Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in Cells

Abstract The cleavage of a protecting group from a protein or drug under bioorthogonal conditions enables accurate spatiotemporal control over protein or drug activity. Disclosed herein is that vinyl ethers serve as protecting groups for alcohol‐containing molecules and as reagents for bioorthogonal bond‐cleavage reactions. A vinyl ether moiety was installed in a range of molecules, including amino acids, a monosaccharide, a fluorophore, and an analogue of the cytotoxic drug duocarmycin. Tetrazine‐mediated decaging proceeded under biocompatible conditions with good yields and reasonable kinetics. Importantly, the nontoxic, vinyl ether duocarmycin double prodrug was successfully decaged in live cells to reinstate cytotoxicity. This bioorthogonal reaction presents broad applicability and may be suitable for in vivo applications.

Bioorthogonal chemistry for covalently conjugating synthetic molecules at ap redefined protein residue has been am ajor focus of research in the past two decades. [1] Very recently,focus has been placed on reactions which can instead cleave specific bonds under bioorthogonal conditions. [2] This strategy holds great potential for the precise spatiotemporal control of protein function in vivo. [1c,2] Fore xample,p hoto-deprotection of agenetically encoded caged cysteinecould be used to reveal the active native protein in live cells. [3] Similarly,p alladium-mediated depropargylation, [4] phosphine-mediated Staudinger reduction, [5] and tetrazine-triggered inverse electron-demand Diels-Alder (IEDDA) elimination reactions [6] were successfully employed to restore the activity of proteins bearing acaged lysine residue in the active site.B ond-cleavage reactions are also attractive for drugdelivery applications.P alladium-catalyzed deprotection of a5 -fluoroacil prodrug was shown as am ethod for controlled drug release in vivo. [7] TheI EDDAr eaction between at etrazine and ac aged doxorubicin derivative efficiently releases the cytotoxic drug. [8] Strategies based on IEDDA elimination reactions with tetrazines are particularly attractive for decaging relevant molecules in cells and interrogating biology,b ecause of the favorable kinetics and the abiotic nature of tetrazines when compared to photo-and metalcatalyzed reactions.O ne limitation, however,h as been the breadth of protecting groups available for stable,y et conditionally reversible linkages.T ypically,I EDDAe limination reactions have been used with strained alkene protecting groups connected through ac arbamate,t hus resulting in ac ascade release of ap rimary amine (Figure 1a). [2,9] Furthermore,the reduced metabolic stability of strained alkenes constitutes am ajor caveat for its utility.F or instance, ciscyclooctene easily isomerizes to the non-reactive trans-cyclooctene,thus limiting the efficiencyofthe decaging process in cells. [10] Herein, we report the development of av inyl ether/ tetrazine system as IEDDAreaction partners for the traceless decaging of alcohol-containing molecules.W ed emonstrate the broad applicability of this reaction on several chemotypes, including the protected amino acids serine and tyrosine,a n 1,6-anhydro sugar, afluorophore,and adrug. Importantly,the reaction proceeds under physiological conditions (aqueous buffer pH 7.4 and 37 8 8C) and was applied to activate apotent toxic derivative of the drug duocarmycin in cancer cells.
To harness the current chemical biology toolbox and develop ab roadly applicable technology for the controlled release of alcohol-containing chemotypes,w es et out to develop bioorthogonal bond-cleavage reactions.Inparticular, we envisaged that vinyl ethers could efficiently mask both aliphatic and aromatic hydroxy groups,a nd be used for traceless release of alcohols through at etrazine IEDDA bond-cleavage reaction. In fact, the reactivity of the vinyl group with tetrazines has been detailed in organic synthesis [11] and such ar eactive pair was very recently employed to visualize and detect RNAunder bioorthogonal conditions. [12] We first used commercial phenyl vinyl ether 1a as am odel compound, and tetrazine 2a to challenge our decaging hypothesis (Figure 1b-d). After reaction with 2a,p henol (3a)and 3,6-di(pyridin-2-yl)pyridazine (4a)were obtained in 49 %and 61 %yield, respectively (Table 1). Thereaction was performed in dichloromethane to ensure that all the reagents were soluble and at room temperature with only two molar equivalents of tetrazine.I ncreasing the amount of tetrazine showed no significant improvement in reaction yield. To assess the scope of the reaction we then synthetized vinyl ether derivatives 1b-e. [13] Ty rosine and serine residues play ap aramount role in the building of binding pocket architecture and controlling catalytic cycles,f or example,a si n tyrosine kinases and serine proteases.I ndeed, bioorthogonal decaging of catalytically crucial residues is emerging as ad isruptive technology in chemical biology. [2] Furthermore chromone-based fluorophores and sugars can efficiently be used to interrogate biological systems. [14] Remarkably,decaging vinyl ether derivatives of such molecules 1b-e with 2a gave the corresponding free hydroxy derivatives in good yields (50-68 %y ields after purification by column chromatography;T able 1). Of note,t he vinyl ether reagents were generally stable under biocompatible conditions (PBS pH 7.4 at 37 8 8C) over 8hours,asassessed by HPLC/UV (Table 1; see the Supporting Information). While some degree of instability was observed for 1c,for instance,the free hydroxy-containing molecule was not detected.
After confirming the stability of the vinyl ether molecules in pH 7.4 buffer and their successful tetrazine-mediated decaging,w ep roceeded to study the reaction mechanism in detail through quantum mechanics at the M06-2X/6-31 + G-(d,p) level of theory (Figure 2a;see the Supporting Information). Unlike tetrazine reactions with highly reactive strained alkenes, [8] our data indicate that the first step,t hat is,t he IEDDAcycloaddition, is the rate-limiting step of the reaction (TS1, DG°% 25 kcal mol À1 ;reaction time ca. 3days) followed by very fast retro-Diels Alder (TS2anti, DG°% 7kcal mol À1 ) and phenoxy group cleavage (TS3, DG°% 11 kcal mol À1 ). Once the 4-phenoxy-4,5-dihydropyridazine (int2)isobtained, it readily tautomerizes into 4-phenoxy-1,4-dihydropyridazine (int4), which can swiftly decage through an elimination reaction. Of note,o ther dihydropyridazine tautomers previously reported to undergo decaging (int3), [6,8] are unreactive in this reaction. To support the theoretical data, we mixed 1a  The initial cycloaddition is the rate-limitingstep. After very fast nitrogen cleavage, different dihidropyridazine tautomers int2-int4 equilibrate before irreversibly decagingtothe experimentally obtained products (3a and 4a). See Figure S4 in the Supporting Informationf or the whole calculated minimum energy pathway.b) 1 HNMR release studies of 1a upon reaction with 2a.The reaction was performed at 3mm of 1a and 2a in 10 %D 2 O/CD 3 CN. The reaction was monitored for 96 h. While the reaction was not always complete at 96 h, the results obtained were consistent with the mechanism supported by the theoretical calculations. Ph = phenyl, Py = pyridine. and 2a in 10 %D 2 Oi nC D 3 CN,a nd recorded the 1 HNMR spectra at selected times to gain insight into the reaction mechanism (Figure 2b). Peaks in the aromatic region assigned to the final products could be identified after 24 hours.C onversely,n op eaks in the d = 2.5-6 ppm region could be attributed to int2,and the intermediate species int3 and int4 were observed over the course of the experiment. Given that no intermediate species were identified through 1 HNMR analysis,unlike amines decaging from carbamates, [8] our data clearly supports that the Diels-Alder cycloaddition is rate-limiting in this case.Afast and irreversible decaging step after IEDDAc ycloaddition is thus responsible for the experimental observations,and fully in line with the predicted reaction coordinate diagram (see Figure S4 in the Supporting Information). Furthermore,o ur spectral data also confirm that no reaction intermediates are trapped, and that all of them evolve to acommon tautomer before the decaging step ( Figure 2b). With adetailed assessment of the reaction mechanism, we then performed kinetic studies by following the decrease of the tetrazine absorbance,that is,the rate-limiting step,inthe visible region. We used 1a and screened tetrazines 2a-c, which included different substituents (Figure 1a nd Table 2).
Stability studies of 1ain the system solvent used (10 %H 2 Oin DMF) showed no significant degradation after 8hours.A s expected, tetrazines bearing electron-withdrawing groups (2a)l ed to faster reactions compared to the one bearing electron-donating ones (2b). Finally, 2c proved to have the fastest kinetics,p robably because of the reduced steric hindrance brought about by hydrogen-substituted tetrazines. [15] We also compared the kinetics of the vinyl ether/ tetrazine decaging with av ery reactive,s trained alkene as reference,5 -norbornen-2-ol. Thes econd-order rate constant determined (k 2 = 0.189 m À1 s À1 )in10%H 2 OinDMF,although lower, compares well with literature values for the same reaction (k 2 = 1.3 m À1 s À1 in H 2 Oa t2 08 8C). [16] Thed ifferences observed are attributed to the faster IEDDAr eactions in polar protic solvents.
To show the potential bioorthogonality of this bondcleavage reaction, we next studied its use for the decaging of av inyl ether fluorogenic probe in live cells (Figure 3a). To this end, we used av inyl ether nonfluorescent coumarin derivative (1d)and 2c,asboth were shown to be nontoxic to HepG2 cells at the concentrations used (Figure 3b;s ee the Supporting Information for A549 cells). In short, after incubation with 25 mm of 1d for 5hours, 2c (10 mm)w as added to the cells for 4hours.Atthis time,cells were imaged using confocal microscopy and the turn-on fluorescence of the released coumarin was recorded as ar esult of the successful tetrazine decaging of the vinyl ether protecting group installed in 1d (Figure 3c). Importantly,t his study highlights the biocompatibility of this approach for turn-on live cell imaging applications.
Thetargeted delivery of drugs to diseased tissues remains at opic of intensive study,a nd an unsolved issue in modern drug discovery.C urrently,a lcohol-containing drugs account for approximately 50 %ofall small FDA-approved chemical entities (cf.DrugBank v5.0;see the Supporting Information), thus providing ample opportunity for the design of innovative drug delivery constructs.A saproof-of-concept for our technology we assessed the spatiotemporal delivery of ad uocarmycin-like natural product (5;F igure 4a). Duorcamycins are isolated from Streptomyces spp.b acteria [17] and have attracted considerable attention as payloads in antibody-drug conjugates,given their potent cytotoxic activity. [18] Interestingly,h alogen-bearing duorcamycin cytotoxics undergo aW instein spirocyclization reaction to afford the bioactive cyclopropanyl, aD NA-alkylating species.T his feature has been explored in antibody-directed enzyme prodrug therapy,w here an ontoxic glycosydic derivative of duocarmycin is activated by ac onjugate of an enzyme and at umor-specific antibody. [19] To demonstrate that our tetrazine-mediated IEDDAc leavage of vinyl ethers could be applied for the traceless release of an alcohol-containing drug, we synthetized the N-Ac-double prodrug 5 in three steps from [a] The reactions were performed in 10 %H 2 OinDMF and were followed by UV through the decay of UV absorption of the tetrazines. An excess of 150-350 fold of 1a was used. In the case of 5-norbornen-2-ol the kinetic rate was determined using the same solvent system with 2awith a20-to 100-fold excess of 5-norbornen-2-ol. the N-Boc-protected 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]-indol-4-one (CBI) starting material (see the Supporting Information for details). [20] We chose this simple duocarmycin analogue featuring only an acetyl group attached to the DNA-alkylating CBI core because it has been shown to be very toxic to rapidly replicating cells. [21] We envisioned that upon tetrazine IEDDAd eprotection of the vinyl ether,t he halogen prodrug 6 would be readily formed and undergo ar apid Winstein spirocyclization reaction to afford active drug 7.T his tetrazine-triggered cascade formation of an active drug through an intermediate prodrug is known as the double prodrug concept. [22] Next we studied the stability of the double prodrug 5 in PBS pH 7.4 at 37 8 8Cusing HPLC.While species 6 and active drug 7 were not formed, we detected some degree of degradation of 5 over time (of note,f ormation of neither 6 or 7 was observed). Importantly, 5 was found to be less toxic when compared with the active drug 7 in both HepG2 and A549 cells (Figure 4d,e;s ee the Supporting Information). Having asuitable masked vinyl ether double prodrug in hand, we performed ad ecaging reaction under physiological conditions (PBS pH 7.4 at 37 8 8C) with 2c.R emarkably,c lose to complete formation of 7 was achieved after 7hours at 37 8 8Cin PBS pH 7.4, with the short-lived species 6 as an intermediate (Figure 4c). After successful demonstration of decaging of 5 under physiologically relevant conditions,wenext proceeded to evaluate the feasibility of this approach for the tetrazinemediated drug-delivery.A549 cells were first incubated with 5 for 24 hours,after which time 2c was added for an additional 46.5 hour period (twice the doubling time of these cells and comparable to our cytotoxicity studies;F igure 4b,e). Satisfyingly,w eo bserved that at 10 mm,t he product formed upon tetrazine-decaging of 5 is as toxic as 7 alone (Figure 4f;s ee the Supporting Information for identical study on HepG2 cells), thus suggesting complete drug activation in cells. Hence,t his data advocates that tetrazine-mediated bond cleavage of vinyl ethers may be used for the traceless release of alcohol-containing drugs.
In summary,w ed escribed av inyl ether/tetrazine pair as IEDDAreaction partners for the efficient traceless decaging of alcohol-containing molecules in live cells.Considering the wealth of hydroxy groups in chemical probes and drugs, coupled to the need of circumventing adverse drug reactions, the spatiotemporal delivery method disclosed herein may find broad applicability in chemical biology and molecular medicine by unraveling new biology and leveraging the controlled modulation of (patho)physiological events.Additionally,and in combination with strategies for the genetic encoding of vinyl-ether-protected tyrosine and serine derivatives,t his tetrazine IEDDAd ecaging reaction is likely to find use for precise control of protein function in vivo.

Experimental Section
Decagingofvinyl ether duocarmycin prodrug in vitro:The N-Ac CBI prodrug 5 was diluted in PBS pH 7.4 to af inal concentration of 100 mm from a1 0mm stock in acetonitrile.T hen the benzoic acid tetrazine 2c was added to af inal concentration of 500 mm from a50mm stock in DMSO.The reaction was performedat378 8Cand was monitored by HPLC/UVatd ifferent times until completion.
Decagingo fv inyl ether duocarmycin double prodrug 5 in cells: Cells were incubated with increasing concentrations of 5 or equivalent vehicle controlsf or 24 h. Thec ulture medium was then exchanged to complete medium supplemented with increasing concentrations of tetrazine 2c,d rug or equivalentv ehicle controls. Cells were incubated for another 46.5 hu ntil proceeding with the CellTiter-Blue Cell Viability Assay (Promega). Relative fluorescence units (R.L.U.) were normalized to the values obtained for the appropriate vehicle controls.B ars represent the average of 3 independent experiments and error bars represent standard error of the mean (SEM).