Abstract
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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