An optimized procedure for preparing fluorine-18 (18F)-labeled peptides by the copper-catalyzed azide-alkyne 1,3-dipolar cyloaddition (CuAAC) is presented here. The two-step radiosynthesis begins with the microwave-assisted nucleophilic 18F-fluorination of a precursor containing a terminal p-toluenesulfonyl, terminal azide and polyethylene glycol backbone. The resulting 18F-fluorinated azide-containing building block is coupled to an alkyne-decorated peptide by the CuAAC. The reaction is accelerated by the copper(I)-stabilizing ligand bathophenanthroline disulfonate and can be performed in either reducing or nonreducing conditions (e.g., to preserve disulfide bonds). After an HPLC purification, 18F-labeled peptide can be obtained with a 31 ± 6% radiochemical yield (n = 4, decay-corrected from 18F-fluoride elution) and a specific activity of 39.0 ± 12.4 Ci μmol−1 within 77 ± 4 min.
Introduced nearly a decade ago1, click chemistry constitutes a set of rapid and selective chemical transformations that facilitate the synthesis of diverse compounds and libraries. The 'click reaction' classification denotes the assembly of modular building blocks in varied reaction conditions (i.e., solvent, temperature) for which limited by-products are formed despite the presence of unprotected functional groups. An important and widely applied click reaction within synthetic chemistry is the copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC)2,3,4,5. With the aforementioned properties of the click reaction (efficiency, kinetics, modularity and chemical orthogonality), the CuAAC also became an important tool for radiochemical synthesis and has been used in the development of diverse positron emission tomography tracers6, including fluorine-18 (18F, 109.5 min half-life)-labeled small molecules7,8, proteins9,10 and peptides11,12,13,14,15.
Technical advancements in the CuAAC-mediated preparation of 18F-labeled peptides
The current protocol, which details the preparation of 18F-labeled peptides from an 18F-fluorinated azide-containing building block and an alkyne-decorated peptide, emerged from advancements in the application of the CuAAC. Initial experiments for preparing 18F-labeled peptides by the CuAAC used copper iodide11 or copper sulfate12 mixed with sodium ascorbate to maintain or generate copper(I), respectively. However, these procedures required a large amount of peptide precursor and, particularly when applied to large peptides14, provided a low CuAAC reaction efficiency. The CuAAC was subsequently improved with the addition of a ligand that stabilizes the oxidation state and promotes the reaction site accessibility of copper(I)16,17. Ligand classes that enhance the CuAAC reaction kinetics include phosphoramadites (e.g., MonoPhos)18, bipyridine/phenanthrolines (e.g., bathophenanthroline disulfonate (BPDS)19) and polytriazoles (e.g., TBTA20 and BTTES21). Notably, a mixture of copper sulfate, BPDS and sodium ascorbate in aqueous conditions markedly enhanced the 18F-labeling of peptides9,13. For peptides prone to reduction (peptides with disulfide bonds), copper sulfate and ascorbate can be replaced with Cu(CH3CN)4PF6 as a source of copper(I)17,22. In either case, BPDS reduces the amount of peptide and time required to complete the CuAAC reaction, thereby improving the radiochemical yield and specific activity of the 18F-labeled peptide.
Comparison with existing methods for preparing 18F-labeled peptides
Site-specific methods for preparing 18F-labeled peptides without the CuAAC include the acylation of protected peptides with activated 18F-fluorinated carboxylic acids during solid-phase synthesis23,24,25, chemoselective modification of thiol groups with 18F-labeled maleimide-bearing prosthetic groups26,27,28,29, conjugation of 18F-fluorinated aldehydes with aminooxy-modified30,31,32,33 or hydrazine-modified34 peptides and the coupling of 18F-labeled N-methylaminooxy-bearing prosthetic groups with maleimide- or vinylsulfone-modified peptides35,36. Additional site-specific methods that directly incorporate 18F-fluoride into peptides in a single step include the 18F-fluorination of silicon-based fluoride acceptor-modified peptides37,38 and the capture of 18F-fluoride in complex with aluminum (Al18F) by peptides conjugated with macrocyclic ligands39,40,41.
Although the current protocol is more challenging to implement than the single-step 18F-fluorination methods, the recommended process provides potentially important advantages. Notably, combining the ligand-accelerated CuAAC with a subsequent HPLC purification provides 18F-labeled peptide with an unparalleled specific activity and radiochemical purity (>99%) while maintaining an acceptable radiochemical yield and reasonable synthesis duration. Furthermore, the CuAAC conditions provided have been used with 18F-labeled building blocks of diverse structural composition from which 18F-labeled peptides with tunable physicochemical and biological properties (e.g., pharmacokinetics, cell membrane penetration and affinity) may be generated.
The assembly of 18F-labeled peptides from an 18F-fluorinated azide-containing building block and an alkyne-decorated peptide is depicted in Figure 1. The alkyne-decorated peptide (1) was prepared by coupling 4-pentynoic acid to the peptide during the solid-phase synthesis. The azide-containing precursor for 18F-fluorination, 11-azido-3,6,9-trioxa-undecanyl p-toluenesulfonate (2), was synthesized from the corresponding ditosylate and an equimolar amount of sodium azide. Executing the protocol entails a two-reaction, two-HPLC synthetic scheme as follows. 18F-fluoride was concentrated in the presence of a phase-transfer catalyst (Steps 1–3) and was reacted with 2 (Step 4) to provide the 18F-fluorinated azide-containing building block, 1-[18F]fluoro-11-azido-3,6,9-trioxa-undecane (3). Compound 3 was subsequently purified by HPLC (Steps 5–7), concentrated by solid-phase extraction (SPE, Steps 8 and 9) and coupled to 1 by the ligand-accelerated CuAAC (Steps 10 and 11). After a second HPLC purification (Steps 12 and 13), the 18F-labeled peptide (4) was concentrated by SPE (Steps 14–16).
The reaction efficiencies when coupling 1 with 3 under varied CuAAC conditions are presented in Supplementary Table 1. These conditions include the ascorbate-based CuAAC (Supplementary Table 1, experiments 1–25) and the ascorbate-free CuAAC for peptides prone to reduction (Supplementary Table 1, experiments 26–35). From these experiments, optimized CuAAC reaction conditions are provided in Step 10 for both reducing conditions (ascorbate-based) and nonreducing conditions (ascorbate-free).
HPLC purification provides 4 with a superior specific activity and radiochemical/chemical purity compared with SPE. However, a single-step SPE purification of 4 from the CuAAC reaction mixture offers process convenience (particularly when automation is required), reduces the synthesis duration by 10–20 min and typically improves the radiochemical yield. SPE purification is also recommended when the HPLC separation of 4 from 1 is limited or when a moderate specific activity (i.e., 1–3 Ci μmol−1) is acceptable. Technical considerations for the SPE purification of 18F-labeled peptides are provided in the Supplementary Methods.
Finally, microwave heating is preferred throughout the protocol; however, conventional heating was also tested, providing a comparable decay-corrected radiochemical yield but a longer synthesis duration (105 min).
Tetrabutylammonium bicarbonate (TBAHCO3) (1.3 M, ABX, cat. no. 816.0000.6)
Kryptofix 222 (Cryptand 222, cat. no. 800.0025)
Acetonitrile (CH3CN, EMD Chemicals, cat. no. AX0143-7)
Milli-Q water (Millipore)
Trifluoroacetic acid, ∼99% (Aldrich, cat. no. 302031)
Formic acid (∼98%, Acros Organics, cat. no. 147930010)
Ethanol (∼100%, Decon Laboratories, cat. no. 2701)
Acetic acid, glacial (Mallinckrodt Chemicals, cat. no. 2504-14)
Dimethylsulfoxide (DMSO, EMD Chemicals, cat. no. MX1457-7)
Copper(II) sulfate pentahydrate (CuSO4·5H2O, Sigma-Aldrich, cat. no. 203165)
L-Ascorbic acid, sodium salt (Alfa Aesar, cat. no. A17759)
Gentisic acid (MP Biomedicals, LLC, cat. no. 02190209)
Tetrakis (acetonitrile)copper(I) hexafluorophosphate (Cu(CH3CN)4PF6, Sigma-Aldrich, cat. no. 346276)
Bathophenanthroline, sulfonated, sodium salt (GFS Chemicals, cat. no. 286)
Sodium phosphate buffer, pH 7, 100 mM
Inert gas (N2, 99.999% purity)
Sodium azide (Aldrich, cat. no. 71290)
Tetraethylene glycol di(p-toluenesulfonate) (Aldrich, cat. no. 341703)
4-Pentynoic acid (Aldrich, cat. no. 232211)
N-hydroxybenzotriazole (HOBt, Aldrich, cat. no. 54804)
N,N′-diisopropylcarbodiimide (DIC; Aldrich, cat. no. D125407)
N,N-dimethylformamide (EMD Chemicals, cat. no. DX1727-7)
Ethyl acetate (EMD Chemicals, cat. no. EX0237-7)
Hexane (EMD Chemicals, cat. no. HX0296-1)
11-azido-3,6,9-trioxa-undecanyl p-toluenesulfonate (2), prepared as described in Box 1
Alkyne-decorated peptide (1), prepared as described in Box 2
18F-fluoride trap-and-release cartridge (8 mg; Oak Ridge Technology Group, cat. no. TR-18-111507)
Phenomenex Luna C18 HPLC column (250 × 10 mm, 5 μm, cat. no. 00G-4041-N0)
Phenomenex Kinetex C18 HPLC column (50 × 2.1 mm, 2.6 μm, cat. no. 00B-4462-AN)
Strata-X Polymeric reversed-phase tube (200 mg in a 3-ml tube, cat. no. 8B-S100-FBJ)
Oasis HLB Plus SPE (Waters, cat. no. 186000132)
Semipreparative HPLC pumps (Knauer, Smartline 1000) with in-line UV monitor (Knauer, Smartline 2500) and sample injector (Valco Instruments, cat. no. EPC6W, SL5KCW)
Analytical HPLC system (Agilent, 1290)
Mass spectrometer (Agilent, 6220)
Microwave generator and reaction cavity (Resonance Instruments, model 521)
Hot plate and stirrer (CAT Ingenieurburo, cat. no. MCS66)
Reaction vial (Wheaton, cat. no. W986297NG)
Teflon/silicone disc (Fisher Scientific, cat. no. PI-12718)
Mass-flow controller (Alicat Scientific, cat. no. MC-1SLPM-D)
Coincidence radiation detector (Bioscan, B-FC-4000 module with two B-FC-4100 detectors)
Radiochemical hotcell (see EQUIPMENT SETUP, Von Gahlen International)
Telemanipulators (Central Research Laboratories, cat. no. G-HD)
RediSep Flash column (120 g; Teledyne ISCO, cat. no. 68-2203-024)
Bruker AVANCE II 400 NMR spectrometer
Prepare a mixture of TBAHCO3 (1.3 M, 10 μl), H2O (400 μl) and CH3CN (500 μl). Freshly prepare before use.
Sodium phosphate (100 mM), pH 7
Dissolve 12.1 g NaH2PO4⋅H2O and 1.75 g Na2HPO4 in 900 ml of water. Adjust to pH 7 with 1 M H3PO4 or 1 N NaOH and add water to a total volume of 1 liter. The solution is stable for at least 1 month when handled with sterile techniques and when stored at 4 °C.
Sodium gentisate stock solution
Prepare a 40 mg ml−1 solution of gentisic acid in 100 mM sodium phosphate, pH 7 (gentle heating required), and neutralize the solution with 2 N sodium hydroxide. Prepare the solution daily and store it away from light. Note that sodium gentisate decreases the formation of radiolytic by-products of 3 and limits the oxidation of 4.
BPDS stock solution
Prepare a 43 mg ml−1 (80 mM) solution of BPDS in 100 mM sodium phosphate, pH 7 (simultaneous heating and vortexing required). Prepare the solution daily.
CuSO4·5H2O stock solution
Prepare a 20.0 mg ml−1 (80 mM) solution of CuSO4·5H2O in 100 mM sodium phosphate, pH 7. If precipitation is observed, thoroughly mix the sample prior to use. Low concentrations of CuSO4·5H2O (e.g., 2 mg ml−1) may form larger particulates, which are difficult to disperse and transfer accurately by pipette. The solution is stable for at least 1 week when handled with sterile techniques and when stored at 4 °C.
Sodium ascorbate stock solution
Prepare a 40 mg ml−1 (200 mM) solution of L-ascorbic acid, sodium salt in 100 mM sodium phosphate, pH 7. Store the resulting solution away from light and use within 2 h of preparation.
Ascorbate-based CuAAC catalyst mixture
Mix 10 μl each of the BPDS and CuSO4·5H2O stock solutions with 30 μl of the sodium ascorbate stock solution. Add 100 μl of the sodium gentisate stock solution.
Cu(CH3CN)4PF6 stock solution
Prepare a 30 mg ml−1 (80 mM) solution of Cu(CH3CN)4PF6 in CH3CN. The solution is stable for over 1 h.
Ascorbate-free CuAAC catalyst mixture
Mix 20 μl of the Cu(CH3CN)4PF6, 10 μl of the BPDS and 100 μl of the sodium gentisate stock solutions.
Solid-phase extraction eluent
For the elution of 3, withdraw 1.5 ml of ethanol into a syringe. For the elution of 4, prepare a mixture of ethanol (3 ml) and glacial acetic acid (30 μl).
1,000–1,800 mCi of 18F-fluoride is produced by the cyclotron irradiation of 2.3–2.8 ml of enriched 18O-H2O with a 40 μA proton current for 120–180 min.
18F-fluoride trap and release cartridge
18F-fluoride is passed directly across the dry trap-and-release cartridge and retained with 75–80% efficiency.
Oasis HLB Plus SPE and Strata-X polymeric reversed-phase tube
Condition the cartridges with 5 ml of ethanol, followed by 5 ml of H2O.
For the reaction vial used during Steps 1–4 (azeotropic removal of H2O and 18F-fluorination), place the Teflon/silicone disc with silicone face into the reaction vial and tightly seal the cap. However, for the subsequent reaction and recovery (Steps 8 and 14), use reaction vials containing a Teflon stir bar.
HPLC system A (Smartline 1000 pump driven)
Use a Phenomenex Luna C18 column with the following parameters: 250 × 10 mm, 5 μm, 18% ethanol, 4–5 ml min−1; perform post-column UV detection (Smartline 2500, UV at 214 nm). Note that 23–25% CH3CN + 0.1% TFA, 7 ml min−1 is an acceptable alternative.
HPLC system B (Agilent 1290)
Use a Phenomenex Kinetex C18 column with the following parameters: 50 × 2.1 mm, 2.6 μm, 5–95% CH3CN/0.1% formic acid, 0.5 ml min−1, 0–3 min; the coincidence radiation detector should be positioned in-line before the mass spectrometer.
HPLC system C (Smart 1000 pump driven)
Use a Phenomenex Luna C18 column with the following parameters: 250 × 10 mm, 5 μm, CH3CN + 0.1% TFA (acetonitrile percentage must be customized to the peptide of interest; 33% CH3CN used for the purification of 4 from 1), 7 ml min−1; perform post-column UV detection (Smartline 2500, UV at 214 nm).
Mass spectrometer (optional)
HPLC system B eluent is directed by electrospray ionization into an Agilent 6220 TOF mass spectrometer (MS) system with acquisition from 200 to 3,200 m/z in high-resolution mode. The fragmentor, capillary, skimmer and octupole radio-frequency (RF) voltages are 120, 3,500, 60 and 250 V, respectively.
The microwave reaction cavity, sample injector, columns associated with HPLC systems A and C, in-line UV monitor and coincidence radiation detector are positioned within the radiochemical hotcell. Samples are handled within the hotcell using telemanipulators.
Preparation of 18F-fluoride
Elute 18F-fluoride from the cartridge with a mixture of TBAHCO3 (1.3 M, 10 μl), H2O (400 μl) and CH3CN (500 μl) into a 3-ml septum-sealed reaction vial equipped with a vent needle.
Evaporate the mixture to dryness with an inert gas (e.g., N2 at 500–800 cm3 min−1) by microwave (50–60 W, 120 °C, 3–5 min) or conventional heating (120 °C, 10 min).
Sequentially add and evaporate three fractions of CH3CN (0.5 ml) to complete dryness and measure the initial activity.
Add 2 (2 mg, 5.3 μmol, in 0.5 ml of CH3CN) to the reaction vial, remove the vent needle and heat the mixture by microwave (40 W, 120 °C, 180 s) or conventional (100 °C, 10 min) heat.
First HPLC purification
Cool the reaction vial to less than 70 °C and evaporate the solvent to near dryness (200 μl is acceptable).
Promptly add H2O (2–3 ml) to the vial, mix and deliver the crude product to the HPLC system A.
Collect the radioactive HPLC peak corresponding to 3 (retention time is ∼15 min), dilute the product with H2O (10 ml), measure the recovered activity and confirm the radiochemical purity by HPLC system B.
Concentration of 3
Deliver the product to a 200-mg Strata-X cartridge. Wash the cartridge with H2O (6 ml), dry it with inert gas (600 cm3 min−1, 10 s) and promptly elute 3 with ethanol (1.5 ml) into a new reaction vial containing a stir bar.
Evaporate the ethanol to near dryness (40–60 W, 90 °C, 180–300 s) and promptly proceed to Step 10.
Promptly add one of the following CuAAC catalyst mixtures to the reaction vial containing 3.
Reaction conditions Catalyst mixture Corresponding experiments in Supplementary Table 1 Reducing conditions Ascorbate-based CuAAC catalyst mixture (0.8 μmol CuSO4, 0.8 μmol BPDS, 6 μmol sodium ascorbate and 4 mg gentisic acid in 100 mM sodium phosphate pH 7) Experiments 1–25 show the effect of temperature, catalyst mixture concentration, pH, organic solvent concentration (DMF, DMSO and ethanol percentage), peptide concentration and time on the ascorbate-based CuAAC reaction efficiency Nonreducing conditions Ascorbate-free CuAAC catalyst mixture (1.6 μmol Cu(CH3CN)4PF6, 0.8 μmol BPDS and 4 mg gentisic acid in 100 mM sodium phosphate pH 7) Experiments 26–35 show the effect of the aforementioned variables on the ascorbate-free CuAAC reaction efficiency
For either condition, promptly add 1 (see Box 2 for the recommended peptide quantity and concentration), remove the vent needle and apply microwave (30W, 60 °C, 10 min) or conventional (preheated vial at 60 °C, 10–20 min) heat. As necessary, monitor the reaction progression by HPLC system B (withdraw a 1-μl aliquot using a 10-μl syringe and dilute 50-fold into an HPLC vial).
Second HPLC purification
Dilute the crude mixture with H2O (1–2 ml) and deliver it to HPLC system C.
Collect the radioactive HPLC peak corresponding to 4, measure the recovered activity and confirm the radiochemical purity by using HPLC system B. Note that SPE may be used instead of the second HPLC purification (Supplementary Methods).
Recovery of 4
Dilute the HPLC fraction(s) containing 4 with H2O (10–20 ml) and deliver the product to an Oasis HLB Plus cartridge. Wash the cartridge with H2O (6 ml), dry with inert gas (600 cm3 min−1, 60 s) and elute 4 with a mixture of ethanol (3 ml) and glacial acetic acid (30 μl) into a new reaction vial containing a stir bar.
Withdraw an aliquot of 4 (e.g., 5 mCi) for quality control assessments, which includes the measurement of radiochemical purity, confirmation of product identity (by mass spectrometry or comparison against cold standard) and determination of specific activity (Box 3).
Evaporate the recovered product to near dryness, neutralize with 0.2–1.0 ml of 0.2 M sterile-filtered sodium phosphate, pH 8 (with 20 mg ml−1 gentisic acid to limit the radiolytically induced oxidation of 4); measure the recovered activity.
Troubleshooting advice can be found in Table 1.
Steps 1–9: The preparation and purification of 3 is typically complete within 40 min (for microwave heating; allow 50 min for conventional heating) after 18F-fluoride elution
Steps 10–16: The coupling of 3 to 1 and subsequent purification and recovery of 4 can be completed within an additional 40 min (allow 55 min for conventional heating)
The 18F-fluorination of 2 to 3 proceeds with an 86 ± 5% efficiency. Subsequent HPLC purification and concentration by SPE provides 3 with a 62 ± 4% (comparable for microwave and conventional heating) decay-corrected yield and a radiochemical purity >99% (Fig. 2). As the radiochemical product is readily separated from the precursor and chemical contaminants, 3 is maintained with a high specific activity through the initial reaction.
Using the reducing conditions (ascorbate-based CuAAC, see Step 10), the conversion of 1 and 3 to 4 occurs with an 89 ± 5% efficiency after 10 min of microwave heating at 60 °C (15–20 min required for a comparable reaction efficiency by conventional heating). As shown in Figure 3, the crude 18F-labeled peptide is primarily contaminated with 3 (1.32 min), whereas radiolytic by-products of 3 (0.3–0.6 min) are negligible because of the presence of 4 mg (20–25 mg ml−1) gentisic acid in the CuAAC reaction mixture. Figure 4 demonstrates that the majority of 1 is not modified during the CuAAC reaction (no oxidation and limited formation of 19F-4 observed).
After a second HPLC purification and recovery by SPE, 4 is obtained with a radiochemical purity greater than 99.5% (Fig. 5), decay-corrected radiochemical yield of 31 ± 5%, specific activity of 39.0 ± 12.4 Ci μmol−1 and total synthesis time of 77 ± 4 min from the elution of 18F-fluoride to the dilution of 4 in formulation buffer. Figure 6 illustrates that 4 is enriched from 1 by HPLC purification. In addition, 19F-4 (and therefore 18F-4) remains intact through the purification and recovery process (although limited oxidation of 1 may be present, this is likely caused by the storage of an aliquot of 4 in an analytical HPLC vial without gentisic acid for more than 12 h).
We thank S. Williams and J.L. Sutcliffe for critical reading of the manuscript and their valuable comments; C. Quan and J. Tom for the peptide synthesis; and J. Tinianow for general assistance with radiolabeling and product characterization.