Tumor targeting using agents with slow pharmacokinetics represents a major challenge in nuclear imaging and targeted radionuclide therapy as they most often result in low imaging contrast and high radiation dose to healthy tissue. To address this challenge, we developed a polymer-based targeting agent that can be used for pretargeted imaging and thus separates tumor accumulation from the imaging step in time. The developed targeting agent is based on polypeptide-graft-polypeptoid polymers (PeptoBrushes) functionalized with trans-cyclooctene (TCO). The complementary 111In-labeled imaging agent is a 1,2,4,5-tetrazine derivative, which can react with aforementioned TCO-modified PeptoBrushes in a rapid bioorthogonal ligation. A high degree of TCO loading (up to 30%) was achieved, without altering the physicochemical properties of the polymeric nanoparticle. The highest degree of TCO loading resulted in significantly increased reaction rates (77-fold enhancement) compared to those with small molecule TCO moieties when using lipophilic tetrazines. Based on computer simulations, we hypothesize that this increase is a result of hydrophobic effects and significant rearrangements within the polymer framework, in which hydrophobic patches of TCO moieties are formed. These patches attract lipophilic tetrazines, leading to increased reaction rates in the bioorthogonal ligation. The most reactive system was evaluated as a targeting agent for pretargeted imaging in tumor-bearing mice. After the setup was optimized, sufficient tumor-to-background ratios were achieved as early as 2 h after administration of the tetrazine imaging agent, which further improved at 22 h, enabling clear visualization of CT-26 tumors. These findings show the potential of PeptoBrushes to be used as a pretargeting agent when an optimized dose of polymer is used.

Bioorthogonal chemistry is bridging the divide between static chemical connectivity and the dynamic physiologic regulation of molecular state, enabling in situ transformations that drive multiple technologies. In spite of maturing mechanistic understanding and new bioorthogonal bond-cleavage reactions, the broader goal of molecular ON/OFF control has been limited by the inability of existing systems to achieve both fast (i.e., seconds to minutes, not hours) and complete (i.e., >99%) cleavage. To attain the stringent performance characteristics needed for high fidelity molecular inactivation, we have designed and synthesized a new C 2 -symmetric trans-cyclooctene linker (C 2 TCO) that exhibits excellent biological stability and can be rapidly and completely cleaved with functionalized alkyl-, aryl-, and H-tetrazines, irrespective of click orientation. By incorporation of C 2 TCO into fluorescent molecular probes, we demonstrate highly efficient extracellular and intracellular bioorthogonal disassembly via omnidirectional tetrazine-triggered cleavage.

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Bioorthogonal ligations have emerged as highly versatile chemical tools for biomedical research. The exceptionally fast reaction between 1,2,4,5-tetrazines and trans-cyclooctenes (TCOs), also known as tetrazine ligation, is frequently used in this regard. Growing numbers of applications for the tetrazine ligation led to an increased demand for TCO compounds, whose commercial availability is still very limited. Reported photochemical procedures for the preparation of TCOs using flow chemistry are straightforward and high yielding but require expensive equipment. Within this contribution, we present the construction and characterization of a low-cost flow photoreactor assembled from readily accessible components. Syntheses of all commonly used trans-cyclooctene derivatives were successfully carried out using the described system. We are convinced that the presented system for photoisomerization will promote access to bioorthogonally reactive TCO derivatives.Graphical abstractElectronic supplementary materialThe online version of this article (doi:10.1007/s00706-016-1668-z) contains supplementary material, which is available to authorized users.

We have investigated the inverse electrondemand Diels−Alder reactions of trans-cyclooctene (TCO) and endo-bicyclo[6.1.0]nonyne (BCN) with a 1,2,4,5-tetrazine, a cyclopentadienone, and an orthobenzoquinone. Tetrazines react significantly faster with TCO compared to BCN because the highest occupied molecular orbital (HOMO) of TCO is significantly higher in energy than the HOMO of BCN and there is less distortion of the tetrazine. Despite the different HOMO energies, TCO and BCN have similar reactivities toward cyclopentadienones, while BCN is significantly more reactive than TCO in the cycloaddition with orthobenzoquinone. We find that the higher reactivity of BCN compared to TCO with ortho-benzoquinone is due to secondary orbital interactions of the BCN HOMO-1 with the diene LUMO.