Trans-cyclooctene-a Swiss army knife for bioorthogonal chemistry: exploring the synthesis, reactivity, and applications in biomedical breakthroughs

Abstract

Background: Trans-cyclooctenes (TCOs) are highly strained alkenes with remarkable reactivity towards tetrazines (Tzs) in inverse electron-demand Diels-Alder reactions. Since their discovery as bioorthogonal reaction partners, novel TCO derivatives have been developed to improve their reactivity, stability, and hydrophilicity, thus expanding their utility in diverse applications.

Main body: TCOs have garnered significant interest for their applications in biomedical settings. In chemical biology, TCOs serve as tools for bioconjugation, enabling the precise labeling and manipulation of biomolecules. Moreover, their role in nuclear medicine is substantial, with TCOs employed in the radiolabeling of peptides and other biomolecules. This has led to their utilization in pretargeted nuclear imaging and therapy, where they function as both bioorthogonal tags and radiotracers, facilitating targeted disease diagnosis and treatment. Beyond these applications, TCOs have been used in targeted cancer therapy through a "click-to-release" approach, in which they act as key components to selectively deliver therapeutic agents to cancer cells, thereby enhancing treatment efficacy while minimizing off-target effects. However, the search for a suitable TCO scaffold with an appropriate balance between stability and reactivity remains a challenge.

Conclusions: This review paper provides a comprehensive overview of the current state of knowledge regarding the synthesis of TCOs, and its challenges, and their development throughout the years. We describe their wide ranging applications as radiolabeled prosthetic groups for radiolabeling, as bioorthogonal tags for pretargeted imaging and therapy, and targeted drug delivery, with the aim of showcasing the versatility and potential of TCOs as valuable tools in advancing biomedical research and applications.

Keywords: Trans-cyclooctene; Bioorthogonal chemistry; In vivo ligation; Pretargeted imaging and therapy.

Scheme 1

Reaction mechanism of inverse electron-demand Diels–Alder [4 + 2] (IEDDA) between a trans-cyclooctene and 1,2,4,5-tetrazine. The cycloaddition is followed by a retro Diels–Alder with expulsion of N₂ forming 1,4-dihydropyridazine isomers which further oxidizes to pyridazine

Fig. 1

Frontier molecular orbital (FMO) model of cycloaddition for a Diels–Alder cycloaddition reaction and IEDDA with ∆E depicting the energy gap between the orbitals. In IEDDA, the energy gap between the highest occupied molecular orbital (HOMO) of the dienophile and the lowest unoccupied molecular orbital (LUMO) of the diene is influenced by the presence of electron-donating groups (EWG) on the diene and electron-withdrawing groups on the dienophile. Adapted from Oliveira et al. (2017) with permission from Royal Society of Chemistry

Fig. 2

Structure and reactivity of tetrazine influenced by electronic, steric, and distortion effects. (a) Tetrazines in increasing order of reactivity. The EWG groups increase the reactivity of the tetrazines. b Repulsive intramolecular interactions reduce the Tz distortion energy and increase reactivity. c The reactivity of 4-substituted tetrazines is FMO-controlled and the reactivity increases in presence of EWG d reactivity of 2-substituted or 3-substituted tetrazines is not FMO-controlled

Fig. 3

Comparison of the reactivities of different TCO derivatives used in ligation reactions. The reactivity is enhanced by increasing the ring strain via cis-ring fusion to the cyclooctene core. Stereochemistry and the inclusion of endocyclic or exocyclic heteroatoms also influence the reactivity

Scheme 2

Multistep synthesis of trans-cyclooctene from cis-cyclooctene via inversion of the alkene stereochemistry

Scheme 3

Scheme depicting the formation of chiral exciplexes when cis-cyclooctene is irradiated in the presence of a chiral sensitizer. Reproduced from Pigga and Fox (2020) Copyright 2019 Wiley–VCH Verlag GmbH & Co. KGaA, Weinheim

Fig. 4

Schematic of Flow-Photo isomerization apparatus for the synthesis of trans-cyclooctene. Adapted with permission from Darko et al. (2018) Copyright 2018 Georg Thieme Verlag KG

Scheme 4

Structure of trans-cyclooct-4-enol and the reaction between thioredoxin-derivatized TCO-OH and 3,6-dipyridyl-s tetrazine. Adapted with permission from Blackman et al. (2008) Copyright 2008, American Chemical Society

Scheme 5

Diastereoselective synthesis of a-TCO via nucleophilic addition is preferred at the equatorial face. Structures of axial and equatorial TCO-OH. Adapted with permission from Pigga et al. (2021) Copyright 2021 Wiley–VCH GmbH

Scheme 6

Synthesis of conformationally strained s-TCO

Fig. 5

Transition-state structures and respective energy barriers for the Diels–Alder reaction of s-tetrazine with crown conformers of trans-cyclooctene, cis-ring-fused s-TCO, and trans-ring-fused s-TCO. Adapted with permission from Taylor et al. (2011) Copyright 2011, American Chemical Society

Scheme 7

Synthesis of double-functionalized s-TCO (Ravasco et al. 2020)

Scheme 8

Diastereoselective synthesis of d-TCO

Scheme 9

Synthetic scheme of aza-TCO

Scheme 10

Synthetic scheme of Ox-TCO

Scheme 11

Synthesis of 4,6-dioxo-TCO (DO-TCO)

Scheme 12

Synthesis of trans-5-oxocene

Fig. 6

Structures of endocyclic heteroatom-containing TCOs

Scheme 13

IEDDA decaging reaction between a TCO-carbamate conjugated to a drug molecule and tetrazine

Fig. 7

a Unsymmetric Tzs containing EWG and a non-EWG promote the elimination efficiency. b The release efficiencies of the symmetric and unsymmetric tetrazines in increasing order. Unsymmetric tetrazines Tzs exhibit superior release efficiency compared to their symmetric counterparts. Hydroxyethyl-substituted Tzs showed a higher release efficiency than methyl-substituted Tzs. Adapted with permission from Fan et al. (2016) Copyright 2016 Wiley–VCH Verlag GmbH & Co. KGaA, Weinheim

Scheme 14

Formation of tricyclic dead-end product via intramolecular cyclization of carbamate amine onto tetrazine, resulting in an incomplete release

Scheme 15

Tautomerization enhancement by acid-functionalized tetrazines via acid-assisted H transfer. The click orientation of the substituted tetrazine determines the pH-dependent drug release. A Head-to-Head orientation favors the formation of a releasing isomer, whereas a head-to-tail orientation favored the formation of a non-releasing isomer

Scheme 16

Schematic representation of TCO-triggered release of payload from tetrazine. The initially formed 4,5-DHP tautomerizes into 1,4-DHP and 2,5-DHP, of which 2,5 DHP liberates the amine-bound payload in conjunction with the formation of pyridazine Adapted from Onzen et al. (2020)with permission. Copyright © 2020, American Chemical Society

Scheme 17

Synthesis of r-TCO

Scheme 18

Synthesis of cTCO

Scheme 19

Synthesis of C₂-TCO

Scheme 20

Synthesis of dcTCO

Scheme 21

a: Synthesis of difunctionalized s-TCO derivative starting from 1,3-cyclooctadiene. b Synthesis of difunctionalized s-TCO derivative starting from 1,5-cyclooctadiene

Scheme 22

Synthesis of difunctionalized s-TCO derivative starting from 1,5-cyclooctadiene

Fig. 8

Representative PET/CT images of PC-3 tumor-bearing mice at 0.5h and 3.5h post-injection with [¹⁸F]F s-TCO, [¹⁸F]F d-TCO, and [¹⁸F]F-OxoTCO-derived NT-analogs. Image-derived tumor-to-liver (T/L) and tumor-to-muscle (T/M) ratios from the conversion of the region of interest to %ID/g. The grey and black bars represent the tumor-to-organ ratios at 0.5h and 3.5h respectively. The tumor-to-background ratio was highest with the oxoTCO-derived NT analog. Reproduced from Wang et al. ( with permission from the Royal Society of Chemistry)

Fig. 9

Representative SPECT/CT image of tumor-bearing mice injected with CC49-TCO, followed by two doses of galactose-albumin-tetrazine clearing agent (30 and 48h post-mAb injection) and ¹⁷⁷Lu-labeled tetrazine at 50h post-mAb injection. The image shows high radioactivity uptake in the tumor and low uptake in non-target organs. Most of the activity is present in the bladder. Adapted from Rossin et al. (2014) with permission Copyright 2014 American Chemical Society

Fig. 10:

¹⁸F-labeled TCO and d-TCO derivatives used in pretargeted imaging