Supplies
4-iodobenzoic acid, N,N’-Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide, tetrakis(triphenylphosphine)palladium (Pd(PPh3)4), tetrakis(pyridine)copper(II)triflate (Cu(OTf)2Py4), tetraethylammonium trifluoromethanesulfonate (N(Et)4(OTf)), and solvents (dimethylacetamide, dichloromethane, ethyl acetate, methanol, heptane, toluene, and acetonitrile) had been obtained from Merck KGaA (Sigma Aldrich model, Darmstadt, Germany). Hexabutylditin was provided from alfa aesar (Haverhill, MA, USA). N-succinimidyl 4-(tri-n-butylstannyl)-benzoate and 1-{[(4-Fluorophenyl)carbonyl]oxy} pyrrolidine-2,5-dione had been bought from abcr GmbH (Karlsruhe, Germany). Oasis WCX Plus Quick Cartridge (225 mg Sorbent per Cartridge, 60 µm), Oasis HLB Plus Quick Cartridge (225 mg Sorbent per Cartridge, 60 µm), Sep-Pak tC18 Plus Quick Cartridge (400 mg Sorbent per Cartridge, 37–55 µm) and Oasis MAX 1 cc Vac Cartridge (10 mg Sorbent per Cartridge, 30 µm) had been purchased from Waters (Milford, MA, USA). PD-10 desalting column was acquired from Cytiva (Marlborough, MA, USA). Skinny-layer chromatography (TLC) (regular section plates, silica gel 60 coated with fluorescent indicator F254s) was obtained from Macherey–Nagel (Duren, Germany).
Devices and software program
Water utilized in all experiments was deionized from Milli-Q Integral Water Purification System. A Bioscan TLC scanner mannequin AR2000 was used to find out the fraction of radioactivity on the TLC-plates akin to the product by Bio-Chrom plus software program v3.3 (LabLogic Methods Ltd., Sheffield, United Kingdom). The purification of [18F]SFB was carried out on a SymmetryPrep C18 column (10 × 150 mm; 5 μm) utilizing an isocratic gradient (Acetonitrile/H2O:36/64) with a move fee of 4 mL/min. UPLC-MS chromatography was carried out on a ACQUITY H-Class sytem with TQD on a UPLC®BEH C18 column (2.1 × 100 mm; 1.7 μm) was analyzed with the gradient move of 0.5 mL/min (Acetonitrile/H2O: 5/95–100/0 from 0 to three min, 100/0–5/95 from 3 to six min). The UPLC chromatograms had been processed with Empower 3 software program (Milford, MA, USA) for analysis. The nanobody was analyzed by Biozen measurement exclusion columns (0.1 M Na2PO4 + 0.025% NaN3, pH 6.8, 0.3 mL/min) from Phenomenex (Torrance, CA, USA).
Synthesis of N-succinimidyl 4-iodobenzoate (2)
N-Succinimidyl 4-iodobenzoate was synthesized from 4-iodobenzoic acid (0.50 g, 2.01 mmol), N-hydroxysuccinimide (0.255 g, 2.21 mmol) and dicyclohexyl carbodiimide (DCC, 0.59 g, 2.85 mmol) dissolved in dichloromethane (15 mL)31. After an in a single day of stirring, the white precipitates had been filtered off, and the filtrate was evaporated utilizing a rotary evaporator. The stable residue was suspended in a 1:1 combination of dichloromethane/hexane and filtered. The insoluble materials was recrystallized from methanol to yield white crystals of N-Succinimidyl 4-iodobenzoate in a yield of 55% (1.11 mmol). Identification and purity of the product had been confirmed by UPLC-MS (calculated: 344.95, obtained: 345.94 (M + H+)) and 1H-NMR (CDCl3): δ 7.9 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 2.9 (s, 4H). Melting level is 138.1–139.3 °C and Rf of 0.55 in TLC (AcOEt/Hexanes 4:6).
Synthesis of N-succinimidyl 4-(tri-n-butylstannyl)-benzoate (3)
N-Succinimidyl 4-iodobenzoate (0.89 g, 2.60 mmol), Pd(PPh3)4 (0.1 g, 0.11 mmol) and hexabutylditin (0.35 mL, 9.24 mmol) had been dissolved in anhydrous toluene (50 mL) underneath a nitrogen environment32. The combination was heated underneath reflux till the answer turned black (24 h) after which the contents of the flask had been concentrated to provide an oily crude combination. The residue was purified by silica gel chromatography utilizing dichloromethane as eluent to yield N-succinimidyl 4-(tri-n-butylstannyl)-benzoate (67%, 1.74 mmol). For affirmation of the purity and identification of the product after purification the remoted materials was analyzed by UPLC-MS (calculated: 508.24, obtained: 527.25 (M + H3O+)) and 1H-NMR (CDCl3): δ 8.039 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 8 Hz, 2H), 2.91 (s, 4H), 0.89–1.55 (m, 27H). Rf of 0.8 in TLC (AcOEt/Hexanes 4:6).
Synthesis of N-benzoyloxysuccinimide (5)
N-hydroxysuccinimide (1.47 g, 12.50 mmol, 1.25 equiv.) was added to an answer of benzoic acid (1.22 g, 10.0 mmol, 1.0 equiv.) and N,N’-dicyclohexylcarbodiimide (DCC, 2.27 g, 11.0 mmol, 1.10 equiv.) in ethyl acetate (40 mL) at room temperature33. After 2 h, 60 mL of ethyl acetate was added into the suspension and the combination was saved at -80 °C for two h. It was filtrated to take away the sideproduct (dicyclohexylurea, DCU) precipitate and washed with chilly ethyl acetate. The filtrate was concentrated underneath decreased stress. White crystal of N-benzoyloxysuccinimide was remoted by crystallization in EtOAc-EtOH (1.91 g, 8.71 mmol, 87%). The product was decided by UPLC-MS and 1H-NMR (CDCl3): δ = 8.13 (m, 2H, Har-meta), 7.68 (m, 1H, Har-para), 7.51 (m, 2H, Har-ortho), 2.90 (s, 4H, CH2). Melting level is 137.1–138.2 °C and Rf of 0.55 in TLC (AcOEt/Hexanes 4:6).
Radiolabeling of [18F]SFB
A compact kit-less synthesizer developed by Neptis® (Fig. S1A,B) was used for the automated manufacturing of fluorine-18 radiotracers. Manufacturing of two.1 ± 0.2 GBq (n = 6) [18F]fluoride ion was achieved by way of the 18O(p,n) 18F response (cyclotron, IBA, Belgium) by irradiation of an isotopically enriched [18O]H2O goal. The exercise was trapped on the Oasis MAX Cartridge and eluted into the reactor with 10 mg (0.04 mmol) of N(Et)4(OTf) in 600 μL of MeOH. The eluate was concentrated at 90 °C underneath a stream of nitrogen for 12 min. Then, 0.7 mL of N-succinimidyl 4-(tri-n-butylstannyl)-benzoate (11 mg, 0.02 mmol) and Cu(OTf)2Py4 (30 mg, 0.04 mmol) in anhydrous DMA was added to the reactor. After labeling for 10 min at 140 °C, the response combination was diluted with water and the combination was handed by means of Oasis WCX and HLB SPE cartridges in sequence. The cartridges had been rinsed with 13% acetonitrile in water to take away impurities. Crude [18F]SFB was eluted from the HLB cartridge with 2 mL acetonitrile and checked for steel residues by ICP-MS. Then it was diluted with 2 mL water and purified with semi-preparative HPLC with a purpose to isolate [18F]SFB from by-products which will intervene with the labeling of the biomolecule. The collected fraction of the purified [18F]SFB with a retention time of 12–14 min was subjected to QC with UPLC. To review molar exercise, the exercise was retrapped on tC18 and eluted with CH3CN It was calculated by assaying radioactivity and its related mass decided by the world underneath UV peak at 240 nm in opposition to a normal mass curve.
Automation of [18F]SFB labeled nanobody
The fraction of purified [18F]SFB from HPLC was collected in a vial containing 60 mL of water. The answer was handed by means of a Sep-Pak tC18 Plus Quick Cartridge and, afterwards, the product was eluted with diethyl ether (1 mL) into the reactor. Lastly, the combination was evaporated to dryness underneath a stream of nitrogen at 40 °C for 10 min and cooled down for five min. Nanobody dissolved in 0.3 mL PBS buffer (cAbBCII10, 1.25 and three.75 mg/mL, pH 8.4) was added to the dried [18F]SFB within the reactor and incubated for 30 min at room temperature. The ultimate purification of [18F]SFB-nanobody was carried out on a PD-10 desalting column carried out on the module. The fraction of purified [18F]SFB-nanobody (3 mL) was eluted with PBS buffer (pH 8.2) and analyzed with measurement exclusion chromatography (SEC). The radiochemical purity (RCP) was additionally decided by acetonitrile precipitation and skinny layer chromatography (TLC). In case of acetonitrile precipitation, the ultimate answer containing (n = 3) the radiolabeled nanobody was incubated for five min in a 1/1 combination with acetonitrile. Subsequent, the samples had been centrifuged, after which the protein-associated radioactivity within the ensuing pellet was measured.
