3-¹⁸F-Fluoropropane-1-thiol and ¹⁸F-PEG₄-1-thiol: versatile prosthetic groups for radiolabeling maleimide functionalized peptides

Abstract

The efficient radiosynthesis of biomolecules utilizing minute quantities of maleimide substrate is important for availability of novel peptide molecular imaging agents. We evaluated both 3-¹⁸F-fluoropropane-1-thiol and 2-(2-(2-(2-¹⁸F-fluoroethoxy)ethoxy)ethoxy)ethane-1-thiol (¹⁸F-fluoro-PEG₄ thiol) as prosthetic groups for radiolabeling under physiological conditions. The precursor employed a benzoate for protection of the thiol and an arylsulfonate leaving group. The radiofluorination was fully automated on an Eckert & Ziegler synthesis system using standard Kryptofix₂₂₂/K₂CO₃ conditions. In order to minimize the amount of biological molecule required for subsequent conjugation, the intermediates, S-(3-¹⁸F-fluoropropyl) benzothioate and ¹⁸F-fluoro-PEG₄ benzothioate, were purified by HPLC. The intermediates were isolated from the HPLC in yields of 37 – 47% and 28 – 35%, respectively, and retrieved from eluate using solid phase extraction. Treatment of the benzothioates with sodium methoxide followed by acetic acid provided the free thiols. The desired maleimide substrate in acetonitrile or phosphate buffer was then added and incubated at room temperature for 15 min. The final radiolabeled bioconjugate was purified on a separate HPLC or NAP-5 column. Maleimides utilized for the coupling reaction included phenyl maleimide, an Evans Blue maleimide derivative, a dimeric RGDfK maleimide (E[c(RGDfK)]₂), two aptamer maleimides, and PSMA maleimide derivative. Isolated radiochemical yields (non-decay corrected) of maleimide addition products based on starting ¹⁸F-fluoride ranged from 6 – 22 % in a synthesis time of about 90 min.

¹⁸F-thiol prosthetic groups were further tested in vivo by conjugation to E[c(RGDfK)]₂ maleimide in a U87MG xenograft model. PET studies demonstrated similar tumor accumulation of both prosthetic groups. ¹⁸F-fluoro-PEG₄-S-E[c(RGDfK)]2 displayed a somewhat favorable pharmacokinetics compared to ¹⁸F-fluoropropyl-S-E[c(RGDfK)]₂. Bone uptake was low for both indicating in vivo stability.

1. Introduction

Despite its favorable chemical properties and decay characteristics (t_1/2 = 109.7 min, 97% β⁺, E(β⁺)max = 634 keV), the use of ¹⁸F for biological molecules is impeded because the incorporation reaction conditions are typically harsh and require protection of many functional groups on biomolecules. Numerous prosthetic groups have been developed, published, and reviewed for labeling biomolecules with ¹⁸F in the last three decades (1–6). The prosthetic groups are small organic molecules that can be easily radiolabeled with ¹⁸F and then conjugated to the biomolecules, mainly by targeting the primary amine of lysine or the thiol of cysteine (1–3).

In 2004, Glaser et al. reported on the labeling of ¹⁸F-fluorothiols and successfully applied to the thiol selective displacement on a 2-chloroacetamide. They utilized model 2-chloroacetamide peptides (3.2 µmol) and, without intermediate purification, conducted the displacement reaction using basic conditions (pH 9–10) and heat (70–80 °C for 30 min) (7). As stated above, most of the biomolecules are unstable under such labeling conditions and attempt by the same group to label a 2-chloroacetamide-RGD peptide using these conditions failed (8).

It is known that the reaction between a thiolated biological molecule and a high molar activity [¹⁸F]fluorinated maleimide worked very effectively (9). We believed that the reverse approach would work as well and thus developed two [¹⁸F]fluorothiol prosthetic groups, similar to those described above by Glaser et al, that were easily purified by HPLC and with an easily cleaved protecting group. Both prosthetic groups were synthesized in good radiochemical yield and high molar activity. The bioconjugation of the [¹⁸F]fluorothiols with maleimide functionalized biomolecules was conducted in phosphate-buffered saline (PBS, pH 7.4) at room temperature (RT) and with small amounts of precursor (Fig. 1). The whole labeling procedure was fully automated in an Eckert & Ziegler modular synthesis system. In order to assess effects of these prosthetic group on biological activity, we conducted in vivo evaluation of an RGD dimer peptide to which [¹⁸F]fluorothiol prosthetic groups had been conjugated.

Fig. 1.

Radiolabeling of various biomolecules containing maleimide via ¹⁸F-thiol prosthetic groups in phosphate buffered saline (PBS) pH 7.4 for 15 min at room temperature.

2. Material and Methods

2.1. General

Solvents and reagents were purchased from Sigma Aldrich or Fisher Scientific. ¹⁸F-fluoride was obtained from NIH Warren Grant Magnuson Clinical Center cyclotron facility. E[c(RGDfK)]₂ was acquired from CS Bio. The ultraviolet absorbance for high-performance liquid chromatography (HPLC) system was monitored at 254 and 210 nm. ¹H, ¹³C and ¹⁹F NMR spectra were obtained on a Bruker Avance 300 MHz NMR spectrometer in CDCl₃. LC-MS were carried out on a Acquity Waters system equipped with an electrospray interface (ESI) similarly to the published protocol (17).

2.2. Chemistry

2.2.1. 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (PEG₄-monotosylate).

(PEG₄ monotosylate) were synthesized according to the published procedure (18).

2.2.2. S-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl) benzothioate (PEG-4-benzothioate).

PEG₄-monotosylate (186 mg, 0.53 mmol) was dissolved in 3 mL CH₃CN. Et₃N (73 µL, 0.53 mmol) was added, followed by thiobenzoic acid (73 mg, 0.53 mmol). TLC (1:1 EtOAc: hexane) was used to follow the reaction. The reaction was stirred for 24 hours, a second portion of 20 mg thiobenzoic acid added, and stirred for three days. The solvent was removed under vacuum and the residue subjected to silica gel chromatography (ISCO system, EtOAc: CH₂Cl₂) using the standard linear gradient. The product was isolated (109 mg, 65%). The product, which contained about 10 mole percent starting material, was used directly in subsequent reactions without additional purification. ¹H-NMR δ 3.34 (t, J = 6.47 Hz, 2H), 3.60–3.67 (m, 2H), 3.67 – 3.81 (m, 12H), 7.44–7.53 (m, 2H), 7.57 – 7.65 (m, 1H), 7.96–8.04 (m, 2H).

2.2.3. S-(2-(2-(2-(2-(((4-nitrophenyl)sulfonyl)oxy)ethoxy)ethoxy)ethoxy)ethyl) benzothioate (nosylate PEG₄ benzothioate).

PEG₄ benzothioate (475 mg ,1.5 mmol) was dissolved in 5 mL ethyl acetate. 4-nitrobenzenesulfonyl chloride (335 mg, 1.5 mmol) was added, followed by Et₃N (209 µL, 1.5 mmol). The mixture was stirred overnight. A precipitate was filtered off and the solvent evaporated. The residue was subjected to silica gel chromatography (CH₂Cl₂ / EtOAc gradient) on a 40-gram cartridge using an ISCO system. The purest fraction was collected for subsequent use (218 mg, 29%). ¹H-NMR δ 3.28 (t, J = 6.2 Hz, 2H), 3.56 (brs, 4H), 3.59–3.75 (m, 8H), 4.24–4.32 (m, 2H), 7.39–7.50 (m, 2H), 7.52–7.62 (m, 1H), 7.90 – 8.00 (m, 2H), 8.13 (dm, J = 8.5 Hz, 2H), 8.39 (dm, J= 8.55, 2H). ¹³C-NMR 191.76, 153.45, 137.11, 133.68, 129.57, 128.85, 127.44, 124.58, 70.94, 70.83, 70.76, 70.59, 70.07,68.76, 28.85.

2.2.4. S-(2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)ethyl) benzothioate (Fluoro-PEG₄-benzothioate).

PEG₄ benzothioate (109 mg, 0.347 mmol) was dissolved in 2 mL CH₂Cl₂. The solution was cooled in ice prior to addition of diethylaminosulfur trifluoride (DAST) (46 µL, 0.347). After 150 min, the reaction was quenched with bicarbonate solution and extracted with CH₂Cl₂. The organic layers were washed with brine and dried. After evaporation, the products were separated by silica gel chromatography (CH₂Cl₂/hexane gradient) to yield the desired product (24 mg, 22%). Products resulting from cleavage of the PEG chain were also observed. ¹H-NMR δ 3.3 (t, J = 6.7 Hz, 2H), 3.67–3.78 (m, 11H), 3.80 (m, 1H), 4.59 (dm, J = 48.3 Hz, 2H), 7.43–7-53 (m, 2H), 7.56–7.65(m, 1H), 7.96–8.05 (m, 1H). ¹⁹F-NMR (external standard) −222.9 (tt, J = 48.4, 29.6 Hz, 1F). GC-MS (CI-CH₄) 105 (100), 149 (25), 165 (50), 257 (5), 297(1), 315(trace), 317 (trace).

2.2.5. S-(3-hydroxypropyl) benzothioate.

Thiobenzoic acid (514 mg, 3.7 mmol), 3-bromopropanol (517 mg, 3.72 mmol), and diisopropylethylamine (480 mg, 3.72 mmol) were combined in 10 mL CH₃CN. The mixture was heated at 80 °C for 5 hours. The CH₃CN was evaporated and the residue partitioned between ether and water. The aqueous layer was extracted with a second portion of ether and the combined ether layers were washed with brine, dried, and evaporated. The residue was chromatographed on silica gel (EtOAc:hexane, ISCO system, standard gradient) to provide the product (313 mg, 42%). GC-MS (EI) TR 6.38 min, m/z 196 (0.28), 123 (22), 105 (100), 77 (45). ¹H-NMR δ 1.92 (quin, J = 6.63 Hz, 2H), 3.21 (t, J = 6.8 Hz, 2H), 3.706 (t, J = 5.8 Hz, 2H), 7.44 (t,J = 7.4 Hz, 2H), 7.57 (tm, J = 7.3 Hz, 1H), 7.91–8.02 (m, 2H). ¹³C-NMR δ 25.40, 32.73, 60.50, 127.44, 128.79, 133.69, 137.02, 193.39.

2.2.6. S-(3-fluoropropyl) benzothioate.

A flask was charged with argon, thiobenzoic acid (1 g, 7.2 mmol) and CH₃CN (20 mL) were added. Then, 3-bromo-1-fluoropropane (1.0 g, 7.2 mmol) and iPr₂NEt (1.26 mL) were added. The reaction was heated at 90 °C for 6 hours, partitioned between water and CH₂Cl₂. The organic layers were dried with Na₂SO₄ and evaporated. The residue was subjected to silica gel chromatography using a gradient of EtOAc and hexane. The product was obtained (846 mg, 59%). GC-MS C₁₀H₁₁FOS: calculated 198.05: observed (EI) 7.03 min, 198(2), 105(100), 77(40). ¹H-NMR δ 2.08 (dm, J = 26 Hz, 2H), 3.19 (t, J = 7 Hz, 2H), 4.55 (d,t, J = 47.7, 5.6 Hz, 2H), 7.92–8.01 (m, 2H), 7.52 – 7.62(m, 1H), 7.4 – 7.51 (m, 2H). ¹³C-NMR 191.82, 137.144, 133.65, 128.83, 127.41, 82.5 (d, J= 166 Hz), 30.76 (J = 20 Hz), 25.06 (d, J + 5.2 Hz). ¹⁹F-NMR (external standard) - 221 (tt, J = 48, 25 Hz).

2.2.7. S-(3-(tosyloxy)propyl) benzothioate.

S-(3-hydroxypropyl) benzothioate (1.3 g, 6.6 mmol) was dissolved in 15 mL of pyridine and cooled in ice. Tosyl chloride (1.26 g, 6.6 mmol) was added. The reaction sat in refrigerator for three days. The solution was poured into water and extracted with 2 × 50 mL ether. The ether layer was washed with portions of 2 N H₂SO₄ until the aqueous layer remained acidic. The ether layer was dried with Na₂SO₄ and evaporated. TLC indicated unreacted starting materials and the chlorinated side product. The residue was subjected to flash chromatography on silica gel (20% EtOAc in hexane) to provide the product (624 mg, 27%). GC-MS 10 min, 105 (100), 77 (18), 178 (4), 277 (trace), 350 (trace). ¹H-NMR δ 2.07 (p, J = 6.4 Hz, 2H), 2.46 (s, 3H), 3.11 (t, J = 7.0 Hz, 2H), 4.18 (t, J = 5.8 Hz, 2H), 7.37 (d, J = 8.2 Hz, 2H), 7.48 (tm, J = 7.1 Hz, 2H), 7.61 (tm, J + 7.5 Hz, 1H), 7.84 (dm, J + 8.4 Hz, 2H), 7.94 (dm, J = 7.1 Hz, 2H).

2.2.8. 3-((3-fluoropropyl)thio)-1-phenylpyrrolidine-2,5-dione.

N-phenyl maleimide (1 g, 5.78 mmol) was treated with 3-mercaptopropanol (532 mg, 5.78 mmol) in 10 mL benzene with a catalytic amount of Et₃N (10 uL). The reaction was stirred overnight, and the solvent evaporated. The residue was subjected to chromatography on silica gel (EtOAc:hexane, ISCO system standard gradient) to provide the product (589 mg, 76%). GC-MS (EI), TR 8.51 min m/z 265 (trace), 175 (100), 119 (95). ¹H-NMR δ 1.74–2.05 (m, 3H), 2.67 (dd, J = 18.59, 3.65 Hz, 1H), 2.86–3.14 (m, 2H), 3.30 (dd, J = 18.5, 9.17 Hz, 1H), 3.75 (brt, J = 5.4 Hz, 2H), 3.89 (dd, J = 9.17, 3.60 Hz, 1H), 7.22–7.33 (m, 2H), 7.36 – 7.53 (m, 3H). ¹³C-NMR 28.69, 31.87, 36.44, 61.17, 126.59, 129.03, 129.42, 131.723, 173.89, 175.95.

3-((3-hydroxypropyl)thio)-1-phenylpyrrolidine-2,5-dione (218 mg, 0.82 mmol) was dissolved in 10 mL CH₂Cl₂, cooled in ice and treated with DAST (108 µL, 0.82 mmol). After 3 hours the reaction was quenched with saturated NaHCO₃, partitioned with CH₂Cl₂, dried and evaporated. The residue was subjected to silica gel chromatography (ISCO system, EtOAc/hexane) using a 12 g silica cartridge. The desired product was obtained (45 mg, 16%). ¹H-NMR δ 1.94–2.22 (m, 2H, alkyl CH2), 2.68 (dd, J =19.4, 3.5, 1H, ring CH2), 2.88–3.02 (m, 1H CH2-S), 3.04 – 3.18 (m, 1H, CH2-S), 3.20 (dd, J = 18.8, 9.2, 1H, ring CH), 3.87 (dd, J = 9.2, 3.6, 1H, ring CH2), 4.56 (dt, J + 46.9, 5.7, 2H, CH2F), 7.24–7.34 (m, 2H), 7.34–7.54 (m, 3H). ¹⁹F-NMR (external standard) −220.78 (tt, J = 47.3, 25.6 Hz). ¹³C-NMR 28.1 (d, J = 4.9 Hz), 30.17 (d, J = 20.4 Hz), 36.20, 39.38, 82.2 (d, J = 166.5 Hz), 125.49, 126.58, 128.42, 129.03, 129.23, 129.44, 173.74, 175.60.

2.2.9. General procedure for conjugation of primary amine to maleimide substrates.

Substrate containing free amine group (1 eq, 40 mg/mL in DMSO was dissolved in 0.5 mL dimethyl sulfoxide (DMSO). Then 1.2 eq of 3-maleimidopropionic acid NHS ester (80 mg/mL in DMSO) were added followed by addition of neat triethylamine (4 eq). The mixture was stirred at RT for 1–2 h. The reaction was diluted with water and purified by HPLC (Higgins C-18 column (5µm, 250 × 20 mm), gradient 95% solvent A [0.1% trifluoroacetic acid (TFA)/H₂0] and 5% solvent B [0.1%TFA/CH₃CN] and changing to 65% solvent B at 35 min, flow rate of 12 mL/min. For aptamers, a concentration of 10 mg/mL aptamer-5AmMC6-modification (free NH₂) in H₂O was used and purification was done on NAP-5 column using phosphate-buffered saline (PBS) as an eluent.

2.2.10. Synthesis of E[c(RGDfK)]₂-maleimide for biological studies.

E[c(RGDfK)]₂-maleimide was prepared similarly to the above procedure, using 1.2 eq of NHS-maleimide and 4 eq of triethylamine, to give 34% isolated product with chemical purity >95%. LC-MS analysis confirmed mass of 1467 [M-H]+.

2.3. Radiolabeling

2.3.1. Radiosynthesis of ¹⁸F-Fluoropropyl benzothioate and ¹⁸F-Fluoro-PEG₄ benzothioate.

Radiosynthesis of both, ¹⁸F-Fluoropropyl benzothioate and ¹⁸F-Fluoro-PEG₄ benzothioate, was done on a modular system (Eckert & Ziegler Eurotope GmbH). 130–270 mCi of ¹⁸F-fluoride were added to a reactor vial contains 12 µmol of K₂CO₃ in 60 µL H₂O and 24 µL K_2.2.2 in 500 µL CH₃CN. Azeotropic drying was done using CH₃CN and heating to 120°C and under a stream of argon and vacuum. Then, 20–24 µmol of the precursors (either tosyloxypropyl benzothioate or nosyl-PEG₄ benzothioate) were added in 500 µL CH₃CN. The reactor was sealed and heated to 95 °C for 10 min. Then after, the reactor was cooled to 40 °C and 1 mL of H₂O was added. The crude mixture was injected into built-in HPLC system using Agilent column (Zorbax 300SB-C18, 5µm, 250 × 20 mm) isocratic conditions of 40 % CH₃CN, 60% H₂O, flow 6 mL/min. ¹⁸F-fluoropropyl benzothioate and ¹⁸F-fluoro-PEG₄ benzothioate were eluted with retention times of 13.8 and 8.2 min, respectively, trapped on Waters light C18 cartridge, and eluted with 500 µL CH₃CN. Quality control and specific activity calculations for radiolabeled compounds were done on a Phenomenex Luna C18 column (5 μm, 4.6×150 mm) using 0.1% trifluoracetic acid (TFA) in water (solvent A) and CH₃CN (solvent B) with a gradient system starting from 80% of solvent A and 20% of for 2 min, changing to 10% solvent A and 90% solvent B at 22 min, with a flow rate of 1 mL/min.

2.3.2. Radiosynthesis of ¹⁸F-Fluoropropyl thiol and ¹⁸F- Fluoro-PEG₄ thiol.

Deprotection of benzothioate group was done as follows; 200 µL of sodium methoxide solution (0.5 M in methanol) were added to a solution of either, ¹⁸F -Fluoropropyl benzothioate and ¹⁸F-Fluoro-PEG₄ benzothioate in 500 µL CH₃CN for 10 min incubation at RT, followed by treatment with 400 µL of 25% acetic acid in H₂O for additional 2 min. The thiolate compounds were used as is for the next step, with no further purification.

2.3.3. Conjugation with maleimide substrates.

To the thiol solutions described above, were added 0.008–3 µmol of maleimide substrate in 400 µL PBS pH 7.4. The reaction mixture was incubated at RT for 15 min, followed by either NAP-5 purification (for ¹⁸F-labeled aptamers) or in all other cases, HPLC purification on Phenomenex Luna C18 column (C-18, 5µm, 250 × 10 mm) using a gradient starting from 95% solvent A and 5% solvent B and changing to 65% solvent B at 35 min at flow rate of 5 mL/min. The labeled compounds were collected, diluted with excess of water, trapped on Waters light C-18 cartridge and eluted with 0.2 mL of 0.5% acetic acid in ethanol solution.

2.4. Biological evaluation of ¹⁸F-Fluoro-propyl-thio-E[c(RGDfK)]₂ and ¹⁸F-Fluoro-PEG₄-thio- E[c(RGDfK)]₂

2.4.1. PBS and serum stability.

20 µL of either ¹⁸F-E[c(RGDfK)]₂ derivative were incubated in 200 µL PBS at RT or mouse serum at 37 °C for 2 h. The stability was tested by analytical HPLC using the system described above. Extraction of radioactivity from mouse serum was done using 2-fold volume excess of CH₃CN, followed by short vortex and centrifugation at 10,600g for 5 min.

2.4.2. Cell lines and mice models.

In vivo studies were conducted in accordance to animal protocol NIBIB 16–01, which had been approved by the Animal Care and Use Committee of the NIH Clinical Center. Athymic nude mice (Envigo) were kept in an animal facility under pathogen free conditions. U87MG cells were purchased from ATCC and cultured in Eagle’s Minimum Essential Medium fortified by addition of 10% fetal bovine serum. Cells were kept in a humidified incubator containing 5% CO₂ in air at 37 °C.

2.4.3. PET imaging studies.

150–170 µCi radiolabeled compounds were diluted with PBS and injected intravenously to U87MG-tumor bearing mice (n = 5). PET scans were acquired on Inveon scanner (Siemens Preclinical Solutions) at 0.5, 1 and 2 h post-injection using acquisition time of 5–10 min. The images were reconstructed using ASIPRO (Siemens Preclinical Solutions) and/or VivoQuant (Invicro).

2.3.4. Statistical Analysis.

Results are reported as mean ± SD. Two-tailed paired and unpaired Student’s t tests were used to determine significance differences.

3. Results

3.1. Chemistry

Synthesis of precursors, tosylate-propyl benzothioate and nosylate-PEG₄ benzothioate was done in two steps and resulted in reasonable yields and high chemical purity (>98%) (Fig. 2). These two examples provide prosthetic groups with differing hydrophilicity that may be of importance for a particular application. ¹⁹F standards for the first labeling step, were synthesized via the hydroxyl group using (Diethylamino)sulfur trifluoride (DAST) as a fluorination reagent. This reaction was more efficient for the propyl benzothioate derivative (59% isolated yield compares to 22% for the PEG₄-benzothioate, Fig. 2). Excluding the commercially available phenyl maleimide, the other maleimide substrates were synthesized by conjugation of free amine to 3-maleimidopropionic acid. ¹⁹F standards reported in this study, were synthesized following the radiolabeling procedure, using ¹⁹F-fluoropropyl benzothioate or ¹⁹F-fluoro-PEG₄ benzothioate. The chemical purities of all fluorinated standards were greater than 95%, as determined by analytical HPLC and LC-MS analysis (Supp. Figs. S1–S7).

Fig. 2.

Synthetic routes for precursors and/or standards. Reagents and yields are indicated in the scheme. The thiol is introduced by nucleophilic reaction of thiobenzoic acid with a mono activated diol. The remaining alcohol is converted to the activated sulfonate ester or to the fluoride using the appropriate reagent.

3.2. Radiolabeling

Synthesis and purification of ¹⁸F-fluoropropyl benzothioate and ¹⁸F-fluoro-PEG₄ benzothioate was done on a modular system using the built-in HPLC module. ¹⁸F-Fluoropropyl benzothioate yields were superior to ¹⁸F-fluoro-PEG₄ benzothioate (37–47% vs. 28–35% non-decay corrected isolated yields, n = 5). Overall synthesis time for this step was 50 min. Both tracers were obtained with high radiochemical purity (>98%) and molar activity of 3.4 ± 1.2 Ci/µmol (n = 5).

Deprotection of the benzothioate group was efficient using excess of sodium methoxide solution, resulted in high conversion to the desired thiol intermediate, followed by addition of acetic acid to quench the sodium methoxide and neutralize the crude mixture before addition of the maleimide biomolecule (Fig. 3). Optimization of the conditions for maleimide conjugation were first done using the commercial phenyl maleimide. Fifteen minutes at RT in the presence of PBS was sufficient to get high conversion to the desired ¹⁸F-phenyl maleimide, therefore, those conditions were used for the other maleimide substrates. We applied this new labeling approach on various maleimide substrates such as small molecule, peptide and aptamers, using small amount of maleimide. Radiochemical yields, based on ¹⁸F-fluoropropyl benzothioate, with various substrates (0.0024 – 3 µmol) are presented in Table 1. The radiochemical yields based on ¹⁸F-fluoro-PEG4-benzothioate with phenyl maleimide, E[c(RGDfK)]₂, and PSMA maleimide were similar to those obtained with ¹⁸F-fluoropropyl benzothioate. In all cases, the reaction was relatively clean and resulted in high conversion to the desired labeled product (Fig. 4).

Fig. 3.

Radiosynthesis of ¹⁸F-thiol prosthetic groups. [¹⁸F]Fluoride displacement is conducted using standard K222/K₂CO₃ conditions in CH₃CN. The intermediate is purified by HPLC. Subsequently, the protective group is cleaved with NaOCH₃.

Table 1.

¹⁸F-Fluoropropyl benzothioate radiolabeling with various maleimides. RCY calculated for isolated product starting from ¹⁸F-Fluoropropyl thiol (n=3).

* n=1.

Fig. 4.

Representative analytical HPLC of crude mixture from conjugation of ¹⁸F-fluoro-propyl-thiol to phenyl maleimide (A) or E[c(RGDfk)]₂ (B) Arrows point to the peak of the desired product.

3.3. Biology

In order to evaluate these two ¹⁸F-thiol synthons for applications in PET imaging, we need to evaluate their in vitro and in vivo stabilities. We chose E[c(RGDfK)]₂ as a model molecule and prepared ¹⁸F-fluoro-propyl-S-E[c(RGDfK)]₂ and ¹⁸F-fluoro-PEG₄-S-E[c(RGDfK)]₂ (Fig. 5). Both ¹⁸F-E[c(RGDfK)]₂ tracers showed high stability in PBS and mouse serum (Fig. 6), with extraction efficiency of 87–92% using CH₃CN. ¹⁸F-fluoro-PEG₄-S-E[c(RGDfK)]₂ displayed a small amount of a new polar peak at 2 h (Fig. 6B). PET studies showed more favorable pharmacokinetics of ¹⁸F-fluoro-PEG₄-S-E[c(RGDfK)]₂ over ¹⁸F-fluoro-propyl-S-E[c(RGDfK)]₂, with less accumulation in the intestine over time (Fig. 7). Although ¹⁸F-fluoro-propyl-S-E[c(RGDfK)]₂ displayed slightly higher tumor accumulation at 0.5- and 1-hpost-injection, those differences were not significant (Fig. 7C). Both compounds seem to be stable in vivo and only low uptake in the bone was observed (0.4 ± 0.3 %ID/g and 0.6 ± 0.1 %ID/g at 2 h post-injection for ¹⁸F-fluoro-propyl-S-E[c(RGDfK)]₂ and ¹⁸F-fluoro-PEG₄-S-E[c(RGDfK)]₂, respectively).

Fig. 5.

Chemical structures of ¹⁸F-fluoro-propyl-S-E[c(RGDfK)]₂ and ¹⁸F-fluoro-PEG₄-S-E[c(RGDfK)]₂ which were used for biological evaluation.

Fig. 6.

Stabilities of ¹⁸F-Fluoro-propyl-S-E[c(RGDfK)]₂ (A) and ¹⁸F-Fluoro-PEG₄-S-E[c(RGDfK)]₂ (B) in ethanol (STD), PBS and mouse serum at 2 h time point.

Fig. 7.

Representative coronal PET images of U87MG tumor mice injected with either ¹⁸F-fluoro-propyl-S-E[c(RGDfK)]₂ (A) or ¹⁸F-fluoro-PEG₄-S-E[c(RGDfK)]₂ (B) over time. White arrows indicate tumor location. (C) Mean tumor uptake calculated from PET images of ¹⁸F-fluoro-propyl-S-E[c(RGDfK)]₂ (red bars) and ¹⁸f-Fluoro-PEG₄-S-E[c(RGDfK)]₂ (blue bars). (D) Biodistribution of ¹⁸F-fluoro-propyl-S-E[c(RGDfK)]₂ (red bars) and ¹⁸F-Fluoro-PEG₄-S-E[c(RGDfK)]₂ (blue bars) at 2 h post-injection by direct tissue sampling.

4. Discussion

Numerous prosthetic groups have been labeled over the past two decades, mainly for conjugation to biomolecules for PET. Fluorination of the prosthetic groups is typically a multi-step process and conjugation to the sensitive biomolecule and is typically done under harsh to moderate conditions. Most of the prosthetic groups take advantage of the abundant amines and thiols that are available in peptides or proteins and are used to conjugate ¹⁸F-prosthetic groups (9–17).

In this paper, we synthesized two ¹⁸F-thiol prosthetic groups which can be easily conjugated to biomolecules, such as aptamers, peptide, etc. under conditions that do not affect biomolecules, namely PBS, pH 7.4 at RT. The prosthetic groups described in this paper, ¹⁸F-fluoropropyl thiol and ¹⁸F-fluoro-PEG4-thiol, were reported by Glaser et al., in 2004 using a different precursor for labeling and different chemistry for conjugation with short peptide (7). In 2009, the same group reported that the ¹⁸F-fluoropropyl thiol prosthetic group failed to label a chloroacetylated RGD peptide (8). Instability of the peptide precursor at high temperature and alkaline conditions were identified as the major challenge.

Our approach was to optimize the radiosynthesis and conjugation reaction of ¹⁸F-fluorothiols with maleimide substrates, which are generally more reactive than chloroacetamides. In order to use small quantities of biomolecule, both ¹⁸F-fluoropropyl benzothioate and ¹⁸F-fluoro-PEG₄ benzothioate, were purified by HPLC to give high specific activity ¹⁸F-intermediates. Our choice of the benzothioate protecting group allowed for rapid deprotection using NaOMe and subsequent bioconjugations could be conducted in the same reaction vessel.

The three-step radiolabeling route we developed provides good yields. The purified F-18 labeled benzothioate precursor is rapidly deprotected and then reacted with the maleimide-tagged target in the same vessel under very mild conditions. We expect that any maleimide-containing biomolecule stable at room temperature and soluble in ethanol PBS mixture would be suitable for this radiolabeling method. We did not evaluate molecules that contain disulfide bonds such as antibodies, therefore we cannot testify for the suitability of the method for such molecules.

A head-to-head comparison of the two prosthetic groups described herein in term of kinetics and yield was done using the small model molecule, N-phenylmaleimide, and gave marginal difference. This multi-step process was converted to fully automated system using two reactors; one for the nucleophilic fluorination and the other for deprotection of the benzothioate and reaction with maleimide substrate.

In vivo ¹⁸F-fluoro-PEG₄-S-E[c(RGDfK)]₂ had favorable pharmacokinetics and less accumulation in metabolic organs than ¹⁸F-fluoro-propyl-S-E[c(RGDfK)]₂. This might be explained by its reduced hydrophobicity.

5. Conclusions

In summary, our benzthioate precursors for preparation of ¹⁸F-mercaptans provided a robust method for radiolabeling of maleimide-containing biomolecules. Both prosthetic groups can be reliably synthesized from the corresponding precursor in high isolated radiochemical yields and can be reacted with maleimide-biomolecules using low molar concentration of the desired biomolecules.