Ethanedithiol

All commercially available materials were used as provided unless noted. All 9-fluorenylmethyloxycarbonyl (Fmoc) protected amino acids were purchased from Novabiochem/EMD Chemicals Inc (La Jolla, CA) or CS Bio (Menlo Park, CA). ¹²⁵I-labeled echistatin was purchased from GE Healthcare Life Sciences (Piscataway, NJ). All other chemicals were purchased from Fisher Scientific (Fair Lawn, NJ) unless otherwise specified. The U87MG human glioblastoma cell line was obtained from American Type Culture Collection (Manassas, VA). Nude mice (nu/nu) were purchased from Charles River Laboratory (Wilmington, MA). Semi-preparative reversed-phase high performance liquid chromatography (RP-HPLC), using a Vydac protein and peptide column (218TP510; 5µm, 250 × 10 mm), was performed on a Dionex 680 chromatography system with a UVD 170U absorbance detector and model 105S single-channel radiation detector (Carroll & Ramsey Associates). The recorded data were processed using Chromeleon version 7.1 software. With a flow rate of 5.0 mL/min, the mobile phase was changed from 95% solvent A [0.1% trifluoroacetic acid (TFA) in water] and 5% B [0.1% TFA in acetonitrile (MeCN)] (0-2 min) to 35% solvent A and 65% solvent B at 32 min. Analytical scale HPLC used the same gradient system except that the flow rate was 1.0 mL/min with a Vydac protein and peptide column (218TP510; 5 µm, 250 × 4.6 mm). UV absorbance was monitored at 218 nm and the identification of the peptides was confirmed based on UV spectrum acquired using a photodiode array detector. Instrumentation used for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) is the same as described in our previous publication 37 (link).
Chemistry and Radiochemistry Peptides corresponding to the sequence of 2.5D and 2.5F were synthesized on a CS Bio CS036 Peptide Synthesizer (Menlo Park, CA) using Fmoc-based solid phase peptide synthesis. Briefly, Rink amide resin was swollen in N,N-dimethylformamide (DMF) for 30 min. Fmoc groups were removed with 20% piperidine in DMF. Aliquots of amino acids (1 mmol) were activated in a solution containing 1 mmol hydroxybenzotriazole (HOBt) and 0.5 M diisopropylcarbodiimide (DIC) in DMF. Following synthesis, side-chain deprotection and resin cleavage were achieved by addition of a 94:2.5:2.5:1 (v/v) mixture of TFA/triisopropylsilane/ethanedithiol/water for 2 h at room temperature. The crude product was precipitated with cold anhydrous ether, and purified using semi-preparative RP-HPLC. Peptide purity was analyzed by analytical scale RP-HPLC.
Large scale folding reactions were performed by incubating the linear peptide with 4 M guanidine, 10 mM reduced glutathione, 2 mM oxidized glutathione, and 0.5 M dimethyl sulfoxide (DMSO) at pH 7.5. The correctly folded peptide was separated from unfolded and partly folded peptides by RP-HPLC, where it appeared as a single peak with a shorter retention time than that of unfolded or misfolded precursors. Following purification, folded peptides 2.5D and 2.5F were lyophilized and stored at room temperature prior to use. Peptide purity and molecular masses were determined by analytical scale HPLC and MALDI-TOF-MS, respectively.
The synthesis of 4-nitrophenyl 2-fluoropropionate (¹⁹F-NFP) is briefly described below. Bis(4-nitrophenyl) carbonate (15.2 mg, 50.0 µmol in 20 µL of diisopropylethylamine (DIPEA)) was added to a solution of 2-fluoropropionic acid (5.0 mg, 54.3 µmol in 200 µL of DMF). After incubating at 60 °C for 3 h, the reaction mixture was cooled to room temperature and diluted with 1 mL 5% acetic acid solution. The product ¹⁹F-NFP was isolated by semi-preparative HPLC. The collected fractions were combined and the solvent was removed under reduced pressure. The product was obtained as white powder (5.9 mg, 56%). ESI-MS: m/z 213.1 [M]⁺; ¹H NMR (CDCl₃, 300 MHz): δ = 8.24 (d, J = 9.0 Hz, 2H), 7.27 (d, J = 9.0 Hz, 2H), 5.21 (m, 1H), 1.70 (dd, J = 6.8 Hz, 23.4 Hz, 3H). ¹³C NMR (CDCl₃, 75 MHz): δ = 18.9 (d, J = 22.5 Hz), 86.0 (d, J = 184.0 Hz), 122.9, 126.0, 146.4, 155.2, 168.5.
¹⁹F-NFP labeled 2.5D and 2.5F (¹⁹F-FP-2.5D, ¹⁹F-FP-2.5F) were prepared as reference standards. Briefly, 2.5D or 2.5F (0.5 mg, 0.15 μmol in 400 μL of DMSO) was mixed with ¹⁹F-NFP (0.3 mg, 1.5 μmol in 100 μL of DMSO and 10 μL of DIPEA) and reacted for 1 h at room temperature. The resulting conjugates, ¹⁹F-FP-2.5D and ¹⁹F-FP-2.5F were then purified by semi-preparative HPLC. Fractions containing the product were collected and lyophilized. The molecular masses of ¹⁹F-FP-2.5D and ¹⁹F-FP-2.5F were confirmed by MALDI-TOF-MS. Folded peptide 2.5D: m/z = 3244.0 for [MH]⁺ (C₁₂₉H₁₈₉N₄₀O₄₇S₆, calculated [MH]⁺ = 3244.5). Retention time on analytical scale HPLC is 16.2 min; Folded peptide 2.5F: m/z = 3292.1 for [MH]⁺ (C₁₃₁H₂₀₁N₄₂O₄₆S₆, calculated [MH]⁺ = 3292.6). Retention time: 15.2 min; ¹⁹F-FP-2.5D: m/z = 3317.5 for [MH]⁺ (C₁₃₂H₁₉₃FN₄₀O₄₈S₆, calculated [MH]⁺ = 3317.4). Retention time: 17.5 min; ¹⁹F-FP-2.5F: m/z 3364.9 for [MH]⁺ (C₁₃₄H₂₀₃FN₄₂O₄₇S₆, calculated [MH]⁺ = 3365.3). Retention time: 18.3 min.
¹⁸F-NFP was prepared and used for knottin radiolabeling based on a previously reported procedure (Figure 1C) 38 (link). ¹⁸F-NFP (specific activity of 40-100 GBq/μmol at the end of synthesis, in 100 μL of DMSO) was added to the 2.5D or 2.5F peptide (100 μg) and 10 μL of DIPEA and reacted for 20 min at 60 °C. After adding 1 mL of water containing 50 μL of TFA to quench the reaction, the resulting conjugates were purified by semi-preparative HPLC using the same elution gradient as described for ¹⁹F-FP-knottin purification. The HPLC fractions containing ¹⁸F-FP-2.5D or ¹⁸F-FP-2.5F were collected, combined, and dried with a rotary evaporator. Radiolabeled peptides were reconstituted in PBS and sterilized using a 0.22 μm filter (Millipore) for in vitro and in vivo experiments.
Cell Culture: U87MG cells were cultured in DMEM containing high glucose (GIBCO, Carlsbad, CA), and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were expanded in tissue culture dishes and kept in a humidified atmosphere of 5% CO₂ at 37 °C, with media changes every other day. Confluent monolayers were detached with 0.05% Trypsin-EDTA, 0.01M PBS (pH 7.4) and dissociated into single-cell suspensions for further cell culture and experimentation.
U87MG Cell Binding Assay: Cell binding assays were performed as previously described 37 (link), 39 (link). Briefly, 2 × 10⁵ U87MG cells were incubated with 0.06 nM ¹²⁵I-labeled echistatin and varying concentrations of peptides (2.5D, 2.5F, ¹⁹F-FP-2.5D or ¹⁹F-FP-2.5F) in integrin binding buffer [25 mM Tris pH 7.4, 150 mM NaCl, 2mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, and 0.1% bovine serum albumin (BSA)] at room temperature for 3 h. The cell-bound radioactivity remaining after washing was measured by gamma-counting. Half-maximal inhibitory concentration (IC₅₀) values were determined by nonlinear regression using GraphPad Prism (GraphPad Software, Inc.). Experiments were performed with quadruplicate samples.
MicroPET Imaging: All animal studies were carried out in compliance with Federal and local institutional regulations for the conduct of animal experimentation. Approximately 10 × 10⁶ U87MG cells were suspended in 100 µL of PBS and subcutaneously implanted in the right shoulders of nude mice. Tumors were grown to a size of 0.5 cm in diameter (approximately 2-3 weeks). MicroPET scans were performed on a microPET R4 rodent model scanner (Concorde Microsystems Inc.). The scanner has a computer-controlled bed and 10.8-cm transaxial and 8-cm axial fields of view (FOVs). It has no septa and operates exclusively in the 3-dimensional (3D) list mode. Mice bearing U87MG xenografts were injected via tail vein with approximately 3.7 MBq (100 μCi) of ¹⁸F-FP-2.5D or ¹⁸F-FP-2.5F, with or without 10 mg/kg mouse body weight c(RGDyK). At 0.5, 1, and 2 h post injection (p.i.) mice were anesthetized with isoflurane (5% for induction and 2% for maintenance in 100% O₂). With the help of a laser beam attached to the scanner, mice were placed in the prone position and near the center of the field of view of the scanner where the image resolution and sensitivity is highest. Three minute static scans were obtained, and images were reconstructed by use of a 2-dimensional ordered-subsets expectation maximization (OSEM) algorithm. No background correction was performed. Region of interests (ROIs) were drawn over the tumor on decay-corrected whole-body coronal images. The maximum counts per pixel per minute were obtained from the ROI and converted to counts per milliliter per minute by using a calibration constant. Based on the assumption of a tissue density of 1 g/ml, ROIs were converted to counts per gram per min. The percent injected dose per gram of tissue (%ID/g) was determined by dividing counts per gram per minute by injected dose. No attenuation correction was performed.
Animal Biodistribution Studies: For biodistribution studies, nude mice bearing U87MG xenografts (n = 3 for each group) were injected via tail vein with approximately 3.7 MBq (100 μCi) of ¹⁸F-FP-2.5D or ¹⁸F-FP-2.5F, with or without 10 mg/kg mouse body weight c(RGDyK). Mice were sacrificed at 2 h p.i., and tumor and normal tissues of interest were removed and weighed, and their radioactivity was measured in a gamma-counter. The radioactivity uptake in the tumor and normal tissues was expressed as % ID/g.
Statistical Method: Statistical analysis was performed using the Student's t-test for unpaired data. A 95% confidence level was chosen to determine the significance between groups, with P < 0.05 being designated as significantly different.