18F-labeling of peptides using FBEM
Lab/Group: Chen Lab (Stanford)
Introduction
Biologically active 18F-labeled molecules are becoming widely used in molecular imaging over the past decades, including some currently undergoing early phase clinical trials. This expansion has created the need for a facile and robust labeling method with 18F. Here we describe the protocol of site-specific labeling of peptides with N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM). Part one of this protocol describes the 18F-FBEM synthesis. From the widely used N-succinimidyl-4-18F-fluorobenzoate (18F-SFB), 18F-FBEM can be obtained in >50% yield by mixing 18F-SFB with N-(2-aminoethyl)maleimide in DMSO for 30 minutes at 40 °C. Part two of this protocol describes the conjugation of 18F-FBEM with peptides. After being treated with TCEP•HCl, a thiolated peptide can be labeled with 18F-FBEM almost quantitatively. The total synthesis time for this protocol is less than 2 h and the decay-corrected radiolabeling yield is more than 40% based on 18F-SFB. In addition, this 18F-FBEM protocol can also be applied to label thiol-containing proteins, antibodies, as well as 5’-thio-functionalized oligonucleotides.
Materials
Reagents
18F-Fluoride (produced by PETtracer cyclotron in-house, GE Healthcare)
!CAUTION: Fluorine-18 emits positron. 18F should be handled by following the guidelines set by the institution and the NRC, as well as consulting with institutional Radiation Safety Office. Proper equipment such as protective gear, shielding, rings, and body dosimetry badge should be obtained.
Trifluoroacetic acid (TFA) (Sigma-Aldrich, 299,537) !CAUTION: Corrosive (see MSDS at http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIAL/299537)
4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclohexacosane (Kryptofix 222) (Sigma-Aldrich, 52910)
Potassium carbonate (anhydrous) (Aldrich, 590681)
Tetrabutylammonium hydroxide solution (40wt. % in H2O) (Sigma-Aldrich, 178780)
N,N′-diisopropylethylamine (DIPEA) (Sigma-Aldrich, 496,219) !CAUTION: Highly flammable and corrosive (see MADS at http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIAL/496219)
N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) (Fluka, 85972) !CAUTION: Irritating to eyes, respiratory system and skin (see MSDS at http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/85972)
N-Succinimidyl S-acetylthioacetate (SATA) (Pierce, 26102)
E[c(RGDyK)]2 (RGD2) (Peptides Int., PCI-3899-PI)
N,N,N-trimethyl-4-(ethoxy)carbonyl benzenaminium trifluoromethane Sulfonate (ABX, 4391)
N-succinimidyl-4-fluorobenzoate (SFB) (ABX, 4392)
N-hydroxysuccinimide (NHS) (Sigma, 130672)
Distilled, deionized water (Milli-Q®; >18 MΩ resistivity)
Acetonitrile (Sigma-Aldrich, 49,444-5)
0.1% TFA in water (Solution A, which has been rigorously degassed for HPLC use.)
0.1% TFA in acetonitrile (Solution B, which has been rigorously degassed for HPLC use.)
N-(2-aminoethyl)maleimide trifluoroacetate salt (Fluka, 42517) !CAUTION: Irritant (see MSDS at http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/42517)
Tris(2-Carboxyethyl) phosphine Hydrochloride (TCEPHCl) (Pierce, 20490)
Phophate buffered saline (PBS), 7.4 (1x) (Invitrogen Corp., 10010)
BupH borate buffer packs (Pierce, 28384)
Methyl sulfoxide (DMSO) (Acros, 61097)
Sodium hydroxide (NaOH) (Fluka, 71695)
Acetic acid, glacial (EMD, AX0073-6)
Silicone oil (Fisher, S159)
Equipment
Digital heating block (VWR, 13259-050)
C18 Sep-Pak solid phase extraction cartridge (Waters Corp., WAT020515)
QMA cartridge (ABX, K-920)
Microcentrifuge tube (E&K Scientific, 695054)
Rotary evaporator (Type:Antrieb VV-Mikro) (Heidolph)
Pear-shaped flask (50 ml) (Chemglass, 24/40)
HPLC needle (22 GA) (Alltech, 90536)
Norm-Ject® Syringe (1 ml) (HSW)
Pipet 20 µl (Biohit, L-20R)
Pipet 200 µl (Biohit, L-200R)
Pipet 1000 µl (Biohit, L-1000R)
Mini-vial (with screw cap) (5 ml) (Alltech, 95050)
PTFE/Silicone liner (Alltech, 95302)
Vydac protein and peptide column (5 µm, 250 × 10 mm) (Vydac, 218TP510)
Dionex 680 chromatography system with a UVD 340U absorbance detector and model 105S single-channel radiation detector (Carroll & Ramsey Associates).
Synthesis module (GE Health, TRACERlab FXFN)
NMR Spectrometer (INOVA, 400 MHz)
Procedure
Initial preparations
1. Heat the oil bath in a well-ventilated, lead-shielding hood to a temperature of 40 °C.
2. Weigh 1.0 mg N-(2-aminoethyl)maleimide and dissolve with 100 µl DMSO in a 5 ml mini-vial and add 20 µl DIPEA to this vial, then cap the vial with a screw cap.
3. Equilibrate the HPLC column by rinsing the column with 5% solution A and 95% solution B at 5 ml/min flow for 5 minutes, then running the column with 95% solution A and 5% solution B at 5 ml/min flow for another 5 minutes.
4. Weigh 200 µg SRGD2 with 50 µl DMSO in a microcentrifuge tube.
5. Weigh 1.0 mg TCEP•HCl and dissolve with 100 µl water in a 5-ml mini-vial and add SRGD2 solution to this vial.
6. Pre-treat C18 Sep-Pak cartridge with 10 ml ethanol and 10 ml water.
Synthesis of 18F-FBEM
7. Transfer crude 18F-SFB (100 mCi) in 2.0 ml acetonitrile to the round-bottom flask containing 20 ml water and 1 ml 5% acetic acid solution.
8. Pass 18F-SFB solution through a pretreated C18 Sep-Pak cartridge, wash the cartridge further with 5 ml water, then wash it with 2 ml acetonitrile and collect the eluent in a 50-ml pear-shaped flask.
9. Attach the flask to a micro rotary evaporator to remove the solvent. Add another 1.5 ml acetonitrile and 0.5 ml DMSO to the flask and concentrate the solvent to less than 0.5 ml.
CRITICAL STEP Evaporation to complete dryness will make the radioactivity stick on the flask and difficult to reconstruct the radioactivity, resulting in the loss of radioactivity.
10. Transfer 100-200 µl 18F-SFB solution to the 5-ml mini-vial with 1.0 mg N-(2-aminoethyl)maleimide and 100 µl DMSO, incubate the solution at 40 °C oil bath for 15 min.
CRITICAL STEP It is most important to make sure that there is no water in the reaction mixture otherwise the pH will be too high for the reaction.
11. Add 600 µl 1.5% TFA water to the mini-vial and withdraw the solution into a 1-ml syringe with HPLC needle. Rinse the vial with 100 µl 1.5% TFA and withdraw the solution into the same syringe. Inject the solution onto the HPLC.
12. Collect the HPLC fractions containing 18F-FBEM (Rt = 13.8-14.2 min) in a test tube. Transfer these fractions to a 50-ml pear-shaped flask and rinse the test tube with 500 µl acetonitrile and transfer the solution to the same flask. Attach the flask to a micro rotary evaporator to concentrate to 100-200 µl.
Synthesis of 18F-FBEM-SRGD2
13. Reconstruct 18F-FBEM in 600 µl PBS and transfer the solution to a 5-ml mini-vial with SRGD2 and TCEP•HCl solution. Adjust pH to 7.2 using 0.2 mol/l NaOH solution and react for 20 min at room temperature (20 °C).
CRITICAL STEP It is pivotal to adjust the pH to around 7.2. If the pH is too high, free thiol is not stable. If the pH is too low, TCEP may not effectively reduce the oxidized disulfide bond.
14. Withdraw the reaction mixture into a 1-ml syringe. Rinse the vial with 100 µl 1% TFA water and withdraw the solution into the same syringe. Inject the solution onto the HPLC.
15. Collect the HPLC fractions containing 18F-FBEM (Rt=15.1-15.5 min) in a test tube. Transfer these fractions to a 50-ml pear-shaped flask and rinse the test tube with 500 µl acetonitrile and transfer the solution to the same flask. Attach the flask to a micro rotary evaporator to remove the solvent and reconstruct with 200 µl PBS (1x, pH 7.4).
Troubleshooting
See Table 1
Critical Steps
Anticipated Results
Typical radiochemical yields.
The decay-corrected radiochemical yields for 18F-FBEM will be more than 50% based on 18F-SFB. For peptide labeling, the coupling reaction is achieved in high yields (more than 85% non-decay corrected), virtually quantitative on the base of 18F-FBEM. The radiochemical purity of the 18F-labeled peptides is >98% according to analytic HPLC. The total yield is more than 40% based on 18F-SFB.
Analytical data
N-[2-(4-fluorobenzamido)ethyl]maleimide. 1H NMR (chloroform-d, 400 MHz): 7.79-7.76 (m,2H, phenyl O-H), 7.26-7.09 (m, 2H, phenyl m-H), 6.75 (2H,CH=CH), 3.86-3.65 (m, 4H, CH2-CH2).
FBEM-SRGD2. C73H98FN21O22S, calculated 1,672.5, observed 1,673.5 ([M+H]+).
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Acknowledgements
We thank Dr. Frederick T. Chin and Dr. David W. Dick from the cyclotron facility for 18F-F− production.
Keywords
positron emission tomography, fluorine-18, peptides, site-specific labeling, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM), N-succinimidyl-4-18F-fluorobenzoate (18F-SFB)
Table 1
Troubleshooting Table
Figure 1
(A) Scheme for the synthesis of N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM). (B) Chemical structure of 18F-FBEM-SRGD2.
Figure 2
Representative radio-HPLC profiles.
(A) 18F-FB after purified by C18 Sep-Pak cartridge, (B) 18F-SFB after purification by C18 Sep-Pak cartridge, (C) the reaction mixture of 18F-FBEM synthesis, (D) the reaction mixture of 18F-FBEM-SRGD2 synthesis showing minimal unreacted 18F-FBEM.
