molecules

Review Activation and Delivery of Tetrazine-Responsive Bioorthogonal Prodrugs

Yayue Wang 1, Chang Zhang 1, Haoxing Wu 1,* and Ping Feng 2,*

1 Huaxi MR Research Center, Department of Nuclear Medicine, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China; [email protected] (Y.W.); [email protected] (C.Z.) 2 Institute of Clinical Trials, West China Hospital, Sichuan University, Chengdu 610041, China * Correspondence: [email protected] (H.W.); [email protected] (P.F.); Tel.: +86-028-65261615 (H.W.); +86-028-85423583 (P.F.)

Academic Editor: Mohammad Najlah



 Received: 25 October 2020; Accepted: 26 November 2020; Published: 30 November 2020

Abstract: Prodrugs, which remain inert until they are activated under appropriate conditions at the target site, have emerged as an attractive alternative to drugs that lack selectivity and show off-target effects. Prodrugs have traditionally been activated by enzymes, pH or other trigger factors associated with the disease. In recent years, bioorthogonal chemistry has allowed the creation of prodrugs that can be chemically activated with spatio-temporal precision. In particular, tetrazine-responsive bioorthogonal reactions can rapidly activate prodrugs with excellent biocompatibility. This review summarized the recent development of tetrazine bioorthogonal cleavage reaction and great promise for prodrug systems.

Keywords: tetrazine; bioorthogonal reaction; prodrug activation

1. Introduction The efficacy of a pharmacotherapy usually depends on whether the drug would achieve its optimal concentration at the specific lesions. Poor physiochemical, biopharmaceutical or pharmacokinetic properties restrict many pharmacologically active compounds from clinical practice [1,2]. One solution to overcome is to administer the drug in a “prodrug” form that remains inert in vitro and converts into its active form in vivo. Prodrugs can provide possibilities to optimize the absorption, distribution, metabolism, excretion and toxicity (ADMET) of the respective parent drugs [3,4]. Prodrug principles as described above, first proposed by Albert in 1958 [5], have been effectively applied to drug design and development [1,6–8]. A classic example is prontosil, an antimicrobial sulfanilamide prodrug that ushered the research and development of antimicrobial agents to a new area [9,10]. In recent years, a growing number of prodrugs have been approved in clinical use [1]. Prodrugs are mainly designed to improve the bioavailability of parent drugs for oral delivery and parenteral administration [1,3,4] by increasing aqueous solubility or membrane permeability, for example, anesthetic fospropofol [11], angiotensin-converting enzyme (ACE) inhibitors enalapril [12], and sofosbuvir for hepatitis C virus infection therapy [13] (Figure1A). On the other hand, prodrugs are playing increasingly important roles in targeted therapy due to either site-directed delivery or site-specific bioactivation [14–17]. Taking capecitabine as an example (Figure1A), it undergoes three enzymatic conversions into active antimetabolite drug 5-fluorouracil in tumor tissue [18].

Molecules 2020, 25, 5640; doi:10.3390/molecules25235640 www.mdpi.com/journal/molecules MoleculesMolecules2020 2020, ,25 25,, 5640 x FOR PEER REVIEW 2 2of of 25 24

Figure 1. (A) Structures of some examples of prodrug (pro-moiety in blue box). (B) The concept of prodrug Figure 1. (A) Structures of some examples of prodrug (pro-moiety in blue box). (B) The concept of activation based on tetrazine-responsive bioorthogonal click-to-release reaction with representative prodrug activation based on tetrazine-responsive bioorthogonal click-to-release reaction with tetrazine triggered release of doxorubicin (Dox) from trans-cyclooctene. representative tetrazine triggered release of doxorubicin (Dox) from trans-cyclooctene. Conventional prodrug activation strategies usually rely on intrinsic physiological changes at the targetConventional site such prodrug as overexpressed activation strategies enzymes usually [19,20 rely], unique on intrinsic microenvironment physiological changes with at lower the pHtarget value site [21 such,22], as and overexpressed higher level of enzymes reactive oxygen[19,20], speciesunique (ROS) microenvironment [23,24] and glutathione with lower (GSH) pH [value25,26 ]. However,[21,22], and these higher endogenous level of reactive changes oxygen may be species too subtle (ROS) to [23,24] activate and prodrug glutathione with high(GSH) selectivity, [25,26]. However, these endogenous changes may be too subtle to activate prodrug with high selectivity, resulting in off-target activation that can give rise to adverse effects [27,28]. An alternative is to activate resulting in off-target activation that can give rise to adverse effects [27,28]. An alternative is to the prodrug with exogenous stimulus such as light [29,30] or robust chemical reactions [2,31]. The latter activate the prodrug with exogenous stimulus such as light [29,30] or robust chemical reactions [2,31]. may be more promising because they are associated with lower cytotoxicity and greater tunability The latter may be more promising because they are associated with lower cytotoxicity and greater than light-based activation. In addition, light usually cannot reach beyond skin to deeper tissues [32]. tunability than light-based activation. In addition, light usually cannot reach beyond skin to deeper Ideal chemical reactant pairs should be stable and avirulent under physiological conditions. tissues [32]. They should react exclusively with each other, but not interact with biological functionalities [33]. Ideal chemical reactant pairs should be stable and avirulent under physiological conditions. Bioorthogonal reactions can be considered as good examples of ideal properties mentioned above, They should react exclusively with each other, but not interact with biological functionalities [33]. and have been widely used in biomedical research [34–38]. The inverse electron demand Diels-Alder Bioorthogonal reactions can be considered as good examples of ideal properties mentioned above, (IEDDA) reactions between 1,2,4,5-tetrazine (Tz) and various dienophiles stand out from bioorthogonal and have been widely used in biomedical research [34–38]. The inverse electron demand Diels-Alder reactions for in vivo applications due to the superfast kinetics and excellent biocompatibility [39–41]. (IEDDA) reactions between 1,2,4,5-tetrazine (Tz) and various dienophiles stand out from The tetrazine-responsive bioorthogonal click-to-release reactions result in the cleavage of carbamates, bioorthogonal reactions for in vivo applications due to the superfast kinetics and excellent esters and ethers [31], thus providing an opportunity to release functional groups for selective biocompatibility [39–41]. The tetrazine-responsive bioorthogonal click-to-release reactions result in activationthe cleavage of proteinsof carbamates, [42,43 esters], fluorogenic and ethers probes [31], thus [44,45 providing] and prodrugs an opportunity [2,27,46] to (Figure release1B) functional in living systems.groups for These selective strategies activation about of tetrazine-responsive proteins [42,43], fluorogenic prodrug activation probes [44,45] mainly and focus prodrugs on the [2,27,46] cytotoxic chemotherapeutics(Figure 1B) in living which systems. usually These associated strategies withabout narrow tetrazine-responsive therapeutic window prodrug and activation severesystemic mainly adversefocus on e fftheects. cytotoxic One of chemotherapeutics the two bioorthogonal which reactants usually associated acts as a pro-moiety with narrow in therapeutic the prodrug, window and the partnerand severe acting systemic as a trigger adverse releases effects. the One active of the drug tw upono bioorthogonal reaction. Only reactants in condition acts as ofa pro-moiety both reactants in comingthe prodrug, into proximity and the partner will the acting prodrug as a betrigger activated. releases And the with active a combinationdrug upon reaction. of targeted Only drug in deliverycondition systems, of both the reactants inactive prodrugscoming into can beproximity selectively will released the prodrug without be causing activated. toxicity And to with normal, a healthycombination tissue. of Lately, targeted the drug FDA delivery has approved systems, a first-in-human the inactive prodrugs Phase I clinical can be trialselectively based onreleased a Click Activatedwithout causing Prodrugs toxicity Against to normal, Cancer (CAPAC)healthy tiss platformue. Lately, [47 ].the In FDA this review,has approved we summarized a first-in-human several prodrug-activationPhase Ⅰ clinical trial chemistries based on a basedClick Activated on tetrazine Prodrugs bioorthogonal Against cleavageCancer (CAPAC) reaction platform and their [47]. further In applicationsthis review, we for summarized in vivo drug severa delivery.l prodrug-activation chemistries based on tetrazine bioorthogonal cleavage reaction and their further applications for in vivo drug delivery. 2. Prodrug-Activation Chemistries 2. Prodrug-ActivationClassical IEDDA bioorthogonalChemistries reactions focus on the formation of chemical bonds which lead toClassical stable ligationIEDDA bioorthogonal products. Tetrazines reactions are focus prone on the to react formation with severalof chemical dienophiles bonds which including lead transto stable-cyclooctenes ligation products. (TCO) Tetrazines [48,49], norbornene are prone to (NB) react [ 50with], bicyclononyneseveral dienophiles (BCN) including [51–53 trans] and- cyclopropanecyclooctenes [(TCO)54,55]. [48,49], With appropriate norbornene structural (NB) [50] modifications,, bicyclononyne an (BCN) elimination [51–53] or rearrangementand cyclopropane step is[54,55]. allowed With to occur appropriate after the structural cycloaddition, modifications, releasing an a fluorophore, elimination proteinor rearrangement residue or activestep is drug allowed with variousto occur functional after the cycloaddition, groups, including releasing amino, a fluoroph hydroxylore, and protein carboxyl. residue In this or active section drug we with discussed various the functional groups, including amino, hydroxyl and carboxyl. In this section we discussed the

Molecules 2020, 25, 5640 3 of 24

Molecules 2020, 25, x FOR PEER REVIEW 3 of 25 chemistry characteristics of the emerging tetrazine-responsive bioorthogonal cleavage reactions and chemistry characteristics of the emerging tetrazine-responsive bioorthogonal cleavage reactions and theirtheir potential potential for for prodrug prodrug activation activation applications. applications. 2.1. Tetrazine-Triggered Cleavage from Trans-Cyclooctene 2.1. Tetrazine-Triggered Cleavage from Trans-Cyclooctene InIn 2013, 2013, Robillard Robillard and and co-workers co-workers developeddeveloped the first first bioorthogonal, bioorthogonal, rapid rapid elimination elimination reaction reaction betweenbetween tetrazine tetrazine and andtrans trans-cyclooctenes-cyclooctenes (TCOs)(TCOs) [[56].56]. They They incorporated incorporated a acarbamate carbamate at atthe the allylic allylic position,position, resulting resulting in thein the cleavable cleavable TCO TCO1. IEDDA 1. IEDDA cycloaddition cycloadditionbetween between TCO 1 TCOand tetrazine1 and tetrazine eliminated N2eliminatedto generate N a 4,5-dihydropyridazine2 to generate a 4,5-dihydropyridazine intermediate 3, which intermediate tautomerized 3, which to 1,4- tautomerized and 2,5-dihydropyridazine to 1,4- and in an2,5-dihydropyridazine aqueous environment. in Thean 1,4-dihydropyridazineaqueous environment.4 Thespontaneously 1,4-dihydropyridazine underwent bond4 spontaneously rearrangement to eliminateunderwent CO bond2 and rearrangement an amino-substituted to eliminate drug CO (Figure2 and2A). an amino-substitutedThis hypothesis was drug confirmed (Figure 2A). byfurther This experimentshypothesis [was57,58 confirmed]. They concludedby further experiments that TCO derivatives [57,58]. They with concluded axial carbamates that TCO derivatives reacted 156-fold with fasteraxial than carbamates the equatorial reacted isomers 156-fold (Figure faster2 B)than [ 56 the,59 ].equatorial Electron densityisomers of(Figure the tetrazine 2B) [56,59]. was alsoElectron found todensity influence of the tetrazine reaction was kinetics also found and the to influence yields of the products. reaction Forkinetics example, and the dipyridyl-tetrazine yields of products. 12 1 1 −1 −1 reactedFor example, rapidly dipyridyl-tetrazine with axial TCO derivates 12 reacted (k 2rapidly= 57.70 with M− axials− )TCO but derivates gave a release (k2 = 57.70 yield M of s only) but 7%. gave a release yield of only 7%. Conversely, dimethyl-tetrazine 11 reacted1 1 more slowly (k2 = 0.54 M−1 Conversely, dimethyl-tetrazine 11 reacted more slowly (k2 = 0.54 M− s− ) but gave a higher release yields−1) ofbut 79% gave [56 a]. higher release yield of 79% [56].

Figure 2. (A) Reaction mechanism between cleavable TCO and tetrazines. (B) TCO isomers and tetrazines Figure 2. (A) Reaction mechanism between cleavable TCO and tetrazines. (B) TCO isomers and toolbox. (C) The mechanism for pH-dependent release of active amine and release profile of 14 at pH 5–7. tetrazines toolbox. (C) The mechanism for pH-dependent release of active amine and release profile Reproduced with permission from [58]; © 2020 American Chemical Society. (D) The mechanism for of 14 at pH 5–7. Reproduced with permission from [58]; © 2020 American Chemical Society. (D) The pH-independent release of active amine and release profile of 15 at pH 3–7.4. Reproduced with permission mechanism for pH-independent release of active amine and release profile of 15 at pH 3–7.4. from [60]; © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Reproduced with permission from [60]; © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Since substituents on the tetrazine affected reaction kinetics and release yields, Chen and Since substituents on the tetrazine affected reaction kinetics and release yields, Chen and co- co-workersworkers systematically systematically studied studied the the structure-activity structure-activity relationship relationship of oftetrazine tetrazine derivatives derivatives [61]. [61 ]. SmallerSmaller alkyl alkyl substituents substituents (such (such as as methyl) methyl) ledled to higher release release yield, yield, while while a abulky bulky terttert-butyl-butyl groupgroup contributedcontributed to to almost almost no no release. release. On On thethe otherother ha hand,nd, electron-withdrawing electron-withdrawing groups groups on on the the tetrazine tetrazine promotedpromoted cycloaddition cycloaddition while while inhibiting inhibiting elimination. elimination. TheThe authors concluded concluded that that the the reaction reaction wouldwould

Molecules 2020, 25, 5640 4 of 24 be faster along with higher release yield if unsymmetric tetrazines bearing an electron-withdrawing group at the 3-position and a small, flexible alkyl group at the 6-position were used. For example, pyrimidine-tetrazine 13 allowed over 80% of the fluorescence to recover upon reaction with TCO-coumarin within 30 min, whereas tetrazine 11 only restored 20% of the fluorescence in the same time interval [61]. Further mechanistic studies showed that 1,4-dihydropyridazine 4, as the key intermediate, underwent an electron-cascade elimination to release the payload, such as a drug. Its rate of formation therefore becomes a limiting factor for the elimination reaction. The 2,5-dihydropyridazine tautomer 5 was slowly oxidized to pyridazine 7 instead of releasing the payload, which limited the overall elimination yield (below 40%) [57]. A detailed investigation of the reaction mechanistic features carried out by the Weissleder group showed that for unsymmetric tetrazines, cycloadditions took place randomly in two orientations (head-to-tail or head-to-head), resulting in different adducts (Figure2C). Head-to-head adduct 16b drove the formation of the releasing tautomer 18 and led to fast release. Head-to-tail adduct 16a, in contrast, contributed to the formation of 2,5-dihydropyridazine, which slowly re-tautomerized to the 1,4-dihydropyridazine tautomer, thereby contributing to release [58]. Acidic conditions accelerate tautomerization and therefore lead to faster and higher release [57,58]. Weissleder and co-workers therefore prepared a plate of acid-functionalized tetrazines and found that the closer the solution pH was to the pKa of the acid, the faster and more complete the release would be. For example, since general acid catalysis facilitates tautomerization, tetrazines 14 bearing a propanoic acid group (pKa = 4.45) were able to release cargos rapidly and with near-quantitative yield at pH 5 (Figure2C) [ 58]. However, an unexpected intramolecular cyclization dead-end product 9 was found (Figure2A). This tricyclic by-product was stable and was slowly oxidized rather than release. But its formation can be blocked by replacing the NH of carbamate with a N-Me group (compound 10) to enable complete release [58]. In an alternative approach, an aminoethyl moiety was decorated resulting in a series of tetrazines that maintained fast elimination kinetics over a broad pH range from 3.5 to 7.5 [60]. The amine on the aminoethyl group existed as cationic ammonium over the whole biologically relevant pH and acted as an intramolecular catalyst of tautomerization (Figure2D). Containing an electron-withdrawing group simultaneously, tetrazine 15 promoted elimination at a rate constant to 120 10 5 s 1 without solvent pH dependence [60]. × − − Reactions to unmask other functional groups have also been developed (Figure3A). Pluth et al. installed a thiocarbamate on TCO (25) which reacted with tetrazines to generate carbonyl sulfide/hydrogen sulfide (Figure3A) [ 62]. The Robillard group demonstrated the TCOs containing a range of axial allylic substituents including aliphatic, benzylic, and aromatic ethers 22a–c, aromatic and aliphatic esters 23a, b and aromatic carbonate 24, could react with tetrazine to release the corresponding alcohols, phenols and carboxylic acids (Figure3A, entries 2–4 in Table1)[ 57]. 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 (Figure3B) [ 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]. Molecules 2020, 25, 5640 5 of 24 Molecules 2020, 25, x FOR PEER REVIEW 6 of 25

Molecules 2020, 25, x FOR PEER REVIEW 5 of 25 than triclosan alone (IC50 = 298 ± 20 nM vs. 122 ± 10 nM). This lower activity was due to the low release yieldMolecules of 39%. 2020 ,They 25, x FOR also PEER built REVIEW a more stable TCO ester prodrug model for the release of carboxylic5 of 25 MoleculesMolecules 2020 2020, 25,, 25x FOR, x FOR PEER PEER REVIEW REVIEW 5 of5 25of 25 acidsMoleculesMolecules (Figure 2020 2020, 25 3C),, 25x FOR, [64].x FOR PEER The PEER REVIEW need REVIEW for bulky substituents to stabilize the ester bond was emphasized.5 of5 25 Aof 25 than triclosan alone (IC50 = 298 ± 20 nM vs. 122 ± 10 nM). This lower activity was due to the low release stablethan TCO-ketoprofen triclosan alone (IC prodrug50 = 298 27 ± 20was nM synthesized, vs. 122 ± 10 andnM). activated This lower rapidly activity by wastetrazine, due to inhibiting the low release the thanthan triclosan triclosan alone alone (IC (IC50 =50 298 = 298 ± 20 ± 20nM nM vs. vs. 122 122 ± 10 ± 10nM). nM). This This lower lower activity activity was was due due to theto the low low release release than Figuretriclosan 3. alone(A) The (IC50 scope50 = 298 of the ± 20 click-to-release nM vs. 122 ± reaction10 nM). between This lower tetrazine activity and was TCO due derivatives. to the low (B release) Biphasic inflammatorythanyieldthan triclosan Figuretriclosan of 39%. aloneresponse3. aloneThey(A) (IC The also(IC50 in =scope50 living 298 built= 298 ± of 20 amacrophages.± the 20morenM nMclick-to-release vs. stable vs. 122 122 ± TCO10 ± 10nM). nM).esterreaction This Thisprodrug lower between lower activity model activity tetrazine wasfor was the due and due release to TCO theto the lowderivatives.of low carboxylic release release (B ) yieldyield ofrelease of39%. 39%. fromThey They TCOalso also derivatesbuilt built a morea22 more–25 stable. Scale: stable TCO 0–1 TCO h ester and ester 0–20 prodrug prodrug h. Reproduced model model for with for the permissionthe release release of from ofcarboxylic carboxylic [57]; © 2020 yieldacidsyield ofBiphasic (Figureof39%. 39%. They 3C)releaseThey also[64]. also from builtThe built needTCO a morea moreforderivates stablebulky stable 22substituenTCO –TCO25 .ester Scale: esterts prodrug to 0–1prodrug stabilize h and model model 0–20the forester h. for theRe bondtheproduced release release was ofemphasized. with ofcarboxylic carboxylic permission A acidsacids (Figure (Figure 3C) 3C) [64]. [64]. The The need need for for bulky bulky substituen substituents tots tostabilizeC stabilize the the ester ester bond bond was was emphasized. emphasized. A A acidsstableacids (FigureWiley-VCH (Figure TCO-ketoprofen 3C) Table3C) [64]. Verlag [64]. 1. The Summary The prodrug GmbH need need for of & 27for tetrazine-responsivebulky Co. was bulky KGaA, synthesized,substituen substituen Weinheimts bi toandtsoorthogonal tostabilize ( activated stabilize) Stable the cleavage TCO therapidly ester ester self-immolative bond reactions. by bond tetrazine, was was emphasized. emphasized. linkerinhibiting and A easterthe A stablestable TCO-ketoprofenfrom TCO-ketoprofen [57]; © 2020 prodrug Wiley-VCH prodrug 27 27was Verlag was synthesized, synthesized, GmbH & andCo. and activatedKGaA, activated Weinheim rapidly rapidly by(C by )tetrazine, Stable tetrazine, TCO inhibiting inhibitingself-immolative the the stablestableinflammatory TCO-ketoprofenprodrug TCO-ketoprofen models. response prodrug prodrug in living 27 27 wasmacrophages. was synthesized, synthesized, and and activated activated rapidly rapidly by by tetrazine, tetrazine, inhibiting inhibiting the the inflammatorylinker and response easter prodrug in living models. macrophages. −1 −1 inflammatoryinflammatory Pro-Moiety response response in inliving living Trigger macrophages. macrophages. k2 (M s ) Release Kinetics Yield of Release (%) Ref inflammatoryinflammatory response response in inliving living macrophages. macrophages. Table 1. Summary of tetrazine-responsive bioorthogonal cleavage reactions. Table 1. Summary of tetrazine-responsive bioorthogonal cleavage reactions. 1 This novelTableTable bioorthogonal 1. Summary1. Summary of tetrazine-responsiveof elimination tetrazine-responsive48.7 ± 1.1 opens bioorthogonal completebi oorthogonalup new < 30 cleavagemin avenue cleavage reactions. forreactions. 79cleavable antibody-drug [56] TableTable 1. Summary1. Summary of tetrazine-responsiveof tetrazine-responsive1 1 bioorthogonal bioorthogonal cleavage cleavage reactions. reactions. Pro-Moiety Trigger k2 (M s−1 )−1 Release Kinetics Yield of Release (%) Ref Pro-Moiety Trigger k2− (M−−1 s−1) Release Kinetics Yield of Release (%) Ref conjugates andPro-Moiety precisely controlled Trigger prodrugk2 (M activati s ) on Release and Kinetics delivery Yield[2,28,44,72]. of Release But(%) rapid Ref reaction 2 2 −1 −1 −1 Pro-MoietyPro-Moiety Trigger Trigger k (Mk −(M1 s−−11) s −1) Release Release Kinetics Kinetics Yield Yield of Releaseof Release (%) (%) Ref Ref kinetics isPro-Moiety aPro-Moiety key consideration Trigger Trigger for implementationk2 (Mk2 −(M1 s−1) s −1) Release in Release vivo Kinetics Kinetics regarding Yield Yield ofthe Releaseof Releasepossible (%) (%) degradation Ref Ref or inactivation2 1 1 of reaction reagents and48.78.5 48.7 ±competitive 0.71.1 ± 1.1 complete complete t1/2 < side3 min< < 30reactions min min in 89 79complex 79 [57]physiological [56] [56] 1 1 48.748.7 ± 1.1 ± 1.1 complete complete < 30 < min30 min 79 79 [56] [56] 1 1 48.748.7 ± 1.1 ± 1.1 complete complete < 30 < min30 min 79 79 [56] [56] environments. For example, if the rate constant is 10 M−1 s−1 and the concentrations in vivo are both

t1/2 = 14.4 h 1 1 10 μM, the half-life is supposedt1/2 = 14.4 to h be 2.8 h [48]. 3 2 15.28.5 ± 0.5 ± 0.7 t1/2 t<1/2 3

conditions is also crucial to drug activation. Dimethyltetrazine 11 was reported to hydrolyze 1 t1/2 = 14.4 h 1 t1/2 = 14.4 h 4 1/2 1/2 1 1 9.4 ± 0.2 t1/2 < 10 min 80~90 [57] approximately3 50% in phosphate-bufferedt =t 14.4= 14.4 h 1 h 1 15.2 saline ± 0.5 (PBS) within t1/2 < 3 min 14 h and dipyridyl-tetrazine 90 [57] 12 with a 3 3 t1/2 =t1/2 14.4 = 14.4 h 1 h 1 15.215.20.5 ± 0.5 t1 t/21/2< < 3 min 90 90 [57] [57] 3 3 15.215.2 ± 0.5 ± 0.5 t1/2 t<1/2 3

relatively stable (maintained over 85% in PBS for 10 h at 37 °C) [73]. Therefore, it is critical to select 4 9.4 ± 0.2 t1/2 < 10 min 80~90 [57] 4 4 9.4 9.40.2 ± 0.2 t1 t/21/2< < 10 min 80~90 80~90 [57] [57] the4 4appropriate and stable tetrazine for9.4 the ±9.4± 0.2 ±practical 0.2 t 1/2click-to-release t<1/2 10 < min10 min system. 80~90 80~90 Furthermore, [57] [57] as small 54 4 2879.4 9.4 ±± 0.210 ± 0.2 t1/2 t1/2 t<1/2 =10 10< min10 h min 80~90 80 80~90 [65][57] [57]

molecules, dialkyl tetrazines are likely to clear from circulation too fast to be quantitatively reacted at the tumor site. Prolonging blood circulation time through proper structural modification without

5 287 ± 10 t1/2 = 10 h 80 [65] reducing5 reactivity or utilizing novel drug287 deliv ± 10 ery systems t1/2 = 10 toh achieve targeted 80 accumulation [65] may 5 5 287287 ± 10 ± 10 t1/2 = t1/2 10 = h10 h 80 80 [65] [65] 5 5 28728710 ± 10 t1 t/21/2= = 10 h h 80 80 [65] [65] become65 5 effective solutions. 3.92287 287 ×± 1010 ±− 4 10 t1/2NA = t1/2 10 2= h10 h 61 80 80 [66] [65] [65]

On the other hand, TCO needs to have sufficient stability to ensure that it maintains enough

reactivity after distribution and metabolism. However, previous studies determined that TCO can 6 3.92 −×3 10−4 NA2 2 61 [66] 7 6 3.3×103.92 × 10−4 NANA 2 <80 61 [67][66] isomerize6 to the unreactive cis-isomer in3.92 the ×− 410 presence−4 ofNA 2thiols 2 [74] and copper-containing61 [66] serum 6 3.92 × 10−4 4 −4 NA 2 22 61 [66] 6 6 6 3.923.923.92 × 10 × 10 NANANA 61 61 61[66][66] [66] proteins [75]. Within 7 h, the TCO almost completely× − converted into cis in 50% fresh mouse serum at

37 °C [75]. Finding ways to avoid contact can be helpful to prevent TCO from deactivation during 7 3.3×10−3 NA 2 <80 [67] 7 3.3×10−3 NA2 2 <80 [67] 8 0.02 −3 −3 NA 2 2 87.5 [68] circulation.7 7 Binding to an antibody with 3.3×10a short3.3×10− 3spacer, forNANA example, 2 TCO moiety<80<80 was shielded[67][67] by the 7 7 7 3.33.3×103.3×1010−3 3−3 NANANA 2 22 <80<80 < 80[67][67] [67] − antibody and stabilized with a half-life of× 5 days in vivo [59]. Additionally, isomerization decreases

reaction efficiency but has the merit of noninterference in payload2 release. Moreover, some highly 8 0.02 NA 2 87.5 [68] 98 8 0.0170.020.02 NANA 2 2 94.787.587.5 [69][68][68] reactive8 and stable cis-dioxolane-fused trans0.02-cyclooctenes NA(d-TCO)2 2 derivatives87.5 have been[68] developed 8 8 0.020.02 NANA 87.587.5 [68][68]

recently [76], which wouldt1/2 = 9.6facilitate h 1 in vivo click-to-release efficiency in the future. However,

synthesis of the TCO derivatives involves a complicated photochemical isomerization followed by 2 9 0.017 NA 2 94.7 [69] 109 9 0.0270.0170.017 NANA 2 2 94.796 94.7 [69][69] 9 0.017 NA2 2 94.7 [69] 9 9 1 0.0170.017 NANA 94.794.7 [69][69] t1/2 = 9.6 h 1 t1/2 = 9.6 h 1/2 1/2 1 1 t =t 9.6= 9.6h 1 h 1 t1/2 =t1/2 9.6 = 9.6h 1 h 1 11 4.0 kelim = 1.6 × 10−4 s−1 99.1 [70] 2 10 0.027 NA 2 96 [69] 2 2 10 10 0.0270.027 NANA 2 2 96 96 [69][69] 1210 10 0.0271.10.027 kelim = NA5 × NA10 2 −5 2s −1 93.596 96 [70][69][69]

−4 −1 11 4.0 kelim = 1.6 × 10−4 s−1 99.1 [70] 13 11 0.301 ± 0.0184.0 kelimk =elim 3.5 = 1.6× 10 ×− 310 s−1 s 10099.1 [71][70] elim elim −4 −−14 −1 11 11 4.0 4.0 k k= 1.6= 1.6× 10 ×− 410 s−−14 s−1 99.199.1 [70][70] 11 11 4.0 4.0 kelimk =elim 1.6 = 1.6× 10 ×− 410 s−−14 s−1 99.199.1 [70][70] −5 −1 12 1.1 kelim = 5 × 10−5 s−1 93.5 [70] 12 1.1 kelim = 5 × 10−5 s−1 93.5 [70] 12 1.1 elim k = 5 × −104 −1 s 93.5 [70] 14 0.344 ± 0.013 k elim= elim3.4 × 10−5 s−−15 −1 100 [71] 12 12 1.1 1.1 k k= 5 =× 510 ×− 510 s−−15 s−1 93.593.5 [70][70] 12 12 1.1 1.1 kelimk =elim 5 =× 510 ×− 510 s−−15 s−1 93.593.5 [70][70] −3 −1 13 0.301 ± 0.018 kelim = 3.5 × 10−3 s−1 100 [71] 13 0.301 ± 0.018 kelim = 3.5 × 10−3 s−1 100 [71] −3 −−13 −1 13 13 0.3010.301 ± 0.018 ± 0.018 kelimk =elim 3.5 = 3.5× 10 × 10 s−3 s−1 100100 [71] [71] 13 0.301 ± 0.018 elimk elim = 3.5 ×− 310−−13 s−1 100 [71] 1513 13 0.3010.3010.25 ± 0.018 ± 0.018 kkelimelim k= =elim 8.05 3.5 = 3.5 ×× 1010 × −106 ss−1 s 80~90100100 [36][71] [71] −4 −1 14 0.344 ± 0.013 kelim = 3.4 × 10−4 s−1 100 [71] 14 0.344 ± 0.013 kelim = 3.4 × 10−4 s−1 100 [71] 14 14 0.3440.344 ± 0.013 ± 0.013 kelimk =elim 3.4 = 3.4× 10 ×− 410 s−−14 s−1 100100 [71] [71] 14 0.344 ± 0.013 kelim = 3.4 ×− 410−−14 s−1 100 [71] 14 14 0.3440.344 ± 0.013 ± 0.013 kelimk =elim 3.4 = 3.4× 10 × 10 s s 100100 [71] [71] 1 Stability assessed in PBS at 37 °C [56]. 2 Elimination kinetics is not assessed since the first step is the −6 −1 15 0.25 kelim = 8.05 × 10−6 s−1 80~90 [36] rate-limiting15 step. NA stands for not assessed. 0.25 kelim = 8.05 × 10−6 s−1 80~90 [36] 15 15 0.250.25 kelimk =elim 8.05 = 8.05 × 10 ×− 610 s−−16 s−1 80~9080~90 [36] [36] 15 0.25 kelim = 8.05 ×− 610−−16 s−1 80~90 [36] 15 15 0.250.25 kelimk =elim 8.05 = 8.05 × 10 × 10 s s 80~9080~90 [36] [36]

1 2 1 Stability assessed in PBS at 37 °C [56]. 2 Elimination kinetics is not assessed since the first step is the 1 1 Stability assessed in PBS at 37 °C [56].2 2 Elimination kinetics is not assessed since the first step is the 1 Stability1 Stability assessed assessed in PBSin PBS at 37at 37°C °C[56]. [56]. 2 Elimination 2 Elimination kinetics kinetics is notis not assessed assessed since since the the first first step step is theis the 1 Stabilityrate-limiting1 Stability assessed assessed step. in NA PBSin PBS stands at 37at 37°C for °C[56]. not [56]. 2assessed. Elimination 2 Elimination kinetics kinetics is notis not assessed assessed since since the the first first step step is theis the rate-limitingrate-limiting step. step. NA NA stands stands for for not not assessed. assessed. rate-limitingrate-limiting step. step. NA NA stands stands for for not not assessed. assessed.

Molecules 2020, 25, x FOR PEER REVIEW 5 of 25 thanMoleculesMolecules triclosan 2020 2020, 25 ,alone, 25x FOR, x FOR (IC PEER PEER50 = REVIEW 298 REVIEW ± 20 nM vs. 122 ± 10 nM). This lower activity was due to the low release5 of5 25of 25 MoleculesMolecules 2020 2020, 25,, 25x FOR, x FOR PEER PEER REVIEW REVIEW 5 of5 25of 25 yieldMoleculesMolecules of 202039%. ,2020 25 They, x, 25FOR, x also FORPEER PEERbuilt REVIEW REVIEWa more stable TCO ester prodrug model for the release of carboxylic5 of 255 of 25 thanthan triclosan triclosan alone alone (IC (IC50 =50 298 = 298 ± 20 ± 20nM nM vs. vs. 122 122 ± 10 ± 10nM). nM). This This lower lower activity activity was was due due to tothe the low low release release acidsthan (Figure triclosan 3C) alone [64]. (ICThe50 need = 298 for ± 20 bulky nM vs. substituen 122 ± 10 tsnM). to stabilize This lower the activity ester bond was was due emphasized.to the low release A thanthan triclosan triclosan alone alone (IC50 (IC = 29850 = ±298 20 ±nM 20 vs.nM 122 vs. ±122 10 ±nM). 10 nM). This This lower lower activity activity was was due dueto the to lowthe lowrelease release stableyieldyield ofTCO-ketoprofen of39%. 39%. They They also also prodrug built built a more27a morewas stable synthesized, stable TCO TCO ester andester prodrug activated prodrug model rapidly model for byfor the tetrazine, the release release inhibitingof ofcarboxylic carboxylic the yieldyieldyield of of39%. of 39%. 39%. They They They also also also built built built a morea amore more stable stable stable TCO TCO TCO ester ester ester prodrug prodrug prodrug model model model for for thefor the releasethe release release of ofcarboxylic of carboxylic carboxylic inflammatoryacidsacids (Figure (Figure 3C) response 3C) [64]. [64]. The in The livingneed need formacrophages. for bulky bulky substituen substituen ts tots tostabilize stabilize the the ester ester bond bond was was emphasized. emphasized. A A acidsacidsacids (Figure (Figure (Figure 3C) 3C) 3C)[64]. [64]. [64]. The The Theneed need need for for bulkyfor bulky bulky substituen substituen substituents totsts tostabilize to stabilize stabilize the the esterthe ester ester bond bond bond was was was emphasized. emphasized. emphasized. A A A stablestable TCO-ketoprofen TCO-ketoprofen prodrug prodrug 27 27was was synthesized, synthesized, and and activated activated rapidly rapidly by by tetrazine, tetrazine, inhibiting inhibiting the the stablestablestable TCO-ketoprofen TCO-ketoprofen TCO-ketoprofen prodrug prodrug prodrug 27 27was 27 was was synthesized, synthesized, synthesized, and and andactivated activated activated rapidly rapidly rapidly by by tetrazine, by tetrazine, tetrazine, inhibiting inhibiting inhibiting the the the inflammatoryinflammatory responseTable response 1. Summaryin inliving living macrophages.of macrophages.tetrazine-responsive bioorthogonal cleavage reactions. inflammatory response in living macrophages. inflammatoryinflammatory response response in living in living macrophages. macrophages. Pro-Moiety Trigger k2 (M−1 s−1) Release Kinetics Yield of Release (%) Ref TableTable 1. Summary1. Summary of tetrazine-responsiveof tetrazine-responsive bioorthogonal bioorthogonal cleavage cleavage reactions. reactions. Table 1. Summary of tetrazine-responsive bioorthogonal cleavage reactions. TableTable 1. Summary 1. Summary of tetrazine-responsive of tetrazine-responsive bioorthogonal bioorthogonal cleavage cleavage reactions. reactions. 2 2 −1 −1 −1 1 Pro-MoietyPro-Moiety Trigger Trigger k48.7(Mk2 ±(M 1.1s−1) s −1) complete Release Release Kinetics< 30 Kinetics min Yield Yield of Releaseof 79 Release (%) (%) [56] Ref Ref Pro-Moiety Trigger k2 −(M1 −−11 s−1) Release Kinetics Yield of Release (%) Ref Pro-MoietyPro-Moiety Trigger Trigger k2 (Mk−12 (Ms−1)− 1 s−1) Release Release Kinetics Kinetics Yield Yield of Release of Release (%) (%) Ref Ref

1 1 48.748.7 ± 1.1 ± 1.1 complete complete < 30 < min30 min 79 79 [56] [56] 1 48.7 ± 1.1 complete < 30 min 79 [56] 1 1 48.7 ±48.7 1.1 ± 1.1 complete complete < 30 min< 30 min 79 79 [56] [56] 2 8.5 ± 0.7 t1/2 < 3 min 89 [57]

1/2 2 2 1/2 1 8.5 8.5± 0.7 ± 0.7 t t<1/2 3

1/2 1/2 1 1 t =t1/2 14.4 = 14.4 h h 1 t1/2 = 14.41 h 1 t1/2 =t 14.4= 14.4 h 1 h 1 3 3 t1/2 = 14.4t1/2 = h14.4 h 15.215.2 ± 0.5 ± 0.5 t1/2 t<1/2 3

4 4 9.4 9.4± 0.2 ± 0.2 t1/2 t<1/2 10 < min10 min 80~90 80~90 [57] [57] 1/2 4 4 9.4 9.4± 0.2 ± 0.2 t1/2 t<1/2 10 < min10 min 80~90 80~90 [57] [57] 4 4 9.4 ± 9.40.2 ± 0.2 t1/2 < t101/2 min< 10 min 80~90 80~90 [57] [57]

5 287 ± 10 t1/2 = 10 h 80 [65]

5 5 287287 ± 10 ± 10 t1/2 = t1/2 10 = h10 h 80 80 [65] [65] 5 287 ± 10 t1/2 = 10 h 80 [65] 5 5 287287 ± 10 ± 10 t1/2 = t 10= h10 h 80 80 [65] [65] 5 5 287 ±287 10 ± 10 t1/2 = 10 t1/2 h = 10 h 80 80 [65] [65]

6 3.92 × 10−4 NA 2 61 [66]

Molecules 2020, 25, 5640 6 of 24 6 3.92 × 10−4 −4 NA 2 2 61 [66] 6 3.92 × 10−4 NA 2 61 [66] 6 3.92 ×− 410 −4 NA2 2 61 [66] 6 6 3.92 ×3.92 10−3 × 10 NA 2NA 61 61 [66] [66] 7 3.3×10 NA <80 [67]

Table 1. Cont. −3 −3 2 2 7 7 3.3×103.3×10−3 NANA 2 <80<80 [67][67] 7 3.3×10−3 −3 NA2 2 <80 [67] 7 7 3.3×103.3×101 −3 1 −3 NANA 2 2 <80<80 [67][67] 7 7 Pro-Moiety Trigger k2 (M3.3×103.3×10s ) ReleaseNA NA Kinetics Yield<80 of <80 Release (%)[67] [67] Ref − − 2 8 0.02 NA 87.5 [68]

2 22 8 8 8 0.020.020.02 NANANA 2 87.587.5 87.5 [68][68] [68] 8 0.02 NA2 2 87.5 [68] 8 8 0.02 0.02 NA 2NA 2 87.5 87.5 [68] [68] 2 9 0.017 NA 94.7 [69]

t t1/2 = 9.69.6 h h 1 1 1/2 2 22 9 9 9 0.0170.0170.017 NANANA 2 94.794.7 94.7 [69][69] [69] 9 0.017 NA2 2 94.7 [69] 9 9 0.0170.017 NA 2NA 2 94.7 94.7 [69] [69] 1 1/2 1/2 1 2 10 t =t1/2 9.6 = 9.6h h 1 0.027 NA 96 [69] t1/2 = 9.61 h 1 t1/2 = 9.6t1/2 =h 9.61 h 1

10 0.027 NA 2 22 96 [69] 1010 0.0270.027 NANA 2 96 96[69] [69] 11 10 4.00.027 kelim = 1.6 ×NA 210 −4 s−1 99.196 [70][69] 10 10 0.0270.027 NA 2NA 2 96 96 [69] [69]

elim −5 −1 12 1.1 k = 5 × 10 − 4s − 1 93.5 [70] 11 11 4.0 4.0 kelimk =elim 1.6 = 1.6× 10 × 10 s−4 s−1 99.199.1 [70][70] elim −44 −1 1 1111 4.04.0 kelimk elim =1.6 1.6 ×− 410−1 ss 99.199.1 [70] [70] 11 4.0 kelim = =1.6elim × 10 s− −4 −−1 99.1 [70] 11 4.0 k = 1.6× × 10 s 99.1 [70] 13 0.301 ± 0.018 kelim = 3.5 × 10−3 s−1 100 [71] elim elim −5 −15 −1 12 12 1.1 1.1 k k=elim 5 =× 510 × 10 s−5 s−1 93.593.5 [70][70] 12 1.1 kelim = 5 ×− 510−−155 s−1 1 93.5 [70] 12 1212 1.1 1.1 kelimk = =5 =×5 510 ×− 10510 s−1 −s5s −1 93.593.5 93.5 [70][70] [70] 12 12 1.1 1.1 kelim =k 5elim × =10 5 × s 10− s− 93.5 93.5 [70] [70] × 14 0.344 ± 0.013 kelim = 3.4 × 10−4 s−1 100 [71] elim elim −3 −13 −1 13 13 0.3010.301 ± 0.018 ± 0.018 k k=elim 3.5 = 3.5× 10 × 10 s−3 s−1 100100 [71] [71] 13 0.301 ± 0.018 kelim = 3.5 ×− 310−−133 s−1 1 100 [71] 13 1313 0.3010.3010.301 ± 0.0180.018 ± 0.018 kelimk = =3.5=3.5 3.5× 10 ×− 310 s−1 −s3s− −1 100100 100 [71] [71] [71] 13 13 0.3010.301 ±± 0.018 ± 0.018 k elim =k 3.5elim =× 3.510× × s 10 s 100 100 [71] [71]

elim elim −4 −14 −1 14 14 0.3440.344 ± 0.013 ± 0.013 k k=elim 3.4 = 3.4× 10 × 10 s−4 s−1 100100 [71] [71] 14 0.344 ± 0.013 kelim = 3.4 ×− −4106 −−−1414 s−1 1 100 [71] 1415 14 0.3440.3440.25 ± 0.0130.013 kkelimelim = = 8.05=3.43.4 × × 10 10−104 s s−1 − 4s−−1 80~90100 100 [71][36] [71] 14 14 0.3440.344 ±± 0.013 ± 0.013 k elim =k 3.4elim =× 3.410× × s 10 s 100 100 [71] [71]

1 2 elim elim −6 −16 −1 15 15 0.250.25 k k=elim 8.05 = 8.05 × 10 × 10 s−6 s−61 80~9080~90 [36] [36] 15 Stability assessed in PBS at 37 °C [56]. Elimination0.25 kineticskelim is = not 8.05 assessed ×− 610−−16 s−1 since the first80~90 step is the [36] 15 15 0.250.25 kelimkelimk = 8.05= 8.058.05 × 10 ×− 610 s10−1 −s−6 −1 80~9080~90 [36] [36] 15 1515 0.250.25 0.25 kelim =k 8.05elim = ×8.05 101 ×× s 10 s 80~9080~9080~90 [36] [36] [36] rate-limiting step. NA stands for not assessed. s−

1 Stability1 Stability assessed assessed in PBSin PBS at 37at 37°C °C[56]. [56]. 2 Elimination 2 Elimination kinetics kinetics is notis not assessed assessed since since the the first first step step is theis the 1 1 Stability assessed in PBS at 37 °C [56].2 2 Elimination kinetics is not assessed since the first step is the 1 Stability1 1 Stability assessed assessed in PBS in PBSat 37 at °C 37 [56]. °C [56].2 2Elimination 2 Elimination kinetics kinetics is not is assessednot assessed since since the first the firststep stepis the is the rate-limitingrate-limitingStability assessedstep. step. NA NA in stands PBS stands at for 37 for ◦notC[ not 56assessed.]. assessed.Elimination kinetics is not assessed since the first step is the rate-limiting rate-limiting step. NA stands for not assessed. rate-limitingstep.rate-limiting NA stands step. step. NA for notstandsNA assessed. stands for not for assessed.not assessed.

To realize the release of alcohols and carboxylic acids, Bernardes’s group applied their insights to the development of stable cleavable predecessors. In 2019, they reported the biocompatible release of alcohols from a TCO-carbamate conjugated with a self-immolative benzyl ether linker (Figure3C) [ 63]. The amine was released from the TCO-carbamate, followed by cascaded 1,6-elimination releasing the alcohol. The linker was highly stable in cell culture media and plasma with no release product detected over 15 h and reacted rapidly with tetrazine: cycloaddition was completed within seconds, and half-life of release was approximately 30 min. Activating the OH-containing prodrug of the antibacterial triclosan efficiently killed E. coli, although much higher concentrations were needed than triclosan alone (IC = 298 20 nM vs. 122 10 nM). This lower activity was due to the low release 50 ± ± yield of 39%. They also built a more stable TCO ester prodrug model for the release of carboxylic acids (Figure3C) [ 64]. The need for bulky substituents to stabilize the ester bond was emphasized. A stable TCO-ketoprofen prodrug 27 was synthesized, and activated rapidly by tetrazine, inhibiting the inflammatory response in living macrophages. This novel bioorthogonal elimination opens up new avenue for cleavable antibody-drug conjugates and precisely controlled prodrug activation and delivery [2,28,44,72]. But rapid reaction kinetics is a key consideration for implementation in vivo regarding the possible degradation or inactivation of reaction reagents and competitive side reactions in complex physiological environments. For example, 1 1 if the rate constant is 10 M− s− and the concentrations in vivo are both 10 µM, the half-life is supposed to be 2.8 h [48]. As a trigger of the click-to-release reaction, the stability of tetrazine under physiological conditions is also crucial to drug activation. Dimethyltetrazine 11 was reported to hydrolyze approximately 50% in phosphate-buffered saline (PBS) within 14 h and dipyridyl-tetrazine 12 with a shorter half-life of 9.6 h [56]. While some other alkyl- or pyridinyl-substituted tetrazines were relatively stable (maintained over 85% in PBS for 10 h at 37 ◦C) [73]. Therefore, it is critical to select the appropriate and stable tetrazine for the practical click-to-release system. Furthermore, as small molecules, dialkyl tetrazines are likely to clear from circulation too fast to be quantitatively reacted at the tumor site. Prolonging Molecules 2020, 25, 5640 7 of 24

blood circulation time through proper structural modification without reducing reactivity or utilizing novel drug delivery systems to achieve targeted accumulation may become effective solutions. On the other hand, TCO needs to have sufficient stability to ensure that it maintains enough reactivity after distribution and metabolism. However, previous studies determined that TCO can isomerize to the unreactive cis-isomer in the presence of thiols [74] and copper-containing serum proteins [75]. Within 7 h, the TCO almost completely converted into cis in 50% fresh mouse serum at 37 ◦C[75]. Finding ways to avoid contact can be helpful to prevent TCO from deactivation during circulation. Binding to an antibody with a short spacer, for example, TCO moiety was shielded by the antibody and stabilized with a half-life of 5 days in vivo [59]. Additionally, isomerization decreases reaction efficiency but has the merit of noninterference in payload release. Moreover, some highly reactive and stable cis-dioxolane-fused trans-cyclooctenes (d-TCO) derivatives have been developed Moleculesrecently 2020 [76,], 25 which, x FOR wouldPEER REVIEW facilitate in vivo click-to-release efficiency in the future. However, synthesis7 of 25 of the TCO derivatives involves a complicated photochemical isomerization followed by tedious tediousisolation isolation of the axial of the isomer, axial isomer, leading leading to low to overall low overall yield [ yield57,77 ].[57,77]. Future Future work work should should address address these thesedeficiencies deficiencies by developing by developing more more suitable suitable synthetic synthetic pathways. pathways.

2.2.2.2. Tetrazine-Triggered Tetrazine-Triggered Cleavage from Vinyl Ether OtherOther dienophiles havehave also also been been developed developed for for tetrazine tetrazine bioorthogonal bioorthogonal cleavage cleavage reactions. reactions. In 2016 In 2016and 2017,and 2017, the groups the groups of Devaraj of Devaraj and Bernardes and Bernardes independently independently reported reported vinyl ether vinyl as a ether temporary as a temporarymask which mask could which be removed could bybe reacting removed with by tetrazine, reacting releasing with tetrazine, compounds releasing containing compounds free OH containinggroup (Figure free4 A)OH [ 45group,66]. IEDDA(Figure cycloaddition4A) [45,66]. IEDDA was the cycloaddition rate-limiting was step, the and rate-limiting after the expulsion step, and of afterN2, aromatizationthe expulsion ofof pyridazineN2, aromatization drove swift of pyridazine release of drove the leaving swift release group [of66 ].the leaving group [66].

Figure 4. (A) Reaction mechanism between vinyl ether derivatives and tetrazines. (B) Structures of vinyl Figureether caged 4. (A) near-infrared Reaction mechanism fluorogenic between probe andvinyl its ether active derivatives form. (C) and The tetrazines. concept of ( prodrug-prodrugB) Structures of vinylactivation. ether ( Dcaged) Reaction near-infrared mechanism fluoro betweengenic vinyl probe ether and self-immolative its active form. linker (C and) The tetrazines. concept (ofE) prodrug- Structures prodrugof PEG-b-Dox activation. and Dox. (D ()F )Reaction Reaction mechanismmechanism between between vinylboronic vinyl ether acids self-immolative and tetrazines. linker and tetrazines. (E) Structures of PEG-b-Dox and Dox. (F) Reaction mechanism between vinylboronic acids andDevaraj tetrazines. et al. used tetrazine to activate a near-infrared cyanine dye caged by vinyl ether (30 to 31 in Figure4B) [ 45]. This chemistry can be combined with specific, sensitive reactions templated by nucleic acidsDevaraj for fluorescent et al. used RNA tetrazine detection. to activate Extending a ne thisar-infrared reaction, cyanine Bernardes dye etcaged al. installed by vinyl aether vinyl (30 ether to 31moiety in Figure on a 4B) range [45]. of This hydroxyl-containing chemistry can be molecules,combined with including specific, amino sensitive acids reactions28b, 28c, atemplated fluorophore by nucleic28d, a monosaccharideacids for fluorescent28e, RNA and an detection. analogue Extending of the cytotoxic this reaction, drug duocarmycin Bernardes et 28fal. installed(Figure4 A)a vinyl [ 66]. ether moiety on a range of hydroxyl-containing molecules, including amino acids 28b, 28c, a fluorophore 28d, a monosaccharide 28e, and an analogue of the cytotoxic drug duocarmycin 28f (Figure 4A) [66]. Most of these vinyl ether derivatives remained intact over 8 h in vitro under conditions similar to physiological ones, and liberated alcohols in yields of 50–68% after reacting with tetrazine. It was demonstrated to be able to activate fluorescence of coumarin and release the drug duocarmycin in live cells. The universality of different chemotypes demonstrated the potential of this reaction for various chemical biology and medicine applications. Interestingly, considering the formation of pyridazine product in tetrazine bioorthogonal reaction, Bradley et al. reported a mutual activation strategy termed as “prodrug-prodrug” activation (Figure 4C) [78]. In this strategy, tetrazine was also designed as a prodrug 32, then reacted with vinyl ether-caged camptothecin 33 to generate the topoisomerase inhibitor camptothecin 35 along with a pyridazine-based microRNA 21 inhibitor 34. Co-treatment of PC3 cells with 10 μM 32 and 0.5 μM 33 showed significant decrease in cell viability compared with the treatment of 33 alone (47 ± 8% vs. 101 ± 10%). Given that pyridazine is a scaffold in many drugs, the “prodrug-prodrug activation” strategy may be widely applicable. This approach may also support theranostics if one of the drugs is replaced

Molecules 2020, 25, 5640 8 of 24

Most of these vinyl ether derivatives remained intact over 8 h in vitro under conditions similar to physiological ones, and liberated alcohols in yields of 50–68% after reacting with tetrazine. It was demonstrated to be able to activate fluorescence of coumarin and release the drug duocarmycin in live cells. The universality of different chemotypes demonstrated the potential of this reaction for various chemical biology and medicine applications. Interestingly, considering the formation of pyridazine product in tetrazine bioorthogonal reaction, Bradley et al. reported a mutual activation strategy termed as “prodrug-prodrug” activation (Figure4C) [ 78]. In this strategy, tetrazine was also designed as a prodrug 32, then reacted with vinyl ether-caged camptothecin 33 to generate the topoisomerase inhibitor camptothecin 35 along with a pyridazine-based microRNA 21 inhibitor 34. Co-treatment of PC3 cells with 10 µM 32 and 0.5 µM 33 showed significant decrease in cell viability compared with the treatment of 33 alone (47 8% ± vs. 101 10%). Given that pyridazine is a scaffold in many drugs, the “prodrug-prodrug activation” ± strategy may be widely applicable. This approach may also support theranostics if one of the drugs is replaced with a diagnostic probe. In a similar manner, traceless Staudinger ligation and other bioorthogonal reactions can liberate phosphoramidate-containing prodrugs [27]. Besides hydroxyl group, amines can also be caged through a self-immolative linker and released by the click-to-release reaction between tetrazine and vinyl ether (Figure4D)[ 79]. Bradley and his team designed a 4-hydroxymethyl phenyl vinyl ether linker, to which they conjugated fluorophores or drugs via a carbamate 36. The 1,6-elimination followed by the liberation of phenolate 38 released amine compounds. Additional functionalization of the doxorubicin (Dox)-connected linker with a methacrylate led to the formation of amphiphilic PEG-b-Dox co-polymer nanoparticles 39, which reacted with tetrazine to activate the cytotoxic Dox. Combining nanoparticles with tetrazine-based prodrug activation may offer new opportunities for targeted and controlled drug delivery. Vinyl ethers are small, stable and easy to prepare, which may be useful for various applications in chemical biology and medicine. But the second-order rate constants (k = 3–5 10 4 M 1 s 1 in 2 × − − − 10% H2O in DMF, entry 6 in Table1)[ 66] are several orders of magnitude lower than those of TCO or other strained alkenes. Although vinyl ethers may function well in proximity-induced reactions such as those involving nucleic acids [80], the sluggish kinetics could be the major obstacle to its application in vivo [80]. Since vinylboronic acids were proven to be serviceable in bioorthogonal conjugation [81–84], Bonger’s group made an attempt to accelerate vinyl ether cycloaddition reactions by adding a boronic acid moiety on vinyl ethers (40 in Figure4F, entry 7 in Table1)[ 67]. Using a tetrazine that bears boron-coordinating ligands, dipyridyl-tetrazine 12 for example, it was found that the reaction rate (k = 3.3 10 3 M 1 s 1 in 75% MeOH in PBS at 20 C) was several times higher than that of vinyl 2 × − − − ◦ ether [67]. Cycloaddition was followed immediately by the release of the alcohol or the amine via a self-immolative carbamate linker (41). When vinylboronic acid ether was employed to protect Dox, the prodrug reacted with tetrazine in HeLa cells, and the released drug inhibited cell growth efficiently [67].

2.3. Tetrazine-Triggered Cleavage from Benzonorbornadiene Derivative The development of tetrazine-mediated removal of TCOs and vinyl ethers expanded the scope of bioorthogonal cleavage reactions, only partially met the requirements for in vivo applications. Exploring other reaction pairs with rapid reaction rate, near-quantitative release and prolonged serum stability is essential for more effective in vivo drug activation. 7-Aza/oxa-benzonorbornadienes 43 reacted with tetrazine through an IEDDA ligation to form dihydropyridazine adducts 44 which spontaneously underwent a retro Diels–Alder cycloreversion to aromatize and generate, isoindoles or isobenzofurans, respectively (Figure5A) [ 85]. This reaction was used in tetrazine-mediated detection of microRNA [86]. What’s more, the reaction was developed into a bioorthogonal cleavage reaction by installing carbamate leaving groups to the bridgehead carbon of aza/oxa-benzonorbornadienes via a methylene linker (47 in Figure5B) [ 68]. Following Molecules 2020, 25, 5640 9 of 24 similar mechanism, isoindoles or isobenzofurans 48 simultaneously release free amines due to the intrinsic lability. Benzonorbornadiene (BNBD) derivatives were highly stable under physiological conditions and reacted moderately with tetrazine to release cargo molecules almost quantitatively [68]. 7-Acetamide-BNBD 51, for example, was stable in PBS-serum (1:1, v/v) for 7 days with no p-nitroaniline 1 1 (compound 53) or associated product observed. It reacted with PEG-tetrazine 52 at a rate of 0.017 M− s− (in 90% DMSO/H2O at 37 ◦C) and released approximately 90% of 53 (Figure5C) [ 68]. Carbonates 47b, esters 47c, and phosphotriesters 47d were released in as high yields as carbamates 47a (Figure5B) [ 87]. NAc-BNBDs showed higher release yields, while O-BNBDs reacted faster with tetrazine but the yields depended on reaction temperature. Reacting a Dox prodrug 50 with PEG-tetrazine 52 was proved to be as toxic as free Dox against A549 cells in culture (EC50 (50) = 96 nM vs. EC50 (Dox) = 88 nM) [68]. Stability of BNBD and facile drug release suggested higher potential for targeted drug delivery. Their reaction kinetics are faster than most bioorthogonal cleavage reactions, except for tetrazine Molecules 2020, 25, x FOR PEER REVIEW 9 of 25 click-to-release reactions with TCO and isocyanides (entries 8–10 in Table1).

Figure 5. (A) Reaction mechanism between benzonorbornadienes and tetrazines. (B) Click-to-release Figurereaction 5. mechanism(A) Reaction of mechanism tetrazine-triggered between release benzonorbornadienes from benzonorbornadiene and tetrazines. derivatives (B) Click-to-release and the range reactionof corresponding mechanism leaving of tetrazine-triggered groups. (C) Examples releas ofe releasing from benzonorbornadienep-nitroaniline and Dox. derivatives (D) Reactivity and the of rangethiol-conjugated of corresponding azanorbornadiene leaving groups. towards (C tetrazines.) Examples of releasing p-nitroaniline and Dox. (D) Reactivity of thiol-conjugated azanorbornadiene towards tetrazines. The long-term stability of benzonorbornadiene derivatives and their straightforward synthesis withoutThe thelong-term need for stability tedious stereoisomerof benzonorbornadiene separations makederivatives them promising and their forstraightforward widespread applications synthesis withoutin chemical the biology.need for And tedious the mutual stereoisomer orthogonality sepa withrations other make bioorthogonal them promising release reactionsfor widespread allow to applicationsliberate two diin ffchemicalerent drugs biology. simultaneously And the mutual [88]. However, orthogonality the kinetics with other of benzonorbornadiene bioorthogonal release may reactionsneed to be allow improved to liberate further two before different being useddrugs for si prodrugmultaneously activation [88]. at However, micromolar the concentration kinetics of benzonorbornadienein vivo. may need to be improved further before being used for prodrug activation at micromolarBernardes concentration et al. have in developed vivo. an azanorbornadiene bromovinyl sulfone reagent for cysteine- selectiveBernardes bioconjugation et al. have (Figure developed5D) [ 89 an]. azanorbornadiene This reagent was demonstrated bromovinyl sulfone to be capable reagent of reactingfor cysteine- with selectivethe cysteine bioconjugation proteome in HeLa(Figure cell 5D) lysates. [89]. TheThis most reag electron-richent was demonstrated double bond to present be capable in [2.2.1] of reacting bicyclic withthiovinyl the cysteine sulfone proteome55 underwent in HeLa rapid cell IEDDAlysates. ligationThe most with electron-rich tetrazine double12 under bond aqueous present conditions, in [2.2.1] bicyclicfollowed thiovinyl by two consecutive sulfone 55retro underwent-Diels-Alder rapid reactions, IEDDA generating ligation with a thiol- tetrazine conjugated 12 under pyrrole aqueous56 along conditions,with a pyridazine followed as a by by-product. two consecutive The initial retro cycloaddition-Diels-Alder step reactions, was rate-limiting, generating and a thiol-conjugated the second-order pyrrole 56 along with a pyridazine as a by-product. The initial cycloaddition step was rate-limiting, and the second-order rate constant was 0.026 ± 0.002 M–1 s–1 (at 37 °C), similar to that of aza/oxabenzonorbornadienes [68]. This bioorthogonal cleavage reaction may lead to new methods for stable, chemoselective bioconjugates.

2.4. Tetrazine-Triggered Cleavage from Isonitrile Isocyanides are another class of reagents that can undergo both bioorthogonal ligation[90] and cleavage reactions [70] with tetrazine. Franzini and co-workers used the removable 3-isocyanopropyl (ICPr, 57) and 3-isocyanopropyl-1-carbamonyl (ICPrc, 58) substituents as masking groups to chemically control the release of bioactive agents and probes (Figure 6A) [70,71]. 57 underwent a [4 + 1] cycloaddition with tetrazine followed by release of N2 and generation of an intermediate that tautomerized to a pyrazole-imine 59. 59 spontaneously hydrolyzed to generate 4-aminopyrazoles 60 and 3-oxopropyl aldehydes 61. Following the β-elimination reactions, 61 released acrolein 62 and hydroxy compound. Correspondingly, 58 linked with carbamates released amines [70].

Molecules 2020, 25, 5640 10 of 24 rate constant was 0.026 0.002 M 1 s 1 (at 37 C), similar to that of aza/oxabenzonorbornadienes [68]. ± − − ◦ This bioorthogonal cleavage reaction may lead to new methods for stable, chemoselective bioconjugates.

2.4. Tetrazine-Triggered Cleavage from Isonitrile Isocyanides are another class of reagents that can undergo both bioorthogonal ligation [90] and cleavage reactions [70] with tetrazine. Franzini and co-workers used the removable 3-isocyanopropyl (ICPr, 57) and 3-isocyanopropyl-1-carbamonyl (ICPrc, 58) substituents as masking groups to chemically control the release of bioactive agents and probes (Figure6A)[ 70,71]. 57 underwent a [4 + 1] cycloaddition with tetrazine followed by release of N2 and generation of an intermediate that tautomerized to a pyrazole-imine 59. 59 spontaneously hydrolyzed to generate 4-aminopyrazoles 60 andMolecules 3-oxopropyl 2020, 25, x FOR aldehydes PEER REVIEW61. Following the β-elimination reactions, 61 released acrolein 6210 andof 25 hydroxy compound. Correspondingly, 58 linked with carbamates released amines [70].

Figure 6. (A) Reaction mechanism between isonitriles and tetrazines. (B) Tetrazine-mediated activation of ICPrc-Dox prodrug. (C) Release of mexiletine in zebrafish embryos. Reproduced with permission Figure 6. (A) Reaction mechanism between isonitriles and tetrazines. (B) Tetrazine-mediated from [70]; © 2020 American Chemical Society. activation of ICPrc-Dox prodrug. (C) Release of mexiletine in zebrafish embryos. Reproduced with permission from [70]; © 2020 American Chemical Society. 1 1 ICPr 57 reacted with PEG-modified tetrazine 63 at a rate of 4.0 M− s− in PBS/DMSO (9:1) at 37 C. With the catalysis of serum albumin, 57 could rapidly and almost quantitatively release the ◦ ICPr 57 reacted with PEG-modified tetrazine 63 at a rate of 4.0 M−1 s−1 in PBS/DMSO (9:1) at 37 active molecules in PBS/serum (1:1), with a half-life of 166 s [70] (entries 11–12 in Table1). Compared °C. With the catalysis of serum albumin, 57 could rapidly and almost quantitatively release the active with dimethyl-tetrazine 11, adding a bulky tert-butyl group on the tetrazine accelerated cycloaddition molecules in PBS/serum (1:1), with a half-life of 166 s [70] (entries 11–12 in Table 1). Compared with without destabilizing it [91]. t-Bu-tetrazine 64 for example, reacted with 2-phenylethyl isonitrile at dimethyl-tetrazine 11, adding a bulky tert-butyl group on the tetrazine accelerated cycloaddition a rate of 0.08 M 1 s 1 in DMSO/H O (4:1) at 37 C, which was 10.8-fold faster than 11 under the without destabilizing− − it [91]. t-Bu-tetrazine2 64 for ◦example, reacted with 2-phenylethyl isonitrile at a same condition. And no decomposition of 64 was observed in DMSO/H2O (4:1) containing 10 mM rate of 0.08 M−1 s−1 in DMSO/H2O (4:1) at 37 °C, which was 10.8-fold faster than 11 under the same glutathione at 37 ◦C for 3 h. Since bulky groups always protect tetrazines from the approach of condition. And no decomposition of 64 was observed in DMSO/H2O (4:1) containing 10 mM dienophiles [61,92], the opposite relation in reaction between bulky tetrazines and isonitriles suggested glutathione at 37 °C for 3 h. Since bulky groups always protect tetrazines from the approach of it may be orthogonal to strain-promoted azide-alkyne cycloaddition and IEDDA reaction between dienophiles [61,92], the opposite relation in reaction between bulky tetrazines and isonitriles TCO and tetrazine, which was demonstrated by the triple-orthogonal labeling of proteins [91]. suggested it may be orthogonal to strain-promoted azide-alkyne cycloaddition and IEDDA reaction Authors also investigated the biocompatibility of this system in living cells and living organisms. between TCO and tetrazine, which was demonstrated by the triple-orthogonal labeling of proteins High-yield drugs (91 4%) were produced by reacting dipyridyl-tetrazine 12 (2 mM, 2 eq) with [91]. ± ICPrc-prodrugs of Dox 65 in PBS/DMSO (1:1, 37 C). Combined treatment of excess 63 with 65 Authors also investigated the biocompatibility of◦ this system in living cells and living organisms. caused cell death in A549 adenocarcinoma cells efficiently (EC (65) = 0.165 0.035 µM vs. High-yield drugs (91 ± 4%) were produced by reacting dipyridyl-tetrazine50 12 (2 mM, 2± eq) with ICPrc- EC (Dox) = 0.144 0.028 µM, Figure6B) [ 70], showing dose-dependent cytotoxicity. This strategy was prodrugs50 of Dox 65± in PBS/DMSO (1:1, 37 °C). Combined treatment of excess 63 with 65 caused cell validated again in vivo by implanting tetrazine-modified beads in zebrafish embryos and incubating death in A549 adenocarcinoma cells efficiently (EC50 (65) = 0.165 ± 0.035 μM vs. EC50 (Dox) = 0.144 ± 0.028 μM, Figure 6B) [70], showing dose-dependent cytotoxicity. This strategy was validated again in vivo by implanting tetrazine-modified beads in zebrafish embryos and incubating with ICPrc- mexiletine 66. The voltage-gated sodium channel blocker mexiletine was released leading to a decreased heart rate, which was consistent with in vitro results above (Figure 6C) [70]. Isocyanides are easy to synthesize and stable in biological fluids. In addition, ICPr and ICPrc are structurally compact, which makes them useful for creating prodrugs with minimal impact on the pharmacokinetics of parent drugs [70]. This innovative chemistry will create new opportunities for biomedical research and drug delivery. However, toxic acrolein as a by-product, may impose restrictions on in vivo applications. Further mechanistic study suggests the elimination reaction is hydrolysis-independent and may avoid the generation of acrolein [93].

2.5. Isonitrile-Triggered Cleavage from Tetrazine Since the 4-aminopyrazole is a spontaneously eliminating functional group [94], the Franzini group reported the first example of release from tetrazine derivatives induced by isonitriles (Figure

Molecules 2020, 25, 5640 11 of 24 with ICPrc-mexiletine 66. The voltage-gated sodium channel blocker mexiletine was released leading to a decreased heart rate, which was consistent with in vitro results above (Figure6C) [70]. Isocyanides are easy to synthesize and stable in biological fluids. In addition, ICPr and ICPrc are structurally compact, which makes them useful for creating prodrugs with minimal impact on the pharmacokinetics of parent drugs [70]. This innovative chemistry will create new opportunities for biomedical research and drug delivery. However, toxic acrolein as a by-product, may impose restrictions on in vivo applications. Further mechanistic study suggests the elimination reaction is hydrolysis-independent and may avoid the generation of acrolein [93].

2.5. Isonitrile-Triggered Cleavage from Tetrazine MoleculesSince 2020 the, 25, 4-aminopyrazole x FOR PEER REVIEW is a spontaneously eliminating functional group [94], the Franzini11 group of 25 reported the first example of release from tetrazine derivatives induced by isonitriles (Figure7A) [ 71]. 7A) [71]. Following the similar mechanism, isonitriles converted tetrazines into 4-aminopyrazoles 70. Following the similar mechanism, isonitriles converted tetrazines into 4-aminopyrazoles 70. Through Through 1,4-elimination reaction of 70, phenols and amines can be obtained from, tetrazylmethyl 1,4-elimination reaction of 70, phenols and amines can be obtained from, tetrazylmethyl (TzMe) (TzMe) 67 and tetrazylmethyloxycarbonyl (Tzmoc) 68 derivatives respectively (Figure 7). The 67 and tetrazylmethyloxycarbonyl (Tzmoc) 68 derivatives respectively (Figure7). The reaction of reaction of isonitrile-induced release is high-speed and near-quantitative in significant measure, isonitrile-induced release is high-speed and near-quantitative in significant measure, especially with especially with (trimethylsilyl)methyl isocyanide 71 (entries 13–14 in Table 1). Serum albumin can (trimethylsilyl)methyl isocyanide 71 (entries 13–14 in Table1). Serum albumin can catalyze the elimination catalyze the elimination step. In addition, the compatibility of this chemistry with live systems was step. In addition, the compatibility of this chemistry with live systems was validated in vitro and in vivo. validated in vitro and in vivo. Upon reaction with 71 (100 μM), a prodrug of Dox (72) restored toxicity Upon reaction with 71 (100 µM), a prodrug of Dox (72) restored toxicity to A549 lung adenocarcinoma to A549 lung adenocarcinoma cells in vitro (EC50 (72) = 0.239 ± 0.014 μM vs. EC50 (Dox) = 0.202 ± 0.025 cells in vitro (EC (72) = 0.239 0.014 µM vs. EC (Dox) = 0.202 0.025 µM) (Figure7B). By reacting μM) (Figure 7B).50 By reacting ±TzMe-fluorescein 5073 with ICPr-resorufin± 74 in zebrafish embryos, TzMe-fluorescein 73 with ICPr-resorufin 74 in zebrafish embryos, double fluorescence turn-on could be double fluorescence turn-on could be simultaneously observed (Figure 7C) [71]. simultaneously observed (Figure7C) [71].

Figure 7. (A) Reaction between isonitriles and tetrazines leading to release of active molecules. Figure(B) Structures 7. (A) Reaction of TMS-NC between and Tzmoc-Dox isonitriles and prodrug, tetrazines and statisticalleading to graph release of cytotoxicityof active molecules. studies with (B) StructuresA549 cells afterof TMS-NC 72 h. Reproduced and Tzmoc-Dox with permission prodrug, and from statistical [71]; © Royal graph Society of cytotoxicity of Chemistry. studies (C) with Dual A549release cells of twoafter fluorophores 72 h. Reproduced from ICPr-with permissi and TzMe-cagedon from [71]; dyes. © Royal Society of Chemistry. (C) Dual release of two fluorophores from ICPr- and TzMe-caged dyes. 2.6. Cyclooctyne-Triggered Cleavage from Tetrazine 2.6. Cyclooctyne-Triggered Cleavage from Tetrazine Chemists have gradually turned their attention to tetrazine as a prodrug linker in view of its two naturalChemists modification have gradually sites at 3 andturned 6 positions. their attention Bioorthogonal to tetrazine cleavage as a prodrug can also linker be achieved in view by of reacting its two naturalcyclooctynes modification with tetrazines sites at through 3 and 6 a so-calledpositions. “click, Bioorthogonal cyclize, and cleavage release” can system also (Figurebe achieved8)[ 36 ,95by]. reactingFor instance, cyclooctynes the cycloaddition with tetrazines reaction, through between a so-called a cyclooctyne “click, with cyclize, a hydroxyl and release” group atsystem the propargyl (Figure 8)position [36,95]. and For a instance, tetrazine the with cycloaddition an amide-connected reaction, leavingbetween group, a cyclooctyne led to lactonization with a hydroxyl with thegroup active at theamine propargyl releasing position [36,96]. and The a regiochemistrytetrazine with an of theamide-connected cycloaddition leaving was that group, the nucleophilic led to lactonization hydroxyl withgroup the was active positioned amine on releasing the same [36,96]. side of theThe amide regiochemistry group (80 vs.of 79thein cycloaddition Figure8), allowing was ethatffective the 1 1 nucleophiliclactonization. hydroxyl R1 groups group can was tune positioned the second on order the ratesame constants side of the in amide a range group of 0.0075 (80 vs. to 0.2579 in M Figure− s− 8),and allowing accelerate effective lactonization lactonization. rate by R adding1 groups conformational can tune the second constrains order (entry rate 15constants in Table in1). a The range slow of 0.0075 to 0.25 M−1 s−1 and accelerate lactonization rate by adding conformational constrains (entry 15 in Table 1). The slow kinetics with a half-life longer than 100 h resulted in almost no possibility for the reaction pairs to click while circulating at low concentrations. However, they can be enriched at the specific area with a targeted modification (82, 83 in Figure 8). Then the locally increased concentrations can accelerate reaction rates and release drugs into targeted areas. Using this “click, cyclize, and release” approach, Wang et al. delivered and activated a Dox prodrug to mitochondria of HeLa cells in this kinetically controlled manner [36].

Molecules 2020, 25, 5640 12 of 24 kinetics with a half-life longer than 100 h resulted in almost no possibility for the reaction pairs to click while circulating at low concentrations. However, they can be enriched at the specific area with a targeted modification (82, 83 in Figure8). Then the locally increased concentrations can accelerate reactionMolecules rates 2020, 25 and, x FOR release PEER drugsREVIEW into targeted areas. Using this “click, cyclize, and release” approach,12 of 25 Wang et al. delivered and activated a Dox prodrug to mitochondria of HeLa cells in this kinetically Molecules 2020, 25, x FOR PEER REVIEW 12 of 25 controlled manner [36].

Figure 8. Reaction mechanism between cyclooctyne containing hydroxyl group and tetrazine. And Figurestructures 8. Reaction of mitochondria-targeted mechanism between cyclooctyne cyclooctyne 82 containing and tetrazine-Dox hydroxyl group prodrug and 83 tetrazine.. And structures ofFigure mitochondria-targeted 8. Reaction mechanism cyclooctyne between82 and cyclooctyne tetrazine-Dox cont prodrugaining 83hydroxyl. group and tetrazine. And 2.7.structures TCO-Triggered of mitochondria-targeted Cleavage from Tetrazine cyclooctyne 82 and tetrazine-Dox prodrug 83. 2.7. TCO-Triggered Cleavage from Tetrazine Robillard and co-workers reported a click-to-release strategy in which tetrazine carried the 2.7.molecule TCO-TriggeredRobillard of interest, and Cleavage co-workers and TCOfrom reportedTetrazineserved as a the click-to-release trigger for bioorthogonal strategy in whichactivation tetrazine [65]. Tetrazine carried the moleculebearingRobillard a of methylene-linked interest, and co-workers and TCO carbamate reported served as reacteda theclick-to-release trigger rapidly for with bioorthogonal strategy TCO andin which a activationsecondary tetrazine [65amine ].carried Tetrazine was the bearingmoleculeliberated. a methylene-linkedof In interest, this elimination, and TCO carbamate 4,5-dihydropyridazine served reacted as the rapidly trigger 85 with fortautomerized TCObioorthogonal and a to secondary 2,5-dihydropyridazine activation amine [65]. was Tetrazine liberated. 86, which led to 1,4-elimination of the carbamate (Figure 9A). Installing a methyl substituent on the Inbearing this elimination, a methylene-linked 4,5-dihydropyridazine carbamate reacted85 tautomerized rapidly with to 2,5-dihydropyridazine TCO and a secondary86, whichamine led was to methylene facilitated cleavage (92 vs. 91), while the bulky N-isopropyl substituent stabilized the 1,4-eliminationliberated. In this of theelimination, carbamate 4,5-dihydropyridazine (Figure9A). Installing a 85 methyl tautomerized substituent to on 2,5-dihydropyridazine the methylene facilitated 86, tetrazine carbamate (93 vs. 92). cleavagewhich led (92 tovs. 1,4-elimination91), while the bulky of theN -isopropylcarbamate substituent (Figure 9A). stabilized Installing the tetrazinea methyl carbamate substituent (93 onvs. 92the). methylene facilitated cleavage (92 vs. 91), while the bulky N-isopropyl substituent stabilized the tetrazine carbamate (93 vs. 92).

Figure 9. (A) Reaction mechanism between methylene carbamate-modified tetrazines and TCO. Figure 9. (A) Reaction mechanism between methylene carbamate-modified tetrazines and TCO. And And structures of tetrazine and TCO derivates. (B) Schematic illustration of the Tz-ADC. Reproduced structures of tetrazine and TCO derivates. (B) Schematic illustration of the Tz-ADC. Reproduced with with permission from [65]; © 2020 American Chemical Society. permission from [65]; © 2020 American Chemical Society. As a result, these cleavable tetrazines maintained stable in serum or in the presence of thiols. FigureAs a 9.result, (A) Reaction these cleavable mechanism tetrazines between maintained methylene ca stablerbamate-modified in serum or tetrazinesin the presence and TCO. of thiols.And Cycloaddition between TCO 84 and dimethylcarbamate-tetrazine 94 proceeded with a rate constant k2 Cycloadditionstructures of between tetrazine1 1 TCOand TCO 84 and derivates. dimethylcarbamate-tetrazine (B) Schematic illustration 94 of proceededthe Tz-ADC. with Reproduced a rate constant with of 3.14 0.10 M− s− (at 20 ◦C in acetonitrile), which was 6-fold faster than cycloaddition between k2 ofpermission 3.14± ± 0.10 from M−1 [65];s−1 (at © 202020 °C Am in ericanacetonitrile), Chemical which Society. was 6-fold faster than cycloaddition between allyl-substituted TCO and 3,6-bisalkyltetrazine (entry 5 in Table1). Using the more reactive sTCO-acid allyl-substituted TCO and 3,6-bisalkyltetrazine (entry 5 in Table 1). Using the more reactive sTCO- acidAs 90 a led result, to a bimolecularthese cleavable rate constanttetrazines up maintained to 420 ± 49 M stable−1 s−1 in in acetonitrile serum or andin the 23,800 presence ± 400 Mof− 1thiols. s−1 Cycloadditionin 25% MeCN/PBS between [97] respectively.TCO 84 and Thesedimethylcarbamate-tetrazine rate constants are several 94orders proceeded of magnitude with a larger rate constant than k2 thatof 3.14 of using ± 0.10 TCO M−1 as s− 1the (at caging 20 °C moleculein acetonitrile), [56]. which was 6-fold faster than cycloaddition between allyl-substituted TCO and 3,6-bisalkyltetrazine (entry 5 in Table 1). Using the more reactive sTCO- acid 90 led to a bimolecular rate constant up to 420 ± 49 M−1 s−1 in acetonitrile and 23,800 ± 400 M−1 s−1 in 25% MeCN/PBS [97] respectively. These rate constants are several orders of magnitude larger than that of using TCO as the caging molecule [56].

Molecules 2020, 25, 5640 13 of 24

90 led to a bimolecular rate constant up to 420 49 M 1 s 1 in acetonitrile and 23,800 400 M 1 s 1 in ± − − ± − − 25% MeCN/PBS [97] respectively. These rate constants are several orders of magnitude larger than that of using TCO as the caging molecule [56]. Then tetrazine was designed as a novel linker for antibody-drug conjugate (ADC). It connected the anti-tumor drug monomethyl auristatin E (MMAE) with a pegylated CC49 diabody which targets tumor associated glycoprotein 72 (TAG72), to form a Tz-ADC. Reaction between the Tz-ADC and TCO afforded 93% release of MMAE, which was greater than release yields of TCO-ADC [72]. The prodrug showed semblable cytotoxicity comparing with the parent drug against human colorectal cancer cells, with an EC50 value of 0.67 nM. In this elimination approach, simpler TCOs that lack allylic substitutions, such as sTCO, can be used to trigger extremely fast reactions and therefore offer great possibilities for prodrug activation in vivo. In addition, acting as a linker, tetrazine maintained high reactivity and adequate stability, which conquered the defects of the primordial TCO-linker. Notwithstanding faster cycloaddition and near-quantitative release were offered, the scope for functional groups of this elimination strategy should be expanded in further research.

3. In Vivo Applications of Prodrug Delivery and Activation For in vivo use, cleavage chemistry should provide high spatiotemporal resolution as well as chemoselectivity. Numerous first-line drugs, such as antitumor drugs, often cause severe toxic effects and show poor efficacy [98]. As an alternative, antitumor drugs could be delivered to tumor sites taking advantage of novel drug delivery systems and activated specifically through tetrazine-responsive bioorthogonal cleavage chemistry, which may reduce the toxicity and improve the efficacy. Consistent with this notion, recent investigations on optimized prodrug delivery and release in vivo are summarized below.

3.1. Targeted Drug Delivery Based on Hydrogels Hydrogels are cross-linked, three-dimensional, macromolecular hydrophilic networks. The high- water content and soft consistency of hydrogels with the similar features of natural extracellular matrix make them suitable for drug delivery [99]. The advance of click-to-release chemistry enables tetrazine to locally trigger and activate the prodrug on delivery. For example, the alginate hydrogel decorated with tetrazine moieties 95 was preinjected into soft tissue sarcoma xenografts in mice through palpation, and TCO-Dox 96 was injected intravenously subsequently (Figure 10)[100]. The hydrogel concentrated and activated the prodrug at the tumor site, leading to greater antitumor efficacy and lower myelosuppression than free Dox control. In another example, Czuban and co-workers developed a reloadable hydrogel platform for antibiotic prodrugs, which was able to inhibit the planktonic and biofilm growth of bacteria efficiently [101]. Injectable hydrogel-based activation may be particularly well suited to cancer therapy because cytotoxic drugs can be concentrated and locally activated at the tumor area. This will allow giving larger therapeutic doses under a better safety profile. Gratifyingly, a first-in-human Phase I clinical trial has been approved by the FDA recently [47]. It’s worth looking forward to whether this new research will bring new opportunities and challenges.

3.2. Targeted Drug Delivery Based on Nano Transporters Prodrugs, like the parent drugs, may suffer from nonspecific distribution, poor water solubility, and low persistence in circulation. Encapsulating prodrugs into nanoparticles can enhance the stability and modulate the biodistribution of the drugs. These compounds are able to reduce systemic toxicity by targeting specific tumor sites [102]. Nanovehicles can also be functionalized in different ways as versatile therapeutic tools. Several nano-platforms based on bioorthogonal click-to-release chemistry have been developed to improve the efficiency of prodrug delivery and activation. For example, the TCO-Dox and tetrazine were packaged separately into micelles and were designed to be dissociated in the presence of matrix metalloproteinase 2 at low pH, which is a characteristic condition of the tumor microenvironment (Figure 11A) [103]. Molecules 2020, 25, x FOR PEER REVIEW 14 of 25

Molecules 2020, 25, 5640 14 of 24 Molecules 2020, 25, x FOR PEER REVIEW 14 of 25

Figure 10. Schematic illustration of the local drug activation approach: a hydrogel comprising alginate monosaccharides modified with tetrazine was co-administered with TCO-Dox. The prodrug conjugate accumulated in the hydrogel where it was also activated by the tetrazine. Reproduced with permission from [100]; © 2020 American Chemical Society.

3.2. Targeted Drug Delivery Based on Nano Transporters Prodrugs, like the parent drugs, may suffer from nonspecific distribution, poor water solubility, and low persistence in circulation. Encapsulating prodrugs into nanoparticles can enhance the stability and modulate the biodistribution of the drugs. These compounds are able to reduce systemic toxicity by targeting specific tumor sites [102]. Nanovehicles can also be functionalized in different ways as versatile therapeutic tools. Several nano-platforms based on bioorthogonal click-to-release Figure 10. Schematic illustration of the local drug activation approach: a hydrogel comprising alginate chemistry have been developed to improve the efficiency of prodrug delivery and activation. monosaccharidesFigure 10. Schematic modified illustration with tetrazine of the local was drug co-administered activation approach: with TCO-Dox. a hydrogel The comprising prodrug conjugate alginate For example, the TCO-Dox and tetrazine were packaged separately into micelles and were accumulatedmonosaccharides in the hydrogelmodified where with ittetrazine was also activatedwas co-administered by the tetrazine. with Reproduced TCO-Dox. with The permission prodrug designed to be dissociated in the presence of matrix metalloproteinase 2 at low pH, which is a fromconjugate [100]; accumulated© 2020 American in the Chemical hydrogel Society. where it was also activated by the tetrazine. Reproduced with characteristicpermission condition from [100]; of © the 2020 tumor American microenvironment Chemical Society. (Figure 11A) [103].

3.2. Targeted Drug Delivery Based on Nano Transporters Prodrugs, like the parent drugs, may suffer from nonspecific distribution, poor water solubility, and low persistence in circulation. Encapsulating prodrugs into nanoparticles can enhance the stability and modulate the biodistribution of the drugs. These compounds are able to reduce systemic toxicity by targeting specific tumor sites [102]. Nanovehicles can also be functionalized in different ways as versatile therapeutic tools. Several nano-platforms based on bioorthogonal click-to-release chemistry have been developed to improve the efficiency of prodrug delivery and activation. For example, the TCO-Dox and tetrazine were packaged separately into micelles and were designed to be dissociated in the presence of matrix metalloproteinase 2 at low pH, which is a characteristic condition of the tumor microenvironment (Figure 11A) [103].

FigureFigure 11. 11.( A(A)) Schematic Schematic illustrationillustration of co-administration co-administration of of two two nanovehicles nanovehicles responsive responsive to the to the tumor tumor microenvironment.microenvironment. (B(B)) ConcentrationsConcentrations of of Dox Dox in in main main organs organs after after two-hour two-hour injection. injection. (C) ( CTumor) Tumor volumes,volumes, (D (D) Tumor) Tumor control control rate rate andand ((E)) Body weight weight of of mice mice under under different different conditions conditions at different at different timetime points. points. Reproduced Reproduced with with permissionpermission from [103]; [103]; ©© RoyalRoyal Society Society ofof Chemistry. Chemistry.

WhenWhen both both TCO-Dox TCO-Dox andand tetrazinetetrazine 97 were present present in in this this microenvironment, microenvironment, activation activation of the of the prodrugprodrug shall shall be be triggered. triggered. This This hierarchically hierarchically regulated regulated strategy strategy inhibited inhibited tumor tumor development development in in 4T1 tumor-bearing mice effectively (Figure 11C–E). This also led to approximately 32.6 times reduction in biodistribution to heart when comparing to Doxorubicin Hydrochloride Liposomal (Doxil) after two-hour systemic administration (Figure 11B). Since cardiotoxicity is one of the main side effects of Dox, this approach with lower toxicity may pave the way to new effective cancer therapy. Gold nanorods (AuNRs) are another desirable vehicle for combination therapy due to contribution in photothermal therapy [104,105]. It was convinced that the combination of chemo- and photothermal therapyFigure inhibited 11. (A) tumor Schematic growth illustration (Figure of 12 co-administration)[106]. The AuNR-Tz of two nanovehicles trigger, prepared responsive by fixing to the PEGylated tumor tetrazinemicroenvironment. onto AuNRs, can(B) Concentrations convert light toof heatDox in and main are organs therefore after useful two-hour for injection. photothermal (C) Tumor therapy as wellvolumes, as optoacoustic (D) Tumor control imaging. rate and Camptothecin (E) Body weight was of maskedmice under by different vinylether conditions (prodrug at different33) and encapsulatedtime points. into Reproduced liposomes. with Upon permission intravenously from [103]; injected © Royal toSociety HeLa-tumor-bearing of Chemistry. mice, the two components accumulated at the site of tumor solely, as revealed by fluorescence imaging and When both TCO-Dox and tetrazine 97 were present in this microenvironment, activation of the prodrug shall be triggered. This hierarchically regulated strategy inhibited tumor development in

Molecules 2020, 25, x FOR PEER REVIEW 15 of 25

4T1 tumor-bearing mice effectively (Figure 11C–E). This also led to approximately 32.6 times reduction in biodistribution to heart when comparing to Doxorubicin Hydrochloride Liposomal (Doxil) after two-hour systemic administration (Figure 11B). Since cardiotoxicity is one of the main side effects of Dox, this approach with lower toxicity may pave the way to new effective cancer therapy. Gold nanorods (AuNRs) are another desirable vehicle for combination therapy due to contribution in photothermal therapy [104,105]. It was convinced that the combination of chemo- and photothermal therapy inhibited tumor growth (Figure 12) [106]. The AuNR-Tz trigger, prepared by fixing PEGylated tetrazine onto AuNRs, can convert light to heat and are therefore useful for photothermal therapy as well as optoacoustic imaging. Camptothecin was masked by vinyl ether Molecules(prodrug2020 33, 25), and 5640 encapsulated into liposomes. Upon intravenously injected to HeLa-tumor-bearing15 of 24 mice, the two components accumulated at the site of tumor solely, as revealed by fluorescence multispectralimaging and optoacoustic multispectral tomography optoacoustic imaging. tomograp Tetrazinehy imaging. on the goldTetrazine nanorods on the activated gold thenanorods prodrug, whileactivated thecombination the prodrug, while of chemo- the combination and photothermal of chemo- therapy and photot inhibitedhermal therapy tumor growthinhibited e fftumorectively, growth effectively, resulting in extremely low relative tumor volume and very high apoptosis level resulting in extremely low relative tumor volume and very high apoptosis level among all treatment among all treatment groups (Figure 12B). groups (Figure 12B).

FigureFigure 12. 12.(A (A) Schematic) Schematic illustration illustration ofof goldgold nanorods supported supported chemotherapy chemotherapy and and photothermal photothermal therapy.therapy. ( B(B)) H H&E & E staining of of tumor tumor biopsies biopsies from from di differentfferent treatment treatment groups. groups. Reproduced Reproduced with with permissionpermission from from [106 [106];]; © ©2020 2020 AmericanAmerican Chemical Society. Society. 3.3. Theranostics 3.3. Theranostics SystemsSystems that that cancan deliverdeliver drugs drugs to to disease disease sites sites wh whileile simultaneously simultaneously allowing allowing those those sites to sites be to beimaged imaged make make it itmuch much easier easier to to examine examine where where drugs drugs are are acting acting in inthe the body body and and how how the the body body respondsresponds to to the the treatment treatment [107[107].]. InIn addition,addition, the the drug drug can can be be administered administered more more selectively selectively under under imagingimaging guidance. guidance. Several Several studiesstudies havehave substantiated the the crucial crucial role role of of nanomaterials nanomaterials for for diagnosis diagnosis andand imaging imaging based based on on tetrazine-responsive tetrazine-responsive bioorth bioorthogonalogonal cleavage cleavage [108–111]. [108–111 ].For For example, example, a vinyl a vinyl ether-cagedether-caged camptothecin camptothecin prodrug prodrug and tetrazine-quenchedand tetrazine-quenched near-infrared near-infrared fluorophore fluorophore were separately were encapsulatedseparately encapsulated into liposomes, into which liposomes, localized which to tumors localized due to to tumors the EPR due eff ectto [the112 ].EPR Reaction effect between[112]. tetrazineReaction and between vinyl ethertetrazine in situ and turned vinyl onether fluorescence in situ turned and inhibited on fluorescence tumor growth.and inhibited tumor growth.In another case, a tetrazine-modified polymer 98 disassembled at pH 6.5 in the tumor microenvironmentIn another case, (Figure a tetrazine-modified 13)[87]. This disassembly polymer 98 process disassembled allowed tetrazineat pH 6.5 to in activate the tumor a vinyl ether-cagedmicroenvironment near-infrared (Figure hemicyanine 13) [87]. This dye disassembly99, which fluoresced process allowed and generated tetrazine singlet to activate oxygen, a vinyl causing tumorether-caged cell death near-infrared through the hemicyanine photodynamic dye eff ect.99, which Tetrazine fluoresced polymers and were generated first intravenously singlet oxygen, injected tocausing 4T1 breast tumor tumor-bearing cell death micethrough followed the byphotodynamic injection of 99effect.two hoursTetrazine later, polymers and lastly were tumors first were intravenously injected to 4T1 breast 2tumor-bearing mice followed by injection of 99 two hours later, exposed to 660 nm laser (0.20 W cm− ) for 20 min. The significant fluorescence and growth inhibition and lastly tumors were exposed to 660 nm laser (0.20 W cm−2) for 20 min. The significant fluorescence of tumor tissues were observed in treatment group (Figure 13B,C). and growth inhibition of tumor tissues were observed in treatment group (Figure 13B,C). 3.4. Enzyme-Instructed Supramolecular Self-Assembly (EISA) Taking advantage of the initiation of the supramolecular self-assembly of small molecules by upregulated phosphatase level in tumor cells, the research group of Chen linked tetrazine to a self-assembly tri-peptide motif KYF to build the prodrug activation trigger 101 (Figure 14)[98]. The phosphorylated tyrosine responded to overexpressed phosphatase, resulting in so-called enzyme-instructed supramolecular self-assembly (EISA) which specifically accumulated tetrazine in 1 cancer cells. TCO-Dox (30 mg kg− ) was intravenously injected in HeLa tumor-bearing mice two hours 1 after the administration of 101 (50 mg kg− ). Activation of TCO-Dox led to greater cytotoxicity than the control group without EISA. This spatiotemporally controlled prodrug activation may serve as a small-molecule strategy for mitigating the adverse effects of chemotherapy. Molecules 2020, 25, x FOR PEER REVIEW 16 of 25

Molecules 2020, 25, 5640 16 of 24 Molecules 2020, 25, x FOR PEER REVIEW 16 of 25

Figure 13. (A) Schematic illustration of activation of vinyl ether-caged fluorogenic probe (CyPVE) by a pH-responsive tetrazine polymer. (B) In vivo fluorescence imaging at different time after injection. (C) Tumor growth curves. ASTNs, pH-sensitive nanoparticles; STNs, pH-insensitive nanoparticles. Reproduced with permission from [87]; © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

3.4.Figure Enzyme-Instructed 13. ((A)) Schematic Schematic Supramolecular illustration illustration ofSelf-Assembly of activation activation of of (EISA) vi vinylnyl ether-caged ether-caged fluoroge fluorogenicnic probe (CyPVE) by a pH-responsive tetr tetrazineazine polymer. ( (BB)) InIn vivo vivo fluorescencefluorescence imaging imaging at at different different time time after after injection. injection. Taking advantage of the initiation of the supramolecular self-assembly of small molecules by (C) TumorTumor growthgrowth curves.curves. ASTNs, pH-sensitive nano nanoparticles;particles; STNs, pH-insensitive nanoparticles. nanoparticles. upregulated phosphatase level in tumor cells, the research group of Chen linked tetrazine to a self- Reproduced with permission from [[87];87]; ©© 2020 Wiley-VCH Verlag GmbHGmbH && Co.Co. KGaA, Weinheim.Weinheim. assembly tri-peptide motif KYF to build the prodrug activation trigger 101 (Figure 14) [98]. 3.4. Enzyme-Instructed Supramolecular Self-Assembly (EISA) Taking advantage of the initiation of the supramolecular self-assembly of small molecules by upregulated phosphatase level in tumor cells, the research group of Chen linked tetrazine to a self- assembly tri-peptide motif KYF to build the prodrug activation trigger 101 (Figure 14) [98].

Figure 14. Synergistic enzymatic and bioorthogonal reaction for prodrug activation in tumor cells. Figure 14. Synergistic enzymatic and bioorthogonal reaction for prodrug activation in tumor cells. ReproducedReproduced with with permission permission from from [ 98[98];]; © © 2020,2020, Springer Nature. Nature. 3.5. Antibody-Drug Conjugates (ADCs)

Drugs can target tumor cells with high selectivity and affinity by conjugating to antibodies targeting specific antigens, which terms as antibody-drug conjugates (ADCs) [113]. Classic activation of ADCs includesFigure several 14. Synergistic processes: enzymatic (1) binding and to bioorthogonal target antigen reacti on theon for tumor prodrug cell surface,activation (2) in internalization tumor cells. by Reproduced with permission from [98]; © 2020, Springer Nature.

Molecules 2020, 25, x FOR PEER REVIEW 17 of 25

The phosphorylated tyrosine responded to overexpressed phosphatase, resulting in so-called enzyme-instructed supramolecular self-assembly (EISA) which specifically accumulated tetrazine in cancer cells. TCO-Dox (30 mg kg−1) was intravenously injected in HeLa tumor-bearing mice two hours after the administration of 101 (50 mg kg−1). Activation of TCO-Dox led to greater cytotoxicity than the control group without EISA. This spatiotemporally controlled prodrug activation may serve as a small-molecule strategy for mitigating the adverse effects of chemotherapy.

3.5. Antibody-Drug Conjugates (ADCs) Drugs can target tumor cells with high selectivity and affinity by conjugating to antibodies Molecules 2020, 25, 5640 17 of 24 targeting specific antigens, which terms as antibody-drug conjugates (ADCs) [113]. Classic activation of ADCs includes several processes: (1) binding to target antigen on the tumor cell surface, (2) receptorinternalization endocytosis by receptor into tumor endocytosis cell, (3) cleavage into tumor of the cell, linker (3) cleavage and release of the of anlinker active and form release of cytotoxic of an moietyactive of form ADCs of [ 114cytotoxic]. However, moiety ine offfi cientADCs internalization [114]. However, limits inefficient ADCs applications, internalization especially limits ADCs in solid applications, especially in solid tumors. In contrast, chemically triggered activation through tumors. In contrast, chemically triggered activation through bioorthogonal cleavage reaction gives bioorthogonal cleavage reaction gives manifold possibilities to deliver drugs actively via internalized manifold possibilities to deliver drugs actively via internalized receptors, non-internalized receptors, receptors, non-internalized receptors, cytoplasmic proteins and even extracellular matrix cytoplasmic proteins and even extracellular matrix components. components. Robillard and co-workers were the first to validate the potential of click-to-release reaction between Robillard and co-workers were the first to validate the potential of click-to-release reaction TCObetween and tetrazine TCO and for tetrazine ADC activation for ADC activation [59]. They [59] linked. They TCO-Dox linked TCO-Dox prodrug prodrug to a monoclonal to a monoclonal antibody (mAbantibody CC49) (mAb against CC49) tumor-associated against tumor-associated glycoprotein-72, glycoprotein-72, which iswhich a non-internalizing is a non-internalizing cancer cancer target (Figuretarget 15 (FigureA). This 15A). model This model ADC ADC showed showed high high tumor tumor uptake uptake of of 30–40% 30–40% ID ID/g/g atat 30 h h after after injection injection 1 (5 mg(5 mg kg kg− −)1) to to LS147T LS147T tumor-bearingtumor-bearing mice and could could be be selectively selectively activated activated by by tetrazine. tetrazine. However, However, releaserelease yields yields of of Dox Dox were were not not satisfactory. satisfactory. ThisThis isis mostmost probably the the result result of of rapid rapid clearance clearance of of tetrazine.tetrazine. Dimethyl-tetrazine Dimethyl-tetrazine11 ,11, for example,for example was, was not ablenot toable activate to activate TCO-Dox TCO-Dox significantly. significantly. Whereas withWhereas a modification with a modification of dextran, of103 dextran,(Figure 103 16 (Figure) gave 51.3%16) gave release 51.3% of release Dox atof 24Dox h afterat 24 injection.h after Therefore,injection. the Therefore, choice of the tetrazine choice of that tetrazine persists that longer persists in circulationlonger in circulation is crucial. is crucial.

Figure 15. “Click-to-release” activation of an antibody-drug conjugate. (A) First demonstration of Figure 15. “Click-to-release” activation of an antibody-drug conjugate. (A) First demonstration of an an ADC in vivo.(B) Chemically triggered drug release from a diabody-based ADC. (C) Hyperbranched ADC in vivo. (B) Chemically triggered drug release from a diabody-based ADC. (C) Hyperbranched polymer-based bioorthogonal approach for drug delivery. (D) Prodrug-antibody conjugates for targeted Moleculespolymer-based 2020, 25, x FOR bioorthogonal PEER REVIEW approach for drug delivery. (D) Prodrug-antibody conjugates for18 of 25 chemotherapy. ADC, antibody-drug conjugate; Dox, doxorubicin; MMAE, monomethyl auristatin E. targeted chemotherapy. ADC, antibody-drug conjugate; Dox, doxorubicin; MMAE, monomethyl auristatin E.

Figure 16. Structures of compounds used in ADCs studies. Figure 16. Structures of compounds used in ADCs studies. The same research group improved the ADC approach by using PEGylated diabody conjugates The same research group improved the ADC approach by using PEGylated diabody conjugates instead of intact antibodies, reducing off-target effect of the ADCs (Figure 15B) [72]. PEG24 residues instead of intact antibodies, reducing off-target effect of the ADCs (Figure 15B) [72]. PEG24 residues were conjugated to CC49, leading to high tumor uptake and fast blood clearance. The tubulin-binding antimitotic monomethyl auristatin E (MMAE), a classic cytotoxic drug for ADCs, was caged by TCO to form the prodrug. Then the prodrug was linked to PEG residues via a lysine-branched spacer to establish linker-drug building block 105. Tetrazine 104 was delivered as a conjugate with tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), which reduced blood clearance rate to allow more complete drug to enrich, and also enabled imaging of the ADC via single-photon emission computed tomography (SPECT). This may provide potential applications in theranostics. Combination of ADC and 104 showed strong antitumor activity in LS174T and OVCAR-3-tumor- bearing mice without observed toxicity. 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 64Cu 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. 24 h later, [64Cu] tetrazine-PEG4-NOTA was injected. The click-to- release reaction released Dox into cancer cells, and the Dox could be quantitated based on 64Cu radioactivity using PET-CT. This system may have several advantages over other ADCs systems: PEGylated HBPs prolonged the circulation time, reduced immunogenicity, and facilitated theranostics. The possibility of real-time imaging can support detailed studies of pharmacokinetics and pharmacodynamics, which can in turn improve future development of nanotherapeutics. This system may be adaptable to treatments of various tumor types by using bispecifics that contain two antibodies targeting different antigens and Fc region creating a third functional binding site to facilitate antibody dependent cell-mediated cytotoxicity (ADCC). ADC-delivered drugs that act on intracellular targets need to diffuse into the target cells, which may be blocked by protonation. One solution is to firstly release a more permeable prodrug instead of the active drug. Lately, the prodrug can be activated by small molecule tetrazine activators. For example, TCO-Dox (106) was linked to Her-2 antibody via a pH-sensitive linker. At low pH, this linker would be cleaved and TCO-Dox was released extracellularly and activated within tumor cells (Figure 15D) [116]. This ADC was intravenously injected in MDA-MB-231-tumor-bearing mice and exerted cytotoxicity after the administration of tetrazines. This combination strategy improves liberation of Dox inside tumors and reduces Dox-related toxicity. However, there is still a risk of off- target toxicity in response to tumor microenvironment since the prodrugs are not completely non- toxic.

Molecules 2020, 25, 5640 18 of 24 were conjugated to CC49, leading to high tumor uptake and fast blood clearance. The tubulin-binding antimitotic monomethyl auristatin E (MMAE), a classic cytotoxic drug for ADCs, was caged by TCO to form the prodrug. Then the prodrug was linked to PEG residues via a lysine-branched spacer to establish linker-drug building block 105. Tetrazine 104 was delivered as a conjugate with tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), which reduced blood clearance rate to allow more complete drug to enrich, and also enabled imaging of the ADC via single-photon emission computed tomography (SPECT). This may provide potential applications in theranostics. Combination of ADC and 104 showed strong antitumor activity in LS174T and OVCAR-3-tumor-bearing mice without observed toxicity. 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 64Cu 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 64 MCF7-tumor-bearing mice. 24 h later, [ Cu] tetrazine-PEG4-NOTA was injected. The click-to-release reaction released Dox into cancer cells, and the Dox could be quantitated based on 64Cu radioactivity using PET-CT. This system may have several advantages over other ADCs systems: PEGylated HBPs prolonged the circulation time, reduced immunogenicity, and facilitated theranostics. The possibility of real-time imaging can support detailed studies of pharmacokinetics and pharmacodynamics, which can in turn improve future development of nanotherapeutics. This system may be adaptable to treatments of various tumor types by using bispecifics that contain two antibodies targeting different antigens and Fc region creating a third functional binding site to facilitate antibody dependent cell-mediated cytotoxicity (ADCC). ADC-delivered drugs that act on intracellular targets need to diffuse into the target cells, which may be blocked by protonation. One solution is to firstly release a more permeable prodrug instead of the active drug. Lately, the prodrug can be activated by small molecule tetrazine activators. For example, TCO-Dox (106) was linked to Her-2 antibody via a pH-sensitive linker. At low pH, this linker would be cleaved and TCO-Dox was released extracellularly and activated within tumor cells (Figure 15D) [116]. This ADC was intravenously injected in MDA-MB-231-tumor-bearing mice and exerted cytotoxicity after the administration of tetrazines. This combination strategy improves liberation of Dox inside tumors and reduces Dox-related toxicity. However, there is still a risk of off-target toxicity in response to tumor microenvironment since the prodrugs are not completely non-toxic. In this case, non-internalizing antigens can be selected as targets, which expands the range of possible target-antigen selections. In addition, further study on enhancing the stability and tumor uptake of ADCs as well as improving the activity and residence time of activators is needed.

4. Summary and Outlook With the understanding of the tetrazine bioorthogonal chemical reaction progress, through rational designs of chemical structures, most of bioorthogonal reaction partners can occur either a ligation or a cleavage reaction with tetrazine. Tetrazines undergo cycloaddition reactions with various reactants, followed by elimination or rearrangement to cleave specific chemical bonds and release active molecules. In these researches discussed above, the stability of substrates, reaction kinetics, release yields and the scope of releasable functional groups have been thoroughly explored and carefully investigated. These diverse reactions can release drugs bearing diversiform functional groups, including amino, hydroxyl and carboxyl groups. Future studies should explore the possibility of liberating other active functional groups, such as sulfonic and phosphoric acids. Due to lower toxicity, prodrugs allow larger administration dosage by comparison to parent drugs and therefore contribute to higher efficacy after activation. Moreover, the employment of Molecules 2020, 25, 5640 19 of 24 exogenous trigger containing a reporter like fluorophore or radioactive isotope for drug activation can simultaneously provide imaging signals to achieve diagnosis and treatment. In addition, recent examples about conditional activation with multiple controls, such as singlet oxygen and light, are expected to provide improved spatial and temporal resolution [117,118]. Furthermore, there are still many challenges in establishing bioorthogonal platforms for multi-drug release and clinical applications in theranostics [119], which will likely require further developments in bioorthogonal chemistry and biomaterials. Improving reaction kinetics and the stability of reactants is also a prerequisite for in vivo applications. This will ensure that the two reactants can still reach effective concentrations at the target site after circulation and distribution so that the reaction can be carried out on a reasonable timescale. Additionally, it is worth noting that giving two molecules rather than one requires stricter safety regulations and both of the two need to meet the FDA approval for clinical treatment. The possibility and effectiveness of clinical transformation are under study. It is a long-term and formidable process from the success of animal experiments to the final clinical use, but we still believe future chemical biology advancements will spur the process onward. Other questions including whether other routes of administration, such as oral delivery, are feasible and whether relevant reagents would survive first pass through the liver and the P450 enzymes, are worthy of further exploration to promote these methods to meet clinic needs imminently. In current tetrazine-responsive bioorthogonal prodrug activation strategies, both tetrazines and dienophiles have the potential to be employed as triggers or molecule cages. Moreover, two natural modifiable positions of the tetrazine molecule make it convenient to modify with functional handles. With the development of tetrazine synthesis methodology, the employment of tetrazine as a cage for proteins, fluorophores, etc., may become a hot topic of research. It is convinced that more chemical biology tools will be developed soon to meet the clinical needs.

Author Contributions: Writing—original draft preparation, Y.W.; writing—review and editing, Y.W., C.Z., H.W. and P.F. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Science and Technology Major Project (2017ZX09304023). Conflicts of Interest: The authors declare no conflict of interest.

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