Efficient ¹⁸F Labeling of Cysteine-Containing Peptides and Proteins Using Tetrazine–Trans-Cyclooctene Ligation

Abstract

18F PET has a number of attributes that make it clinically attractive, including nearly 100% positron efficiency, very high specific radioactivity, and short half-life of ~110 min. However, the short half-life of 18F and the poor nucleophilicity of fluoride introduce challenges for the incorporation of 18F into complex molecules. Recently, the tetrazine-trans-cyclooctene ligation has been introduced as a novel 18F labeling method that proceeds with fast reaction rates without catalysis. Herein, we report an efficient method for 18F-labeling of free cysteines of peptides and proteins based on sequential ligation with a bifunctional tetrazinyl-maleimide and an 18F-labeled trans-cyclooctene. The newly developed method was tested for site specific labeling of both c(RGDyC) peptide and VEGF-SH protein. Starting with 4 mCi of 18F-trans-cyclooctene and only 10 μg of tetrazine-RGD (80–100 μM) or 15 μg of tetrazine-VEGF (6.0 μM), 18F labeled RGD peptide and VEGF protein could be obtained within five minutes in 95% yield and 75% yield, respectively. The obtained tracers were then evaluated in mice. In conclusion, a highly efficient method has been developed for site-specific 18F labeling of cysteine containing peptides and proteins. The special characteristics of the tetrazine-trans-cyclooctene ligation provide unprecedented opportunities to synthesize 18F-labeled probes with high specific activity for PET applications.

Improved Metabolic Stability for 18F PET Probes Rapidly Constructed via Tetrazine trans-Cyclooctene Ligation

“…27 As shown in Figure 1, 18 F-labeled TCO 2 could be obtained in high radiochemical yield (71%) by combining nosylate 1 with 18 F-fluoride (100 mCi). 18 F- 2 is an effective reagent for creating 18 F-labeled probes within seconds at low micromolar concentrations, and we have used this reagent to make cyclic RGD (cRGD) and VEGF protein conjugates for cancer imaging 28, 29 and exendin-4 conjugates for applications in insulinoma imaging and diabetes monitoring. 30 Notably, these conjugates were synthesized without using a large excess of the peptidic labeling precursor (Figure 1b).…”

Two Rapid Catalyst-Free Click Reactions for In Vivo Protein Labeling of Genetically Encoded Strained Alkene/Alkyne Functionalities

“…Applications of these two reactions include labeling biomolecules in cells and cancer diagnostics. 22−24 Here, we report kinetic investigations of IEDDAC and NADC reactions that involve strained alkene/alkyne functionalities including norbornene, trans -cyclooctene, and cyclooctyne, and their applications in labeling proteins that are genetically modified with these functional groups.…”

Enhanced Aqueous Suzuki–Miyaura Coupling Allows Site-Specific Polypeptide 18F-Labeling

“…7 Protein-labeling using prosthetic 18 F-synthons has been developed and applied to a variety of substrates, generally targeting either lysine 8−11 or cysteine 12−14 residues. Some variants employ heterobifunctional cross-linkers to Lys/Cys, allowing a two-step process combining other modification chemistries 15−21 or even complexation. 22,23 However, these protein-labeling techniques can result in nonspecific labeling, which may affect the biological activity and typically generate mixtures of differently labeled proteins as product.…”