¹⁸F labeling of cysteine containing peptides (RGD and Exendin-4) and protein (VEGF) with high yield and specific activity using tetrazine-trans-cyclooctene ligation

Objectives The tetrazine-trans-cyclooctene ligation has been developed as a novel ¹⁸F labeling method that proceeds with fast reaction rates without catalysis. The aim of this study was to develop an efficient method for ¹⁸F-lableing of free cysteines of peptide and protein based on sequential ligation with a bifunctional tetrazinyl-maleimide and an ¹⁸F-labeled trans-cyclooctene.

Methods The newly developed method was tested for site-specific labeling of c(RGDyC), Exendin-4-SH, and VEGF-SH protein. The resulting ¹⁸F probes were tested in U87MG glioblastoma model, insulinoma mouse model, and normal nude mice.

Results Starting with 4 mCi of ¹⁸F-trans-cyclooctene and only 10 μg of tetrazine-RGD (80-100 µM), 5µg of Exendin-4-SH (10µM), or 15 μg of tetrazine-VEGF (6.0 µM), ¹⁸F labeled RGD peptide (¹⁸F-TTM-RGD), Exendin-4 peptide (¹⁸F-TTM-Exendin-4) and VEGF protein (¹⁸F-TTM-VEGF) could be obtained within five minutes in 95%, 80%, and 75% yield, respectively. The specific activity of ¹⁸F-TTM-RGD was estimated to be approximately 3-6 Ci/µmol. The tumor uptake of ¹⁸F-TTM-RGD in U87MG tumor reached 1.98 ± 0.33, 1.80 ± 0.15, and 1.27 ± 0.33 % ID/g at 0.5, 1, and 2 h p.i., respectively. ¹⁸F-TTM-VEGF was mainly accumulated in kidneys, which correlated well with previous report.

Conclusions 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