Tetrazine‐Triggered Release of Carboxylic‐Acid‐Containing Molecules for Activation of an Anti‐inflammatory Drug

Abstract

In addition to its use for the study of biomolecules in living systems, bioorthogonal chemistry has emerged as ap romising strategyt oe nable protein or drug activation in as patially and temporally controlled manner.T his study demonstrates the application of ab ioorthogonal inverse electron-demand Diels-Alder (iEDDA) reaction to cleave trans-cyclooctene (TCO) and vinyl protecting groups from carboxylic acid-containing molecules. The tetrazine-mediated decaging reactionp roceeded under biocompatiblec onditions with fast reaction kinetics (< 2min). The anti-inflammatory activity of ketoprofen was successfully reinstated after decaging of the nontoxic TCOprodrug in live macrophages. Overall, this work expandst he scope of functional groups and the application of decaging reactions to an ew class of drugs.Early research in the fieldo fb ioorthogonal chemistry focused on ligation reactions such as the Staudinger reaction, [1] coppercatalysed azide-alkyne 1,3-dipolar cycloaddition (CuAAC), [2,3] palladium-catalysed cross-couplings, [4] ruthenium-catalysed olefin metatheses, [5] strain-promoted azide-alkyne cycloaddition (SPAAC), [6] tetrazole photoinduced 1,3-dipolar cycloadditions, [7,8] and inverse electron-demandD iels-Alder (iEDDA) tetrazine ligation. [9,10] Of these, the iEDDA reactionb etween transcyclooctene (TCO) and at etrazine is one of the more selective and fastestb ioorthogonal reactions to date. [11] Since it was first introduced by Fox et al., [9] this reactionh as been used in numerous biological applicationss uch as cell and in vivo pretargeting imaging. [12][13][14] Recently,b ioorthogonal cleavage reactions have emerged as promising strategies to control the activation of caged proteins,f luorophores, and small-molecule drugs in living systems. [15] The TCO-tetrazine iEDDA ligation can be re-engineered into ac leavage reaction by placing a leaving group at the allylic positiono fT CO. After the initial cycloaddition and elimination of nitrogen, the 4,5-dihydropyridazine now contains an appropriatelyp laced substituent that eliminates upon tautomerisation. [10] Robillard'sg roup reported the first use of the TCO-tetrazine reaction for bioorthogonal decaging to release amine-containing drugs ( Figure 1A), in which they demonstrated the release of doxorubicin (Dox) from aT CO carbamate prodrug in vitro. [16] They then applied this "click-to-release" strategy to successfullytrigger the release of Dox and monomethyl auristatin E( MMAE) from an antibody-drug conjugate (ADC). [17,18] Mejia Oneto and co-workers also reported targeted in vivo activation of aD ox-TCO carbamate prodrug by injecting an alginate hydrogel modified with tetrazines near the tumour site. [19] Al imitation of the click-toreleases trategy is the need for delivery,a nd therefore optimisation of the pharmacokinetic properties, of both the prodrug and the tetrazine. [20,21] However,t he previously mentioned approaches demonstrate the potential of bioorthogonal decaging reactions for targeted drug activation in vivo.Bioorthogonal chemist...

Activation and Delivery of Tetrazine-Responsive Bioorthogonal Prodrugs

“…Cycloaddition consumed these TCOs within minutes, and the release exhibited biphasic kinetics profiles with a fast release up to approximately 70-85% within 10-30 min, followed by slower re-tautomerization of 2,5-dihydropyridazine to release the rest within 20 h ( Figure 3B) [57]. Despite the broad scope of these reactions, not all chemical groups are useful for in vivo applications: ester bonds (compound 23) and carbonate (compound 24), for example, are unstable in plasma and cell medium [57,63,64]. The ether bond (compound 22), however, is relatively stable in physiological condition: after reacting with tetrazine, TCO caged tyrosine 22d released the free amino acid for controllable cell growth as a proof of concept verification [57].…”

“…This lower activity was due to the low release yield of 39%. They also built a more stable TCO ester prodrug model for the release of carboxylic acids ( Figure 3C) [64]. The need for bulky substituents to stabilize the ester bond was emphasized.…”

“…The toolbox of TCO moieties that can be directly attached to an antibody without interfering its bio-function is to be expanded. Thurecht's group developed a novel polymer-drug conjugate for a three-component system ( Figure 15C) [115]: (1) a TCO-Dox prodrug conjugated to a PEGylated hyperbranched polymer (HBP); (2) a PEGylated tetrazine trigger functionalized with the 64 Cu chelator NOTA; (3) a bispecific antibody (BsAb) that recognized tumor-associated glycoprotein-72 as well as PEGylated nanoparticles via strong non-covalent interactions. Firstly, the HBP-TCO-Dox was incubated with BsAb (at a ratio of 1:1) for one hour and then the conjugate was injected intravenously to MCF7-tumor-bearing mice.…”

Synthetic methodology towards allylictrans-cyclooctene-ethers enables modification of carbohydrates: bioorthogonal manipulation of thelacrepressor

“…1A, green). Recently, Robillard 8 and Bernardes 20,21 showed that release of other functional groups, such as carboxylic acids (from TCO-esters) and alcohols (from TCOcarbonates or TCO-ethers), is indeed possible (Fig. 1A, orange and red).…”

“…1A, orange and red). Whilst TCO-esters and carbonates suffer from reduced hydrolytic stability in biological systems, 8,20,21 TCO-ethers are particularly appealing due to their high stability and surprisingly fast decaging kinetics when compared to vinyl ether analogues. 8,[22][23][24] Unfortunately, widespread use of TCO-ethers is constricted by their challenging synthesis.…”