Oxalic Acid Supported Si - ACS Publications - American Chemical

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Oxalic Acid Supported Si−18F-Radiofluorination: One-Step Radiosynthesis of N-Succinimidyl 3-(Di-tert-butyl[18F]fluorosilyl) benzoate ([18F]SiFB) for Protein Labeling Alexey P. Kostikov,*,† Joshua Chin,† Katy Orchowski,† Sabrina Niedermoser,§ Miriam M. Kovacevic,† Antonio Aliaga,† Klaus Jurkschat,‡ Bjoern Wan̈ gler,§ Carmen Wan̈ gler,§ Hans-Jürgen Wester,∥ and Ralf Schirrmacher*,† †

McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montreal, Canada; Lehrstuhl für Anorganische Chemie II, Technische Universität Dortmund, Dortmund, Germany; § Department of Nuclear Medicine, Hospital of the Ludwig-Maximilians-University, Munich, Germany, ∥ Technical University Munich, Pharmaceutical Radiochemistry, Munich, Germany ‡

ABSTRACT: N-Succinimidyl 3-(di-tert-butyl[18F]fluorosilyl)benzoate ([18F]SiFB), a novel synthon for one-step labeling of proteins, was synthesized via a simple 18F−19F isotopic exchange. A new labeling technique that circumvents the cleavage of the highly reactive active ester moiety under regular basic 18F-labeling conditions was established. In order to synthesize high radioactivity amounts of [18F]SiFB, it was crucial to partially neutralize the potassium oxalate/hydroxide that was used to elute 18 − F from the QMA cartridge with oxalic acid to prevent decomposition of the active ester moiety. Purification of [18F]SiFB was performed by simple solid-phase extraction, which avoided time-consuming HPLC and yielded high specific activities of at least 525 Ci/mmol and radiochemical yields of 40−56%. In addition to conventional azeotropic drying of 18F− in the presence of [K+⊂2.2.2.]C2O4, a strong anion-exchange (SAX) cartridge was used to prepare anhydrous 18F− for nucleophilic radio-fluorination omitting the vacuum assisted drying of 18F−. Using a lyophilized mixture of [K+⊂2.2.2.]OH resolubilized in acetonitrile, the 18F− was eluted from the SAX cartridge and used directly for the [18F]SiFB synthesis. [18F]SiFB was applied to the labeling of various proteins in likeness to the most commonly used labeling synthon in protein labeling, N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). Rat serum albumin (RSA), apotransferrin, a β-cell-specific single chain antibody, and erythropoietin were successfully labeled with [18F]SiFB in good radiochemical yields between 19% and 36%. [18F]SiFB- and [18F]SFB-derivatized RSA were directly compared as blood pool imaging agents in healthy rats using small animal positron emission tomography. Both compounds demonstrated identical biodistributions in healthy rats, accurately visualizing the blood pool with PET.

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INTRODUCTION [18F]Fluorine is one of the most useful radioisotopes in positron emission tomography (PET) due to its favorable decay characteristics. The relatively long half-life (110 min) allows for multistep syntheses of radiopharmaceuticals and longer PET scans, while the low positron energy (635 keV) allows for highresolution imaging with the newest generation of PET cameras. A significant number of 18F-radiotracers have been proposed and synthesized for clinical PET, but very few are widely used in nuclear medicine, with the only notable exception being 2-[18F]Fluoro-2-deoxy-D-glucose ([18F]FDG).1 Harsh reaction environments, such as high temperatures and strong basic labeling conditions, are commonly required for the incorporation of anionic 18F into organic molecules by means of nucleophilic substitution. These requirements limit the range of possible precursors and decrease the yield of the desired product through the formation of unwanted side products. Moreover, the formation © 2011 American Chemical Society

of byproducts necessitates the use of time-consuming purification procedures. These complications become especially pronounced when labeling larger biomolecules such as peptides and proteins, where the labeling process relies on prosthetic 18F-labeling agents. In this approach, a prosthetic group (labeling synthon) is synthesized in one or more steps via nucleophilic 18F-fluorination and then reacted at mild conditions with the biomolecule intended for PET imaging. Numerous 18F-labeled precursors have been conjugated to biomolecules by means of amidation, imidation, alkylation, and Michael addition, among others.2−4 However, N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), which readily acylates proteins in aqueous media, remains the standard method for protein 18F-labeling since it was first Received: September 28, 2011 Revised: December 7, 2011 Published: December 8, 2011 106

dx.doi.org/10.1021/bc200525x | Bioconjugate Chem. 2012, 23, 106−114

Bioconjugate Chemistry

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reported in 1992.5,6 A variety of peptides and proteins have been labeled with [18F]SFB, including monoclonal antibodies, albumin, transferrin, and annexin V.7−9 Protein labeling is usually carried out in borate buffer at pH 8−9 upon incubation for 15−30 min, and the labeled protein is purified using sizeexclusion chromatography. Despite the ease of the bioconjugation step, preparation of [18F]SFB requires time- and laborconsuming procedures, including a three-step synthesis and HPLC purification.10 Starting from 18F−, the overall labeling of the peptide is carried out in four steps and two HPLC purifications, which takes over two hours to complete. Consequently, novel methods for the fast production of 18 F-labeling agents of both small and larger biomolecules, ideally under mild conditions, are highly sought after. Several groups have recently started to investigate the potential of “nonconventional” silicon,11−20 phosphorus,21 boron,22−24 and aluminum chelate25−27 containing precursors for 18F-labeling of organic vectors. These approaches deviate from the conventional formation of a carbon-18F bond by nucleophilic substitution. We have investigated a new 18F-labeling method based on the 18F−19F isotopic exchange at the silicon atom using no-carrier-added 18F− and nanomolar amounts of parasubstituted di-tert-butylphenylfluorosilanes (SiFA compounds) at room temperature.12−15 We have previously shown that 18 F-labeled SiFA compounds can be obtained in high radiochemical yields (RCYs) and specific activities (SAs) in a short amount of time due to the efficiency of the isotopic exchange reaction at the Si−F bond, in contrast to isotopic exchange reactions that involve a carbon−fluorine bond. Successful application of [18F]SiFA-isothiocyanate ([18F]SiFA−ITC) as a synthon for the labeling of proteins has been previously reported;28 however, this compound suffers from somewhat low hydrolytic stability that limits the amount of 18F incorporated into it in a one-batch synthesis or routine production. In this report, the high-yield, oxalic acid supported synthesis of N-succinimidyl 3-(di-tert-butyl[18F]fluorosilyl)benzoate ([18F]SiFB), a SiFA analogue of the [18F]SFB labeling synthon, via isotopic exchange and application of this synthon for the labeling of various proteins are described. Furthermore, the utilization of a novel SAX cartridge-based drying method for anhydrous basic 18F− in polar aprotic solvents (hereafter referred as to the Munich-method)29 for nucleophilic fluorination has been exploited and adapted to the efficient production of [18F]SiFB. A similar 18F drying strategy using organic bases has recently been reported.30

The labeling precursor N-succinimidyl 3-(di-tert-butylfluorosilyl)benzoate (SiFB) was synthesized as described below. Radiosynthesis of [18F]SFB was performed using a Scintomics Hotbox III module, whereas labeling of SiFB was carried out manually. Radio TLCs (silicagel-60 plates) were monitored using an Instant Imager (Packard) or Mini Gita (Raytest). Analytical HPLC and labeled protein purification was performed on an Agilent Technologies 1200 system equipped with a Gabi radioactivity detector (Raytest). For [18F]SFB and [18F]SiFB labeled protein purifications, the following HPLC conditions were applied: Phenomenex Biosep-SEC-S4000, 0.1 M sodium phosphate buffer pH 7.2, flow 0.7 mL/min, Rt (protein) = 15 min. Animal experiments were performed using a Concorde Micro-PET camera (Siemens). The NMR experiments were carried out with Varian Mercury 300 spectrometers. The chemical shifts δ are given in ppm and referenced to the solvent peaks with the usual values calibrated against tetramethylsilane (1H, 13C). Synthesis of N-Succinimidyl 3-(Di-tert-butylfluorosilyl)benzoate (SiFB). (3-Bromobenzyl)oxy(tertbutyl)dimethylsilane (1). Imidazole (4.53 g, 66.5 mmol, 2.5 equiv) and tert-butyl dimethylsilyl chlorid (TBSCl) (4.93 g, 32.7 mmol, 1.25 equiv) were sequentially added to a solution of 3-bromobenzyl alcohol (4.98 g, 26.6 mmol) in DMF (15 mL) at 25 °C, and the mixture was stirred at room temperature overnight. The reaction was quenched by addition of H2O (100 mL), and the mixture was extracted with Et2O (3 × 75 mL). The combined organic phase was washed with H2O (50 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (neat hexanes) to afford 1 as a colorless liquid (7.63 g, 95%). 1H NMR (300 MHz, CDCl3) δ 7.48 (s, 1H), 7.37 (d, 1H, J = 7.2 Hz), 7.24 (d, 1H, J = 7.8 Hz), 7.19 (t, 1H, J = 7.5 Hz), 4.71 (s, 2H), 0.95 (s, 9H), 0.11 (s, 6H). 13C NMR (300 MHz, CDCl3) δ 143.8, 129.9, 129.8, 129.0, 124.4, 122.4, 64.2, 25.9, 18.4, −5.3. Di-tert-butyl(3-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)fluorosilane (2). A solution of tert-butyl lithium in pentanes (1.7 M, 22.0 mL, 2.1 equiv) was added dropwise to a solution of 1 (5.36 g, 17.8 mmol) in anhydrous Et2O (20 mL) over a period of 15 min at −78 °C. After the solution had been stirred at the same temperature for additional 15 min, a solution of di-tert-butyldifluorosilane (3.85 g, 21.4 mmol, 1.2 equiv) in anhydrous Et2O (5 mL) was added dropwise over a period of 15 min at −78 °C. The reaction mixture was allowed to warm to room temperature overnight. The reaction was quenched by addition of saturated aqueous sodium chloride solution (100 mL), and the product was extracted with Et2O (3 × 75 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated in vacuo to afford 2 as a pale-yellow liquid (7.33 g) that was carried forward without further purification. (3-(Di-tert-butylfluorosilyl)phenyl)methanol (3). A concentrated aqueous hydrochloric acid solution (37 wt %, 0.5 mL) was added dropwise to a solution of crude 2 (6.77 g) in MeOH (50 mL) at 25 °C, and the mixture was stirred at room temperature overnight. After all volatiles have been removed under reduced pressure, the residue was redissolved in ether (100 mL), and the solution washed with saturated aqueous sodium bicarbonate solution (50 mL). The aqueous phase was extracted with ether (3 × 50 mL), and the combined organic phase was washed with H2O (50 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was

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EXPERIMENTAL SECTION Materials and Methods. Highly enriched [18O]water (>97%) was purchased from Rotem. (4-Ethoxycarbonylphenyl) trimethylammonium triflate, a precursor for the radiosynthesis of [18F]SFB, as well as nonradioactive standard, were purchased from ABX (Germany). All other commercially available chemicals, such as Kryptofix 2.2.2, acetonitrile, potassium oxalate, oxalic acid, tetrapropylammonium hydroxide, and N,N,N′,N′tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate, and RSA, apo-transferrin and IgG, were purchased at the highest available purity from Sigma-Aldrich. Recombinant erythropoitin (EPO) was purchased from Creative Biomart (US). The beta cell SCA was provided by Dr. S. Schneider (Innere Medizin II, Diabetologie und Endokrinologie St. Vinzenz-Hospital, Universität zu Köln). The SAX cartridges (Sep-Pak Light (46 mg) Accell Plus QMA Carbonate) were purchased from Waters (USA). 107



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Tetrapropylammonium hydroxide (TPAH, 20 μL of 1 M aq. solution in 2 mL MeCN) was then added, and the reaction mixture was heated to 120 °C for 3 min to hydrolyze the ethyl ester. Finally, after complete evaporation of MeCN to dryness, N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU, 12 mg, 40 μmol)dissolved in 1 mL of MeCN was added, and the reaction mixture was heated to 90 °C for 5 min. The resulting solution of crude [18F]SFB was cooled by a stream of air to room temperature and diluted with 1.5 mL of HPLC eluent for direct injection onto the HPLC system (μBondapak C-18 column, MeCN/0.01 M H3PO4, 40:60; flow 2 mL/min; Rt = 11 min). The radioactivity peak corresponding to the [18F]SFB was collected in a 50 mL round-bottom flask, and the eluent was carefully evaporated in vacuo under very gentle heating, as the product was volatile. The 18F-labeling synthon was redissolved in borate buffer pH 9 or water to be used in the bioconjugation reaction. The radiochemical purity of [18F]SFB was confirmed by radio-TLC (hexane/ethyl acetate 1:1, Rf = 0.5). Protein Labeling with [18F]SFB. An aliquot (0.5 mL) of the solution described above was added to the solution of protein (0.5 mL, 2 mg/mL, cf. Table 1) in borate buffer (pH 9),

purified by flash column chromatography on silica gel (9:1 to 7:1 hexanes/EtOAc) to afford 3 as a white solid (3.02 g, 64% over 2 steps). 1H NMR (300 MHz, CDCl3) δ 7.58 (s, 1H), 7.53 (d, 1H, J = 6.9 Hz), 7.44 (d, 1H, J = 7.5 Hz), 7.38 (t, 1H, J = 7.4 Hz), 4.71 (s, 2H), 1.86 (br s, 1H), 1.06 (d, 18H, J(1H, 19 F) = 1.2 Hz). 13C NMR (300 MHz, CDCl3) δ 139.9, 134.0 (J(13C, 19F) = 53 Hz), 133.3 (J(13C, 19F) = 17 Hz), 132.5 (J(13C, 19F) = 17 Hz), 128.3, 127.8 (J(13C, 19F) = 5 Hz), 65.5, 27.3 (J(13C, 19F) = 3 Hz), 20.2 (J(13C, 19F) = 50 Hz). 19F NMR (300 MHz, CDCl3) δ −188.84. HRMS (ESI, m/z): [M+Na]+ calc.: 291.1551; found: 291.1545. 3-(Di-tert-butylfluorosilyl)benzaldehyde (4). A solution of 3 (2.50 g, 9.31 mmol) in anhydrous CH2Cl2 (50 mL) was added dropwise to a solution of pyridinium chlorochromate (PCC) (5.02 g, 23.3 mmol, 2.5 equiv) in anhydrous CH2Cl2 (250 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and then allowed to warm to room temperature for 2.5 h, diluted with ether (200 mL), and filtered. The precipitate was washed with anhydrous Et2O, and the combined organic phase was concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (19:1 hexanes/ EtOAc) to afford 4 as a yellow oil (2.28 g, 92%). 1H NMR (300 MHz, CDCl3) δ 10.05 (s, 1H), 8.11 (s, 1H), 7.93 (dt, 1 H, J = 7.5, 1.5 Hz), 7.87 (dt, 1H, J = 7.5, 1.2 Hz), 1.07 (d, 18H, J(1H, 19F) = 1.2 Hz). 13C NMR (300 MHz, CDCl3) δ 192.6, 139.8 (J(13C, 19F) = 17 Hz). 135.5 (J(13C, 19F) = 18 Hz), 130.5, 128.3 (J(13C, 19F) = 4 Hz), 27.2 (J(13C, 19F) = 3 Hz), 20.2 (J(13C, 19F) = 48 Hz). 19F NMR (300 MHz, CDCl3) δ −188.43. HRMS (ESI, m/z): [M+H]+ calc.: 267.1575; found: 267.1571. N-Succinimidyl 3-(di-tert-butylfluorosilyl)benzoate (5). The N-succinimidyl ester was synthesized according to the procedure reported by Glaser31 for the synthesis of [18F]SFB. N-Hydroxysuccinimide (115 mg, 1 mmol) and 4 (53.3 mg, 0.2 mmol) were mixed in ethyl acetate (4 mL) in a 5 mL vial, and the mixture was cooled to 0 °C. Iodobenzene diacetate (PhI(OAc)2) (80.5 mg, 0.25 mmol) was added in one portion, and the reaction mixture was stirred for 15 min at 0 °C and then allowed to warm to room temperature for 1 h. After dilution with hexane (4 mL), the crude product was purified by column chromatography (hexane/ether 1:1). The N-succinimidyl ester 5 (64 mg, 0.17 mmol, 84% yield) was obtained as an oil that solidified upon standing. 1H NMR δ 8.37 (s, 1H), 8.19 (d, 1H), 7.90 (d, 1H), 7.52 (t, 1H), 2.90 (s, 4H), 1.03 (s, 18H). 13C NMR δ 169.3, 162.0, 140.2 (d), 135.7, 135.3 (d), 131.6, 128.2, 124.6, 27.2, 25.7, 20.2 (d). 19F NMR (300 MHz, CDCl3) δ −188.42. HRMS (ESI, m/z): [M+Na]+ calc.: 402.1507; found: 402.1510. Fully Automated Synthesis of N-Succinimidyl-4-[18F]fluorobenzoate([18F]SFB). A one-pot procedure reported by Tang et al.10 was used for the synthesis of [18F]SFB. Briefly, no-carrier-added aqueous [18F]fluoride/H2[18O]O (300−450 mCi, 11.1−16.6 GBq, ∼2.2 mL), generated using an IBA cyclotron (Cyclon 18/9) by the 18O(p,n)18F nuclear reaction on an enriched [18O]water target, was passed through a QMA cartridge. The 18F− was then eluted from the QMA cartridge with a solution of Kryptofix2.2.2 (10 mg, 27 μmol) and K2CO3 (13.5 μmol) in CH3CN/H2O (96:4, 1.5 mL). The residual water was removed by coevaporation to dryness with CH3CN using a stream of argon at 105 °C and a gentle vacuum. This step was repeated twice more with 0.5 mL CH3CN. (4-Ethoxycarbonylphenyl) trimethylammonium triflate (5 mg, 14 μmol) dissolved in 1 mL of MeCN was added to the “dried” [K+⊂2.2.2.][18F]F− cryptate and heated to 90 °C for 10 min.

Table 1. 18F-Labeled Proteins Using [18F]SiFB and [18F]SFBa RCY (by HPLC) 18

protein

[ F]SiFB

RSA (n = 3) Apo-transferrin (n = 3) β-cell SCA (n = 2)32 Erythropoietin (n = 2)

70 21 18 63

± ± ± ±

9% 2% 1% 5%

18

RCY (isolated)

[ F]SFB

[ F]SiFB

[18F]SFB

± ± ± ±

35 ± 8% n.i.b n.i. 19 ± 4%

27 ± 3% n.i. n.i. 19 ± 2%

38 33 14 27

3% 4% 3% 3%

18

a

RCYs refer to the starting activity of the labeling synthon. bn.i.: not isolated.

and the mixture was incubated for 20 min at room temperature. The entire batch was then injected into an HPLC system, and the collected 18F-labeled protein fraction was used in animal PET studies without further reformulation. Small Scale Synthesis of Succinimidyl 3-(Di-tertbutyl[18F]fluorosilyl)benzoate ([18F]SiFB)([18F]5) with aliquots of azeotropically dried 18F−. [18F]fluoride/H2[18O] O (370−1850 MBq, 10−50 mCi) was passed through a QMA cartridge preconditioned with 10 mL 0.5 M K2CO3, followed by 10 mL H2O. The 18F− was then eluted from the QMA cartridge with a solution of Kryptofix2.2.2 (7.5 mg, 20 μmol) and K2C2O4 (10 μmol) in CH3CN/H2O (96:4, 1 mL). The residual water was removed by coevaporation to dryness with CH3CN using a stream of argon at 105 °C and a gentle vacuum. This step was repeated twice more with 1 mL CH3CN. The residue containing the [K+⊂2.2.2.][18F]F cryptate was redissolved in MeCN (1 mL), and oxalic acid (0.1 M, 5 μmol) in anhydrous acetonitrile (50 μL) was added. SiFB (130 nmol, 1 μg/μL in MeCN) was added, and the reaction mixture was kept at room temperature for 5 min, diluted with distilled water (10 mL), and passed through a C-18 light cartridge (SepPak, Waters) preconditioned with 10 mL MeOH followed by 10 mL water. The cartridge was washed with 10 mL water to remove all traces of residual [K+⊂2.2.2.][18F]F. The desired [18F]SiFB was eluted from the cartridge with diethyl ether (3 mL) into an Eppendorf vial (5 mL), and the solvent was removed by a gentle argon stream at room temperature. Both aqueous and organic eluates were analyzed by means of radio-TLC (hexane/ 108



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ethyl acetate 1:1, Rf = 0.7) to confirm the quantitative separation of unreacted 18F− anion and [18F]SiFB. Synthesis of [18F]SiFB ([18F]5) Using the Munich Method (A: using whole 18F batch; B: using 18F aliquots). Preparation of [K+⊂2.2.2.]OH. Kryptofix2.2.2 (41 mg, 110 μmol) was dissolved in aqueous potassium hydroxide (1 M, 100 μL; 100 μmol). The resulting solution was further diluted with water (∼1 mL), lyophilized to dryness overnight, and dissolved in anhydrous acetonitrile (0.5 mL) immediately before use. Retention and Elution of 18F− from Anion Exchange Cartridge. Bombarded water from the target ( 18 F − / [18O]H2O) was passed through a SAX cartridge (Sep-Pak Light (46 mg) Accell Plus QMA Carbonate) preconditioned with water (10 mL). The cartridge was flushed with air (20 mL), rinsed with anhydrous MeCN (5 mL) to remove traces of water, and flushed again with air (20 mL). 18F− was eluted with the previously described [K+⊂2.2.2.]OH solution in acetonitrile (0.5 mL). Radiolabeling A. A solution of oxalic acid in anhydrous acetonitrile (0.5 M, 150 μL, 75 μmol) was added to the eluate containing [K+⊂2.2.2.][18F]F− (0.5 mL, 100 μmol), followed by a solution (5 × 10−3 M) of SiFB in acetonitrile (40 μL, 200 nmol). The reaction was kept at room temperature for 30 min. Starting from 263 mCi of 18F−, [18F]SiFB (105 mCi) was synthesized in 40% isolated yield (EOS), with a specific activity of 525 Ci/mmol. Radiolabeling B. The eluate containing [K+⊂2.2.2.][18F]F− (0.5 mL, 400 mCi) was aliquotized in 100 μL portions, each containing 20 μmol of KOH. A solution (0.5 M) of oxalic acid in anhydrous acetonitrile (0−40 μL, 0−20 μmol; Figure 4) was added, followed by a solution of SiFB in acetonitrile (5 × 10−3 M, 20−40 μL, 100−200 nmol), and the reaction was kept at room temperature for 5−15 min. Radiochemical yields were determined by radio-TLC (hexane/EtOAc 3:1). Three independent reaction parameters were optimized: n(KOH) = 20 μmol; n(H2C2O4) = 15 μmol; n(SiFB) = 100 nmol; t = 15 min; rt. Starting from 80 mCi of 18F−, [18F]SiFB (45 mCi) with a specific activity of 450 Ci/mmol (calculated lower limit) was synthesized in 56% yield (EOS). Purification of [18F]SiFB. The reaction mixture was diluted with an excess of aqueous H3PO4 (0.01 M, 3−5 mL) and passed through a preconditioned (10 mL MeOH, followed by 10 mL H2O) C18 cartridge. The cartridge was washed with water (5 mL) to remove traces of K2.2.2 and unreacted 18F−, and the desired product was eluted with diethyl ether (3 mL). The ether was removed within 5−10 min at room temperature using a stream of nitrogen. The [18F]SiFB was subsequently used for protein labeling as described below. The purity of [18F]SiFB was confirmed by radio-TLC and HPLC to be >98%. Protein Labeling with [18F]SiFB ([18F]5). A freshly prepared solution of the protein (e.g., RSA: 3.25 mg, 50 nM) in borate buffer (1 mL, pH 9) was added to an Eppendorf vial containing dried [18F]SiFB. The mixture was incubated for 30 min at room temperature. The entire batch was then injected into an HPLC system, and the collected protein was used directly for animal PET studies (Phenomenex BiosepSEC-S4000, 0.1 M sodium phosphate buffer pH 7.2, flow 0.7 mL/min, Rt (protein) = 15 min, k′(proteins) = 3). Small Animal PET Studies. Rats for small animal PET studies were housed in a 12 h light/dark cycle at 21 °C with access to food and water ad libitum, treatment was according to the Guide to the Care and Use of Experimental Animals (Ed2)

of the Canadian Council on Animal Care. The MicroPET imaging protocol was approved by the Animal Care Committee of McGill University (Montreal, Canada). Respiration rate, heart rate, and body temperature were monitored throughout the scan (Biopac systems MP150, Goleta, CA, USA). PET images of [18F]SiFB-RSA (11.7 MBq, 0.31 mCi) and 18 [ F]SFB-RSA (11.25 MBq, 0.3 mCi) were obtained from male Sprague−Dawley rats. Maximum intensity projections were obtained after the injection of [18F]SiFB- and [18F]SFB-RSA into a healthy rat. Data were acquired 5 s post tracer administration over 3600 s with the Concorde MicroPET R4 small animal tomograph. All images were reconstructed using the filtered back projection after applying normalized scatter correction for attenuation and radioactive decay. The PET images were analyzed using ASIPRO software (Concorde Microsystems) with 3D regions of interest (RIO) drawn around areas of increased tracer accumulation. The time−activity curves (TAC) were obtained from ROIs in heart (LV), artery, liver, kidney, and bone. Uptake of [18F]SiFB-RSA and [18F]SFB-RSA in the tissues were expressed as standard uptake value (%SUV) calculated as average tissue concentration (MBq/cc) divided by the ratio of injected dose (MBq) over subject mass multiplied by 100%.

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RESULTS AND DISCUSSION Design and Radioactive Labeling of [18F]SiFB. Nonsite-specific acylation with [18F]SFB remains the most widely used method of 18F protein labeling (Figure 1). The radiochemical

Figure 1. Radioactive labeling of various proteins using [18F]SiFB ([18F]5) and the most commonly applied synthon in protein labeling

synthesis of this prosthetic group has been improved over the years. Recently, a relatively simple one-pot procedure was reported by Tang et al.;10 however, this procedure required a three-step synthesis and a final HPLC purification in order to obtain highly pure [18F]SFB. The goal of the present study was to develop a novel 18F protein labeling prosthetic group based on SiFA group radiochemistry, which could be (1) synthesized in one simple step (e.g., 19F for 18F isotopic exchange), (2) purified using fast, solid-phase extraction technique, and (3) used like [18F]SFB for protein labeling. Since the labeling and conjugation reactions should be each one-step reactions, we investigated the direct isotopic 19F-for-18F exchange reaction on SiFB (5). This SiFA labeling precursor consists of a highly reactive group, e.g., the succinimidyl ester for the conjugation 109



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Figure 2. Synthetic scheme for SiFB (5) and its 18F-fluorination: (a) TBSCl/imidazole, DMF, rt; 95%; (b) t-BuLi/(t-Bu)2SiF2, ether, −78 °C → rt; (c) HCl, MeOH, rt; 64% over 2 steps; (d) PCC, DCM, −0 °C → rt; 92%; (e) NHS/PhI(OAc)2, EtOAc, 0 °C → rt; 84%; (f) 18F−/K2C2O4/K2.2.2/ H2C2O4, MeCN, rt (note: no product formation without oxalic acid).

reactions) to avoid formation of the aforementioned byproduct. Under these conditions, the reaction mixture had to be cooled down to a minimum of 0 °C to prevent fast hydrolysis of the initially formed [18F]SiFB under nucleophilic fluorination conditions. This short shelf-life in even slightly basic solution necessitated the immediate use of [18F]SiFB for protein labeling. Furthermore, utilizing a full batch of azeotropically dried 18F− did not give a high radioactivity amount of [18F]SiFB. The labeling procedure was substantially improved by addition of oxalic acid to the basic labeling cocktail before the isotope exchange reaction was initiated. Oxalic acid was selected over inorganic acids for two reasons: First, it contains the same anion as potassium oxalate, which is already present in the reaction mixture, thus limiting the possibility of anioninduced side reactions; and second, oxalic acid is soluble in anhydrous acetonitrile and therefore could be added to the [K+⊂2.2.2.][18F]F cryptate complex after azeotropic drying. The dependence of the incorporation yield of 18F into SiFB on the ratio of K2C2O4 used for 18F− elution from QMA to H2C2O4 was thoroughly investigated (Figure 3). The addition of an

to a protein and a di-tert-butylfluorosilyl group for highly efficient isotope exchange (Figure 2). Although in conventional 18 F radiochemistry, like the [18F]SFB synthesis, a succinimidyl ester moiety results in the formation of the corresponding acid [18F]fluorides that hydrolyze upon analyses, we assumed that the SiFA approach might be able to overcome this limitation. Most recently, Olberg et al. reported the elegant one-step synthesis of 6-[18F]fluoronicotinic acid 2,3,5,6-terafluorophenyl ester ([18F]F-Py-TFP), another new prosthetic group for protein labeling.33 The tetrafluorophenyl ester moiety of this reported labeling synthon survived the basic reaction conditions for 18F-labeling at 40 °C. The [18F]SFB synthon contains 18F in the para-position to the succinimidyl group because the aromatic nucleophilic 18F fluorination of the precursor (4-ethoxycarbonylphenyl) trimethylammonium triflate is facilitated by an electron-withdrawing ester group in the para-position. However, for highly reactive SiFA compounds, the relative orientation of the SiFA group to the other substituents in the molecule does not play a significant role in the isotope exchange reaction, making an activated aromatic system unnecessary. On the contrary, preliminary hydrolysis studies gave reason to expect a faster hydrolysis rate for para-substituted SiFA compounds compared to meta-substituted ones. For this reason, we designed N-[18F]succinimidyl 3-(di-tert-butylfluorosilyl)benzoate ([18F]SiFB) as a novel prosthetic molecule for protein labeling, bearing the SiFA building block in meta-position to the active ester moiety (Figure 2). Of primary concern was the inability to label SiFB in one step without hydrolyzing the N-succinimidyl ester under typical basic labeling conditions (Figure 2f). Optimization of the Reaction Conditions for the Synthesis of [18F]SiFB and Its Purification. The base used for the azeotropic drying of 18F−appeared to have a crucial influence on 18F-incorporation yields into SiFA compounds. Utilizing potassium carbonate as a base in this particular 18 F-fluorination led to a side product that did not correspond to the desired [18F]SiFB. Thus, a milder base, potassium oxalate, was used in all of the subsequent isotope exchange reactions leading to [18F]SiFB. However, even with this very mild base, the concentration of the [K+⊂2.2.2.]2C2O4 had to be kept at the very low level (1 mg/mL or 2.7 μmol/mL, approximately 10-times more dilute than for conventional SN2 fluorination

Figure 3. Using azeotropic 18F− drying ([K+⊂2.2.2.]2C2O42−): Dependence of incorporation yield of 18F− into SiFB (5) (50 μg, 130 nmol) by isotopic exchange on the amount of added oxalic acid (range: 0−10 μmol), keeping a constant K2C2O4 amount (10 μmol) after 5 and 20 min reaction time (n = 10 for each point, errors are expressed as SEM).

excess of H2C2O4 led to a decrease in RCY, which was probably due to a formation of H18F, which is not as reactive as nucleophilic 18F−. The optimal molar ratio for obtaining highest 110



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RCYs with a full batch of azeotropically dried 18F− was found to be ν([K+⊂2.2.2.]2C2O42‑):ν(H2C2O4) = 2:1. These conditions provided the stability of the N-succinimidyl ester group over time up to 30 min without compromising incorporation yields of the isotope exchange. Addition of the optimum amount of H2C2O4 was a rather delicate task due to unpredictable surface adsorption effects of the [K+⊂2.2.2.]2C2O42−cryptate on the walls of the azeotropic drying receptacle (Wheaton vial). To control the true amount of basic oxalate in the labeling solution, the most recently described cartridge-based procedure, developed by Wester et al. (Munich-Method),29 was employed to obtain anhydrous 18F− in polar aprotic solvents without azeotropic drying. In this procedure, lyophilized [K+⊂2.2.2.]OH cryptate, resolubilized in dry acetonitrile, is used to elute 18F− from the SAX cartridges of different sizes in acetonitril. Here, a solution of [K+⊂2.2.2.]OH cryptate formed by lyophilization of 110 μmol K2.2.2 and 100 μmol KOH and redissolved in 0.5 mL of anhydrous acetonitrile was used to elute the retained 18F− from the SAX cartridge. When the labeling reaction was carried out using a 100 μL aliquot of the 0.5 mL full batch solution of [K+⊂2.2.2.]18F (containing 100 μmol potassium hydroxide or potassium carbonate, respectively), 15 μmol of oxalic acid had to be added to the 20 μmol of KOH in order to get the required incorporation yields (Figure 4). This is consistent with a 2:1 ratio of oxalate to

reduced to 5 min when the Munich method was used. This was four times faster than conventional azeotropic drying and only required a single anion exchange cartridge. When a full batch of 18 − F containing 100 μmol of KOH in 0.5 mL acetonitrile was used, the isotopic exchange reaction took 30 min to obtain incorporation yields of 18F− of 50−70%. This was a result of the larger reaction volume. Smaller aliquots of 100 μL from the 0.5 mL stock solution required a reaction time of only 5−10 min to ensure sufficient 18F− incorporation. In one experiment, a starting activity of 400 mCi [K+⊂2.2.2.]18F in 0.5 mL of acetonitrile was used. From this stock solution, a 100 μL aliquot containing 80 mCi yielded 45 mCi of [18F]SiFB after 20 min. Next, optimized conditions for the purification of [18F]5 were developed. A main advantage of [18F]SiFA compounds is their relatively simple purification without use of HPLC. Due to the aforesaid optimized mild reaction conditions, no radioactive side-products other than unreacted 18F− were observed. As a result of their very different lipophilicities, the separation of [18F]5 from 18F− was achieved by simple solid-phase cartridge extraction. The crude reaction mixture was diluted with aqueous H3PO4 (0.01 M, 1:10 v/v) and passed through a C18 cartridge. [18F]5 was quantitatively retained on the solidphase material, whereas the 18F− and Kryptofix2.2.2 fraction were discarded. It is possible to easily elute [18F]5 from the cartridge using common organic solvents, such as MeOH, MeCN, THF, or ether. Since the organic solvent had to be completely removed without overheating the N-succinimidyl ester in order for a successful subsequent bioconjugation reaction to occur, volatile diethyl ether was chosen as an eluent. The radiochemical purity (RCP) of [18F]5 was usually >99%, as determined by radio-TLC. In direct comparison with the [18F]SFB synthesis, no HPLC purification was needed, and the total preparation time for [18F]5 could be reduced to 55 min if a 100 μL 18F− aliquot is used (Figure 5A,D). Besides saving valuable synthesis time, the technical effort to obtain [18F]5 as a labeling synthon for proteins is marginal. Even laboratories with limited equipment would be able to obtain 18F-labeled proteins by minimizing the synthetic effort. Bioconjugation of [18F]SiFB with Proteins. Bioconjugation of [18F]SFB to proteins is usually carried out in aqueous borate buffers at pH 8−9 in the absence of organic solvents. The rate of N-acylation under these conditions is considered comparable to or better than that of ester hydrolysis and typically yields 30−50% within 15−30 min. These same conditions were applied for coupling of [18F]SiFB with proteins, as it follows the same functional group chemistry. In a previous publication,34 we reported an average half-life of SiFA compounds of 2 h at pH 10. Therefore, the extent of hydrolysis at pH 9 within 30 min was not expected to be very significant and within acceptable limits. After 20−30 min reaction time, the purification of the labeled protein was carried out on HPLC, using a size-exclusion column and sodium phosphate buffer as an eluent. Since neither organic solvents nor potassium salts were used in the HPLC solvent, the eluate (pH 7.4) was suitable for animal injection. As expected, three competing pathways during the [18F]5 protein labeling were observed: (i) bioconjugation of [18F]5 to the protein; (ii) hydrolysis of the N-succinimidyl ester; and (iii) hydrolysis of di-tert-butyl[18F]fluorosilyl group yielding anionic 18F−. The predominant pathway appeared to be the bioconjugation of [18F]5 to the protein. Typically, four major peaks were observed on the chromatogram: (i) labeled protein (up to 80%); (ii) unreacted [18F]SiFB (∼10%); (iii) unknown byproduct (∼5%, most likely 3-di-tert-butyl[18F]fluorosilylbenzoic

Figure 4. Using the Munich method for 18F− drying: Radiochemical incorporation yields of 18F into [18F]SiFB versus amount of oxalic acid after 5 (red line) and 10 (blue line) minutes reaction time ([Kryptofix]OH = 20 μmol, n(SiFB) = 100 nmol, n = 10 for each point, errors are expressed as SEM).

oxalic acid. Any deviation from this ratio led to a significant decrease of RCYs. The Munich method reduced the overall synthesis time by 20 min and significantly decreased the technical complexity for preparing anhydrous 18F−, for which no heating, vacuum, or gas flow was needed. The amount of the SiFA-compound 5 also had to be optimized in order to obtain the maximal specific activity of [18F]5 without lowering its radiochemical yield. The acceptable incorporation yield of 50−70% was achieved using as little as 38 μg (100 nmol) of the precursor. Figure 5 summarizes the different compounds and methods used in this investigation. The synthesis time for [18F]SiFB varied between 20 and 50 min depending on the method used to obtain anhydrous 18 − F (either azeotropic drying or the Munich cartridge method) and whether the full 18F− batch or a smaller 18F− aliquot was used (Figure 5). The preparation time for anhydrous 18F− was 111



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Figure 5. Synthetic time line of stepwise syntheses of [18F]SFB and [18F]SiFB. (A) [18F]SFB synthesis: (a) reaction of (4-ethoxycarbonylphenyl) trimethylammonium triflate with 18F; (b) hydrolysis of ethyl ester with TPAH; (c) evaporation of solvent and coupling with TSTU; (d) HPLC purification; (e) evaporation of solvent and reformulation. (B) [18F]SiFB synthesis using an 18F‑ aliquot obtained by azeotropic drying of 18F−: (a′) K2.2.2/K2C2O4/18F− cooled to rt; (b′) addition of oxalic acid and reaction of 5 with 18F−; (c′) cartridge purification of [18F]5; (d′) evaporation of ether and reformulation in buffer. (C) [18F]SiFB synthesis using a full batch of 18F−obtained by the Munich method: (a″) addition of oxalic acid reaction of 5 with 18F− (0.5 mL); (b″) cartridge purification of [18F]5; (c″) evaporation of ether and reformulation in buffer. [18F]SiFB (D) [18F]SiFB synthesis using an aliquot of 18F− batch in 0.5 mL acetonitrile obtained by the Munich method: (a‴) addition of oxalic acid reaction of 5 with aliquot of 18F− (0.1 mL); (b‴) cartridge purification of [18F]5; (c‴) evaporation of ether and reformulation in buffer.

acid); and (iv) 18F− (∼ 5%). In a typical experiment starting from 7.5 mCi [18F]5, 1.7 mCi of labeled RSA in 1.5 mL of phosphate buffer was collected, and the resulting solution was used for animal experiments without further reformulation. The results of protein labeling with [18F]SiFB ([18F]5) and [18F]SFB are summarized in Table 1. Only labeled RSA and erythropoietin were purified via HPLC and prepared as injectable solutions. The overall preparative RCYs in these two cases were comparable. The obvious discrepancy between the RCYs determined via HPLC and the de facto isolated RCYs in the case of [18F]SiFB might be explainable by a higher absorption of unreacted [18F]SiFB (and/or hydrolysis products) on the column material in comparison to the less lipophilic [18F]SFB. The absorption might take place at the very beginning of the column preventing a later detection via UV and radioactivity detection. [18F]SiFB to Protein Ratio. In order to preserve the biological activity of the protein, the ratio of the labeling synthon to the molecule to be labeled should not exceed two to three.35,36 Proteins, especially antibodies, lose their biological integrity when there are higher amounts of derivatization sites. In this study, the initial labeling procedure required 100 nmol of SiFB as a starting material, whereas the amount of labeled RSA (1 mg, MW ≈ 65 kDa) was 15.4 nmol. Therefore, the resulting ratio of 6.5:1 was not ideal. In attempts to reduce the ratio, the amount of nonradioactive SiFB was decreased and the mass of RSA remained constant. However, this led to significantly reduced incorporation yields of 18F into the SiFA molecule. In the next attempt, the amount of protein was increased to 50 nmol (3.25 mg), while the concentration of SiFB remained at 100 nmol, thus reaching a 2:1 ratio. This approach proved to be more successful, as the desired ratio was

achieved without compromising the yield of the labeled bioconjugate, and a full batch of [18F]SiFB could be used. Regarding the other proteins employed for labeling, the following molar amounts were used for the labeling with [18F]SiFB: transferrin (1 mg, 12.5 nmol), EPO (1 mg, 50 nmol), and a β-cell SCA (1 mg, 32 nmol). The amount of [18F]SiFB (from a stock solution containing 100 nmol SiFB) used for labeling was adjusted in order to not exceed the targeted SiFB:protein ratio of 2:1. In order to permit a direct comparison between the labeling efficiency of [18F]SiFB and [18F]SFB, the molar amounts of protein were kept the same for both labeling synthons. Animal Experiments. Blood pool imaging in planar or single photon emission computer tomography is capable of determining left and right ventricular volumes and ejection fractions in both humans and animals. It has been shown that radiolabeled serum albumins are excellent tracers for small animal PET blood pool studies. In this experiment, rat serum albumin was labeled with either [18F]SFB or [18F]SiFB according to the above-described procedures. The labeled compounds were then injected into healthy rats (n = 3 for each labeled RSA), and the biodistribution was studied for 60 min from the time of injection. Images revealed that the blood pool in the heart could be well delineated. Tracer accumulation was clearly observed in large arteries, the liver, and the left ventricle of the heart. Furthermore, the right ventricle and atria were also noticeable. No significant bone uptake was observed, proving the stability of the labeled protein in vivo toward defluorination (Figure 6). The time activity curves depicting the radioactivity amount in different organs (heart, liver, and kidney), the main artery, and bone, indicated that there were no major differences in 112



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CONCLUSIONS A novel prosthetic group for non-site-specific protein labeling was designed and synthesized in one step by simple 19F−18F isotopic exchange. Addition of oxalic acid was crucial to prevent decomposition of the active ester moiety. Pure (>99%) [18F]SiFB was obtained in up to 56% yield (EOS, non-decaycorrected) within 20−55 min depending on the synthetic method employed. The technical effort to obtain [18F]SiFB is incomparably smaller than for [18F]SFB. The resulting [18F]SiFB was used for labeling of various proteins, such as RSA in RCYs comparable to those obtained with [18F]SFB. RSA labeled with [18F]SiFB showed the same biodistribution as [18F]SFB-labeled RSA in healthy rats, accurately depicting blood flow using small animal PET. The number of introduced labeling sites to the protein using [18F]SiFB is uncritical, guaranteeing an uncompromised biological integrity of the given protein. This conclusively demonstrates the applicability of [18F]SiFB as a novel labeling synthon for protein labeling. [18F]SiFB has the potential to become a valuable, easily accessible alternative to [18F]SFB as a result of its easy preparation and similar reactivity toward protein labeling.

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AUTHOR INFORMATION Corresponding Author *Alexey Kostikov: Tel: 514 398 8527, [email protected]. Ralf Schirrmacher: Tel: 514 398 1857, Fax: 514 398 1392, ralf. [email protected].

Figure 6. Biodistribution of labeled RSA in rat [(a + a′): [18F]SFB-RSA; (b + b′): [18F]SiFB-RSA; coronal plane sum images t = 5−60 min].

distributions between SFB and SiFB labeled RSA (Figure 7). The time activity curves corroborated that both labeled RSAs

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ACKNOWLEDGMENTS The authors wish to acknowledge financial support from the following agencies: Bayern-Quebec-Allianz to S.N.; DFG grant no. WA 2132/3-1 to B.W.; Canada Foundation for Innovation Project#: 203639 to R.S.; Canadian Institutes of Health Research (CIHR), Operating Grant #97970 to R.S.

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Figure 7. Time−activity curves for small bone, vessel, spleen, aorta, and liver extracted from in vivo PET scan for up to 60 min post injection of [18F]SiB-RSA (A) and [18F]SFB-RSA (B). Each point represents the mean of three independent animal experiments and errors are expressed as SEM (number of animals: n = 3).

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