Tetrazine‐Triggered Bioorthogonal Cleavage of trans‐Cyclooctene‐Caged Phenols Using a Minimal Self‐Immolative Linker Strategy**

Abstract

Bond-cleavage reactions triggered by bioorthogonal tetrazine ligation have emerged as strategies to chemically control the function of (bio)molecules and achieve activation of prodrugs in living systems. While most of these approaches make use of caged amines, current methods for the release of phenols are limited by unfavorable reaction kinetics or insufficient stability of the Tz-responsive reactants. To address this issue, we have implemented a self-immolative linker that enables the con-nection of cleavable trans-cyclooctenes (TCO) and phenols via carbamate linkages. Based on detailed investigation of the reaction mechanism with several Tz, revealing up to 96 % elimination after 2 hours, we have developed a TCO-caged prodrug with 750-fold reduced cytotoxicity compared to the parent drug and achieved in situ activation upon Tz/TCO clickto-release.

Oxidative Desymmetrization Enables the Concise Synthesis of a trans‐Cyclooctene Linker for Bioorthogonal Bond Cleavage

“…For initial assessment of the release efficiency, ax‐8 was reacted with selected Tz ( 9 – 13 ) in PBS at 37 °C and the reaction mixtures were analyzed after 48 h by HPLC (Figure 4c). We observed a dcTCO‐release of 89 % with DMT ( 9 ), 53 % with the alkyl‐aryl‐Tz MeBA ( 10 ), and 62 % with PymK ( 11 ), similar to previous findings [15] and matching very recently reported data for the release of rTCO‐DMEDA‐conjugates [16] . As indicated by LCMS data, and in line with previous reports, [5a,8,16] the non‐released by‐products were assigned to an oxidized pyridazine dead‐end, formed upon click with DMT ( 9 ), and stable dihydropyridazine tautomers with the aryl‐substituent of MeBA ( 10 ) and PymK ( 11 ), respectively, in a head‐to‐head position [5a,8] relative to the dcTCO‐carbamate.…”

“…We observed a dcTCO‐release of 89 % with DMT ( 9 ), 53 % with the alkyl‐aryl‐Tz MeBA ( 10 ), and 62 % with PymK ( 11 ), similar to previous findings [15] and matching very recently reported data for the release of rTCO‐DMEDA‐conjugates [16] . As indicated by LCMS data, and in line with previous reports, [5a,8,16] the non‐released by‐products were assigned to an oxidized pyridazine dead‐end, formed upon click with DMT ( 9 ), and stable dihydropyridazine tautomers with the aryl‐substituent of MeBA ( 10 ) and PymK ( 11 ), respectively, in a head‐to‐head position [5a,8] relative to the dcTCO‐carbamate. In contrast, the reaction of ax‐8 with the monosubstituted aryl‐Tz HBA ( 12 ) and 2Pyr 2 ( 13 ) resulted in only <30 % release (Figure 4c), consistent with reported data for rTCO and other cleavable TCOs [5a,7,13,15] .…”

“…While we have recently reported a 750‐fold reduced cytotoxicity of an analogous cTCO‐caged CA4 prodrug, the observed “turn‐on” in toxicity upon reaction with DMT ( 9 ) was limited to a factor of 220. [16] In contrast, cytotoxicity could be fully restored via bioorthogonal activation of dcTCO‐prodrug 15 by in‐situ reaction with DMT ( 9 ), thus providing a therapeutic window of three orders of magnitude (>1000‐fold, Figure 5 c). Moreover, this number notably exceeds the reported data for cTCO‐caged SQP33 (a doxorubicin‐prodrug with 83‐fold reduced cytotoxicity), currently being evaluated in a phase I study.…”

“…To demonstrate bioorthogonal cleavage of a dcTCO linker in a biological environment, we designed a prodrug of the antimitotic agent combretastatin A‐4 ( CA4 ) [17] . Analogous to our recently reported approach for the Tz‐triggered release of phenols, [16] we conjugated dcTCO to CA4 through DMEDA as a self‐immolative linker. dcTCO‐DMEDA‐CA4 ( 14 ) was obtained by reacting ax‐6 with DMEDA and PNP‐CA4, followed by removal of the TIPS protecting group (for details see the Supporting Information).…”

“…Therefore, we monitored the reaction of 14 with DMT (9) by serial HPLC measurements. In comparison to rTCO-DMEDA-CA4 (16), we observed a moderately faster elimination, leading to 85 % release of CA4 within 4 h (Figure S1). We then investigated prodrug 15 in HT1080 human fibrosarcoma cancer cells, revealing a remarkable > 1000-fold reduced cytotoxicity compared to the parent drug.…”