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Bioconjugate Chem. 2009, 20, 2254–2261

Direct One-Step18F-Labeling of Peptides via Nucleophilic Aromatic Substitution Jessica Becaud,† Linjing Mu,† Myle`ne Karramkam,† Pius A. Schubiger,† Simon M. Ametamey,*,† Keith Graham,‡ Timo Stellfeld,‡ Lutz Lehmann,‡ Sandra Borkowski,‡ Dietmar Berndorff,‡ Ludger Dinkelborg,‡ Ananth Srinivasan,*,‡ Rene´ Smits,§ and Beate Koksch§ Center for Radiopharmaceutical Science of ETH, PSI and USZ, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland, Bayer Schering Pharma AG, Global Drug Discovery, D-13342 Berlin, Germany, Department of Chemistry and Biochemistry - Organic Chemistry, FU Berlin, Takustraβe 3, D-14195 Berlin, Germany. Received June 3, 2009; Revised Manuscript Received October 30, 2009

Methods for the radiolabeling molecules of interest with [18F]-fluoride need to be rapid, convenient, and efficient. Numerous [18F]-labeled prosthetic groups, e.g., N-succinimidyl 4 [18F]-fluorobenzoate ([18F]-SFB), 4-azidophenacyl[18F]-fluoride ([18F]-APF), and 1-(3-(2-[18F]fluoropyridin-3-yloxy)propyl)pyrrole-2,5-dione ([18F]-FpyMe), for conjugating to biomolecules have been developed. As the synthesis of these prosthetic groups usually requires multistep procedures, there is still a need for direct methods for the nucleophilic [18F]-fluorination of biomolecules. We report here on the development of a procedure based on the trimethylammonium (TMA) leaving group attached to an aromatic ring and activated with different electron-withdrawing groups (EWGs). A series of model compounds containing different electron-withdrawing substituents, a trimethylammonium leaving group, and carboxylic functionality for subsequent coupling to peptides were designed and synthesized. The optimal model compound, 2-cyano-4-(methoxycarbonyl)-N,N,N-trimethylbenzenaminium trifluoromethanesulfonate, was converted to carboxylic acid and coupled to peptides. The results of the one-step [18F]-fluorination of tetrapeptides and bombesin peptides show that the direct 18F-labeling of peptides is feasible under mild conditions and in good radiochemical yields.

18

INTRODUCTION Positron emission tomography (PET) is of particular interest in diagnostic nuclear medicine as it can track positron emitting probes in a repetitive and noninvasive manner (1, 2). 18F is considered an ideal PET isotope because it has the best imaging characteristics due to the low positron energy (0.64 MeV). In addition, the 110 min half-life of 18F is sufficiently long to permit complex or multistep organic syntheses, commercial distribution to other imaging centers, and extended in ViVo investigations. In general, large biomolecules such as peptides or oligonucleotides are not readily amenable to direct fluorination with no-carrier-added (nca) 18F-fluoride. As a result, 18F has been incorporated into many biologically active molecules such as peptides (3-6), proteins (7, 8), and oligonucleotides (9, 10) via indirect methods using as building blocks so-called prosthetic groups. These prosthetic groups serve to enhance the efficiency and site-specificity of labeling. The first report on protein labeling with 18F-fluoride was published by Mueller-Platz et al. (11) who tagged urokinase with 18F-acetate. Herman et al. (12) successfully labeled human serum albumin (HSA) protein using two pentafluorophenyl derivatives as prosthetic groups. The well-known 18F-SFB (13, 14) has been employed by several research groups to label bioactive molecules. Besides the aforementioned 18F labeling synthons, a large number of other * Corresponding author. Center for Radiopharmaceutical Science of ETH, PSI and USZ, ETH Ho¨nggerberg D-CHAB IPW HCI H427, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland. Tel.: +41 44 633 74 63; fax: +41 44 633 13 67. E-mail address: [email protected] (S. Ametamey). † ETH Zurich. ‡ Bayer Schering Pharma AG. § FU Berlin.

F-labeled prosthetic groups have been investigated (12, 15, 16) and prominent routes for their conjugation to macromolecules have been mainly via 18F-fluoroalkylation (16, 17), 18F-fluoroacylation (18, 19), 18F-fluoroamidation (20, 21), thiol-coupling (22, 23), or photochemical conjugation (8, 24). Among the above-mentioned methods, nucleophilic aromatic fluorination with nca 18F-fluoride has been well-established. The 18F-for+ N(CH3)3 substitution was first reported more than two decades ago, and a large variety of 18F-labeled aromatic radiopharmaceuticals have been reported (25). A disadvantage, however, of the established methods is the fact that the synthetic methods entail multistep syntheses and the 18F label is incorporated very early in the synthesis sequence. In order to circumvent this shortcoming, research groups are focusing on new synthetic strategies that avoid the use of prosthetic groups. Recent studies have demonstrated the feasibility of labeling organoboron and organosilicon bioconjugates with 18F in a single step (26-28). The one-step process can easily be automated for routine production. The aim of this work was to develop a direct onestep method for the nca fluorine-18 labeling of peptides.

EXPERIMENTAL PROCEDURES General. All chemicals unless otherwise stated were purchased from Sigma-Aldrich or Merck and used without further purification. Fmoc-amino acids were purchased from IRIS Biotech except Fmoc-statine and FA01010, which were purchased from NeoMPS (now Polypeptide). The solvents were of HPLC quality. Peptide syntheses were carried out using Rink amide resin (0.68 mmol/g) following the standard Fmoc strategy (29). All amino acid residues were, if not further specified, L-amino acid residues. FA01010 denotes (4R,5S)-4-amino-5methylheptanoic acid, statine (Sta) (3S,4S)-4-amino-3-hydroxy6-methylheptanoic acid, and Ava 5-aminopentanoic acid. High-

10.1021/bc900240z  2009 American Chemical Society Published on Web 11/18/2009

Direct 18F-Labeling of Peptides

performance liquid chromatography (HPLC) analyses were performed using either a Altech Econsphere C18 RP column (53 × 7 mm, 3 µm) or an ACE C18 column (50 × 4.6 mm, 3 µm) under the indicated conditions. Analytical HPLC chromatograms were obtained using an Agilent 1100 system with Gina software, equipped with UV multiwavelength and Raytest Gabi Star detectors. Semipreparative HPLC purifications were carried out using a semipreparative ACE C18 column (250 × 10 mm, 5 µm) under the indicated conditions. Semipreparative HPLC system used was a Merck-Hitachi L6200A system equipped with Knauer variable wavelength detector and Eberline radiation detector. TMA-Based Peptides Syntheses. Peptide Synthesis (General Procedure). The resin-bound Fmoc peptide was treated with 20% piperidine in DMF (v/v) for 5 min. This step was repeated with a reaction time of 20 min, and thereafter, the resin was washed with DMF (2×), CH2Cl2 (2×), and DMF (2×). A solution of Fmoc-Xaa-OH (Xaa ) amino acid, 4 equiv), HBTU (O(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 4 equiv), HOBT (1-hydroxybenzotriazole, 4 equiv), DIPEA (N,N′-di-iso-propylethylamine, 8 equiv) in DMF was added to the resin-bound free amine peptide and shaken for 90 min at room temperature. This step was repeated with a reaction time of 60 min, and the resin was washed with DMF (2×), CH2Cl2 (2×), and DMF (2×). The two steps of Fmocdeprotection and amino acid addition were then repeated until the final elongation of the desired peptide was achieved. The peptides were typically prepared starting with 147 mg (0.1 mmol) of the resin. The amounts of reagents and building blocks in all subsequent reactions were calculated on the basis of this amount. Procedure for the Synthesis of (4-Trimethylammonium-3-cyanobenzoyl)-Functionalized Peptides Trifluoroacetate Salts (Precursor Compounds 6a-11a). Representative procedure: N-methylmorpholine (NMM, 22 µL, 0.2 mmol) was added to a solution of 2-cyano-4-carboxylphenyl)trimethylammonium triflate salt (71 mg, 0.2 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin2-yl)-4-methylmorpholinium tetrafluoroborate (66 mg, 0.2 mmol) in DMF (2 mL). The mixture was added to the resin-bound, side-chain-protected, Fmoc-deprotected peptide (0.1 mmol, based on the initial resin loading), which was prepared by following the general procedures described above. The reaction mixture was shaken intensively for 4 h. The resin was then filtered and washed with DMF (3 × 4 mL) and CH2Cl2 (3 × 4 mL). The coupling step was repeated. Thereafter, the resin was treated with a mixture of trifluoroacetic acid, distilled water, phenol, and triisopropylsilane (85/5/5/5, 1.5 mL) for 3 h. The mixture was added to ice-cold methyl tert-butyl ether, and the precipitate was separated by centrifugation. Water was added to the pellet, and the supernatant was lyophilized. The residue was purified by preparative RP-18 HPLC-MS with a gradient of 5-30% acetonitrile in 20 min and 0.1% trifluoroacetic acid as cosolvent. The desired fraction was collected and lypophilized, and the product was analyzed by HPLC-MS. 3-Cyano-4-trimethylammonium-benzoyl-Val-βAla-Arg-GlyNH2-trifluoroacetate salt (tetrapeptide 6a, 17 mg, 24%): m/z M+ calcd 587.3; found 587.4. 3-Cyano-4-trimethylammonium-benzoyl-Val-βAla-Phe-GlyNH2-trifluoroacetate salt (tetrapeptide 7a, 25 mg, 36%): m/z M+ calcd 578.3; found 578.6. 3-Cyano-4-trimethylammonium-benzoyl-Val-βAla-Lys-GlyNH2-trifluoroacetate salt (tetrapeptide 8a, 19.5 mg, 29%): m/z M+ calcd 559.3; found 559.1. 3-Cyano-4-trimethylammonium-benzoyl-Val-βAla-Met-GlyNH2-trifluoroacetate salt (tetrapeptide 9a, 19 mg, 28%): m/z M+ calcd 562.3; found 562.5.

Bioconjugate Chem., Vol. 20, No. 12, 2009 2255

3-Cyano-4-trimethylammonium-benzoyl-Ava-Gln-Trp-AlaVal-Gly-His-FA01010-Leu-NH2-trifluoroacetate salt (10a, 0.03 mmol scale, 15 mg, 37%): m/z M+ calcd 1235.7; found 1235.8. 3-Cyano-4-trimethylammonium-benzoyl-Arg-Ava-Gln-TrpAla-Val-NMeGly-His-Sta-Leu-NH2-trifluoroacetate salt (11a, 10 mg, 7%): m/z [M+H]2+/2 calcd 711.4; found 711.9. Syntheses of Fluorinated TMA-Based Peptides (Reference Compounds 6b-11b). Representative procedure: A solution of 3-cyano-4-fluorobenzoic acid (66 mg, 4 equiv), HBTU (O(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 151 mg, 4 equiv), HOBT (1-hydroxybenzotriazole, 61 mg, 4 equiv), and DIPEA (N,N′-di-iso-propylethylamine, 70 µL, 4 equiv) in DMF (2 mL) was added to the resin-bound, side-chain-protected peptide (0.1 mmol, based on the initial resin loading), which was prepared by following the general procedures described above. The reaction mixture was shaken intensively for 4 h. The resin was then filtered and washed with DMF (3 × 4 mL) and CH2Cl2 (3 × 4 mL). Thereafter, the resin was treated with a mixture of trifluoroacetic acid, distilled water, phenol, and triisopropylsilane (85/5/5/5, 1.5 mL) for 3 h. The mixture was added to ice-cold methyl tert-butyl ether, and the precipitate was separated by centrifugation. Water was added to the pellet, and the supernatant was lyophilized. The residue was purified by preparative RP-18 HPLC-MS with a gradient of 5-50% acetonitrile in 20 min and 0.1% trifluoroacetic acid as cosolvent. The desired fraction was collected and lypophilized, and the product was analyzed by HPLC-MS. 3-Cyano-4-fluorobenzoyl-Val-βAla-Arg-Gly-NH2 (tetrapeptide 6b, 32 mg, 58%): m/z [M+H]+ calcd 548.3; found 548.0. 3-Cyano-4-fluorobenzoyl-Val-βAla-Phe-Gly-NH2 (tetrapeptide 7b, 18 mg, 33%): m/z [M+H]+ calcd 539.2; found 539.1. 3-Cyano-4-fluorobenzoyl-Val-βAla-Lys-Gly-NH2 (tetrapeptide 8b, 16.5 mg, 32%): m/z [M+H]+ calcd 520.3; found 520.1. 3-Cyano-4-fluorobenzoyl-Val-βAla-Met-Gly-NH2 (tetrapeptide 9b, 17 mg, 33%): m/z [M+H]+ calcd 523.2; found 523.0. 3-Cyano-4-fluorobenzoyl-Ava-Gln-Trp-Ala-Val-Gly-HisFA01010-Leu-NH2 (10b, 0.03 mmol scale, 9 mg, 25%): m/z [M+H]+ calcd 1196.6; found 1196.7. 3-Cyano-4-fluorobenzoyl-Arg-Ava-Gln-Trp-Ala-Val-NMeGlyHis-Sta-Leu-NH2 (11b, 0.03 mmol scale, 6.5 mg, 16%): m/z [M+2H]2+/2 calcd 692.9; found 692.4. 18 F-Radiolabeling of TMA-Based Model Compounds and Peptides. General. No-carrier-added [18F] fluoride was produced via the 18O (p,n) 18F nuclear reaction by irradiation of enriched [18O] water. 18F-fluoride was trapped on an anion-exchange resin cartridge (Sep-Pak QMA light, Waters). The cartridge was eluted with a solution of Kryptofix (5 mg), potassium carbonate (1 mg), or Cs2CO3 (2.4 mg) in water (500 µL) and MeCN (1 mL). The solvent was removed by azeotropic drying at 110 °C under vacuum for 10 min with a stream of nitrogen. Anhydrous MeCN (1 mL) was added and evaporated. This step of adding acetonitrile (1 mL) was repeated two more times. The tested precursor was dissolved in DMSO and added to the dry K[18F]F/ K2.2.2. or Cs[18F]F/K2.2.2 complex. The reaction mixture was then heated at 50-70 °C for 5-15 min. An aliquot was taken from the reaction mixture and analyzed by analytical HPLC. The 18Flabeled product was confirmed by coinjection with the nonradioactive reference compound on the same analytical HPLC using either ACE C18 column (50 × 4.6 mm, 3 µm) or Altech Econsphere C18 RP column (53 × 7 mm, 3 µm). Elute conditions with ACE C18 column: 10 mM K2HPO4 in water (solvent A), 10 mM K2HPO4 in water/acetonitrile 3/7 (solvent B); 0 min, 5% B; 0-7.00 min, 5f95% B; 7.00-7.10 min, 95f100% B; 7.10-8.80 min, 100% B; 8.80-9.00 min, 100f5% B. The flow rate was 2 mL/min. Elute conditions with Altech Econsphere C18 RP column: 0.1% aq TFA (solvent A), 0.1% TFA in acetonitrile/water 9/1 (solvent B); 0 min, 5% B;

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Figure 1. Structures of synthesized TMA-based model precursor compounds 1a-5a and their corresponding fluorinated compounds 1b-5b.

0-7 min, 5f95% B; 7.00-7.10 min, 95f100% B; 7.10-8.80 min, 100% B; 8.80-9.00 min, 100f5% B; flow rate: 2 mL/ min. 18 F-Radiolabeling of TMA-Based Model Compounds. TMAbased bifunctional model precursor compounds (1a-5a, Figure 1) were synthesized and characterized (see Supporting Information). Their corresponding reference compounds (1b-5b) are, in most cases, commercially available. Typical experimental conditions for the 18F-radiolabeling of precursor compounds 3a and 5a are described below. Methyl-3-cyano-4-[18F]fluorobenzoate ([18F]3b). Compound 3a (4.2 mg) in anhydrous DMSO (300 µL) was added to a reaction vial containing dry K[18F]F/K2.2.2 complex (6.57 GBq). After heating at 50 °C for 15 min, an aliquot was taken for HPLC analysis (ACE C18 column), and more than 90% 18Fincorporation was obtained. The reaction mixture was diluted with water (1.5 mL), and this solution (3.86 GBq) was injected onto an ACE semipreparative HPLC column (0.1% TFA in water (solvent A), 0.1% TFA in acetonitrile/water 9/1 (solvent B), isocratic 30% B, flow rate: 4 mL/min). The decay-corrected radiochemical yield of the isolated product (2.89 GBq) was around 77% and radiochemical purity was greater than 99%. Specific activity was 95 GBq/µmol at the end of the synthesis. Methyl 2-(3-cyano-4-[18F]fluoro-N-methyl-phenyl-sulfonamido)acetate ([18F]5b). A solution of compound 5a (1 mg) in anhydrous DMSO (100 µL) was added to a reaction vial containing dry K[18F]F/K2.2.2 complex. After heating at 70 °C for 5 min, an aliquot of the reaction mixture was taken for HPLC analysis (ACE C18 column). The 18F-incorporation was 80%. 18 F-Radiolabeling of TMA-Based Peptides. 18F-labeling of the TMA-based peptides (Figure 2) was performed under similar reaction conditions as described above. 3-Cyano-4-[18F]fluorobenzoyl-Val-βAla-Arg-Gly-NH2 18 ([ F]6b). A solution of compound 6a (2 mg) in anhydrous DMSO (300 µL) was added to a reaction vial containing dry K[18F]F/K2.2.2 complex. After heating the reaction mixture at 50 °C for 15 min, the crude reaction mixture was analyzed using an Altech Econsphere C18 RP column (53 × 7 mm, 3 µm) with the following elute: 0.1% aq TFA (solvent A), 0.1% TFA in acetonitrile/water 9/1 (solvent B); 0 min, 5% B; 0-7 min, 5f95% B; 7.00-7.10 min, 95f100% B; 7.10-8.80 min, 100% B; 8.80-9.00 min, 100f5% B; flow rate: 3 mL/min.). The 18Fincorporation determined by HPLC was 92%. The similar procedure described for [18F]6b was used for the preparation of 3-cyano-4-[18F]fluorobenzoyl-Val-βAla-Phe-GlyNH2 ([18F]7b) and 3-cyano-4-[18F]fluorobenzoyl-Val-βAla-LysGly-NH2 ([18F]8b), and resulted in 80% and 25% 18Fincorporation, respectively. 3-Cyano-4-[18F]fluorobenzoyl-Val-βAla-Met-Gly-NH2 18 ([ F]9b). A solution of compound 9a (2 mg) in anhydrous

Becaud et al.

DMSO (300 µL) was added to a reaction vial containing dry K[18F]F/K2.2.2 complex. After heating at 90 °C for 15 min, the crude reaction mixture was analyzed under the same analytical HPLC conditions as described for [18F]6b. The 18F-incorporation determined by HPLC was 89%. The reaction mixture was diluted with water and injected onto an ACE semipreparative HPLC column (0.1% TFA in water (solvent A), B: 0.1% TFA in acetonitrile/water 9/1 (solvent B), isocratic, 35% B, flow rate: 4 mL/min) and the product was collected. The decay-corrected radiochemical yield of the isolated product was around 57% and radiochemical purity was greater than 99%. Specific activity was 74 GBq/µmol at the end of the synthesis. 3-Cyano-4-[18F]fluorobenzoyl-Ava-Gln-Trp-Ala-Val-Gly-HisFA01010-Leu-NH2 ([18F]10b). A solution of compound 10a (2 mg) in DMSO (150 µL) was added to a reaction vial containing dry Cs[18F]F/K2.2.2 complex. The reaction vessel was sealed and heated at 70 °C for 15 min. Analysis of an aliquot of the crude reaction mixture afforded an 18F-incorporation of 77% with ACE C18 column. Thereafter, the reaction mixture was transferred to a vial containing water (4 mL) and then injected onto a semipreparative ACE column (0.1% aq TFA (solvent A); 0.1% TFA in acetonitrile/water 9/1 (solvent B), gradient 0-5 min, 30% B, flow rate 2 mL/min; 5-15 min, 30f70% B, flow rate: 3 mL/min). The desired 18F-labeled product was collected (253 MBq, 20% d.c.), and the specific activity was around 75 GBq/ µmol at the end of the synthesis. 3-Cyano-4-[18F]fluorobenzoyl-Arg-Ava-Gln-Trp-Ala-ValNMeGly-His-Sta-Leu-NH2 ([18F]11b). A solution of compound 11a (2 mg) in DMSO (150 µL) was added to a reaction vial containing dry CS[18F]F/K2.2.2 complex. The reaction vessel was sealed and heated at 70 °C for 15 min. An aliquot was taken from the crude reaction mixture and analyzed by HPLC (ACE C18 column); the 18F-incorporation was 74%. The reaction mixture was then transferred to a vial containing water (4 mL). This product was purified using a semipreparative HPLC column (ACE C18, 0.1% aq TFA (solvent A); 0.1% TFA in acetonitrile/ water 9/1 (solvent B), 0-5 min, 29% B; 5-25 min, 29f34% B, flow rate: 3 mL/min). The desired 18F-labeled product was collected (150 MBq, 21% d.c.), and the specific activity obtained was 73 GBq/µmol at the end of the synthesis. In Vitro Binding Affinity Assay. The receptor affinities of two bombesin (BBN) analogues 10b and 11b were determined in quadruplicate in a scintillation proximity assay (SPA) using cellular membranes transfected with human bombesin 2 receptors (gastrin-releasing peptide receptors) from PerkinElmer (RBHBS2M). The membranes and agglutinin-coupled SPA beads type A (PVT PEI Treated Wheatgerm, Amersham Bioscience) were mixed in assay buffer (50 mM Tris/HCl pH 7.2; 5 mM MgCl2; 1 mM EGTA, protease inhibitor (Roche Diagnostics GmbH; 1 tablet/50 mL) and 0.3% polyethylenemine) to give final concentrations of approximately 20 µg/mL protein and 8 mg/mL PVT-SPA beads. The ligand [125I]-Tyr4bombesin (PerkinElmer; specific activity: 81.4 TBq/mmol) was diluted to 0.2 nM in assay buffer. The test compounds were dissolved in DMSO to give 1 mM stock solutions. They were further diluted in assay buffer to 2 pM to 300 nM. Unspecific binding was determined by an excess of 10 000 nM Tyr4bombesin (Sigma). The assay was then performed as follows: First, 10 µL of compound solution to be tested for binding was placed in white 384 well plates (Lumitrac 200, Greiner). Next, 20 µL GRPR/ PVT-SPA bead mixture and 20 µL of the ligand solution were added. After 120 min incubation at room temperature, another 50 µL of assay buffer was added; the plate was sealed and centrifuged for 10 min at 520 × g at room temperature. Signals were measured in a TopCount (Perkin-Elmer) for 1 min integration time per well. Unspecific binding determined by an

Direct 18F-Labeling of Peptides

Bioconjugate Chem., Vol. 20, No. 12, 2009 2257

Figure 2. Structures of TMA-based peptides.

excess of Tyr4-bombesin was subtracted from total binding to yield the specific binding at each concentration. The IC50 and Ki values were calculated by nonlinear regression using GraFit 5 data analysis software (Erithacus Software Ltd.). Plasma Stability Studies. The stability of the radiolabeled compound [18F]10b and [18F]11b was investigated in mouse plasma at different time points (from 0 to 90 min) at 37 °C. Phosphate-buffered saline (PBS) was used as a control. After incubation, plasma proteins were precipitated with acetonitrile followed by centrifugation (10 min, 13 000 rpm) at 4 °C. The PBS controls were diluted with the same volume of acetonitrile. The supernatants and PBS controls were analyzed by HPLC (Column Ecosphere C18, 53 × 7 mm, 3 mm, flow rate: 1.8 mL/min). The percentage of intact compound was determined at different incubation times.

RESULTS AND DISCUSSION Syntheses and 18F-Labeling of TMA-Based Model Compounds. Precursor compounds 1a-5a were obtained in two steps with moderate to good yields (Supporting Information). In most cases, the displacement of fluoride with dimethylamine was carried out via nucleophilic aromatic substitution under basic conditions. The quarternization of dimethylamine intermediate with methyl triflate was performed under normal or increased pressure (1-10 bar). The crude products were purified by reversed-phase column chromatography. All the corresponding

reference compounds 1b-5b are commercially available except 3b. Compound 3b was obtained in two steps starting from commercially available 2-fluoro-5-formylbenzonitrile, which was converted to the corresponding acid by classical oxidation method followed by acid esterification. Since nucleophilic aromatic substitutions of 18F-for-+N(CH3)3 typically require high temperatures (>100 °C) which are not suitable for radiolabeling of peptides, electron withdrawing groups such as -CF3, -CN, and -F and a carbonyl functionality have been introduced to the aromatic ring in order to increase the reactivity of the trimethylammonium leaving group. To demonstrate the feasibility of direct 18F-labeling of peptides, a series of trimethylammonium-based model compounds 1a-5a (Figure 1) bearing electron-withdrawing groups (-CN, -CF3, -F) were tested. The carbonyl functionality has the added attraction that, via conversion to the corresponding acid, these trimethylammonium building blocks can be coupled to peptides. There are only a few publications on nucleophilic aromatic substitution of a TMA group by 18F-fluoride ion in aromatic compounds containing two or more substitutents besides the trimethylammonium moiety (30, 31). The radiosynthetic route is depicted in Scheme 1, and the radiolabeling results are shown in Table 1. From the results shown in Table 1, high 18F-incorporation was obtained with all the model compounds under mild radiolabeling conditions. The radiolabeling efficiency was not

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Becaud et al.

Scheme 1. General Radiosynthetic Pathway to TMA-Based Model Compounds (1a-5a)

Table 1.

F-Radiolabeling of TMA-Based Model Compoundsa

18

entry

compd.

precursor amount

temp (°C)

reaction time

conversion(%)

1 2 3 4 5 6 7 8

1a 2a 3a 3a 3a 4a 5a 5a

1 mg 1 mg 4 mg 2 mg 1 mg 2 mg 1 mg 1 mg

70 50 50 50 50 50 50 70

15 min 15 min 15 min 15 min 15 min 15 min 5 min 5 min

79 ( 7 (n ) 3) 82 ( 4 (n ) 3) 89 ( 4 (n ) 3) 83 ( 15 (n ) 2) 80 ( 2 (n ) 2) 79 ( 10 (n ) 2) 65 ( 4 (n ) 3) 80 ( 4 (n ) 2)

a 18 F-labeling was carried out in DMSO with K2CO3 as the base. Conversion was determined from radio-HPLC chromatogram representing the percentage of radioactivity area of product related to the total radioactivity area.

dramatically influenced by the amount of precursor (Table 1, entries 3-5). However, by increasing the reaction temperature from 50 to 70 °C, the 18F-incorporation of compound [18F]5b increased from 65% to 80% (Table 1, entries 7 and 8). Although no detailed study on the influence of reaction time was performed, it seems that 5 min reaction time is sufficient for a good conversion. The combined effect of the electron-withdrawing group and the carbonyl/sulfonyl on the phenyl ring facilitated an easy 18F-for-+N(CH3)3 substitution. This would explain why no decomposition of the ammonium precursors and the formation of volatile methyl [18F]fluoride were observed with precursor compounds 1a-5a under the indicated radiolabeling conditions (Table 1) compared to published results (32). Furthermore, no 18F-19F isotopic exchange was observed for compounds [18F]1b and [18F]2b under the mild radiolabeling conditions (50 and 70 °C). The large polarity difference between the TMAprecursor and fluorinated product allowed an easy semi-HPLC separation of the 18F-labeled products from their corresponding precursors. For example, the radiochemical purity of compound [18F]3b was greater than 99% after semi-HPLC purification, and the specific activity obtained was around 95 GBq/µmol at the end of the synthesis. Among the model compounds studied, compound [18F]3b with the cyano-activating group gave the best radiochemical yield of 77%. Syntheses and 18F-Labeling of TMA-Based Peptides. On the basis of the radiolabeling results of model compounds 1a-5a, compound 3a was converted to its corresponding acid 4-carboxy-2-cyano-N,N,N-trimethylbenzenaminium trifluoromethanesulfonate (Supporting Information) for coupling to target peptides using standard solid-phase peptide synthesis protocols (29). For TMA-based tetrapeptides, amino acids such as Arg, Phe, Lys, and Met were introduced in order to study their influence on 18F-labeling efficiency. Two bombesin analogues were synthesized with modifications of the amino acid sequence of BBN(7-14)-NH2. Non-natural amino acid such as FA01010 or Sta were incorporated into the bombesin peptide sequence for increasing its metabolic stability; Ava or Arg-βAla was used as a linker for attaching the aryltrimethylammonium building block and the N-terminal bombesin peptide. The corresponding standard references were synthesized by the conjugation of 3-cyano-4-fluorobenzoic acid with the same peptides applying similar protocols used for the syntheses of the precursors.

Table 2.

F-Radiolabeling of TMA-Based Peptidesa

18

entry

compd.

temp (°C)

base

reaction time

conversion (%)

1 2 3 4 5 6 7 8

6a 7a 8a 8a 9a 9a 10a 11a

50 50 90 90 50 90 70 70

K2CO3 K2CO3 K2CO3 Cs2CO3 K2CO3 K2CO3 Cs2CO3 Cs2CO3

15 min 15 min 15 min 15 min 15 min 15 min 15 min 15 min

92 (n ) 1) 80 (n ) 1) 0 (n ) 2) 24 ( 8 (n ) 3) 19 ( 4 (n ) 3) 89 ( 3 (n ) 3) 70 ( 7 (n ) 3) 51 ( 5 (n ) 2)

a 18 F-labeling was carried out in DMSO (200-300 µL) with 2 mg of precursor. Conversion was determined from radio-HPLC chromatogram representing the percentage of radioactivity area of product related to the total radioactivity area.

The direct one-step 18F-labeling was applied to four tetrapeptides (Table 2, entries 1-6) differing only in one amino acid in the peptide sequence. Using similar radiolabeling conditions as applied to the model compounds, tetrapeptides 6a and 7a gave 18 F-incorporation of 92% and 80%, respectively. In the case of the lysine-containing tetrapeptide 8a (entry 3), there was no product formation when potassium carbonate was used as the base; however, a moderate 18F-incorporation (24%) was achieved with cesium carbonate as the base at 90 °C. The low 18Fincorporation might be due to the presence of free amino functionality. Nevertheless, the improvement is remarkable. With the methionine-containing tetrapeptide 9a, the temperature effect for the 18F-incorporation is worth mentioning. The 18Fincorporation was only 19% after 15 min at 50 °C; however, by increasing the temperature to 90 °C, a dramatic increase of 18 F-incorporation (89%) was achieved (Table 2, entries 5 and 6). Because these results were encouraging, this one-step 18Flabeling approach was applied to two bombesin peptides 10a and 11a. As shown in Table 2, more than 70% 18F-incorporation was found for [18F]10b when an aliquot was directly removed from the reaction mixture and analyzed by HPLC. A typical radio-HPLC profile of compound [18F]10b is shown in Figure 3. Only two radioactive peaks were observed from the analytical HPLC chromatogram of the reaction mixture. Apart from free 18 F-fluoride and the desired 18F-labeled compound (retention time ) 7.2 min), no other radioactive compound was observed. After adding HPLC eluent to the reaction vial and purifying the mixture by semi-HPLC, around 20% (decay-corrected) radiochemical yield of the final product was obtained. The relatively large difference between isolated radiochemical yield and 18F-incorporation of compound [18F]10b (20% vs 70%) is due to the fact that Cs[18F]F/K2.2.2 complex can be absorbed on the surface of the reaction vial (33). This absorbed 18F-fluoride activity is, however, not taken into account when an aliquot of the reaction mixture is taken for analysis. The radiochemical purity was 99% and specific activity was around 79 GBq/µmol at the end of synthesis. With respect to the stereochemistry of the peptides, similar peptides have been labeled under basic conditions (pH 8-10) and even at higher or elevated temperatures (75-100 °C), and no racemization of the peptides was observed (34-36). Because our radiolabeling conditions (pH < 8, temp 50-70 °C) are even milder than those previously reported, we do not expect that racemization will occur with any of the peptides reported in this manuscript. In Vitro Binding Affinity Assay. The in Vitro binding affinity of the fluorinated bombesin peptides 10b and 11b to the gastrinreleasing peptide receptor were assessed via a competitive displacement assay using [125I]-Tyr4-bombesin as the radioligand. Both peptides demonstrated high binding affinity to the gastrin-releasing peptide receptor with IC50 values of 9.20 nM and 2.71 nM (Figure 4). The KD value of the radioligand I-125Tyr4-bombesin was determined in former assays to be ap-

Direct 18F-Labeling of Peptides

Bioconjugate Chem., Vol. 20, No. 12, 2009 2259

Figure 3. Radio-HPLC profiles of 3-cyano-4-[18F]fluorobenzoyl-Ava-Gln-Trp-Ala-Val-Gly-His-FA01010-Leu-NH2 ([18F]10b): (a) reaction mixture; (b) purified compound.

Figure 4. Competitive binding of (a) 3-cyano-4-fluorobenzoyl-Ava-Gln-Trp-Ala-Val-Gly-His-FA01010-Leu-NH2 (10b) and (b) 3-cyano-4fluorobenzoyl-Arg-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2 (11b) versus [125I]-Tyr4-bombesin on human bombesin 2 receptor membranes in a scintillation proximity assay.

Figure 5. Radiochemical stability studies of [18F]10b and [18F]11b at 37 °C. 2, with PBS; 9, with mouse plasma.

proximately 0.06 nM. Therefore, the normalized binding affinities represented excellent Ki values of 1.90 nM and 0.70 nM for 10b and 11b, respectively (Figure 4). For comparison, human GRP serving as a positive control showed a Ki of 5-10 nM, whereas Neuromedin B serving as a negative control had a Ki of >500 nM (see Supporting Information). Plasma Stability Studies. Plasma stability was evaluated for both bombesin peptides [18F]10b and [18F]11b in mouse plasma at time points of 0, 30, 60, 90, and 120 min at 37 °C, with PBS as control. As indicated in Figure 5, [18F]10b showed excellent

plasma stability over the tested time period. [18F]11b, however, was slightly degraded to 18F-fluoride, but is stable enough to allow for in ViVo studies. There are no significant differences between plasma results and PBS findings of compounds [18F]10b and [18F]11b.

CONCLUSION In conclusion, a series of trimethylammonium-based model compounds containing different electron-withdrawing substituents, a trimethylammonium (TMA) leaving group, and a linker

2260 Bioconjugate Chem., Vol. 20, No. 12, 2009

for coupling to peptides were designed and synthesized. The appropriate model compounds were converted to their corresponding carboxylic acids and coupled to model tetrapeptides containing different amino acids and bombesin peptides. High 18 F-incorporation was obtained with all the model compounds under mild radiolabeling conditions. The results of the onestep [18F]-fluorination of the tetrapeptides and bombesin peptides show that the direct 18F-labeling of peptides is feasible under mild conditions and in good radiochemical yields. This method might not be applicable in all cases. However, in our hands, we were able to achieve a 24% radiochemical yield of one of the peptides bearing a free amino group in lysine. Peptides containing histidine, tryptophan, and arginine amino acids are also amenable to fluorine-18 labeling using this direct approach. Under normal circumstances, these amino acids would require protection with appropriate protecting groups. We are currently working on more peptides which contain cysteine and tyrosine amino acids. In conclusion, this work demonstrates that a rational approach to peptide design can be useful for the direct 18 F labeling of peptides and other large biomolecules.

ACKNOWLEDGMENT We gratefully acknowledge Katrin Dinse, Peter Friese, Selahattin Ede, Marion Zerna, Jo¨rg Pioch, Ingo Horn, Rene´ Zernicke and Cindy Fischer for technical support. Dr. Detlev Suelzle for fruitful discussions and computational chemistry. Supporting Information Available: Analytical data for compounds 1a-5a and experimental parts. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) Kilbourn, M. R., and Welch, M. J. (1986) 18F-labeled receptor based radiopharmaceuticals. Appl. Radiat. Isot. 37, 677–683. (2) McGuire, A. H., Dehdashti, F., Siegel, B. A., Lyss, A. P., Brodack, J. W., Mathias, C. J., Mintun, M. A., Katzenellenbogen, J. A., and Welch, M. J. (1991) Positron tomographic assessment of 16-R-[18F]-fluoro-17-β-estradiol uptake in metastatic breast carcinoma. J. Nucl. Med. 32, 1526–1531. (3) Vaidyanathan, G., and Zalutsky, M. R. (1997) Fluorine-18labeled [Nle4, D-Phe7]-R-MSH, an R-melanocyte stimulating hormone analogue. Nucl. Med. Biol. 24, 171–178. (4) Vaidyanathan, G., and Zalutsky, M. R. (1995) 18F-Labeled chemotactic peptides - a potential approach for the PET imaging of bacterial-infection. Nucl. Med. Biol. 22, 759–764. (5) Guhlke, S., Wester, H. J., Bruns, C., and Stoecklin, G. (1994) (2-[18F]Fluoropropionyl-(D)phe1)-octreotide, a potential radiopharmaceutical for quantitative somatostatin receptor imaging with PET - synthesis, radiolabeling, in-vitro validation and biodistribution in mice. Nucl. Med. Biol. 21, 819–825. (6) Moody, T. W., Leyton, J., Unsworth, E., John, C., Lang, L. X., and Eckelman, W. C. (1998) (Arg15, Arg21)VIP: Evaluation of biological activity and localization to breast cancer tumors. Peptides 19, 585–592. (7) Vaidyanathan, G., Bigner, D. D., and Zalutsky, M. R. (1992) Fluorine-18-labeled monoclonal antibody fragments - a potential approach for combining radioimmunoscintigraphy and positron emission tomography. J. Nucl. Med. 33, 1535–1541. (8) Wester, H. J., Hamacher, K., and Stoecklin, G. (1996) A comparative study of n.c.a. fluorine-18 labeling of proteins via acylation and photochemical conjugation. Nucl. Med. Biol. 23, 365–372. (9) Dolle, F., Hinnen, F., Vaufrey, F., Tavitian, B., and Crouzel, C. (1997) A general method for labeling oligodeoxynucleotides with 18F for in vivo PET imaging. J. Labelled Compd. Radiopharm. 39, 319–330. (10) Kuhnast, B., Dolle, F., Terrazzino, S., Rousseau, B., Loc’h, C., Vaufrey, F., Hinnen, F., Doignon, I., Pillon, F., David, C.,

Becaud et al. Crouzel, C., and Tavitian, B. (2000) General method to label antisense oligonucleotides with radioactive halogens for pharmacological and imaging studies. Bioconjugate Chem. 11, 627– 636. (11) Mueller-Platz, C. M., Kloster, G., Legler, G., and Stoecklin, G. (1982) 18F-Fluoroacetate - an agent for introducing no-carrieradded fluorine-18 into urokinase without loss of biological activity. J. Labelled Compd. Radiopharm. 19, 1645–1646. (12) Herman, L. W., Fischman, A. J., Tompkins, R. G., Hanson, R. N., Byon, C., Strauss, H. W., and Elmaleh, D. R. (1994) The use of pentafluorophenyl derivatives for the 18F labeling of proteins. Nucl. Med. Biol. 21, 1005–1010. (13) Lang, L. X., and Eckelman, W. C. (1994) One-step synthesis of 18F labeled [18F] N-succinimidyl 4-(fluoromethyl)benzoate for protein labeling. Appl. Radiat. Isot. 45, 1155–1163. (14) Lang, L., and Eckelman, W. C. (1997) Labeling proteins at high specific activity using N-succinimidyl 4-[18F](fluoromethyl) benzoate. Appl. Radiat. Isot. 48, 169–173. (15) Okarvi, S. M. (2001) Recent progress in fluorine-18 labelled peptide radiopharmaceuticals. Eur. J. Nucl. Med. 28, 929–938. (16) Kilbourn, M. R., Dence, C. S., Welch, M. J., and Mathias, C. J. (1987) Fluorine-18 labeling of proteins. J. Nucl. Med. 28, 462–470. (17) Block, D., Coenen, H. H., and Stoecklin, G. (1988) N.c.a. 18F-Fluoroalkylation of H-Acidic Compounds. J. Labelled Compd. Radiopharm. 25, 201–216. (18) Block, D., Coenen, H. H., and Stoecklin, G. (1988) N.c.a. 18F-fluoroacylation via fluorocarboxylic acid-esters. J. Labelled Compd. Radiopharm. 25, 185–200. (19) Jacobson, K. A., Furlano, D. C., and Kirk, K. L. (1988) A prosthetic group for the rapid introduction of fluorine into peptides and functionalized drugs. J. Fluorine Chem. 39, 339– 347. (20) Shai, Y., Kirk, K. L., Channing, M. A., Dunn, B. B., Lesniak, M. A., Eastman, R. C., Finn, R. D., Roth, J., and Jacobson, K. A. (1989) 18F-Labeled insulin - a prosthetic group methodology for incorporation of a positron emitter into peptides and proteins. Biochemistry 28, 4801–4806. (21) Jelinski, M., Hamacher, K., and Coenen, H. H. (2002) C-Terminal 18F-fluoroethylamidation exemplified on [Gly-OH9] oxytocin. J. Labelled Compd. Radiopharm. 45, 217–229. (22) Shiue, C. J., Wolf, A. P., and Hainfeld, J. F. (1988) 18F labeled N-(p-(18F)fluorophenyl)maleimide and its derivatives for labelling monoclonal antibodies with 18F. J. Labelled Compd. Radiopharm. 26, 287–289. (23) Toyokuni, T., Walsh, J. C., Dominguez, A., Phelps, M. E., Barrio, J. R., Gambhir, S. S., and Satyamurthy, N. (2003) Synthesis of a new heterobifunctional linker, N-[4-(Aminooxy)butyl]maleimide, for facile access to a thiol-reactive 18F-labeling agent. Bioconjugate Chem. 14, 1253–1259. (24) Wester, H. J., Hamacher, K., Guhlke, S., and Stoecklin, G. (1994) Simple and fast 18F-labeling of proteins by coupling with photogenerated [18F]-arylnitrene. J. Nucl. Med. 35, P73–P73. (25) Coenen, H. H. (2007) in PET chemistry - the driVing force in molecular imaging (Schubiger, P. A. L., L., and Friebe, M., Eds.) Springer-Verlag, Berlin. (26) Ting, R., Adam, M. J., Ruth, T. J., and Perrin, D. M. (2005) Arylfluoroborates and alkylfluorosilicates as potential PET imaging agents: High-yielding aqueous biomolecular 18F-labeling. J. Am. Chem. Soc. 127, 13094–13095. (27) Mu, L. J., Hoehne, A., Schubiger, R. A., Ametamey, S. M., Graham, K., Cyr, J. E., Dinkelborg, L., Stellfeld, T., Srinivasan, A., Voigtmann, U., and Klar, U. (2008) Silicon-based building blocks for one-step 18F-radiolabeling of peptides for PET imaging. Angew. Chem., Int. Ed. 47, 4922–4925. (28) Schirrmacher, R., Bradtmoller, G., Schirrmacher, E., Thews, O., Tillmanns, J., Siessmeier, T., Buchholz, H. G., Bartenstein, P., Waengler, B., Niemeyer, C. M., and Jurkschat, K. (2006) F-18-labeling of peptides by means of an organosilicon-based fluoride acceptor. Angew. Chem., Int. Ed. 45, 6047–6050.

Direct 18F-Labeling of Peptides (29) Fields, G. B., and Noble, R. L. (1990) Solid-phase peptidesynthesis utilizing 9-fluorenylmethoxycarbonyl amino-acids. Int. J. Pept. Protein Res. 35, 161–214. (30) Oya, S., Choi, S. R., Coenen, H., and Kung, H. F. (2002) New PET imaging agent for the serotonin transporter: [18F]ACF (2-[(2-amino-4-chloro-5-fluorophenyl)thiol-N, N-dimethyl-benzenmethanamine). J. Med. Chem. 45, 4716–4723. (31) Li, Z. Z., Ding, Y. S., Gifford, A., Fowler, J. S., and Gatley, J. S. (2003) Synthesis of structurally identical fluorine-18 and iodine isotope labeling compounds for comparative imaging. Bioconjugate Chem. 14, 287–294. (32) Gail, R., and Coenen, H. H. (1994) A one-step preparation of the n.c.a. 18F labeled synthons - 4-fluorobromobenzene and 4-fluoroiodobenzene. Appl. Radiat. Isot. 45, 105–111. (33) Hoehne, A., Mu, L., Honer, M., Schubiger, P. A., Ametamey, S. M., Graham, K., Stellfeld, T., Borkowski, S., Berndorff, D., Klar, U., Voigtmann, U., Cyr, J. E., Friebe, M., Dinkelborg, L., and Srinivasan, A. (2008) Synthesis, F-18-labeling, and in vitro and in vivo studies of bombesin peptides modified with silicon-based building blocks. Bioconjugate Chem 19, 1871–1879.

Bioconjugate Chem., Vol. 20, No. 12, 2009 2261 (34) Boswell, C. A., Regino, C. A. S., Baidoo, K. E., Wong, K. J., Bumb, A., Xu, H., Milenic, D. E., Kelley, J. A., Lai, C. C., and Brechbiel, M. W. (2008) Synthesis of a cross-bridged cyclam derivative for peptide conjugation and Cu-64 radiolabeling. Bioconjugate Chem. 19, 1476–1484. (35) Van Domselaar, G. H., Okarvi, S. M., Fanta, M., Suresh, M. R., and Wishart, D. S. (2000) Synthesis and 99(m)Tc-labelling of bz-MAG3-triprolinyl-peptides, their radiochemical evaluation and in vitro receptor-binding. J. Labelled Compd. Radiopharm. 43, 1193–1204. (36) Smith, C. J., Sieckman, G. L., Owen, N. K., Hayes, D. L., Mazuru, D. G., Kannan, R., Volkert, W. A., and Hoffman, T. J. (2003) Radiochemical investigations of gastrin-releasing peptide receptor-specific [Tc-99m(X)(CO)(3)-Dpr-Ser-Ser-Ser-Gln-TrpAla-Val-Gly-His-Leu-Met-(NH2)] in PC-3, tumor-bearing, rodent models: Syntheses, radiolabeling, and in vitro/in vivo studies where Dpr)2,3-diaminopropionic acid and X ) H2O or P(CH2OH)(3). Cancer Res. 63, 4082–4088. BC900240Z