1140
Bioconjugate Chem. 2002, 13, 1140−1145
A Trithiolate Tripodal Bifunctional Ligand for the Radiolabeling of Peptides with Gallium(III) Leonard G. Luyt and John A. Katzenellenbogen* Department of Chemistry, University of Illinois, Urbana, Illinois 61801. Received May 20, 2002; Revised Manuscript Received July 2, 2002
A tripodal bifunctional chelator for gallium has been prepared with a chelation core consisting of three thiols and a tertiary amine. The synthesis proceeds in 13 steps with an overall yield of 22%. An aromatic amine is available for conjugation to peptides through carbodiimide coupling. Gallium(III) complexes were readily prepared from both the bifunctional chelator and a phenylalanine-conjugated system. These complexes underwent stability evaluation and were found to be stable to ligand exchange and enzymatic hydrolysis. This bifunctional chelator appears to be suitable for conjugation to peptides for the preparation of gallium radiopharmaceuticals.
INTRODUCTION
Gallium-68 (t1/2 ) 68 min) is considered to be a promising radionuclide for use in positron emission tomography (PET) and has been used in a number of clinical studies. A particular convenience associated with the use of 68Ga is that it can be obtained from a 68Ge/68Ga generator system; thus, PET imaging can be done with this isotope without the need for an on-site cyclotron (1). Gallium-67 (t1/2 ) 3.3 days) is a gallium isotope that has been used for single-photon emission computed tomography (SPECT) for many years, even though its long half-life limits its usefulness as an imaging radionuclide. Current gallium-based radiopharmaceuticals are, for the most part, simple metal-ligand complexes, such as Ga-citrate.(2) Recently, however, a number of gallium radiopharmaceuticals have been successfully developed by the conjugation of a gallium chelation unit to a large biomolecule (3-6). We are interested in developing methods for radiolabeling large biomolecules, such as peptides and proteins, with gallium for use in receptorbased imaging. Ideally, a chelation unit for labeling biomolecules with gallium for in vivo use should have the following characteristics: (a) it should bind the metal with high kinetic stability, (b) it should have a single site for chelation to facilitate complete spectroscopic characterization of a single product, and (c) and it should have bifunctional character that will allow its conjugation to a variety of biomolecules through a single linking site. A 4-coordinate tripodal S3N ligand for gallium(III) was previously described by Koch and co-workers and was found to be stable, neutral, and uniquely, for a gallium(III) complex, able to pass through the blood-brain barrier (7-10). In contrast to indium and iron complexes with the same chelate system, the gallium(III) complex retains its tetrahedral geometry in the presence of water and DMF. This tripodal ligand appeared to be a suitable candidate for the radiolabeling of peptides with gallium, through a pendant approach as depicted in Figure 1. * Corresponding author: Department of Chemistry, University of Illinois, 600 S. Mathews Ave., Urbana, IL 61801. Telephone 217-333-6310. Fax 217-333-7325. E-mail jkatzene@ uiuc.edu.
Figure 1. A tripodal gallium(III) bioconjugate.
Herein is a description of the synthesis of this bifunctional chelator, its conjugation to a biomolecule model, and a discussion of the stability of the resultant gallium(III) complexes. EXPERIMENTAL SECTION
Reagents and solvents were purchased from Acros, Aldrich, Fisher, Strem, or TCI. [GaCl4][PPh4] was prepared as previously reported (11). Diethyl ether, methylene chloride, and tetrahydrofuran were dried by a solvent delivery system (using activated neutral alumina columns) designed by J. C. Meyer. All air-sensitive reactions were carried out under a nitrogen or argon atmosphere, using flame- or oven-dried glassware. Reaction progress was monitored by analytical thin-layer chromatography (silica). Flash chromatography was performed using Woelm silica gel (0.040-0.063 mm) packing. Hexane for chromatography was distilled prior to use. 1 H and 13C NMR spectra were obtained using a Unity 400, Unity 500, or Unity INOVA 500NB Varian FT-NMR spectrometer. Chemical shifts (δ) are reported in parts per million downfield from internal tetramethylsilane and referenced from solvent resonances. NMR coupling constants are reported as absolute values in Hertz. Lowresolution electron impact (EI) mass spectra and highresolution EI mass spectra (HRMS) were obtained on Micromass 70-VSE, or Micromass 70-SE-4F spectrometers. Both low- and high-resolution fast atom bombardment (FAB) mass spectra were obtained on Micromass ZAB-SE and 70-SE-4F spectrometers, respectively. Melting points were determined on a Thomas-Hoover melting point apparatus and are uncorrected. HPLC analysis was performed with a ThermoQuest Spectrasystem P4000/AS4000/UV1000, with the UV detector set to 254 nm. A Microsorb-MV C18 reversed-phase column (5 µm, 100 Å) was used, with an isocratic eluent of 80% MeOH/20% water at 1.0 mL/min.
10.1021/bc025552g CCC: $22.00 © 2002 American Chemical Society Published on Web 08/24/2002
Bifunctional Gallium(III) Ligand
(S-Benzylthio)salicylic Acid. Thiosalicylic acid (6.33 g, 41.1 mmol) was dissolved in 100 mL of ethanol and 100 mL of 1 M NaOH solution. The solution was cooled to 0 °C; then benzyl bromide (5.13 mL, 43.1 mmol) was added, and the solution was warmed to room temperature and stirred for three h. A 1 M HCl solution was added to acidify the reaction, and the crude product was collected by filtration and then recrystallized from benzene to yield 9.95 g (99%) of off-white needles: mp 186-187 °C. 1H NMR (d6-acetone): δ 4.21 (s, 2H), 7.20 to 7.53 (m, 8H), 8.01 (m, 1H), 11.33 (br s, 1H). 13C NMR (d6-acetone): δ 37.24, 124.66, 126.68, 127.98, 128.21, 129.29, 130.00, 132.11, 133.24, 137.64, 143.26, 167.55. MS (EI, 70 eV): m/z 244 (15.9%, M+), 91 (100%, SBn). 2-(Benzylthio)benzyl Alcohol (1). A three-necked, flame-dried flask was filled with 30 mL of THF and 16 mL of 1.0 M BH3‚THF and then cooled on an ice-bath (0 °C). To the flask was added (benzylthio)salicylic acid (2.53 g, 7.85 mmol) dissolved in 30 mL of THF, and the reaction was stirred at 0 °C for 1 h and then at RT for 3 h. To this was carefully added 40 mL water, and stirring was continued for 1 h. Solid K2CO3 was added until the solution was saturated, and the layers were separated. The organic layer was evaporated to dryness, partioned between ether and water, and extracted into ether (×2). The original aqueous layer was extracted with ether (×2), and all organics were combined, washed with 1 M NaOH, water, and brine, dried over MgSO4, evaporated, and dried under vacuum. The solid product was recrystallized from diethyl ether/petroleum ether (37-54 °C) to give colorless needles (1.77 g, 97%): mp 50 °C (lit. 48.5-49.5 °C) (10). 1H NMR (CDCl3): δ 1.97 (br s, 1H, OH), 4.05 (s, 2H, CH2S), 4.61 (s, 2H, CH2O), 7.17 to 7.40 (m, 9H). 13C NMR (CDCl3): δ 39.88, 63.58, 127.29, 127.34, 128.27, 128.41, 128.50, 128.75, 131.84, 134.05, 137.36, 141.76. MS (EI, 70 eV): m/z 230 (7.6%, M+), 139 (61.9%, M-SBn), 91 (100%, SBn). 2-(Benzylthio)benzyl Bromide (2). A flame-dried flask was charged with benzylic alcohol 1 (1.71 g, 7.43 mmol) dissolved in 100 mL of diethyl ether. After cooling to 0 °C, 0.78 mL of freshly distilled PBr3 (8.18 mmol) was added, and the reaction was stirred at 0 °C for 15 min. then at RT for 1 h. The reaction was quenched with methanol; then water was added, and the organic layer was washed with sat. NaHCO3, water, brine, dried over MgSO4, evaporated and dried under vacuum yielding 2.12 g (97%) of a clear, colorless oil. 1H NMR (CDCl3): δ 4.14 (s, 2H, CH2S), 4.61 (s, 2H, CH2Br), 7.20 to 7.39 (m, 9H). 13C NMR (CDCl3): δ 32.08, 39.65, 127.29, 127.39, 128.48, 128.92, 129.14, 130.58, 132.11, 135.95, 137.09, 138.66. MS (EI, 70 eV): m/z 294 (5.1%, M+), 292 (5.0%, M+), 213 (21.9%, M-Br), 91 (100%, SBn); HRMS calcd C14H13BrS, 291.9921, found, 291.9924. 4-Nitro-2-thiocyanatobenzoic Acid (3). This material was prepared from 2-amino-4-nitrobenzoic acid by diazotization followed by treatment with copper thiocyanate, as previously described (yield 86%) (12). 1H NMR (CD3OD): δ 8.29 (dd, 1H, J ) 8.4, 1.9 Hz), 8.36 (d, 1H, J ) 8.4 Hz), 8.68 (d, 1H, J ) 1.9 Hz). 13C NMR (CD3OD): δ 112.11, 123.13, 123.54, 133.36, 134.26, 134.77, 151.99, 167.95. MS (EI, 70 eV): m/z 224 (17.9%, M+), 197 (100%, M - CN); HRMS calcd C8H4N2O4S, 223.9892, found, 223.9888. 2,2′-Dithiobis(4-nitrosalicylic Acid) (4). This material was prepared from 3 as previously described in 86% yield (12). 1H NMR (d6-DMSO): δ 8.14 (dd, 2H, J ) 2.4, 8.8 Hz), 8.26 (d, 2H, J ) 8.8 Hz), 8.38 (d, 2H, J ) 2.4 Hz). 13C NMR (d6-DMSO): δ 119.94, 121.33, 133.31, 133.61, 140.69, 150.25, 166.56.
Bioconjugate Chem., Vol. 13, No. 5, 2002 1141
4-Nitrothiosalicylic Acid. Disulfide 4 (2.05 g, 5.16 mmol) was dissolved in 150 mL of abs ethanol and heated to 70 °C. Sodium borohydride (0.78 g, 20.6 mmol) was added portionwise. The reaction was heated at reflux for 90 min (TLC, 40% MeOH/60% CHCl3), cooled, poured into 300 mL of water, and acidified with concentrated HCl. The product was extracted into diethyl ether (×3), washed with water, dried over Na2SO4, and filtered, the solvent was evaporated, and the product was dried under vacuum to yield 1.91 g (93%) of yellow crystals: mp 267268 °C. 1H NMR (CD3OD): δ 7.95 (dd, 1H, J ) 2.1, 8.6 Hz), 8.20 (d, 1H, J ) 8.6 Hz), 8.33 (d, 1H, J ) 2.1 Hz). 13C NMR (CD OD): δ 119.74, 126.14, 132.55, 134.00, 3 142.64, 150.81, 168.20. MS (EI, 70 eV): m/z 199 (9.1%, M+), 181 (100%, M-OH2); HRMS calcd C7H5NO4S, 198.9939, found, 198.9937. 2-(Benzylthio)-4-nitrobenzoic Acid (5). To a solution of 4-nitrothiosalicylic acid, prepared above, (1.81 g, 9.08 mmol) in 60 mL of abs ethanol were added 30 mL of 1 M NaOH and 1.1 mL (9.25 mmol) of benzyl bromide. The solution was stirred at 0 °C for 1 h and then at RT for 2 h (TLC, 30% MeOH/70% CHCl3). Water (30 mL) was added, followed by 32 mL of 1 M HCl. The mixture was cooled to 0 °C, and the product, which precipitated, was collected by filtration to yield, after drying, 2.12 g (81%) of a yellow crystalline solid: mp 194-196 °C. 1H NMR (CD3OD): δ 4.30 (s, 2H), 7.25 (m, 1H), 7.32 (m, 2H), 7.47 (m, 2H), 7.97 (dd, 1H, J ) 2.2, 8.5 Hz), 8.12 (d, 1H, J ) 8.5 Hz), 8.25 (d, 1H, J ) 2.2 Hz). 13C NMR (CD3OD): δ 37.88, 119.43, 121.66, 128.56, 129.68, 130.26, 133.38, 134.30, 137.17, 145.61, 151.05, 168.03. MS (EI, 70 eV): m/z 289 (6.3%, M+), 91 (100%, Bn); HRMS calcd C14H11NO4S, 289.0409, found, 289.0408. 2-(Benzylthio)-4-nitrobenzamide (6). In a flamedried, N2-purged flask, a slurry of the nitrobenzoic acid 5 (1.92 g, 6.64 mmol) in 150 mL of CH2Cl2 and 2 drops DMF, was cooled to 0 °C. Oxalyl chloride (1.2 mL, 13.3 mmol) was added, the solution was warmed to RT and stirred for 2 h (gas evolution had ceased). The solvent was evaporated, and the resultant yellow solid was dissolved in 100 mL of CH2Cl2. Ammonium hydroxide solution (10 mL) was added, and the reaction mixture was stirred at RT for 40 min. The mixture was diluted with water, and the organic layer was separated and washed with sodium bicarbonate, water, and brine. The organic phase was dried over magnesium sulfate and filtered, and the solvent was evaporated to leave 1.81 g (94%) of a yellow solid: mp 171-173 °C. 1H NMR (CD3OD): δ 4.28 (s, 2H), 7.22 (m, 1H), 7.28 (m, 2H), 7.38 (m, 2H), 7.62 (m, 1H), 8.02 (m, 1H), 8.17 (m, 1H). 13C NMR (CD3OD): δ 38.12, 120.58, 123.23, 128.19, 129.35, 129.58, 129.96, 137.33, 140.01, 142.46, 149.33, 168.70. MS (EI, 70 eV): m/z 288 (134%, M+), 197 (22.3%, M - SBn), 91 (100%, Bn); HRMS calcd C14H12N2O3S, 288.0569, found, 288.0574. 2-(Benzylthio)-4-nitrobenzylamine (7). A flamedried flask with attached condenser was charged with 40 mL of THF and 30 mL of 1.0 M BH3‚THF. To this was slowly added nitro amide 6 (1.76 g, 6.11 mmol) dissolved in 60 mL of THF, and the solution was heated at reflux overnight. After cooling to room temperature, 40 mL of 6.0 M HCl was carefully added, the mixture was heated at 50 °C for 90 min and then cooled, and the THF was removed using a rotary evaporator. HCl (1 M) was added to the aqueous solution, followed by ether extraction (×2), with the combined organics being washed with additional 1 M HCl. The combined aqueous layers were basified with solid NaOH, extracted with ether (×3), washed with brine, dried over MgSO4, evaporated, and
1142 Bioconjugate Chem., Vol. 13, No. 5, 2002
dried under vacuum to give a yellow solid (0.96 g, 57%). 1 H NMR (CDCl3): δ 3.90 (s, 2H), 4.21 (s, 2H), 7.24 to 7.34 (m, 5H), 7.53 (d, 1H, J ) 8.4 Hz), 8.00 (dd, 1H, J ) 8.4, 2.3 Hz), 8.16 (d, 1H, J ) 2.1 Hz). 13C NMR (CDCl3): δ 38.06, 43.98, 120.85, 122.69, 127.73, 128.01, 128.72, 128.87, 135.73, 137.39, 147.01, 148.86. MS (EI, 70 eV): m/z 274 (1.3%, M+), 183 (100%, M - SBn); HRMS calcd C14H14N2O2S, 274.0776, found, 274.0775. Bis((2-(benzylthio)benzyl)(2-(benzylthio)-4-nitrobenzyl)amine (8). 2 (1.92 g, 6.55 mmol) and the nitrobenzylamine 7 (0.90 g, 3.28 mmol) were dissolved in 100 mL of acetonitrile and then stirred at RT overnight. The solids were removed by filtration, and the solvent was removed using a rotary evaporator to give 2.40 g of crude material. Flash column purification (silica, 1:1/CH2Cl2:hexane) yielded the product 8 as a yellow oil (1.73 g, 75%). 1H NMR (CDCl3): δ 3.53 (s, 2H, NCH2), 3.56 (s, 4H, N(CH2)2), 4.01 (s, 4H, S(CH2)2), 4.11 (s, 2H, SCH2), 7.10 to 7.30 (m, 21H), 7.48 (m, 2H), 7.71 (d, 1H, J ) 7.5 Hz), 7.81 (dd, 1H, J ) 8.5, 2.2 Hz), 8.04 (d, 1H, J ) 2.2 Hz). 13C NMR (CDCl3): δ 38.19, 39.09, 55.34, 56.34, 120.18, 122.04, 126.16, 127.21, 127.49, 127.62, 128.42, 128.66, 128.82, 128.96, 129.53, 129.56, 129.88, 135.81, 136.25, 137.13, 138.20, 138.60, 145.95, 146.78. MS (FAB, +): m/z 699 (100%, M+ + H); HRMS calcd C42H38N22O2S3, 699.2174, found, 699.2169. Bis(2-(benzylthio)benzyl)(2-(benzylthio)-4-aminobenzyl)amine (9). The benzylamine 8 (1.72 g, 2.46 mmol) was dissolved in 50 mL of abs ethanol and 50 mL of 1,2-dichloroethane. To this solution was added 0.5 mL of hydrazine hydrate followed by 0.1 mL of 50% Raney nickel slurry. These additions were repeated twice until the reaction was complete by TLC (1 h). The slurry was filtered through Celite, evaporated, and dried under vacuum to yield 1.57 g (95%) of clear, pale yellow viscous oil. 1H NMR (CDCl3): δ 3.53 (s, 2H), 3.61 (s, 4H), 4.02 (s, 2H), 4.05 (s, 4H), 6.49 (dd, 1H, J ) 8.1, 2.4 Hz), 6.64 (d, 1H, J ) 2.4 Hz), 7.13 to 7.31 (m, 21 H), 7.38 (d, 1H, J ) 8.1 Hz), 7.65 (m, 2H). 13C NMR (CDCl3): δ 39.05, 39.09, 55.20, 55.50, 113.48, 115.91, 126.19, 126.94, 127.08 (2 peaks), 128.39 (2 peaks), 128.89, 128.91, 129.25, 129.38, 129.52, 130.62, 135.70, 136.82, 137.31, 137.33, 139.74, 145.28. MS (FAB, +): m/z 669 (100%, M+ + H); HRMS calcd C42H40N2S3, 669.2432, found, 669.2431. Bis(2-thio)benzyl-(2-thio-4-aminobenzyl)amine gallium(III) (11). A flame-dried three-necked flask with attached dry ice condenser was cooled to -78 °C and filled with 15 mL of anhydrous ammonia. To this was added the chelate system 9 (143 mg, 0.214 mmol) dissolved in 2 mL of THF, followed by small pieces of sodium. The mixture was warmed to -33 °C to improve solubility and allowed to gently reflux for 45 min. The reaction was quenched with solid NH4Cl, and the ammonia was evaporated under argon flow, followed by vacuum-drying. The crude trithiol was dissolved in 8 mL of degassed methanol, and [PPh4][GaCl4] (118 mg, 0.214 mmol) dissolved in 2 mL of degassed acetonitrile was then added. The pH was adjusted to basic with 0.8 mL of 1 M KOH solution (degassed); the mixture was stirred at room temperature for 30 min, and the solvents were evaporated and dried under vacuum. The crude product was triturated with chloroform, and the solvent was removed by rotary evaporation and dried under vacuum to yield a yellow solid (40 mg, 40%). 1H NMR (CDCl3): δ 3.27 (d, 1H, J ) 12.5 Hz to 4.54), 3.32 (d, 1H, J ) 12.5 Hz to 4.70), 3.36 (d, 1H, J ) 12.5 Hz to 4.59), 3.76 (br s, 2H, NH2), 4.54 (d, 1H, J ) 12.2 Hz), 4.59 (d, 1H, J ) 12.2 Hz), 4.70 (d, 1H, J ) 12.2 Hz), 6.44 (dd, 1H, J ) 2.0, 8.1 Hz), 6.86 (d, 1H, J ) 2.2 Hz), 6.94 (d, 1H, J )
Luyt and Katzenellenbogen
8.1 Hz), 7.16 (m, 4H), 7.24 (m, 2H), 7.52 (m, 2H). MS (FAB, +): m/z 467 (11%, 71Ga), 465 (13%, 69Ga); HRMS calcd C21H19N2S369Ga, 465.0044, found, 465.0043. Bis(2-(benzylthio)benzyl)(2-(benzylthio)-4-aminobenzyl)amido(tert-butoxycarbonyl)phenylalanine (10). The chelate system 9 (213 mg, 0.318 mmol) was combined with N-(tert-butoxycarbonyloxy)phenylalanine (89 mg, 0.334 mmol), 1,3-dicyclohexylcarbodiimide (66 mg, 0.334 mmol), 1-hydroxybenzotriazole (43 mg, 0.334 mmol), and 111 µL of N,N-diisopropylethylamine (0.637 mmol) in a flask with 20 mL of methylene chloride. The mixture was stirred at RT for 24 h and then filtered and washed with 0.1 M NaOH, water, and brine. The organic layer was filtered through cotton wool and then evaporated and dried under vacuum. Purification was carried out by flash chromatography (silica, 30% ethyl acetate/ 70% hexanes) to yield 10 (259 mg, 89%). 1H NMR (CDCl3): δ 1.43 (s, 9H), 3.15 (m, 2H), 3.48 (s, 2H), 3.55 (s, 4H), 4.01 (s, 4H), 4.03 (s, 2H), 4.45 (br, 1H), 5.16 (br, 1H), 7.01 (m, 1H), 7.10 to 7.33 (m, 27H), 7.49 (m, 1H), 7.57 (m, 2H). 13C NMR (CDCl3): δ 28.21, 38.35, 38.67, 39.05, 55.20, 55.62, 56.62, 80.55, 117.45, 119.80, 126.23, 127.06, 127.09, 127.17, 128.38, 128.40, 128.44, 128.81, 128.85, 129.01, 129.26, 129.29, 129.56, 129.67, 134.99, 135.72, 136.16, 136.58, 136.83, 136.94, 137.24, 139.42, 155.77, 169.40. MS (FAB, +): m/z 916 (100%); HRMS calcd C56H57N3O3S3, 916.3640, found, 916.3643. Bis(2-thio)benzyl-(2-thio-4-aminobenzyl)amido(tert-butoxycarbonyl)phenylalanine gallium(III) (12). A flame-dried three-necked flask with attached dry ice condenser was cooled to -78 °C and filled with 15 mL of anhydrous ammonia. To this was added the chelate system 10 (57 mg, 0.062 mmol) dissolved in 1.5 mL of THF, followed by small pieces of sodium until a blue color persisted, and the reaction was stirred at -78 °C for 1 h. The reaction was quenched with solid NH4Cl, and then the ammonia was evaporated under nitrogen flow, followed by vacuum-drying. The crude trithiol was dissolved in 10 mL of degassed methanol, and then [GaCl4][PPh4] (34 mg, 0.062 mmol) was added dissolved in 2 mL of degassed acetonitrile. The pH was adjusted to basic with 0.2 mL of 1 M KOH solution (degassed) and then stirred at room temperature for 35 min, after which the solvents were evaporated and the product was dried under vacuum. The crude material was purified by flash chromatography (silica, 50% ethyl acetate/50% hexanes) to yield 25 mg (57%) of 12 as a colorless solid. 1H NMR (CDCl3): δ 1.41 (s, 9H), 3.12 (m, 2H, CH2 of Phe), 3.37 (m, 3H, N-CH(H) × 3), 4.42 (m, 1H, CH), 4.55 (d, 1H, J ) 12.2 Hz coupled to 3.37 ppm, N-CH(H)), 4.60 (d, 1H, J ) 12.4 Hz coupled to 3.37 ppm, N-CH(H)), 4.63 (d, 1H, J ) 12.4 Hz coupled to 3.37 ppm, N-CH(H)), 5.08 (m, 1H, NH(BOC)), 7.11 to 7.83 (12H, ar and NH). MS (FAB, +): m/z 714 (3%), 713 (6%), 712 (4%), 711 (6%); HRMS calcd C35H35N3O3S369Ga, 711.1175, found, 711.1174. Histidine and Cysteine Challenge. A conical vial containing a stir bar was charged with 900 µL of test solution (10 mM histidine or cysteine in a buffered saline solution) and 100 µL of substrate (1 mM of 11 or 12 dissolved in methanol). The vial was sealed with a Teflon lined cap and maintained with stirring at 37 °C, with periodic analysis by HPLC. Enzymatic Hydrolysis. A conical vial containing a stir bar was charged with 800 µL buffered saline solution, 100 µL of substrate solution (1 mM 12 in DMF), 20 µL of Tween solution (1% in water), and 100 µL of R-chymotrypsin solution (approximately 0.7 units in a buffered saline solution). The vial was sealed with a Teflon-lined
Bifunctional Gallium(III) Ligand
Bioconjugate Chem., Vol. 13, No. 5, 2002 1143 Scheme 2
a
Figure 2. Retrosynthetic analysis. Scheme 1
a
a Reagents and conditions (yield): (a) BnBr, NaOH, EtOH (76%); (b) BH3‚THF, THF (97%); (c) PBr3, ether (97%).
cap and maintained with stirring at 37 °C, with periodic analysis by HPLC (retention time for 11 ) 4.4 min, retention time for 12 ) 15.5 min). The activity of the R-chymotrypsin was confirmed by the hydrolysis of N-benzoyl-L-tyrosine ethyl ester.
a Reagents and conditions (yield): (a) NaNO , HCl; (b) 2 CuSCN, KSCN (86%, two steps); (c) NH4OH (86%); (d) NaBH4, EtOH (94%); (e) BnBr, NaOH, EtOH (76%); (f) oxalyl chloride, DMF, CH2Cl2; (g) NH4OH, CH2Cl2 (94% two steps); (h) BH3‚THF, THF (88%).
Scheme 3
a
RESULTS AND DISCUSSION
The conjugation of the S3N tripodal chelate system to a peptide (Figure 2, R ) peptide) requires a point of attachment on one of the mercaptobenzene groups of the ligand. Since the other two mercaptobenzenes will be identical, this design permits the bifunctional tertiary amine tripodal chelate system to be prepared by a 2 + 1 approach, as depicted in Figure 2. Scheme 1 outlines the preparation of the benzyl bromide derivative 2 starting from thiosalicylic acid, an approach modified from that described earlier for the preparation of the chloride analogue of 2 (13). Thiol protection with a benzyl group proceeds in 76% yield, and reduction to the alcohol 1 in 97% yield using borane-THF complex. The benzyl bromide 2 was obtained through reaction with PBr3 in 97% yield. The preparation of the benzylamine portion of the tripodal ligand is described in Scheme 2. Prior to developing this route, a more direct approach was taken whereby 4-nitroanthranilic acid was diazotized and then reacted with benzyl mercaptan under basic conditions. Although this produced (benzylthio)nitrobenzoic acid 5 in two steps, the yield was poor (20%) and the chromatographic purification difficult. The improved route proceeds in five steps also starting with 4-nitroanthranilic acid, with the first three steps following a literature precedent (12). Formation of the diazonium salt followed by Sandmeyer-type reaction with copper thiocyanate produced the aryl thiocyanate 3 in 86% yield. Disulfide 4, prepared by ammonolysis followed by spontaneous oxidation of the thiolate, was rereduced with sodium borohydride, and the resultant thiol was protected with a benzyl group. This five-step transformation to the acid 5 was carried out in 53% yield, a significant improvement over the initial direct approach, with only simple crystallizations required for product recovery. The remaining three steps to the desired benzylamine derivative involve formation
a Reagents and conditions (yield): (a) K CO , acetone (75%); 2 3 (b) Raney Ni, hydrazine, EtOH, 1,2-dichloroethane (95%); (c) Phe(BOC), DCC, 1-HOBT, DiPEA, CH2Cl2 (89%).
of the acid chloride with oxalyl chloride and subsequent reaction with ammonia to generate benzamide 6 in 94% yield. Reduction with borane-THF complex proceeds in 88% yield giving the benzylamine 7. Construction of the tripodal ligand is illustrated in Scheme 3. Initial attempts were carried out using the chloride equivalent of the benzylic bromide 2, but reaction ceased after formation of the secondary amine and could not be driven further to the tertiary amine. The addition of sodium iodide produced byproducts due to sulfonium salt formation. The benzyl bromide 2 provided the correct reactivity, and with it the tertiary amine 8 could be formed at room temperature in 75% yield. The aromatic nitro substituent was readily reduced with hydrazine/ Raney nickel to give the aniline 9 in excellent yield. This amino group is available for attachment of peptides. To model the attachment to peptides, BOC-protected phenylalanine was conjugated via carbodiimide amide formation, to give 89% of 10. Deprotection of the thiol groups was carried out using sodium in liquid ammonia, as indicated in Scheme 4. It also proved possible to proceed directly from 8 to the deprotected trithiol, using sodium in liquid ammonia, thus carrying out both the nitro reduction and the thiol deprotection in one step. Due to the air sensitivity of the trithiol compounds, complexation with [PPh4][GaCl4] was
1144 Bioconjugate Chem., Vol. 13, No. 5, 2002 Scheme 4
Luyt and Katzenellenbogen
a
a Reagents and conditions: (a) NH , THF, -78 °C; (b) 3 [PPh4][GaCl4], KOH, MeOH, CH3CN.
protecting group. However, complex 11 was found to be much less stable under strongly alkaline conditions, as compared to complex 12. Under neutral conditions, both complexes are stable for long time periods in an aqueous medium. We were also interested in determining the stability of conjugates with an amide attachment to this gallium core, such as represented by 12. Thus, the susceptibility of 12 to enzymatic hydrolysis was studied (which would produce 11 as the product), following the hydrolysis by HPLC analysis. With both the phenylalanine substrate specific hydrolase chymotrypsin, and the more general amide hydrolyzing enzyme subtilisin, there was an insignificant level enzymatic hydrolysis of 12. Thus, the bulky chelate must hinder access of the complex to the active site of these enzymes. This suggests that conjugation of a biomolecule at this location through an amide linkage will provide a complex that will be stable in vivo. CONCLUSIONS
A tripodal S3N chelate system has been developed for the radiolabeling of large biomolecules with gallium radioisotopes. The conjugation of this chelator to phenylalanine demonstrates the appropriateness for utilizing this system in the production of gallium-labeled peptides. The bioconjugate complex was found to be stable to ligand exchange and enzymatic hydrolysis, providing initial evidence of its suitability for in vivo use. Figure 3. 1H NMR (CDCl3, 500 MHz) of 11 from δ 8.0 to 3.0 with inset 1H-1H COSY. Table 1. a Stability of Complexes 11 and 12 against Ligand Exchange and Enzymatic Hydrolysis 11
12
conditions
1h
4h
24 h
1h
4h
24 h
aqueous histidine cysteine R-chymotrypsin
>99 >99 97
>99 98 94
>99 >99 91
>99 99 98 97
>99 94 92 98
>99 55 44 93
a Percent of complex remaining at the indicated time. Conditions tested: aqueous medium at room temperature; ligand exchange with histidine; ligand exchange with cysteine; enzymatic hydrolysis with R-chymotrypsin. See Experimental Section for details.
carried out immediately, without purification of the trithiol intermediates. This two-step procedure for the aniline derivative 9 resulted in a 40% yield of the gallium complex 11. For the phenylalanine conjugate 10, a 57% yield was achieved in forming the complex 12. These four-coordinate gallium complexes have a propeller-type conformation, suggesting that the six benzylic protons (all diastereotopic pairs) would exist as four distinct doublets in the 1H NMR spectrum. Figure 3 shows the 1H NMR spectrum of 11, with an inset COSY (1H-1H correlation) for the relevant aliphatic region. The six benzylic protons appear as six distinct doublets, suggesting that in solution complex 11 exists as a twisted propeller, resulting in all six protons being nonequivalent. To test the applicability of this system for use as an in vivo imaging agent, we analyzed the stability of the gallium complexes 11 and 12 to ligand exchange, as presented in Table 1. In the presence of histidine and cysteine, both complexes appear relatively stable over 24 h. Thus, even with the increased electron density from the free amino group in 11, there is no significant loss in complex stability at a neutral pH. The decreased stability of the phenylalanine conjugate may be due to the BOC
ACKNOWLEDGMENT
This research has been supported through a grant from the Department of Energy (DE FG02 86ER60401). NMR spectra were obtained in the Varian Oxford Instrument Center for Excellence in NMR Laboratory. Funding for this instrumentation was provided in part from the W.M. Keck Foundation and the National Science Foundation (NSF CHE 96-10502). Mass spectra were obtained on instruments supported by grants from the National Institute of General Medical Sciences (GM 27029), the National Institute of Health (RR 01575), and the National Science Foundation (PCM 8121494). LITERATURE CITED (1) Green, M. A. (1990) The Potential for Generator-Based PET Perfusion Tracers. J. Nucl. Med. 31, 1641-1645. (2) Green, M. A., and Welch, M. J. (1989) Gallium Radiopharmaceutical Chemistry. Nucl. Med. Biol. 16, 435-448. (3) Wang, S., Lee, R. J., Mathias, Green, M. A., and Low, P. S. (1996) Synthesis, Purification, and Tumor Cell Uptake of 67Ga-Deferoxamine-Folate, a Potential Radiopharmaceutical for Tumor Imaging. Bioconjugate Chem. 7, 56-62. (4) Wu, C., Jagoda, E., Brechbiel, M., Webber, K. O., Pastan, I., Gansow, O., and Eckelman, W. C. (1997) Biodistribution and Catabolism of Ga-67-Labeled Anit-Tac dsFv Fragment. Bioconjugate Chem. 8, 365-369. (5) Sun, Y. S., Martell, A. E., Motekaitis, R. J., and Welch, M. J. (1998) Synthesis and Stabilities of the Ga(III) and In(III) Chelates of a New Diaminodithiol Bifunctional Liagnd. Tetrahedron 54, 4203-4210. (6) Heppeler, A., Froidevaux, S., Ma¨cke, H. R., Jermann, E., Be´he´, M., Powell, P., and Hennig, M. (1999) RadiometalLabeled Macrocyclic Chelator-Derivatised Somatostatin Analogue with Superb Tumour-Targeting Properties and Potential for Receptor-Mediated Internal Radiotherapy. Chem. Eur. J. 5, 1974-1981. (7) Govindaswamy, N., Quarless, Jr., D. A., and Koch, D. A. (1995) New Amine Trithiolate Tripod Ligand and Its Iron(II) and Iron(III) Complexes. J. Am. Chem. Soc. 117, 84688469.
Bifunctional Gallium(III) Ligand (8) Motekaitis, R. J., Martell, A. E., Koch, S. A., Hwang, J., Quarless, Jr., D. A., and Welch, M. J. (1998) The Gallium(III) and Indium(III) Complexes of Tris(2-mercaptobenzyl)amine and Tris(2-hydroxybenzyl)amine. Inorg. Chem. 37, 5902-5911. (9) Cutler, C. S., Giron, M. C., Reichert, D. E., Snyder, A. Z., Herrero, P., Anderson, C. J., Quarless, D. A., Koch, S. A., and Welch, M. J. (1999) Evaluation of Gallium-68 Tris(2-mercaptobenzyl)amine: A Complex with Brain and Myocardial Uptake. Nucl. Med. Biol. 26, 305-316. (10) Sun, Y., Cutler, C. S., Martell, A. E., and Welch, M. J. (1999) New Multidentate Ligands Containing Mercaptobenzyl Functional Groups, and Biodistribution of Gallium-67-TACNHSB. Tetrahedron 55, 5733-5740.
Bioconjugate Chem., Vol. 13, No. 5, 2002 1145 (11) Maelia, L., and Koch, S. A. (1986) Gallium Analogues of Iron-Sulfide-Thiolate Compounds. Analysis of the Structural Parameters in Gallium(III) and Iron(III) Chalcogenide Compounds. Inorg. Chem. 25, 1896-1904. (12) van der Stelt, C., van der Lugt, W., and Nauta, W. T. (1951) The Synthesis of 2-Mercapto-4-aminobenzoic Acid. Recueil. Trav. Chim. 70, 285-288. (13) Stacy, G. W., Villaescusa, F. W., and Wollner, T. E. (1965) 2-Aminobenzo[b]thiophene. An Aromatic Ring Tautomer. J. Org. Chem. 30, 4074-4078.
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