Fast coupling was also observed by radio-HPLC: labeling of the peptide was finished in less than 15 min. The radiolabeled peptide was trapped on a t-C18 cartridge, washed with water, and eluted with TFA. Deprotection of the tyrosine residue was accomplished in less than 3 min, and the targeted labeled peptide was obtained with a yield of 90% (coupling step + deprotection, decay corrected) and a radiochemical purity >95%. We would also like to point out that these results were obtained at room temperature with very diluted solutions of alkyne (0.003 mmol/mL), which is about 10 times less than that used by Sirion, Ramenda, or Glaser. Similar results have been reported by Marik and Sutcliffe who also used a copper (I) salt and not the CuSO4/sodium ascorbate catalytic system used by the other groups. The low quantity of peptide required facilitates the HPLC separation of the labeled peptide from the unlabeled peptide precursor, which is essential to obtain suitable chemical purity.

CONCLUSIONS A novel strategy for the preparation of clickable peptides has been developed. It can be applied to any peptides modified beforehand to contain a non-natural iodophenyl group. As demonstrated here on a model peptide, this moiety can be simply introduced during the synthesis of the peptide by substituting the modified amino acid 4-iodophenylalanine for a phenylalanine residue. Furthermore, as the selective monoiodination of tyrosine residue in peptides with IPy2BF4 has been reported by Espun˜a et al., our approach can also been extended to directly label tyrosine containing peptides (37). In cases where the peptide does not contain any Phe or Tyr groups, 4-iodobenzoic acid can be used to N-terminally modify the peptide eventually through an appropriate linker (19). A new labeling agent 1-(azidomethyl)-4-[18F]-fluorobenzene 18 [ F]5 has been produced in 75 min with a 34% radiochemical

822 Bioconjugate Chem., Vol. 20, No. 4, 2009

yield (decay corrected). These satisfying results for a four-step procedure have been obtained thanks to the exploitation of solid phase supported reactions and the absence of the solvent evaporation process that allow one to minimize the losses of time and radioactivity during reaction workup and purification. Moreover, this radiosynthesis has been fully automated in our laboratory. Although all of these advantages are present, this click labeling agent is obtained in a less straightforward manner than others produced in a single step (8, 10, 11). However, the radiocompound [18F]5 is advantageous in that it is nonvolatile and bears an aromatic bound fluorine, which is known to be less susceptible to in ViVo radiodefluorination. For these reasons, it will be very interesting to produce 1-(azidomethyl)-4-[18F]fluorobenzene [18F]5 in one or two fast steps. Work in this direction is in progress in our laboratory. Furthermore, this compound has been bound very rapidly (10 min), at room temperature, with a very diluted solution of alkyne (0.003M), in mild conditions and with an excellent yield to a model alkyne-peptide.

ACKNOWLEDGMENT We acknowledge the financial support from the Biowin Project of the Walloon Region (NeoFor and KeyMarker).

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