Design, Synthesis, Conjugation, and Reactivity of Novel trans,trans-1,5-Cyclooctadiene-Derived Bioorthogonal Linkers

Abstract

The tetrazine/trans-cyclooctene (TCO) inverse electron-demand Diels−Alder (IEDDA) reaction is the fastest bioorthogonal "click" ligation process reported to date. In this context, TCO reagents have found widespread applications; however, their availability and structural diversity is still somewhat limited due to challenges connected with their synthesis and structural modification. To address this issue, we developed a novel strategy for the conjugation of TCO derivatives to a biomolecule, which allows for the creation of greater structural diversity from a single precursor molecule, i.e., trans,trans-1,5-cyclooctadiene [(E,E)-COD] 1, whose preparation requires standard laboratory equipment and readily available reagents. This two-step strategy relies on the use of new bifunctional TCO linkers (5a−11a) for IEDDA reactions, which can be synthesized via 1,3-dipolar cycloaddition of (E,E)-COD 1 with different azido spacers (5−11) carrying an electrophilic function (NHS-ester, N-succinimidyl carbonate, pnitrophenyl-carbonate, maleimide) in the ω-position. Following bioconjugation of these electrophilic linkers to the nucleophilic residue (cysteine or lysine) of a protein (step 1), the resulting TCO-decorated constructs can be subjected to a IEDDA reaction with tetrazines functionalized with fluorescent or near-infrared (NIR) tags (step 2). We successfully used this strategy to label bovine serum albumin with the TCO linker 8a and subsequently reacted it in a cell lysate with the fluorescein-isothiocyanate (FITC)derived tetrazine 12. The same strategy was then used to label the bacterial wall of Gram-positive Staphylococcus aureus, showing the potential of these linkers for live-cell imaging. Finally, we determined the impact of structural differences of the linkers upon the stability of the bioorthogonal constructs. The compounds for stability studies were prepared by conjugation of TCO linkers 6a, 8a, and 10a to mAbs, such as Rituximab and Obinutuzumab, and subsequent labeling with a reactive Cy3-functionalized tetrazine.

SQ3370 Activates Cytotoxic Drug via Click Chemistry at Tumor and Elicits Sustained Responses in Injected and Non‐Injected Lesions

“…[16] The reaction between tetrazines and transcyclooctene (TCO) is one of the most efficient click reactions reported to date, with demonstrated utility in animal models systems. [17][18][19][20][21][22][23][24] Tetrazine and TCO react irreversibly and specifically in vivo with a high thermodynamic driving force, with minimal interaction with the surrounding biological mileu. [25,26] The CAPAC Platform uses a tetrazine-modified biopolymer injected at the tumor that can capture a systemically circulating TCO-modified protodrug, to precisely release the active drug at the tumor.…”

IEDDA: An Attractive Bioorthogonal Reaction for Biomedical Applications

“…The chemistry allowed less-tedious chemical modification of TCO and was exploited by Longo et al to prepare several TCO derivatives for live-cell fluorescent imaging. However, the kinetics of 17 were too slow for an in vivo pretargeting study [ 80 ]. So far, and to the best of our knowledge, no other dienophiles than 5-OH-TCO have been successfully advanced to in vivo pretargeting studies.…”

“… ( A ) Represented dienophiles for IEDDA reaction: 11 [ 75 ], 12 – 15 [ 77 ], 16 [ 78 ], 17 [ 80 ]. ( B ) Modified dienophiles for antibody-based pretargeting studies.…”

The Collective Power of Genetically Encoded Protein/Peptide Tags and Bioorthogonal Chemistry in Biological Fluorescence Imaging

“…The second order rate constant of the IEDDA between TCO and Tz could be over 10 5 M −1 s −1 , [135b] making it the fastest type of bioorthogonal reactions reported to date. A TCO analog that is derived from trans , trans ‐1,5‐cyclooctadiene [(E,E)‐COD] was recently reported, [136] which, other than being competent in bioconjugation, offers an appealing new synthesis to the core structure of TCO. In addition to strained alkenes, vinyl boronic acid (VBA) could engage in a bioorthogonal IEDDA with Tz (Figure 11c) [137] .…”