# A novel 18F-labelled tetrazine ester prosthetic group for improved radiolabelling and in vivo stability of proteins and peptides

## TL;DR

This paper introduces a new radiolabeling method for proteins and peptides using a fluorine-18 tetrazine ester, improving stability and ease of synthesis for medical imaging.

## Contribution

A novel single-step synthesis of [18F]TzE2 that combines the benefits of prior tetrazine prosthetic groups for radiolabeling.

## Key findings

- [18F]TzE2 was synthesized in 13 minutes with a 30.4% radiochemical yield.
- [18F]TzE2-Z09591 showed improved plasma stability and PDGFRβ targeting compared to [18F]TzE-Z09591.
- The radiolabeled Affibody retained specific binding to PDGFRβ-expressing tissues.

## Abstract

Monoclonal antibodies, engineered protein scaffolds, and peptides are increasingly important in therapy and diagnostics owing to their high specificity and affinity. Recent advances have enabled radiolabelling of such compounds with fluorine-18 in aqueous solution at room temperature via conjugation of [18F]tetrazines with trans-cyclooctene (TCO) moieties through the inverse electron-demand Diels–Alder (IEDDA) reaction. To simplify fluorine-18 labelling of large molecules, a novel tetrazine: 4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl 6-[fluoro-18F]nicotinate ([18F]TzE2), was here developed and synthesized in a single-step procedure directly on a standard QMA cartridge used for trapping [18F]fluoride. The QMA containing [18F]fluoride was eluted over 2 min with N,N,N-trimethyl-5-(((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)oxy)carbonyl)pyridin-2-aminium chloride in acetonitrile, forming [18F]TzE2 instantaneously as it passed through the cartridge. [18F]TzE2 was designed to combine the most favourable features of the previously described tetrazine prosthetic groups, [18F]TzAm and [18F]TzE, specifically, the superior in vivo performance and stability of [18F]TzAm, and the efficient and practical radiosynthesis of [18F]TzE. [18F]TzE2 was evaluated biologically as a reagent for direct labelling of the TCO-conjugated Affibody molecule Z09591, a high affinity marker for PDGFRβ.

The radiochemical yield for [1⁸F]TzE2 was 30.4 ± 3% (n = 4), corresponding to an activity yield of 5.8 GBq starting from 20 GBq [1⁸F]fluoride, with a total synthesis time of 13 min. [18F]TzE2 demonstrated greater stability in human plasma than the previous ester tetrazine [18F]TzE, but was significantly less stable than [18F]TzAm. The Affibody molecule Z09591 was successfully radiolabelled, giving an activity yield of 410 ± 160 MBq and > 90% radiochemical purity within a total synthesis time of 30 min from [18F]fluoride. [18F]TzE2-Z09591 exhibited improved plasma stability relative to [18F]TzE-Z09591, though lower than [18F]TzAm-Z09591. [18F]TzE2-Z09591 retained specific blockable binding to PDGFRβ-expressing human and murine tissues as demonstrated by in vitro autoradiography. Biodistribution of [18F]TzE2-Z09591 was rapid, with predominantly renal clearance, and showed improved targeting of PDGFRβ-expressing tissues compared with [18F]TzE-Z09591.

The novel tetrazine prosthetic group [18F]TzE2 enabled straightforward radiolabelling of Affibody molecule Z09591 without affecting its binding properties. [18F]TzE2-Z09591 showed improved stability and in vivo targeting of PDGFRβ compared with the previously reported tetrazine ester [18F]TzE-Z09591. [18F]TzE2 represents a promising tetrazine prosthetic group for labelling of proteins and peptides with fluorine-18.

The online version contains supplementary material available at 10.1186/s41181-026-00430-6.