**4. Conclusions**

We synthesized a series of tetrazines combining radiolabeling for PET with optical NIR imaging on a single scaffold. A Sulfo-Cy7 conjugate was shown to have suboptimal properties but versions based on Sulfo-Cy5 and IRDye800CW revealed specific binding to TCO-modified targets in vitro and specific accumulation in vivo exemplified by imaging. A dimeric version showed enhanced and more specific signal in vivo, making this a promising candidate to further investigate multimodality imaging techniques based on pretargeting strategies.

**Supplementary Materials:** The following are available online, Figure S1: OI and PET Imaging of <sup>68</sup>Ga-labeled IRDdye800CW-MAFC-PEG<sup>5</sup> -Tz in mice.

**Author Contributions:** Conceptualization, C.D. and D.S.; methodology, D.S., M.P., M.H., and C.R.; validation, S.M., P.K., and M.K.; formal analysis, D.S., J.P., S.M., and P.K.; investigation, J.P., S.M., D.S., M.P., M.K., and P.K.; resources, H.H., M.P., C.D., C.R., and M.H.; data curation, D.S., C.D., M.P., and M.H.; writing—original draft preparation, D.S.; writing—review and editing, C.D. and C.R.; visualization, D.S., M.P., and M.H.; supervision, C.D. and H.H.; project administration, C.D. and M.P.; funding acquisition, C.D. and M.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Austrian Science Foundation (FWF) grant P 25899-B23 to D.S. and C.D. and the European Regional Development Fund - Project ENOCH (No. CZ.02.1.01/0.0/0.0/16\_019/0000868) to M.P. Open Access Funding by the Austrian Science Fund (FWF).

**Acknowledgments:** Claudia Manzl is acknowledged for providing Raji cells.

**Conflicts of Interest:** The authors declare no conflict of interest.
