Controlled release of covalently conjugated molecules using host-guest interactions

Abstract

An approach using supramolecular gated self-immolative chemistry for the control of covalent bond scission through noncovalent host-guest interactions via an ethylenediamine-based self-immolative entity (SIE) in combination with a bioorthogonal cucurbituril-meditated host-guest system. The entity engages in supramolecular complexation with cucurbit[7]uril (CB) host molecules, which effectively masks its reactivity and prevents premature initiation of the self-immolative cyclization–elimination cascade. The introduction of a competitive guest molecule, such as 1-adamantylamine, triggers the removal of the supramolecular CB capping group from the CB-SIE complex, revealing the nascent self-immolative structure. This decomplexation initiates a rapid cyclization-elimination cascade that releases the loaded cargo molecules from the complex

Description

TITLECONTROLLED RELEASE OF COVALENTLY CONJUGATED MOLECULES USING HOST-GUEST INTERACTIONSBACKGROUND OF THE INVENTION1. FIELD OF THE INVENTION

[0001] The present disclosure relates to compound delivery and release mechanisms and, more specifically, to a guest compound that will self-immolate in response to a triggering competitive guest molecule to release a conjugated cargo compound.2. DESCRIPTION OF THE RELATED ART

[0002] The concept of self-immolative linkers was first introduced byKatzenellenbogen and colleagues and has gained significant attention in diverse fields such as medicinal chemistry, polymer chemistry, and materials science.1-3 Self-immolative entities (SIEs) have evolved from traditional protecting group strategies where reactive functionalities were temporarily masked to modify reactivity, subsequently unmasked through selective cleavage under desired triggering environment.4-7 Although the terminology varies, a typical SIE comprises a trigger, a linker, and a cargo moiety.5 Triggering the protecting group causes bond fragmentation in the SIE through mechanisms such as electronic cascade8- or cyclization elimination- ultimately releasing the cargo molecules. While physical stimuli such as temperature, light, mechanical force-, and ultrasound- are useful in spatiotemporally regulating self-immolative chemistry in different applications, their use in biological settings can be limited by poor tissue penetration capabilities and needs for specialized equipment. On the other hand, chemical stimuli such as redox, pH changes, and enzymes have been used to achieve smart systems for targeted delivery and controlled release. However, off-target expression of these chemical signals in healthy tissues compromises the selectivity of chemical stimuli-responsive strategies, potentially leading to undesired drug leakage and adverse side effects. To address these challenges, the utilization of bioorthogonal chemistry to regulate self-immolation presents a promising venue for precise control of cargo release.

[0003] Bioorthogonal chemistry refers to highly specific chemical reactions that can occur inside living systems without interfering with native biochemical processes and has been coupled with controlled release systems. Non-covalent chemistry is emerging as an alternative approach to covalency for forming unnatural complexes in living systems for applications such as cell labeling. Besides its potential for reversibility, non-covalent chemistry benefits from not being dependent on second-order rate constants or highly122365586. v1 -10 / 1 / 25activated reagents, which are major challenges present in developing bioorthogonal reactions. Instead, the emphasis is on achieving high binding affinities (Ka). A group of synthetic pumpkin-shaped macrocyclic molecules known as cucurbit[n]urils (n = 5-8) have attracted increasing attention in medicinal chemistry and biomedical applications due to their high affinity and highly selective binding interactions which typically do not interfere with biological systems. These characteristics have rendered cucurbit-mediated host-guest chemistry promising for a range of biological applications, such as proteins and biomolecule labeling, cellular and animal imaging- modulating dynamic DNA chemistry, and protein aggregation, controlling bioorthogonal catalysis, and protein separation.

[0004] Of particular interest are cucurbit[n]uril-mediated supramolecular drug delivery systems encapsulating physically entrapped therapeutics. A popular strategy involves encapsulating engineered small molecule drugs in the cucurbit cavity through non- covalent host-guest interactions to down-regulate their bioactivity, while introducing a competitive guest molecule with a higher binding affinity enables the on-demand release of the ‘caged’ therapeutic molecule and activation of its therapeutic effects. Moreover, cucurbit[n]uril-based chemistry has been innovatively used to construct self-assembling DNA-small molecule chimeras, nanomaterials,, and hydrogel networks for the delivery of therapeutics. Beyond conventional chemotherapy, cucurbit-mediated supramolecular chemistry has also been innovatively applied to regulate advanced therapeutic modalities such as photodynamic therapy and photothermal therapy, gene therapy, and supramolecular nanoassemblies. However, the scope of therapeutic guests whose therapeutic effect can be con-trolled by host-guest interactions remains limited. Their structures usually need to be engineered to simultaneously achieve high binding affinity and maintain bioactivity. A general approach that enables the supramolecular regulation of diverse therapeutic structures and allows for the release of drugs in their native forms remains an undeveloped yet transformative research avenue.BRIEF SUMMARY OF THE INVENTION

[0005] The present invention introduces a new strategy to regulate self-immolative chemistry and molecular release using bioorthogonal cucurbit-mediated host-guest interactions for delivering a cargo molecule. In one embodiment, the present invention includes an ethylenediamine-based self-immolative entity bound to a cucurbit[7]uril host molecule, along with a cargo molecule coupled to the described configuration. When exposed to a competitive guest molecule, a rapid cyclization-elimination cascade occurs, resulting in the release of the cargo molecule from the configuration. The self-immolative entity bears222365586. v1 -10 / 1 / 25one or a pair of benzylamine mo ie ties for binding to the cucurbit[7]uril host, optionally connected by a 4-hydroxybenzyl alcohol linker.

[0006] In a further embodiment, the preset invention includes a method for on- demand activation of a therapeutic cargo molecule in a subject, comprising administering the complex coupled to a cargo molecule to a subject in need of treatment with the cargo molecule and then administering the competitive guest molecule, such as 1-adamantylamine, in an amount effective to competitively displace the cucurbit[7]uril host and trigger the cyclization-elimination cascade that releases the cargo molecule.

[0007] These and other features will become more fully apparent from the following detailed description and the accompanying drawings.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0008] The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:

[0009] FIG. 1 is a schematic of an approach to regulate self-immolative chemistry and molecular release using bioorthogonal cucurbit-mediated host-guest interactions according to the present invention.

[0010] FIG. 2 is a series of graphs of (a) Structure of SIE1, SIE2, and SIE3 molecules, (b)1H NMR spectra of SIE1 in D2O before and after adding CB[7]. ([SIE1 ]o = 2 mM, D2O with 10% DMSO-d6) (c) ITC assays carried out by titrating 50 pM of SIE1 in deionized water with 0.5 mM CB in water at 25 °C. (d) The effect of added CB on the halflives of cargo release from SIE1, SIE2, and SIE3 molecules under our experimental conditions, (e) NMR-measured percentage of coumarin released from SIE1 over time as a function of the CB stoichiometry. 2 mM SIE1 in 90% pH 6.5 phosphate buffer in DaO and 10% DMSO-de. (g) ITC assays carried out by titrating a 50 pM solution of SIE2 in deionized water with 1 mM CB in water at 25 °C. (h) Left: NMR-measured percentage of coumarin released from SIE2 over time as a function of the CB stoichiometry. 2 mM SIE1 in 90% pH 6.5 phosphate buffer in DaO and 10% DMSO-de. Right: Fluorescence-monitored coumarin release from SIE2 over time as a function of the CB stoichiometry. Error bars represent the standard deviations for three trials. 10 pM SIE2 in pH 6.5 MES buffer in H2O. All experiments were conducted at room temperature.

[0011] FIG. 3 is a schematic of (a) The structure of a protonated model molecule SIE2modei. (b) Optimized structure of SIE2modei in complex with two CB, highlighting the formation of a hydrogen bond (orange) interaction between the acidic nitrogen of SIE2modei322365586. v1 -10 / 1 / 25and the carbonyl oxygen of the CB molecule. Oxygen, nitrogen, and hydrogen atoms are shown in red, blue, and white, respectively. The carbon atoms of CB and SIE2modei are shown in cyan and grey, respectively.

[0012] FIG. 4 is a series of graphs and charts of aupramolecular activation and deactivation of coumarin release from SIE2. (a) Schematic illustration for the inhibition of self-immolation reactivity in a CB-SIE supramolecular complex, and the reactivation of cyclization-elimination triggered by a competitive adamantane guest, (b) Fluorescence intensity of respective solutions as a function of incubation time. [SIE2]o = 10 pM, [CB] = 100 pM, [Ad] = 200 pM. (c) Fluorescence spectroscopy monitoring the reversible activation and deactivation of coumarin release. An initial solution of 10><CB / SIE2 ([SIE2]o = 10 pM) exhibited suppressed release rates. The addition of 20 x adamantane at 1.5 h significantly accelerated cargo release. Subsequent addition of 20xCB at 2.5 h paused the reactivity, while a further introduction of 20xadamantane at 4.5 h reactivated the self-immolative release. Error bars represent the standard deviations for three trials. Xex= 365 nm. All experiments were conducted at 25 °C in pH 6.5 MES buffer solutions.

[0013] FIG. 5 is a series of diagrams and graphs of supramolecular activation and deactivation of MB release from the prodrug molecule SIE5. (a) Structures of SIE4 and SIE5 molecules, (b) The self-immolation mechanism for SIE5, and its decomposition products that were experimentally confirmed, (c) Time-course UV-vis spectra (solid curves) of a 16 pM solution of SIE5 incubated at 37 °C. The dashed curve corresponds to the absorbance spectrum of a separately prepared 16 pM solution of MB. (d) The left y-axis shows the peak absorbance of a 16 pM SIE5 solution as a function of incubation time. The absorbance results were converted to the concentration of released MB (right y-axis). Error bars represent the standard deviations for three trials, (e) Determination of the concentration of photo-generatedXO2 in different SIE systems ([SOSG] = 5 pM in all samples, [SIE5]o =16 pM, 4 h incubation) using SOSG assay. All experiments were conducted at 37 °C in pH 6.5 MES buffer solutions. Irradiation was conducted using a 3000K LED light equipped with a 600 nm long-pass filter (light intensity = 2.2 mW / cm2). Excitation wavelength for SOSG: = 488 nm.

[0014] FIG. 6 is a series of schematic and graphs of supramolecular activation and deactivation of PDT was demonstrated in vitro using HeLa cells, (a) Schematic illustration of the ex-situ (top) and in-situ (bottom) cellular experiments, (b, c) MTT cell viability assays show significant photo-induced cytotoxicity for cells co-incubated with SIE5 (solid bars);422365586. v1 -10 / 1 / 25This PDT cytotoxicity is absent in the CB-gated 10><CB / SIE5 group (striped); The PDT effect is reactivated in the 10xCB / SIE5 / 20xAd group (crosshatched) with added adamantane trigger molecules. The concentration of SIE5 in cell media in both experiments (b and c) is 16 pM. (d) Control experiment showing high cell viability as assessed by MTT assay for cells treated with CB (160 pM), adamantane (320 pM), or both, with or without light exposure. Irradiation was conducted using a 3000K LED light equipped with a 600 nm long-pass filter (10 min, light intensity = 13.2 mW / cm2). Error bars represent the standard deviations for three trials.

[0015] FIG. 7 is a graph of the structure of SIE2-TFA.

[0016] FIG. 8 is a graph of the structure of SIE3-TFA.

[0017] FIG. 9 is the NMR-measured percentage of coumarin released from SIE3 over time, as a function of the CB stoichiometry. The experiments were conducted with 2 mM SIE3 in 90% pH 6.5 phosphate buffer in DaO with 10% DMSO-de at room temperature.

[0018] FIG. 10 is the NMR-measured percentage of coumarin released from the respective solutions over time. [SIE2]o = 2 mM, [CB] = 8 mM, and [Ad] = 16 mM. The experiments were conducted in 90% pH 6.5 phosphate buffer in DaO with 10% DMSO-de at room temperature.

[0019] FIG. 11 is a schematic of SIE4.

[0020] FIG. 12 is a schematic of an unexpected byproduct (BP1) from the self- immolation of SIE5. Self-immolation reaction condition: A 3.7 mL DMSO solution of SIE5- TFA (39.4 mg, 37.4 pM) was added to 370 mL of 1XMES buffer, yielding a final SIE concentration of 100 pM. The mixture was incubated at 37 °C over three days and then concentrated to 50 mL under reduced pressure. The concentrated solution was extracted with DCM (150 mL) three times, and the organic phase was washed with brine (50 mL), dried over NaaSCL, filtered, and concentrated under reduced pressure. The crude was separated by column chromatography (hexanes / ethyl acetate) to yield BP1 (13 mg, 37%) and the cyclization product (5 mg, 51%), the structures of both were confirmed by NMR. Additionally, the identity of BP1 was confirmed by HRMS (ESI, m / z): calcd for [C48H50N5O5S]+(M+H)+, 808.3527; found, 808.3499.

[0021] FIG. 13 is a schematic illustration of the ex-situ cellular PDT experiments. CB / SIE5 / Ad group was used as a representative example to demonstrate the process. The SIE5 or CB / SIE5 group followed a similar procedure, with the absence of CB / Ad or Ad, respectively.522365586. v1 -10 / 1 / 25

[0022] FIG. 14 is a schematic illustration of the in-situ cellular experiments conducted with the CB / SIE5 / Ad group.

[0023] FIG. 15 is a series of graphs of the photo-generation of102 in (a) MB ([MB] = 1 pM), (b) lOxCB / MB (([MB] = 1 pM, [CB] = 10 pM), and (c) 10><CB / MB / 20xAd ([MB] = 1 pM, [CB] = 10 pM, [Ad] = 20 pM) systems was monitored using the SOSG fluorescence assay, (d) The pho to luminescence intensities at 530 nm over irradiation time, as shown in plots a, b, and c, are summarized. All three systems showed a similar irradiation-dependent increase in fluorescence, indicating comparable efficiency in photo-generating102. This suggests that the addition of CB and adamantane does not affect the PDT effect of MB. Irradiation was conducted using a 3000K LED light equipped with a 600 nm long-pass filter (light intensity = 2.2 mW / cm2). [SOSG] = 5 pM in all samples. Excitation wavelength for fluorescence measurements: X = 488 nm.

[0024] FIG. 16 is a graph of MTT viability assays demonstrated MB concentrationdependent PDT cytotoxicity in cells. Irradiation was conducted using a 3000K LED light equipped with a 600 nm long-pass filter (light intensity = 13.2 mW / cm2).

[0025] FIG. 17 is a schematic of (a) Optimized reactant and the transition state (TS) for model molecule SIE2modei. (b) Optimized reactant ground state and SIE2modei rearranged state.

[0026] FIG. 18 is a schematic of equilibrium calculations.

[0027] FIG. 19 is a schematic of the synthesis of SIE1 in the form of a TFA salt.

[0028] FIG. 20 is a schematic of the synthesis of SIE2 in the form of a TFA salt.

[0029] FIG. 21 is a schematic of the synthesis of SIE3 in the form of a TFA salt.

[0030] FIG. 22 is a schematic of the synthesis of SIE4 in the form of a TFA salt.

[0031] FIG. 23 is a schematic of the synthesis of SIE5 in the form of a TFA salt.DETAILED DESCRIPTION OF THE INVENTION

[0032] Referring to the figures, wherein like numerals refer to like parts throughout, there is seen in FIG. 1, an approach for controlled delivery of a cargo molecule, such as a drug, using a self-immolative guest molecule (SIE) that form a complex with a bioorthogonal cucurbit[7]uril (CB) host molecule.

[0033] In order to illustrate this generally applicable strategy, a subset of self- immolative guest molecules SIE1, SIE2, and SIE3 were rationally designed to form complexes with the bioorthogonal cucurbit[7]uril (CB) host molecules. The reactivity of CB- capped SIE2 — referred to as CB / SIE2, where CB essentially serves as a ‘supramolecular622365586. v1 -10 / 1 / 25protecting group’ masking the reactive functional groups in SIE2 — was suppressed up to over 200 times under our experimental conditions, effectively preventing premature payload release through the cyclization-elimination cascade. The introduction of a competitive guest molecule, the FDA-approved 1-adamantylamine, triggers the disassembly of CB / SIE2 complex. The removal of the supramolecular CB protecting group reveals the nascent self- immolative structure, initiating a rapid cyclization-elimination cascade that releases the loaded cargo molecules. As a proof-of-concept, a self-immolative methylene blue prodrug SIE5 whose reactivity is suppressed when caged in the form of a CB / SIE5 supramolecular complex was used. The addition of 1-adamantylamine decomplexes the CB / SIE5 and triggers the self-immolation of SIE5, leading to the release of the photosensitizer payload and activating photodynamic therapy on demand. This mechanistically novel strategy of regulating covalent bond scission through noncovalent and bioorthogonal host-guest interactions holds promise for various applications in targeted drug delivery, controlled release, self-immolative materials, and beyond.

[0034] Note that this general strategy of using host-guest interactions to control self- immolative covalent bond scission is not limited to the proof-of-concept SIE1-3 molecules. The SIE structures can be replaced by other guest structures that form host-guest complexes with diverse types of host molecules. The host molecules are not limited to CB but could be other host molecules such as cucurbit[5]uri. cucurbit[6]uril, or pillararenes. The convalently conjugated cargo molecule is not limited to methylene blue, but could be changed to different types of drugs including chemo therapeutics and immunotherapeutic agents. The competitive guest molecule is not limited to 1-adamantylamine but could be replaced by structures such as ferrocene.

[0035] The reactivity of CB-capped SIE2 (referred to as CB / SIE2), where CB essentially serves as a ‘supramolecular protecting group’ masking the reactive functional groups in SIE, was suppressed up to over 200 times under experimental conditions, effectively preventing premature payload release through the cyclization-elimination cascade. The introduction of a competitive guest molecule, the FDA-approved 1- adamantylamine for example, triggers the disassembly of CB / SIE2 complex. The removal of the supramolecular CB protecting group reveals the nascent self-immolative structure, initiating a rapid cyclization-elimination cascade that releases the loaded cargo molecules.

[0036] Benzyl amine and similar structures are well-known to form supramolecular complexes with CB with a high binding affinity of about 105to 107M’1. An initially designed self-immolative linker SIE1 (FIG. 2a) incorporating a benzylamine group at the nitrogen722365586. v1 -10 / 1 / 25atom distal from the cargo loading site was tested first. SIE1 was readily synthesized in two simple steps from commercially available starting materials. Its protonated form, SIE1-TFA, is a gummy solid that is stable in the refrigerator for over a year. SIE1-TFA also exhibited good stability in deionized water and D2O, while it quickly released the coumarin cargo through a well-understood cyclization-elimination reaction once the pH of aqueous solution was raised by adding buffering salts. The half-life (ti / 2) of cargo release was estimated to be 0.45 h in pH 6.5 phosphate buffer according to NMR measurements (FIGS. 2d and e).

[0037] The complexation between CB and SIE1 was investigated by introducing 1-2 equivalents of CB to a solution of SIE1-TFA in D2O.1H NMR demonstrated a notable shift in the benzyl signals, while protons on the coumarin motif remained unaffected even at increased CB concentrations (FIG. 2b). This observation suggests that CB binds selectively and efficiently to the benzylamine moiety in SIE1. The formation of supramolecular complexes was further supported by isothermal titration calorimetry (ITC) measurements. The isotherm displayed a sharp transition, consistent with a 1:1 CB:SIE1 binding stoichiometry and revealing a binding affinity Ka = 5.4><105M-1. With the formation of CB / SIE1 complex confirmed, the effect of the CB cap on the self-immolative reactivity of SIE1 (FIG. 2d) was examined. The introduction of one equivalent of CB — referred to as 1 xCB — into a 2 mM solution of SIE1 in pH 6.5 phosphate buffer increased the ti / 2 of coumarin release to 1.22 h (FIG. 2e), as compared to ti / 2 = 0.45 h for free SIE1. Increasing the stoichiometry of CB to two and four equivalents slightly increased the ti / 2 further to 1.58 h and 1.61 h, respectively. This weak dependence on CB stoichiometry is expected as the high binding affinity between CB and SIE1 ensures near-complete complexation across the concentrations studied. Although the increase of ti / 2 to 1-2 hours was modest, these proof-of- principle results encouraged the design of the next generation of CB-binding linker, SIE2.

[0038] The self-immolative intramolecular cyclization involves the nucleophilic secondary amine attacking the carbonyl group. Introducing an additional benzyl group at the carbamate nitrogen atom in compound SIE2 was rationalized to increase the steric hindrance around the reactive carbonyl group, thereby further discriminating the cyclization reactivity in the presence of CB. The formation of a supramolecular complex between SIE2 and CB in D2O was supported by significant shifts in the NMR signals of SIE2 before and after introducing CB (FIG. 7), as well as ITC measurements (FIG. 2g). However, ITC results unexpectedly revealed two distinct binding events between CB and SIE2 (Kai = 6.3 x 106M-1and Ka2 = 3.7 x 104M-1), each corresponding to the binding of approximately one equivalent of CB. This 2:1 stoichiometry between CB and SIE2 is attributed to the positively822365586. v1 -10 / 1 / 25charged secondary amine promoting the caging of both benzyl groups in SIE2 by CB. A control molecule SIE3 featuring a single benzylamine substituent at the carbamate nitrogen also demonstrated strong binding affinity with CB (Ka = 5.3 x 106M-1), supporting that the secondary amine effectively facilitates CB binding to the benzyl group at the carbamate nitrogen, although they are relatively remote.

[0039] To highlight the structure of the dual-complex of SIE2, we optimized the geometry of a positively charged model molecule SIE2modei with two CB molecules at o)B97x-d / 6-31G* level of theory. Note that both benzyl binding sites in SIE2modei are fully inserted inside the CB cage in the bound complex (FIG. 3). The CB cages create high steric hindrance around the secondary amine and carbonyl reactive groups, sequestering the reactive centers. The protonated SIE2modei molecule is also stabilized by a hydrogen bond interaction with the CB cage (FIG. 3b).

[0040] The self-immolation chemistry of SIE2 was then investigated to confirm its molecular releasing capability (FIG. 2f). A 2 mM SIE2 solution was incubated at room temperature overnight (90% pH 6.5 phosphate buffer in DaO and 10% DMSO-de) and purified the reaction mixture using silica gel flash chromatography. More specifically, the mixture was incubated overnight at room temperature, then extracted with DCM (50 mL) three times. The combined organic phases were washed with brine (50 mL), dried over NaaSCh, fdtered, and concentrated under reduced pressure. The crude product was separated by column chromatography (hexanes / ethyl acetate) to yield the cyclization product (9.5 mg, 80%) and 4-hydroxy-7-methylcoumarin (5.5 mg, 70%). Additionally, the identity of the cyclization product was further supported by HRMS (ESI, m / z): calcd for [C17H19N2O1]+(M+H)+, 267.1482; found, 267.1489. The hypothesized cyclization product and the released coumarin cargo were successfully recovered in high yields, with their identities confirmed by NMR and mass spectrometry.

[0041] After confirming the CB-SIE host-guest interactions and the cyclizationelimination chemistry of SIE2, we systematically investigated the ‘gating’ effect of the supramolecular CB protecting group on the rate of self-immolative release. We initiated the self-immolation by adding pH 6.5 PBS buffering salts into solutions of SIE linkers (2 mM) with varying equivalents of CB, and monitored the release of coumarin by NMR (the1H NMR spectra of 2mM SIE1 (a), SIE2 (b), and SIE3 (c) was determined after 2 hours of incubation at room temperature without CB or with lx and 2x CB with experiments were conducted in 90% pH 6.5 phosphate buffer in DaO with 10% DMSO-de).922365586. v1 -10 / 1 / 25

[0042] SIE1-3 all underwent rapid self-immolation, releasing > 90% of coumarin payloads within 2 h. The introduction of 1XCB slowed their release rates to 58%, 33%, and 78% at 2h, respectively. Further increasing the CB stoichiometry to 2x and 4x only marginally enhanced the suppression of SIE1 and SIE3, as 1XCB already enabled nearcomplete complexation of those high-affinity self-immolative structures (FIG. 2e and FIG. 9). In contrast, linker SIE2 showed a significantly suppressed reactivity when the CB equivalence was increased from lx to 2 x and then to 4x; the percentage of released coumarin was estimated to be about 33%, 6%, and 3% after 2 h incubation, respectively. By fitting the time-dependent coumarin release results to a first-order rate expression (FIG. 2h, left), the introduction of lx, 2x, and 4XCB extended the ti / 2 of coumarin release from SIE2 to 4.8h, 44.5 h, and 88.4 h, respectively, corresponding to a 14-, 131-, and 260-fold decrease in pseudo-first-order reaction rates compared to free SIE2 (FIG. 2d). The CB-stoichiometry- dependent reactivity of SIE2 is attributed to the dual-binding interactions at both benzyl substituents, characterized by a high binding constant of 6.26 x 106M-1and a second, more moderate constant of 3.73 x 104M-1. Equilibrium calculations based on ITC-determined binding constants indicate that 89% of SIE2 molecules are dual-capped in the presence of 2XCB under our experimental conditions ([SIE2]o = 2 mM). This percentage increases further to > 99% when 4XCB is used. Although the proof-of-concept SIE2 requires excess CB for maximized ‘gating’, our future work is focusing on increasing the binding affinity at both sites to ensure more complete complexation. When both benzyl groups of SIE2 were masked by the CB cages, the resulting steric hindrance around the secondary amine and carbonyl reactive groups essentially prevented the intramolecular cyclization in the dual-binding complex. Note that for the reaction to occur, the SIE2 must undergo a structural rearrangement involving multiple rotations around single bond, which can readily occur in the absence of the CB cages. The free energy barrier was calculated for the reaction of SIE2modei without the CB cages to be 17.24 kcal / mol. In contrast, the CB cages make the SIE2modei rearrangement unfavorable. The structurally rearranged reactant is 25.14 kcal / mol above the reactant in the presence of CB. Therefore, even the structural rearrangement of the reactant requires much higher energy when the CB cages are present.

[0043] The fluorogenic coumarin cargo provides a convenient fluorescence readout for monitoring the cyclization-elimination cascade at lower concentrations ([SIE2]o = 10 pM). Because coumarin is weakly fluorescent in PBS buffer, we conducted this set of experiments in a pH 6.5 MES buffer and monitored coumarin release by fluorescence spectroscopy. The ti / 2 of free SIE2 was determined to be 1.5 h by fitting the time-dependent1022365586. v1 -10 / 1 / 25coumarin release results to a first-order rate expression (FIG. 2h, right). The introduction of lx, 2x, 4x, and lOxCB to the SIE2 solution extended the ti / 2 of coumarin release to 3.5 h, 6.0 h, 10.8 h, and 82.8 h respectively, corresponding to approximately 2-, 4-, 7-, and 55-fold decrease in reaction rates com-pared to the free SIE2. The CB-dependent reactivity trends from these fluorescence-monitored experiments agree with the NMR-measured trends described in the earlier section, while the slightly faster release rates in the fluorescence- monitored group are attributed to the micromolar concentration used in fluorescence studies and the replacement of pH 6.5 phosphate buffer by pH 6.5 MES.

[0044] Triggered reactivation of the self-immolative reactivity from the gated CB- SIE2 complex was investigated using a competitive guest molecule 1-adamantylamine (FIG. 4a). 1-adamantylamine is known for its high binding affinity with CB (Ka > 1011M-1) and its excellent safety profile as evidenced by its approval by the Food and Drug Administration (FDA) as a prophylactic for influenza A and to treat Parkinson's disease. We added 200 pM adamantane into a solution of the 10xCB / SIE2 complex (pH 6.5 MES, [SIE2]o = 10 pM). The introduction of adamantane essentially restored the rate of coumarin release as shown by fluorescence measurements (FIG. 4b, blue).

[0045] As SIE2 was explored as a platform for the controlled release of therapeutic agents, it was found that this platform necessitates a phenol group as the leaving group. A model compound SIE4 containing an alcoholic leaving group was essentially inert under our experimental conditions (FIG. 5a, 11). The difference in reactivity between SIE2 and SIE4 can be explained by the electron-withdrawing effect of the benzene that stabilizes the putative phenolate anion, lowering the energy barrier of the reaction. Consequently, we advanced the SIE structure by incorporating a 4-hydroxybenzyl alcohol linker as a leaving group (as illustrated by SIE5, FIG. 5a), allowing for the modular conjugation of different functional molecules and their release through a well-established 1,6-elimination mechanism. As a proof-of-concept demonstration, methylene blue (MB, an FDA-approved agent for methemoglobinemia and a well-known photosensitizer) was encapsulated as a therapeutic payload in SIE5 (FIG. 5b).

[0046] SIE5 was designed to undergo a cyclization-elimination cascade and release an MB-precursor (FIG. 5b), which spontaneously oxidizes to form MB. The successful MB release from SIE5 (16 pM in pH 6.5 MES, 37°C) was supported by the emergence of its characteristic absorption peak around 665 nm (FIG. 5c), with an estimated reaction half-life of 9 h (FIG. 5d, square). However, we found that, quantitatively, the concentration of released MB was limited to approximately 60% of the theoretical yield even after prolonged1122365586. v1 -10 / 1 / 25incubation. To fully understand the reaction, a MES solution of SIE5 was incubated at 37 °C for three days, extracted by copious dichloromethane, and then the extract was concentrated and separated through silica gel chromatography. We analyzed the reaction products by NMR and mass spectrometry (FIG. 5b, 12) and identified a major byproduct BP1 (isolated yield = 37%), which could be explained as the 1,6-elimination intermediate of the 4-hydroxybenzyl alcohol linker, the quinone methide, reacted with the nucleophilic amine group in SIE5.

[0047] Having elucidated the self-immolative release of MB from SIE5, we explored its modulation through CB-adamantane supramolecular interactions. The introduction of 10xCB to a 16 pM solution of SIE5 in pH 6.5 MES resulted in a reduction in MB release rate by over ninefold (FIG. 5d, triangle) compared to free SIE5 (FIG. 5d, square). Adding 20xadamantane (320 pM) to the solution of 10xCB / SIE5 restored the rate of MB release (FIG. 5d, dot). The attenuation of the release rate from SIE5 by CB was less pronounced in this experiment compared to that observed for SIE2 (FIG. 4), which can be ascribed to the incomplete CB-SIE5 complexation at 37 °C under the used concentrations, and to structural differences in the leaving groups. While our future research aims to enhance CB-SIE complexation, the reactivity of the SIE5 system was significantly modulated by the CB- adamantane supramolecular chemistry — after 4-hour incubation, 1.88 pM, 0.43 pM, and 2.15 pM of MB were released from SIE5, 10xCB / SIE5, and 10xCB / SIE5 / 20xAd, respectively. This level of suppression is sufficient to distinguish the photodynamic cell-killing effects between the gated CB-SIE5 complex and the active forms of SIE5 (the therapeutically effective threshold MB concentration was established to be 2 pM under our experimental conditions; see FIG. 16).

[0048] Singlet Oxygen Sensor Green (SOSG) was employed to evaluate the1O2- generation ability of SIE5, 10xCB / SIE5, and 10xCB / SIE5 / 20xAd. Each sample was incubated at 37 °C for 4 h in pH 6.5 MES, followed by the addition of SOSG. After 5 min of light irradiation, the emission from ^-activated SOSG was measured by fluorescence spectroscopy. The SIE5 solution showed significant fluorescence from the ^-activated SOSG (FIG. 5e, red), consistent with its confirmed reactivity toward photosensitizer release. The fluorescence from the 10xCB / SIE5 group was more than 3 times lower compared to SIE5 (FIG. 5e, blue) as a result of CB-inhibited MB release. Importantly, the irradiated 10xCB / SIE5 / 20xAd sample exhibited comparable fluorescence (FIG. 5e, green) as the free SIE5, demonstrating that the adamantane trigger effectively restored MB release and activated its photodynamic effect.1222365586. v1 -10 / 1 / 25

[0049] We further investigated the supramolecular modulation of photodynamic therapy (PDT) through an ex-situ cell-based assay as illustrated in FIG. 6a (top schematic). (1) We first incubated a solution of SIE5 (80 pM in pH 6.5 MES buffer) for 8 h at 37 °C in a vial, which resulted in the release of 7.75 pM MB as quantified by UV-vis measurements. The baseline-subtracted peak absorbance at 665 nm for SIE5, 10><CB / SIE5, and 10xCB / SIE5 / 20xAd solutions as a function of incubation time. [SIE5]o = 80 pM, [CB] = 800 pM, [Ad] = 1.6 mM. All experiments were conducted at 37 °C in pH 6.5 MES buffer solutions. The observed baseline drift is attributed to light scattering caused by partially undissolved SIE5 aggregates, and this baseline drift was mitigated over time as more materials were dissolved. The drift in SIE5 is more severe than in 10xCB / SIE5 / 20xAd, although the concentrations of SIE5 are the same in those experiments. We attribute this difference to the sequence of adding adamantane into well-dispersed 10xCB / SIE5 (CB complexation promotes the dissolution of SIE5), which facilitates the release of SIE5 from CB / SIE5 in a better-dispersed state compared to directly dissolving SIE5 into MES buffer.

[0050] An aliquot of this solution was diluted 5-fold with MES buffer, then added to 96-well plate containing HeLa cells (100 pL per well), achieving a final MB concentration of 1.57 pM in the cell culture medium. Subsequently, cells were incubated in this solution at 37 °C for 1 h and then subjected to 10 min photo-irradiation. An MTT colorimetric assay showed <10% cell viability following this PDT treatment (FIG. 6b, solid bars). (2) In contrast to the free SIE5 group, PDT treatment using the 10xCB / SIE5 complex under otherwise identical conditions exhibited minimal photo-induced cytotoxicity (FIG. 6b, striped bars), as the concentration of released MB in the CB-inhibited group is below the therapeutic dose. This result underscores the “gating” effect of the supramolecular CB protecting group on the therapeutic effect of the MB-prodrug SIE5. (3) Further, we studied the adamantane-triggered reactivation of the therapeutic PDT effect: 20xadamantane (1.6 mM) was added into a 10xCB / SIE5 solution in a vial to liberate SIE5 through the competitive binding between CB and adamantane, initiating the self-immolation and MB release (9.90 pM MB released for 8 h at 37 °C). This solution was subjected to the same incubation conditions as described above, added to cell media, and the co-incubated cells were subjected to identical photo-irradiation conditions with the cell viability evaluated by MTT. Indeed, this 10xCB / SIE5 / 20xAd group exhibited a recovered PDT cell-killing effect, as indicated by < 20% cell viability (FIG. 6b, crosshatched bars). Control experiments where cells received identical treatment but without photo-irradiation showed > 60% cell viability (FIG. 6b, bars with white base color), confirming that the therapeutic cytotoxic effects observed in the treated cancer cells arise1322365586. v1 -10 / 1 / 25from PDT. The observed viability of 60-80% across the dark-control groups suggests moderately good biocompatibility of SIE5 and its degradation products, while treatments involving only the supramolecular entities without SIE5 demonstrated no cytotoxic effects in HeLa cells (FIG. 6d). Future efforts will aim to mitigate the background toxicity associated with SIE5 and its products.

[0051] Finally, the supramolecular regulation of PDT was demonstrated through a series of in-situ cellular experiments under optimized conditions. SIE5, 10><CB / SIE5, or 10xCB / SIE5 / 20xAd were introduced into the HeLa cell culture media to achieve a final SIE5 concentration of 16 pM. After 4 h of co-cultivation, cells were exposed to 10 min photoirradiation, followed by viability assessment using the MTT assay. Consistent with the ex-situ findings, (1) the SIE5 group exhibited significant PDT cytotoxicity (FIG. 6c, solid), (2) the PDT effect was essentially eliminated in the CB-gated 10xCB / SIE5 group (FIG. 6c, striped), and (3) the PDT cell-killing effect was restored in the 10xCB / SIE5 / 20xAd group (FIG. 6c, crosshatched), which included the adamantane as a supramolecular trigger.

[0052] In summary, this study introduces a new concept of regulating self-immolation and cargo release using the bioorthogonal cucurbit-mediated supramolecular chemistry. Through a structure-activity relationship study of a subgroup of rationally designed self- immolative linkers, we identified the SIE2 scaffold featuring two CB-binding benzyl substitutes. When forming a dual-binding supramolecular complex with CB, the cyclizationelimination rate of SIE2 was downregulated up to 260-fold under our experimental conditions. Importantly, this rate can be reversibly restored through the addition of a competitive adamantane guest molecule, allowing for the triggered release of covalently bound cargo molecules from the CB / SIE2 supramolecular complex. Leveraging insights from the SIE2 design, we developed a SIE5 scaffold that enables the modular incorporation of different functional groups. An MB photosensitizer was incorporated into the SIE5 prodrug for a proof-of-concept demonstration. Material characterizations and in-vitro cellular experiments confirmed the deactivation of MB release and PDT effect in the CB-SIE5 supramolecular complex, which could be reactivated by the introduction of the supramolecular trigger adamantane. This supramolecular strategy to regulate self-immolative chemistry provides a general bioorthogonal approach for prodrug activation and controlled release and finds promising applications in medicinal chemistry, drug delivery, materials science, and beyond.

[0053] EXAMPLE1422365586. v1 -10 / 1 / 25

[0054] All reactions were conducted under standard air-free conditions under an atmosphere of nitrogen gas with magnetic stirring unless otherwise mentioned. All reactants and solvents were purchased from commercial suppliers and used without further purification unless otherwise noted. Flash chromatography was performed on a Biotage Isolera System with Yamazen Corp, universal silica gel columns (Pore Size 60 angstroms, Particle Size 40- 63 microns).

[0055] NMR spectra were acquired on a Bruker Avance III HD 400 MHz spectrometer.1H NMR spectra are reported relative to residual protonated solvent (7.26 ppm for CHCI3, 3.31 ppm for CD3OD, 4.79 ppm for D2O).13C NMR spectra are reported relative to residual protonated solvent (77.16 ppm for CHCI3, 49.00 ppm for CD3OD). Multiplicity abbreviations are as follows: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, ABq = AB quartet, m = multiplet, br = broad.

[0056] Isothermal titration calorimetry (ITC): The affinity and stoichiometry between SIE1 or SIE2 or SIE3 with CB were measured by ITC using a Nano ITC Calorimeter (TA Instruments, New Castle, DE). All titrations were performed at 25 °C and in deionized water. The CB (0.5 mM for SIE1 and SIE3, 1 mM for SIE2) was titrated in 42 injections of 1.2 pL each to SIE1 or SIE2 or SIE3 (50 pM, cell volume 170 pL) with an injection spacing of 200 seconds. The obtained thermogram was baseline-corrected and the binding signals were corrected by the end injections after saturation. The analysis was conducted using NanoAnalyze (v4.0.2) (TA Instruments, New Castle, DE).

[0057] Mass spectra were acquired on a DART-SVP (Direct Analysis in Real Time) ion source (lonSense, Saugus, MA) coupled to an Exactive Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) at the Cornell Chemistry Mass Spectrometry Facility.

[0058] All solution optical spectra were acquired of samples in quartz cuvettes. Electronic absorbance spectra were acquired with an Evolution 201 UV-visible spectrophotometer in double-beam mode using a solvent-containing cuvette for background subtraction spectra of solution samples. Fluorescence spectra were measured with an Agilent Cary Eclipse G9800A Fluorescence Spectrophotometer.

[0059] Light irradiations were conducted using (1) a 3000K LED light equipped with a 600 nm long-pass filter with an intensity of 2.2 mW / cm2for SOSG experiments or (2) a 3000K LED light equipped with a 600 nm long-pass filter with an intensity of 13.2 mW / cm2for in-vitro photodynamic therapy (PDT) experiments. Light intensity was measured using a digital photometer (Industrial Fiber Optics, #IF PM).1522365586. v1 -10 / 1 / 25

[0060] A deuterated phosphate buffer for the NMR assay was prepared by dissolving 380 mg of potassium phosphate monobasic and 210 mg of potassium phosphate dibasic in 4 mL of D2O, resulting in a 1 M pH 6.5 deuterated phosphate buffer. For the NMR-based coumarin release assay, SIE molecules were initially dissolved in DMSO-de to create a 20 mM stock solution. CB and adamantane were separately dissolved in D2O at final concentrations of 10 mM and 20 mM, respectively. For the NMR kinetics study, the stock solutions of SIE molecules, CB, and adamantane were diluted directly into D2O in the order of the compounds as listed by name (e.g., the sequence of addition for the CB / SIE3 / Ad group is CB->SIE3->Ad). The total volume of the mixture in D2O was 900 pL, to which 100 pL of 1 M pH 6.5 deuterated phosphate buffer was added, resulting in a final buffer concentration of 100 mM. NMR kinetics measurements were then carried out.

[0061] pH 6.5 5 xMES (2-(N-morpholino)ethanesulfonic acid)buffer was purchased from Fisher Scientific. For the kinetics study in MES buffer, the stock solutions of SIE molecules, CB, and adamantane were diluted directly into DI water in the order of the compounds as listed by name (e.g., the sequence of addition for the CB / SIE3 / Ad group is CB->SIE3->Ad). The total volume of the mixture in DI water was 4 mL, to which 1 mL of the pH 6.5 5XMES buffer was added, resulting in a final concentration of IxMES buffer. Fluorescence-based kinetics measurements were then carried out.

[0062] Stock solutions of methylene blue (MB, 10 mM) and SIE5-TFA (10 mM) were separately prepared by dissolving each sample in DMSO. Stock solutions of CB (10 mM) and adamantane (20 mM) were separately prepared in deionized water. HeLa cells were incubated in DMEM media, with 10% fetal bovine serum at 37 °C and 5% CO2.Approximately 5,000 HeLa cells suspended in 100 pL of media were seeded into each well of a 96-well plate 12 hours prior to PDT experiments.

[0063] A solution containing an appropriate amount of each compound in MES buffer was prepared by adding corresponding stock solutions in a sequence as listed by compound names (e.g., the sequence of addition for the CB / SIE5 / Ad group is CBDSIE5 D Adamantane). After 8 hours of incubation at 37°C in a vial, each sample was diluted 5 times with MES buffer. We replaced the cell culture media in a 96-well plate with the corresponding diluted MES buffer solutions (MB, SIE5, CB / SIE5, or CB / SIE5 / Ad, depending on the specific assay, 100 pL / well). Cells were incubated in this solution for 1 h at 37°C, and then treated with 10 minutes of photo-irradiation using a 3000K LED light equipped with a 600 nm long-pass filter (light intensity = 13.2 mW / cm2). After this PDT treatment, we replaced the supernatant in the 96-well plate with 100 pL fresh DMEM and cultured the cells for 24 hours at 37 °C in1622365586. v1 -10 / 1 / 25an atmosphere containing 5% CO2. Cell culture supernatant was removed, followed by the addition of MTT (0.5 mg-mL-1 in DMEM media, 100 pL / well) to each well. After 4 hours of incubation at 37 °C, the supernatant was removed, followed by the addition of 100 pL DMSO to each well to dissolve the formazan product. The plates were shaken for 60 s and the absorbance at 490 nm in each well was recorded by a microplate reader.

[0064] The cell culture supernatant in a 96-well plate was removed and replaced with 100 pL corresponding solutions (SIE5 (16 pM) or CB / SIE5 (160 pM / 16 pM) solutions, depending on the specific assay). After 4 hours of co-incubation, cells were irradiated for 10 minutes using a 3000K LED light equipped with a 600 nm long-pass filter (light intensity = 13.2 mW / cm2). After PDT treatment, the supernatant in the 96-well plate was replaced with fresh DMEM, and cells were incubated for 24 hours at 37 °C in an atmosphere containing 5% CO2. Then, the supernatant was replaced with MTT (0.5 mg-mL-1 in DMEM media, 100 pL / well). After 4 hours of incubation at 37 °C, the supernatant was removed, followed by the addition of 100 pL DMSO to each well to dissolve the formazan product. The plates were shaken for 60 s and the absorbance at 490 nm in each well was recorded by a microplate reader.

[0065] The cell culture media in a 96-well plate was replaced with 100 pL of an MES solution of CB / SIE5 (160 pM / 16 pM). Subsequently, an MES solution of 1-adamantylamine (1.6 pL, 20 mM) was added to each well, resulting in a final adamantane concentration of 320 pM. Cells were incubated in this solution for 4 hours at 37 °C in an atmosphere containing 5% CO2, followed by 10 minutes of photo-irradiation using a 3000K LED light equipped with a 600 nm long-pass filter (light intensity = 13.2 mW / cm2). After PDT treatment, the cell culture media was replaced with fresh DMEM, and cells were incubated for 24 h at 37 °C in an atmosphere containing 5% CO2. Then, the supernatant was replaced with MTT (0.5 mg-mL-1 in DMEM media, 100 pL / well). After 4 hours of incubation at 37 °C, the supernatant was removed, followed by the addition of 100 pL DMSO to each well to dissolve the formazan product. The plates were shaken for 60 s and the absorbance at 490 nm in each well was recorded by a microplate reader.

[0066] Photo-generation of singlet oxygen (102) by MB containing different supramolecular entities was investigated using the commercial Singlet Oxygen Sensor Green (SOSG) fluorescent assays. All SOSG experiments were conducted in pH 6.5 MES buffer solutions with 5 pM of SOSG using a 3000K LED light equipped with a 600 nm long-pass filter (light intensity = 2.2 mW / cm2). FIG. 15 shows the fluorescence of activated SOSG in a 1 pM solution of MB gradually increased over the course of photo-irradiation, confirming the1722365586. v1 -10 / 1 / 25photo-induced generation of 102. To elucidate the influence of supramolecular entities, we introduced an excess of 10><CB or 10><CB / 20xAd ([CB] = 10 pM, [Ad] = 20 pM) into a 1 pM solution of MB and monitored the production of 102 using the same SOSG assay (FIG. 15b and c). The addition of supramolecular entities showed negligible effect on the generation of 102 from MB (FIG. 15d).

[0067] Following the confirmation of 102 generation from MB, we assessed its PDT cell-killing efficacy through a cellular experiment, with the viability of HeLa cells subjected to different PDT conditions accessed by the colorimetric MTT assay (FIG. 16). All in-vitro PDT experiments were conducted using a 3000K LED light equipped with a 600 nm long- pass filter (light intensity = 13.2 mW / cm2), and two irradiation doses (i.e., 5 and 10 min) were tested at different concentrations of MB. Photo-irradiation alone without MB did not induce significant toxicity in HeLa cells (MTT viability ~ 100%). Increasing the MB concentration up to 2-4 pM did not cause significant dark cytotoxicity (MTT viability >70 %). However, MB at concentrations higher than 4 pM started to compromise the cell viability in the dark. Importantly, 10 min of photo-irradiation showed significant PDT cell-killing effects when the concentration of MB is 1 pM or higher.

[0068] From this initial condition screening, we identified an optimized PDT condition for our supramolecularly regulated PDT systems: (1) 10 min photo-irradiation, (2) targeted dosage of released MB is around 2 pM for the “unmasked” 10xCB / SIE5 / 20xAd group. Under this optimized condition, (i) the concentration of released MB (2 pM) from the “unmasked” 10xCB / SIE5 / 20xAd group is sufficient to induce significant photo-cytotoxicity, while exhibiting limited dark cytotoxicity; and (ii) The “CB-gated” 10xCB / SIE5 group releases MB approximately 5 times slower than the “unmasked” group, corresponding to <0.5 pM released MB which is significantly lower than the therapeutically necessary MB dosage (FIG. 16).

[0069] Geometry optimization of the model molecule SIE2model (deprotonated) + CB7 complex is performed at < B97xd / 6-31+G* level of theory. The initial geometry of CB7 was taken from reference (J. Am. Chem. Soc. 2012, 134, 37, 15318-15323). Then, the complex was reoptimized in the protonated SIE2model form, where the CB7 cages were fixed, and the SIE2model was allowed to relax. To minimize the computational cost and compare results at the same level of theory, all the results are finally reported at the o)B97xd / 6-31G level of theory (Phys. Chem. Chem. Phys., 2008,10, 6615-6620). Since the cyclization and deprotonation of SIE2 are two different steps, the transition state calculations were performed starting from the deprotonated SIE2. The activation free energy barrier for1822365586. v1 -10 / 1 / 25the cyclization of SIE2 is calculated to be 17.24 kcal / mol at the <*)B97xd / 6-31G level of theory. All calculations were performed in Gaussian 16 using the CPCM solvation model, where water was used as a solvent.

[0070] Equilibrium Calculations

[0071] (1) When [SIE2-TFA]0 = [Bnl]0 = [Bn2]0 = 2 mM, [CB]0 = 4 mM:

[0072] Complexation of Bnl groups (the high-affinity binding site, Kai = 6.255 x106 M-l):Bnl + CB CB-Bnl where CB-Bnl refers to the CB-caged Bnl groups both in the mono-complex and the dualcomplex. Since the Bnl group has a much higher binding affinity with CB than Bn2, we ignored the presence of Bn2 in this section for simplicity.

[0073] At equilibrium,Kai x ([Bnl]0 - [CB-Bnl]) x ([CB]0 - [CB-Bnl]) = [CB-Bnl]Solving this equation,[CB-Bnl] ~ 1.9998 x 10-3 MThus, the high-affinity Bnl binding sites are near quantitatively (> 99.9%) capped under the experimental conditions.

[0074] Complexation of Bn2 groups (with lower affinity binding, Ka2 = 3.73 x 104 M-l)Bn2 + CB CB-Bn2 where CB-Bn2 refers to the CB-caged Bn2 groups both in the mono-complex and the dualcomplex. Since the Bnl group (2 mM) is nearly fully complexed at the experimental concentration by an equal amount of CB, we approximated that the concentration of CB available for the host-guest interaction with the Bn2 binding group is 2 mM and ignored the CB-capped Bnl in this calculation section for simplicity.

[0075] At equilibrium,Ka2 x ([Bn2]0 - [CB-Bn2]) x ([CB] - [CB-Bn2]) = [CB-Bn2]Solving this equation,[CB-Bn2] ~ 1.78 x 10-3 MThus, the Bn2 binding sites are 89% capped under the experimental conditions.(2) When [SIE2-TFA]0 = [Bnl]0 = [Bn2]0 = 2 mM, [CB]0 = 8 mM:Complexation of Bnl groups (the high-affinity binding site, Kai = 6.255 x 106 M-l): Bnl + CB CB-Bnl1922365586. v1 -10 / 1 / 25Since the Bnl group has a much higher binding affinity with CB than Bn2, we ignored the presence of Bn2 in this section for simplicity.

[0076] At equilibrium,Kai x ([Bnl]0 - [CB-Bnl]) x ([CB]0 - [CB-Bnl]) = [CB-Bnl] Solving this equation, [CB-Bnl] ~ 1.9999 x 10-3 MThus, the high-affinity Bnl binding sites are near quantitatively (> 99.9%) capped under the experimental conditions.

[0077] Complexation of Bn2 groups (with lower affinity binding, Ka2 = 3.73 x 104 M-l)Bn2 + CB CB-Bn2Since the Bnl group (2 mM) is nearly fully complexed at the experimental concentration by an equal amount of CB, we approximated that the concentration of CB available for the hostguest interaction with the Bn2 binding group is 6 mM and ignored the CB-capped Bnl in this calculation section for simplicity.

[0078] At equilibrium,Ka2 x ([Bn2]0 - [CB-Bn2]) x ([CB] - [CB-Bn2]) = [CB-Bn2]Solving this equation,[CB-Bn2] ~ 1.99 x 10-3 MThus, the Bn2 binding sites are 99% capped under the experimental conditions.

[0079] Synthetic Details

[0080] Tert-butyl benzyl (2-((((4-methyl-2-oxo-2H-chromen-7- yl)oxy)carbonyl)amino)ethyl) carbamate (1).

[0081] A round bottom flask with a stir bar was charged with tert-butyl (2- aminoethyl)(benzyl)carbamate (0.2 g, 0.8 mmol) and 1 mL ethyl acetate. The solution was cooled to 0 °C in an ice bath. Coumarin chloroformate (0.381 g, 1.6 mmol) (Ref : J. Am. Chem. Soc. 2019, 141, 15018-15023) was dissolved into another 1 mL of ethyl acetate followed by the dropwise addition into a round bottom flask. Potassium carbonate (0.28 g, 2 mmol) was dissolved into 2 mL water and added into the mixture dropwise. After stirring the mixture for 40 min at room temperature, the mixture was extracted with DCM (50 mL). The organic fraction was washed with 10% NH4C1 (50 mL), water (50 mL), and brine (50 mL). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The product was separated by column chromatography (5-25 % ether / DCM) to provide the title compound as a light-yellow liquid (220 mg, 61%). Rf = 0.18 (DCM : ether2022365586. v1 -10 / 1 / 2510 : 1). 1H NMR (400 MHz, Chloroform-d) 8 7.57 (d, J = 8.4 Hz, 1H), 7.47 - 7.20 (m, 6H), 7.18 - 7.07 (m, 2H), 6.24 (d, J = 1.3 Hz, 1H), 4.48 (s, 2H), 3.75 - 3.23 (m, 4H), 2.42 (d, J = 1.3 Hz, 3H), 1.52 (s, 9H). 13C NMR (100 MHz, CDC13) 8 160.80, 154.19, 153.78, 152.18, 137.99, 128.76, 127.56, 127.35, 125.27, 118.05, 117.27, 114.14, 110.13, 51.22, 45.77, 40.54, 28.46, 18.77. HRMS (ESI, m / z): calcd for [C25H32N3O6]+ (M+NH4)+, 470.2286; found, 470.2288.

[0082] TFA salt of N-benzyl-2-((((4-methyl-2-oxo-2H-chromen-7- yl)oxy)carbonyl)amino)ethan-l-aminium (M-l), 2,2,2-trifluoroacetaldehyde (SIE1).

[0083] A 20 mL vial equipped with a stir bar was charged with 1 (0.1 g, 0.8 mmol) and DCM (2 mL) at room temperature. 2 mL Trifluoroacetic acid (TFA) was added into the mixture dropwise. After 1 h reaction at room temperature, the product was obtained as a light-yellow liquid by removing all solvent under reduced pressure (>99%, 106 mg). 1H NMR (400 MHz, Chloroform-d) 8 7.52 (d, J = 9.4 Hz, 1H), 7.45 - 7.30 (m, 5H), 7.09 - 7.02 (m, 2H), 6.68 (t, J = 5.9 Hz, 1H), 6.21 (d, J = 1.3 Hz, 1H), 4.22 - 3.84 (m, 4H), 3.76 - 3.42 (m, 2H), 3.34 - 3.00 (m, 2H), 2.39 (d, J = 1.3 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) 8 161.04, 154.45, 154.05, 153.42, 152.49, 130.11, 130.07, 129.84, 129.50, 125.42, 118.13, 117.55, 114.24, 110.26, 51.74, 46.96, 37.87, 18.83. HRMS (ESI, m / z): calcd for [C20H21O4N2]+ (M)+, 353.1496; found, 353.1471.

[0084] Tert-butyl (4-methyl-2-oxo-2H-chromen-7-yl) ethane-1,2- diylbis(benzylcarbamate) (2) was prepared following a similar procedure as that for compound 1. A flame-dried round bottom flask equipped with a stir bar was charged with tert-butyl benzyl(2-(benzylamino)ethyl)carbamate (0.1 g, 0.294 mmol) and 1 mL ethyl acetate. The solution was cooled to 0 °C in an ice bath. Coumarin chloroformate (0.140 g, 0.587 mmol) was dissolved into another 1 mL of ethyl acetate followed by the dropwise addition into round bottom flask. Potassium carbonate (0.1 g, 0.735 mmol) was dissolved into 2 mL of water and added into the mixture dropwise. After stirring the mixture for 40 min at room temperature, the mixture was extracted with DCM (50 mL). The organic fraction was washed with 10% NH4C1 (50 mL), water (50 mL), and brine (50 mL). The organic phase was dried over Na2SO4, fdtered, and concentrated under reduced pressure. The product was separated by column chromatography (5-15 % ether / DCM) to provide the title compound as a light-yellow liquid (60 mg, 38%). Rf = 0.33 (DCM : ether 10 : 1). 1H NMR (400 MHz, Chloroform-d) 8 7.59 (t, J = 9.1 Hz, 1H), 7.44 - 6.97 (m, 12H), 6.26 (d, J = 6.8 Hz, 1H), 4.75 - 4.31 (m, 4H), 3.66 - 3.21 (m, 4H), 2.51 - 2.35 (m, 3H), 1.47 (s, 9H). 13C NMR (100 MHz, Chloroform-d) 8 160.71, 155.87, 154.25, 153.92, 152.08, 138.32, 137.89, 137.23, 136.97,2122365586. v1 -10 / 1 / 25128.89, 128.75, 128.66, 128.41, 128.23, 128.03, 127.61, 127.40, 125.31, 118.44, 118.25, 117.50, 114.33, 110.62, 110.43, 80.45, 51.71, 51.35, 51.19, 50.87, 50.46, 45.66, 44.93, 44.76, 44.51, 43.99, 43.65, 28.54, 18.82. HRMS (ESI, m / z): calcd for [C32H35N2O6]+ (M+H)+, 543.2490; found, 543.2492.

[0085] TFA salt of N -benzyl -2-(benzyl(((4-methyL2-oxo-2H-chromen-7- yl)oxy)carbonyl)amino) ethan-l-aminium, 2,2,2-trifluoroacetaldehyde (SIE2) was prepared following a similar procedure as that for SIE1. A 20 mL vial equipped with a stir bar was charged with 2 (60 mg, 0.111 mmol) and DCM (1 mL) at room temperature. 1 mL TFA was added into the mixture dropwise. After 1 h reaction at room temperature, the product was obtained as a light-yellow liquid by removing all solvent under reduced pressure (>99%, 62 mg). 1H NMR (400 MHz, Methanol-d4) 8 7.67 (d, J = 8.6 Hz, 1H), 7.45 - 7.20 (m, 10H), 7.18 - 7.01 (m, 2H), 6.19 (d, J = 1.5 Hz, 1H), 4.69 - 4.46 (m, 2H), 4.16 (s, 2H), 3.75 - 3.57 (m, 2H), 3.34 - 3.06 (m, 4H), 2.36 (d, J = 1.3 Hz, 3H). 13C NMR (100 MHz, Methanol-d4) 8 162.65, 156.33, 155.20, 155.07, 154.99, 138.03, 137.89, 132.33, 131.05, 130.82, 130.37, 130.11, 129.11, 128.57, 127.13, 119.68, 119.43, 118.99, 114.79, 111.39, 111.22, 52.60, 52.44, 52.25, 46.70, 46.09, 45.04, 44.28, 18.71. HRMS (ESI, m / z): calcd for [C27H27O4N2]+ (M)+, 443.1965; found, 443.1963.

[0086] 4-methyl-2 -oxo-2H-chromen-7-yl benzyl(2-((tert- butoxycarbonyl)(methyl)amino)ethyl) carbamate (3) was prepared following a similar procedure as that for compound 1. A flame-dried round bottom flask equipped with a stir bar was charged with tert-butyl (2-(benzylamino)ethyl)(methyl)carbamate (0.1 g, 0.378 mmol) and 1 mL ethyl acetate. The solution was cooled to 0 °C in an ice bath. Coumarin chloroformate (0.135 g, 0.567 mmol) was dissolved into another 1 mL of ethyl acetate followed by the dropwise addition into round bottom flask. Potassium carbonate (0.1 g, 0.735 mmol) was dissolved into 2 mL of water and added into round bottom flask dropwise. After stirring the mixture for 40 min at room temperature, the mixture was extracted with DCM (50 mL). The organic fraction was washed with 10% NH4C1 (50 mL), water (50 mL), and brine (50 mL). The organic phase was dried over Na2SO4, fdtered, and concentrated under reduced pressure. The product was separated as a light-yellow liquid by column chromatography (5- 25 % ether / DCM) to provide the title compound as a light-yellow oil (92 mg, 52%). Rf = 0.37 (DCM : ether 5 : 1). 1H NMR (400 MHz, Chloroform-d) 8 7.59 (t, J = 10.8 Hz, 1H), 7.44 - 7.27 (m, 5H), 7.12 - 7.00 (m, 1H), 7.08 (d, J = 1.9 Hz, 1H), 6.30 - 6.20 (m, 1H), 4.81 - 4.47 (m, 2H), 3.60 - 3.33 (m, 4H), 2.89 (d, J = 7.6 Hz, 3H), 2.43 (dd, J = 5.4, 1.3 Hz, 3H), 1.54 - 1.38 (m, 9H). 13C NMR (100 MHz, Chloroform-d) 8 160.68, 155.56, 154.23, 153.91,2222365586. v1 -10 / 1 / 25152.08, 128.91, 128.30, 128.05, 127.28, 125.33, 118.42, 118.24, 117.51, 114.31, 110.39, 79.67, 53.53, 51.15, 50.97, 47.11, 46.60, 45.76, 44.66, 44.25, 35.20, 34.85, 34.66, 28.53, 18.79. HRMS (ESI, m / z): calcd for [C26H31N2O6]+ (M+H)+, 467.2177; found, 467.2177.

[0087] TFA salt of 4-methyl-2-oxo-2H-chromen-7-yl benzyl(2- (methylamino)ethyl)carbamate, 2,2,2-trifluoroacetaldehyde (SIE3) was prepared following a similar procedure as that for SIE1. A 20 mL vial equipped with a stir bar was charged with 4 (92 mg, 0.195 mmol), and DCM (1 mL) at room temperature. 1 mL TFA was added into the mixture dropwise. After 1 h reaction at room temperature, the product was obtained as a light-yellow liquid by removing all solvent under reduced pressure (>99%, 96 mg). 1H NMR (400 MHz, Methanol-d4) 8 8.68 - 8.52 (m, 1H), 8.35 - 7.93 (m, 7H), 7.12 (d, J = 1.4 Hz, 1H), 5.68 - 5.37 (m, 2H), 4.68 - 4.44 (m, 2H), 4.21 - 4.04 (m, 3H), 3.67 - 3.49 (m, 3H), 3.39 - 3.18 (m, 3H). 13C NMR (100 MHz, Methanol-d4) 8 162.63, 161.35, 160.97, 156.35, 155.53, 155.15, 155.06, 154.95, 138.00, 137.75, 130.07, 129.09, 128.54, 127.06, 119.70, 119.44, 118.93, 118.65, 115.78, 114.75, 112.92, 111.43, 111.22, 52.24, 51.84, 45.16, 44.25, 34.02, 18.67. HRMS (ESI, m / z): calcd for [C21H23O4N2]+ (M)+, 367.1652; found, 367.1652.

[0088] Tert-butyl (4-nitrophenyl) ethane- 1 ,2-diylbis(benzylcarbamate) (4).

[0089] A round bottom flask equipped with a stir bar was charged with tert-butyl benzyl(2-(benzylamino)ethyl)carbamate (0.1 g, 0.294 mmol) (1 g, 2.938 mmol), 4- nitrophenyl chloroformate (0.533 g, 2.643 mmol), and anhydrous THF (20 mL). The mixture was cooled to 0 °C in an ice bath before adding triethylamine (TEA, 0.297 g, 2.97 mmol) dropwise. After overnight reaction at room temperature, the mixture was extracted with EtOAc (150 mL). The organic fraction was washed with 10% NH4C1 (150 mL), water (150 mL), and brine (150 mL). The organic phase was dried over Na2SO4, fdtered, and concentrated under reduced pressure. The product was separated by column chromatography (5-15 % EtOAc / hexanes) to provide the title compound as a light-yellow oil (1.06 g, 72%). Rf = 0.50 (hexanes : EtOAc 5 : 1). 1H NMR (400 MHz, Chloroform-d) 8 8.25 (t, J = 9.4 Hz, 2H), 7.46 - 7.06 (m, 12H), 4.79 - 4.30 (m, 4H), 3.61 - 3.33 (m, 4H), 1.57 - 1.36 (m, 9H). 13C NMR (100 MHz, Chloroform-d) 8 128.98, 128.82, 128.73, 128.44, 128.28, 128.07, 127.69, 127.50, 127.36, 125.24, 122.70, 122.34, 53.56, 51.45, 28.58. HRMS (ESI, m / z): calcd for [C28H32N3O6]+ (M+H)+, 506.2286; found, 506.2285.

[0090] Tert-butyl prop-2-yn-l-yl ethane- 1 ,2-diylbis(benzylcarbamate) (5)

[0091] A 20 mL vial equipped with a stir bar was charged with NaH (60 % dispersion in mineral oil, 18.8 mg, 0.471 mmol) and anhydrous DMF (2 mL) at room temperature. The2322365586. v1 -10 / 1 / 25propargyl alcohol (30.4 mg, 0.543 mmol) was added to the mixture dropwise. After stirring at room temperature for 30 min, compound 4 (183mg, 0.362 mmol) dissolved in anhydrous DMF (2 mL) was dropwise added into the mixture. After reaction at 110 °C for 2 h, 1 mL water was added in to quench the reaction. The mixture was extracted with EtOAc (100 mL). The organic fraction was washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to yield a crude mixture. The crude product was purified by column chromatography (3-15% EtOAC / hexanes) to provide the title compound as a colorless liquid (0.1 g, 65%). Rf = 0.40 (hexanes : EtOAc 10 : 1). 1H NMR (400 MHz, Chloroform-d) 8 7.42 - 7.02 (m, 10H), 4.76 - 4.69 (m, 2H), 4.55 - 4.30 (m, 4H), 3.50 - 3.14 (m, 4H), 2.49 - 2.42 (m, 1H), 1.52 - 1.42 (m, 9H). 13C NMR (100 MHz, Chloroform-d) 8 155.78, 138.48, 138.24, 137.53, 137.35, 128.75, 128.66, 128.14, 127.69, 127.51, 125.22, 80.19, 53.25, 53.15, 51.41, 51.11, 50.54, 45.22, 44.65, 44.41, 44.03, 28.55. HRMS (ESI, m / z): calcd for [C25H31N2O4]+ (M+H)+, 423.2278; found, 423.2276.

[0092] TFA salt of N-benzyl-2-(benzyl((prop-2-yn- 1 -yloxy)carbonyl)amino)ethan- 1 - aminium, 2,2,2-trifluoroacetaldehyde (SIE4) was prepared following a similar procedure as that for SIE1. A 20 mL vial equipped with a stir bar was charged with 5 (100 mg, 0.245 mmol) and DCM (2 mL) at room temperature. 2 mL TFA was added into the mixture dropwise. After 1 h reaction at room temperature, the product was obtained as a light-yellow liquid by removing all solvent under reduced pressure. (>99%, 104 mg). 1H NMR (400 MHz, Chloroform-d) 8 7.47 - 7.04 (m, 10H), 4.84 - 4.65 (m, 2H), 4.53 - 4.35 (m, 2H), 3.60 - 3.42 (m, 2H), 3.11 - 2.78 (m, 2H), 2.56 - 2.36 (m, 1H). 13C NMR (100 MHz, Chloroform-d) 8 161.41, 161.02, 157.71, 135.79, 130.07, 129.92, 129.51, 129.32, 129.15, 128.55, 128.38, 127.86, 125.26, 117.07, 114.22, 54.27, 51.69, 46.53, 43.98. HRMS (ESI, m / z): calcd for [C20H23O2N2]+ (M)+, 323.1754; found, 323.1751.

[0093] tert-butyl (4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl) ethane- 1 ,2-diy Ibis-10094] (benzylcarbamate) (6) was prepared following a similar procedure as that for compound 4. A 20 mL vial equipped with a stir bar was charged with NaH (60 % dispersion in mineral oil, 48 mg, 1.19 mmol) and anhydrous DMF (2 mL) at room temperature. The 4- (((tert-butyldimethylsilyl)oxy)methyl)phenol (283 mg, 1.19 mmol) dissolved in anhydrous DMF was added to the mixture dropwise. After stirring at room temperature for 30 min, compound 5 (0.5g, 0.989 mmol) dissolved in anhydrous DMF (4 mL) was dropwise added into the mixture. After reacting at 110 °C for 2 h, 1 mL water was added in to quench the reaction. The mixture was extracted with EtOAc (150 mL). The organic fraction washed with brine (150 mL). The organic fraction was dried over Na2SO4, filtered, and concentrated2422365586. v1 -10 / 1 / 25under reduced pressure to yield a crude mixture. The crude product was purified by column chromatography (5-25% EtOAC / hexanes) to provide the title compound as a colorless liquid (0.38 g, 64%). Rf = 0.15 (hexanes : EtOAc 10 : 1). 1H NMR (400 MHz, Chloroform-d) 8 7.40 - 7.13 (m, 12H), 7.10 - 6.99 (m, 2H), 4.81 - 4.37 (m, 6H), 3.56 - 3.27 (m, 4H), 1.53 - 1.42 (m, 9H), 0.99 - 0.88 (m, 9H), 0.14 - 0.05 (m, 6H). 13C NMR (100 MHz, Chloroform-d) 8 155.91, 155.34, 150.25, 138.70, 128.82, 128.72, 128.47, 128.29, 128.13, 127.52, 127.04, 121.65, 121.50, 80.15, 64.62, 44.66, 28.57, 26.07, 18.53, -5.11. HRMS (ESI, m / z): calcd for [C35H49N2O5Si]+ (M+H)+, 605.3405; found, 605.3408.

[0095] tert-butyl (4-(hydroxymethyl)phenyl) ethane- l,2-diylbis(benzylcarbamate) (7)

[0096] A flame-dried round bottom flask equipped with a stir bar was charged with 6 (0.38 g, 0.628 mmol) and 4 mL anhydrous THF. The mixture was cooled to 0 °C in an ice bath, followed by the dropwise addition of TBAF (1 M in THF, 0.754 mL, 0.754 mmol). The stirred reaction was allowed to warm up to room temperature. After one hour, the reaction was quenched with NH4C1 (10 mL) and extracted with EtOAc (100 mL). The organic fraction was washed with water (50 mL) and brine (50 mL). The organic fraction was dried over Na2SO4, fdtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (15-35% EtOAc / hexanes) to yield the title compound as a white solid (204 mg, 66%). Rf = 0.23 (hexanes : EtOAc 5 : 1). 1H NMR (400 MHz, Chloroform-d) 8 7.41 - 7.14 (m, 12H), 7.15 - 7.04 (m, 2H), 4.76 - 4.34 (m, 6H), 3.56 - 3.25 (m, 4H), 1.52 - 1.38 (m, 9H). 13C NMR (100 MHz, Chloroform-d) 8 155.93, 155.24, 150.88, 138.20, 128.85, 128.74, 128.46, 128.28, 128.11, 127.84, 127.51, 122.06, 121.91, 64.95, 53.56, 28.58. HRMS (ESI, m / z): calcd for [C29H35N2O5]+ (M+H)+, 491.2541; found, 491.2540.

[0097] 4-((benzyl(2-(benzyl(tert-butoxycarbonyl)amino)ethyl)carbamoyl)oxy)benzyl3,7-

[0098] bis(dimethylamino)- lOH-pheno thiazine- 10-carboxylate (8).

[0099] A flame-dried round bottom flask equipped with a stir bar was charged with 7 (0.111 g, 0.226 mmol), MB-chloro formate (0.095g, 0.294 mmol) (Ref : J. Am. Chem. Soc. 2022, 144, 37, 16799-16807) and 3 mL anhydrous THF. DMAP (35.9 mg, 0.294 mmol) dissolved in anhydrous THF (1 mL) was dropwise added into the mixture. After reaction at 55 °C for 1 day, the mixture was extracted with EtOAc (50 mL). The organic fraction was washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to yield a crude mixture. The crude product was purified by column chromatography (50-100% Ether / hexanes) to provide the title compound as a colorless liquid (0.105 g, 58%).2522365586. v1 -10 / 1 / 25Rf = 0.15 (hexanes : ether 1 : 1). 1H NMR (400 MHz, Chloroform-d) 8 7.56 - 7.15 (m, 18H), 7.15 - 7.04 (m, 2H), 5.29 - 5.14 (m, 2H), 4.75 - 4.35 (m, 4H), 3.58 - 3.25 (m, 4H), 3.00 (s, 12H), 1.54 - 1.39 (m, 9H). 13C NMR (100 MHz, Chloroform-d) 8 155.92, 155.14, 154.52, 151.04, 148.95, 133.77, 132.94, 129.10, 129.07, 128.85, 128.74, 128.37, 128.13, 127.53, 127.11, 121.90, 121.77, 111.23, 110.49, 67.16, 50.50, 44.73, 44.47, 40.84, 28.58. HRMS (ESI, m / z): calcd for [C46H52N5O6S]+ (M+H)+, 802.3633; found, 802.3617.

[0100] TFA salt of N -benzyl -2-(benzyl((4-(((3,7-bis(dimethylamino)-10H- phenothiazine- 10-carbonyl)oxy)methyl)phenoxy)carbonyl)amino)ethan- 1 -aminium (SIE5) was prepared following a similar procedure as that for SIE1. A 20 mL vial equipped with a stir bar was charged with 8 (30 mg, 0.037 mmol) and DCM (1 mL) at room temperature. 1 mL TFA was added into the mixture dropwise. After 1 h reaction at room temperature, the product was obtained as a light-blue liquid by removing all solvent under reduced pressure. (>99%, 30 mg). 1H NMR (400 MHz, Chloroform-d) 8 7.53 - 7.44 (m, 2H), 7.41 - 7.28 (m, 10H), 7.25 - 7.20 (m, 2H), 7.17 - 7.02 (m, 6H), 5.22 (s, 2H), 4.61 (s, 2H), 4.15 (s, 2H), 3.65 - 3.57 (m, 2H), 3.23 - 3.13 (m, 2H), 3.00 (s, 12H). 13C NMR (100 MHz, Chloroform-d) 8 161.46, 161.07, 160.69, 151.12, 142.35, 137.46, 133.95, 130.09, 130.03, 129.65, 129.46, 129.26, 128.74, 128.44, 127.66, 122.19, 118.61, 117.20, 114.33, 87.04, 53.57, 51.79, 46.41, 45.81, 27.72. HRMS (ESI, m / z): calcd for [C41H44O4N5S]+ (M)+, 702.3109; found, 702.31002622365586. v1 -10 / 1 / 25

Claims

CLAIMSWhat is claimed is:

1. A system for delivering a therapeutic agent, comprising: a complex of an ethylenediamine-based self-immolative entity and a cucurbit[7]uril host molecule; and a cargo molecule coupled to the complex of the ethylenediamine-based self- immolative entity and the cucurbit[7]uril host molecule; wherein a competitive guest molecule is capable of triggering a rapid cyclizationelimination cascade that will release the cargo molecule from the complex.

2. The system of claim 1, wherein the ethylenediamine-based self-immolative entity includes at least one benzylamine moiety for binding to the cucurbit[7]uril host molecule.

3. The system of claim 1, wherein the ethylenediamine-based self-immolative entity includes a pair of benzylamine moieties for binding to the cucurbit[7]uril host molecule.

4. The system of claim 3, wherein the ethylenediamine-based self-immolative entity includes a 4-hydroxybenzyl alcohol linker.

5. The system of claim 1, wherein the complex has a formula selected from the group consisting of:

6. The system of claim 1, wherein the cargo molecule is a therapeutic drug.The system of claim 1, wherein exposure of the complex to 1-adamantylamine releases the cargo molecule.2722365586. v1 -10 / 1 / 258. A method for on-demand release of a therapeutic payload, the method comprising: administering a complex of an ethylenediamine -based self-immolative entity and a cucurbit[7]uril host molecule that has a cargo molecule coupled to the complex of the ethylenediamine-based self-immolative entity and the cucurbit[7]uril host molecule; and administering 1-adamantylamine in an amount effective to competitively displace the cucurbit[7]uril host molecule and trigger a cyclization-elimination cascade within the ethylenediamine-based self-immolative entity that releases the cargo molecule.

9. The method of claim 8, wherein the ethylenediamine-based self-immolative entity includes at least one benzylamine moiety for binding to the cucurbit[7]uril host molecule.

10. The method of claim 9, wherein the ethylenediamine-based self-immolative entity includes a pair of benzylamine moieties for binding to the cucurbit[7]uril host molecule.

11. The method of claim 10, wherein the ethylenediamine-based self-immolative entity includes a 4-hydroxybenzyl alcohol linker.

12. The method of claim 8, wherein the complex has a formula selected from the group consisting of:

13. The method of claim 8, wherein the cargo molecule is a therapeutic drug.

14. The method of claim 8, wherein exposure of the complex to 1- adamantylamine releases the cargo molecule.2822365586. v1 -10 / 1 / 25

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