18F-labeling of peptides using FBEM

doi:10.1038/nprot.2007.286

Method Article

¹⁸F-labeling of peptides using FBEM

https://doi.org/10.1038/nprot.2007.286

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Biological techniques

Biochemistry

positron emission tomography

PET

fluorine-18

peptides

site-specific labeling

N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM)

N-succinimidyl-4-18F-fluorobenzoate (18F-SFB)

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.

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":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":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":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":http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/42517)

Tris(2-Carboxyethyl) phosphine Hydrochloride (TCEPHCl) (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)

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)

Initial preparations

Heat the oil bath in a well-ventilated, lead-shielding hood to a temperature of 40 °C.
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.
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.
Weigh 200 µg SRGD2 with 50 µl DMSO in a microcentrifuge tube.
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.
Pre-treat C18 Sep-Pak cartridge with 10 ml ethanol and 10 ml water.

Synthesis of ¹⁸F-FBEM

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.
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.
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.
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.
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.
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

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.
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.
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).

120 min

Steps 1-7, 15 min

Steps 8-10, 10 min

Step 11, 20 min

Steps 12 and 13, 25 min

Steps 14-10, 25 min

Steps 15 and 16, 25 min

See Table 1

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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We thank Dr. Frederick T. Chin and Dr. David W. Dick from the cyclotron facility for ¹⁸F-F− production.

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¹⁸F-labeling of peptides using FBEM