Activity-Based Optical Sensing Enabled by Self-Immolative

Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Activity-Based Optical Sensing Enabled by Self-Immolative Scaffolds: Monitoring of Release Events by Fluorescence or Chemiluminescence Output Published as part of the Accounts of Chemical Research special issue “Activity-Based Sensing”. Samer Gnaim and Doron Shabat*

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School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel Aviv 69978 Israel

CONSPECTUS: Functional molecular scaffolds comprised of self-immolative adaptors are being used in widespread applications in the fields of enzyme activity analyses, signal amplification, and bioimaging. Optically detected chemical probes are very promising compounds for sensing and diagnosis, since they present several attractive features such as high specificity, low detection limits, fast response times, and technical simplicity. During the last two decades, we have developed several distinct molecular scaffolds that harness the self-immolative disassembly feature of these adaptors to amplify chromogenic output for diagnosis and drug delivery applications. In order to study the molecular behavior of the various amplification systems, an optical output, used to monitor the progress of the disassembly pattern, was required. Therefore, over the course of our research, diverse molecular scaffolds that produce an optical signal in response to a disassembly step, were evaluated. These optically active scaffolds have been incorporated into self-immolative dendrimers and self-immolative polymers to implement unique disassembly properties that result with linear and exponential signal amplification capabilities. In addition, some scaffolds, aimed for linker technology, were used in delivery systems to monitor release of drug molecules. The optical signal used to monitor the release event could be produced by analysis of reporter molecules with chromogenic or fluorogenic properties. Recently, we have also developed molecular scaffolds modified to produce a chemiluminescent signal to monitor the self-immolative disassembly step. The main advantage of these scaffolds over others is the use of chemiluminescence as an output signal. It is well-known that chemiluminescence is considered as one the most sensitive diagnostic methods due to its high signal-to-noise ratio. The unique structures of the self-immolative chemiluminescence scaffolds have been used in the design of three different distinctive concepts: self-immolative chemiluminescence polymers, auto-inductive amplification systems with chemiluminescence signal and monitoring of drug release by a chemiluminescence output. Furthermore, we reported the design and synthesis of the first theranostic prodrug for the monitoring of drug release achieved by a chemiluminescence mode of action. Quinone-methide elimination has proven to serve as a valuable functional tool for composing molecular scaffolds with self-immolative capabilities. Such scaffolds function as molecular adaptors that can almost simultaneously release a target molecule with an accompanied emission of a light signal that is used to monitor the release event. We anticipate that these self-immolative scaffolds will continue to find utility as functional linkers in various chemical and biological research areas such as drug delivery, theranostic applications, and as molecular sensors with signal amplification.

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ctivity-based sensing systems1,2 are now commonly used both in academic and in industrial research for monitoring of diverse biological, clinical, and environmental processes.3−5 In order to develop such tools, there is an obvious need to design molecular probes with high selectivity and sensitivity.6 Optically detected chemical probes are a very promising compounds for sensing and diagnosis, since they present several attractive features such as high specificity, low © XXXX American Chemical Society

detection limits, fast response times, and technical simplicity.7−9 Functional molecular structures comprised of self-immolative adaptors have found widespread applications in the fields of enzyme diagnosis, signal amplification, and bioimaging.10−14 Received: June 26, 2019

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DOI: 10.1021/acs.accounts.9b00338 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research

Figure 1. Disassembly mechanisms of (A) 1,6-quinone-methide elimination and (B) 1,4-quinone-methide elimination. PG, protecting group.

Figure 2. General structure and disassembly pathway of self-immolative polymers. PG, protecting group.

One of the widely used self-immolative scaffolds is the 4hydroxy benzyl alcohol.15 As illustrated in Figure 1, removal of a protecting group from probe I results in formation of phenolate derivative II, which undergoes rapid 1,6-elimination to release an active reporter molecule. The elimination reaction generates a reactive quinone-methide species that is usually trapped by an available nucleophile (e.g., H2O). The elimination reaction occurs with both the widely used 4hydroxy benzyl alcohol scaffold (Figure 1A) and a 2-hydroxy benzyl alcohol scaffold (Figure 1B).11 During the past two decades, we have developed several molecular scaffolds with self-immolative disassembly features that have been incorporated into probes with amplified chromogenic output for diagnosis and that have been used in drug-delivery applications.16−19 We first developed unique molecular platforms that are known today as self-immolative dendrimers.20−23 These dendrimers disassemble upon a single triggering event in a domino-like manner from the focal point to the periphery with the consequent release of multiple end groups. When incorporated into self-immolative dendritic prodrugs and diagnostic probes, a single activation event results in amplified disassembly.24,25 Self-immolative polymers that act as molecular amplifiers have also been synthesized that disassemble from head to tail upon a single triggering event.26,27 Another amplification technique developed by our group uses a simple self-immolative dendron that upon triggered disassembly results in exponential evolution of diagnostic signal.28−32 The analyte of interest reacts with the corresponding trigger of the dendron probe to release a reporter unit and a reagent molecule or molecules. Upon their release, the reagents acquire the chemical reactivity of the analyte and thus can activate two additional probe molecules. The process proceeds exponentially to achieve complete disassembly of the dendrons yielding an amplified diagnostic signal. This signal amplification approach is termed as dendritic chain reaction.

In order to monitor the progress of the disassembly of the various amplification systems described here, we sought to develop diverse molecular scaffolds that produce an optical signal in response to a disassembly step. The optical signal used to monitor the release event could be produced by reporter molecules or molecular variations based on either chromogenic or fluorogenic response. Recently, we have also successfully developed molecular scaffolds that can integrate a chemiluminescence reporter to monitor the disassembly of self-immolative probe systems. In this account, we summarize the rationale behind a number of the self-immolative scaffolds that are used to generate optical signal in order to monitor an amplification cycle or a release event of a target molecule.

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MOLECULAR SCAFFOLDS FOR COMPOSING SELF-IMMOLATIVE POLYMERS Self-immolative polymers are unique materials that disassemble in a domino-like mechanism from head to tail upon a triggering event induced by an external stimulus.26,33 This class of polymers directly addressed challenges associated with selfimmolative dendritic analogues; specifically their time-consuming stepwise synthesis and steric hindrance limitations. The first design of a self-immolative polymer was based on a polycarbamate backbone (Figure 2). Removal of the end-cap from the self-immolative polycarbamate unmasks an aniline and initiates a series of consecutive 1,6-quinone-methide elimination and decarboxylation reactions, leading to total disassembly of polymer 1 into carbon dioxide and azaquinonemethide units. When fragmentation of the polymer takes place in an aqueous medium, the highly reactive azaquinone-methide reacts rapidly with a water molecule to generate the 4aminobenzyl alcohol. Self-immolative polymers have been used as sensory materials, drug-release platforms, and green plastics.34,35 These polymers act as unique molecular platforms for signal amplification. By incorporation of suitable monomeric units B


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Figure 3. (A) Structure and disassembly of self-immolative polymer 2 under aqueous conditions. (B) Structure and disassembly of self-immolative polymer 3, prepared with a 4-nitroaniline tail unit.

Figure 4. Protein labeling using self-immolative polymers 4 and 5.

β-elimination reaction catalyzed by bovine serum albumin. Incubation of the polymer with bovine serum albumin results in the generation of fluorescent signal due to disassembly of the polymeric backbone into its fluorescent monomers. In order to verify the head-to-tail depolymerization, we synthesized polymer 3, which has a 4-nitroaniline reporter as the terminal tail group (Figure 3B). Monitoring of the release of 4-nitroaniline confirmed that the polymer indeed disassembled from head to tail. The disassembly of self-immolative polymers is accompanied by the release of azaquinone-methide intermediates. In aqueous medium, these highly reactive species react with water molecules to generate the fluorogenic aniline building blocks. When the polymer disassembly is triggered by reaction of a protein or an enzyme, a nucleophilic protein residue can react with the electrophilic azaquinone-methide units, to generate a covalent linkage between the protein and the fluorescent building blocks. To demonstrate such a polymer-

with the desired triggering moiety, these systems can be used to detect a variety of chemical and biological activities with high sensitivity and low background.26 In order to create a detectable signal that would allow monitoring of polymer degradation, we designed and synthesized polymer 2 (Figure 3A). The monomeric unit of polymer 2 is composed of 4aminobenzyl alcohol equipped with an ortho-acrylic acid substituent. When free in solution, this molecule produces fluorescent light with maximum emission at a wavelength of 510 nm due to generation of donor−acceptor. The light emission of the fluorescent aniline is quenched in the polymer because the amine group is protected in the form of a carbamate bond. Removal of the head-trigger produces signal amplification resulting from the release of numerous building blocks that can then fluoresce (Figure 3A). Under physiological conditions, the carboxyl group of the monomeric unit is ionized, and the polymer is soluble. Polymer 2 also contains a 4-hydroxy-2-butanone end-cap that can be activated through a C


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Figure 5. General structure and activation pathway of Schaap’s dioxetane-based probes. PG, protecting group.

Figure 6. Structure and degradation of probe VII, which combines the turn-ON mechanism of Schaap’s adamantylidene-dioxetane with 1,6quinone-methide elimination. ET, electron transfer. PG, protecting group.

Figure 7. (A) Molecular structures and activation mechanisms of chemiluminescence self-immolative polymers. (B) Molecular structures of selfimmolative monomer, dimer, and trimer probes sensitive to fluoride. (C) Chemiluminescence kinetic profiles of polymer 6 and its oligomers after activation with tetra-n-butylammonium fluoride (TBAF; 1 equiv).

D


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Figure 8. (A) Structure of chemiluminescence probe 9 and mechanism of disassembly triggered by fluoride. (B) Chemiluminescence kinetic profiles and total light emission profiles of probe 9 activated by various equivalents of TBAF.

dioxetane into the quinone-methide elimination. Such integration could be achieved in a molecular structure like probe VII (Figure 6).46 Removal of the substrate from probe VII triggers the quinone-methide elimination to release quinone-methide VIII. The quinone-methide then reacts with an accessible nucleophile (e.g., H2O) to generate phenolate IX. The latter can then decompose through chemiexcitation process to produce benzoate X, adamantanone, and a blue photon (detected at 499 nm). The disassembly mechanism of the self-immolative compound VII was confirmed by 1H NMR and by monitoring the kinetics of chemiluminescence.

based protein labeling approach, we prepared polymers 4 and 5 (Figure 4).36 Polymer 4 was capped with a 4-hydroxy-2butanone head trigger, which is a substrate of catalytic antibody 38C2. Removal of the trigger by the catalytic antibody generates the azaquinone-methide species, which reacts with the antibody structure to produce the labeled protein. We also demonstrated labeling of the enzyme penicillin-G-amidase (PGA) by polymer 5, which contains a phenyl acetamide that is cleaved by PGA.

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SELF-IMMOLATIVE CHEMILUMINESCENT SCAFFOLD The phenomenon of light production as a result of a simple chemical reaction is known as chemiluminescence.37−40 Assays based on chemiluminescence are utilized in various chemical and biomedical applications due to high sensitivity and a high signal-to-noise ratio.41,42 Usually, two chemicals react to form an excited (high-energy) intermediate that breaks down to release its energy as light. In 1987, Schaap and co-workers described the chemiexcitation pathway of 1,2-dioxetane with a protected phenolic substituent at the meta position (Figure 5).43−45 Schaap’s dioxetane IV can be equipped with an enzyme- or an analyte-responsive protecting group. Removal of the triggering substrate releases the phenolate-dioxetane intermediate V, which decomposes through a chemically initiated electron exchange luminescence process to produce the electronically excited benzoate ester VI. The decay of VI to its ground state results in emission of a photon in the blue range of the visible spectrum. In a recent work, we have reported the assimilation of the distinct turn-ON mechanism of Schaap’s adamantylidene-

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SELF-IMMOLATIVE CHEMILUMINESCENCE POLYMERS Chemiluminescence assays are exceptionally sensitive methods for determination of enzyme activity and analyte concentrations due to the high signal-to-noise ratio.47 In a design similar to those of the self-immolative polymers presented in Figure 3, we used molecular unit VII as a building block for construction of self-immolative polymers with a mode of action that amplifies the chemiluminescence signal (Figure 7A).46 These self-immolative polymers have polycarbonate backbones with molecular unit VII as the monomer building block and appropriate responsive substrates as head groups. Removal of the substrate by an analyte of interest initiates the selfimmolative disassembly through multiple quinone-methide eliminations and decarboxylations (Figure 7A). The released quinone-methide units react with available nucleophiles, such as water molecules, to regenerate the phenolate. The latter E


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Understanding the relationship between structure and function can assist in design of new amplification systems with improved sensitivity. Using the same phenoxy-dioxetane described for probe 9 (Figure 8A), we have designed a new signal amplification system with a linear chain reaction (LCR) amplification mechanism and chemiluminescence output.49 Probe 10, designed to detect the analyte hydrogen peroxide, consists of phenoxy 1,2-dioxetane as a chemiluminescent reporter and phenylboronic ester as a triggering substrate for hydrogen peroxide (Figure 9).49 The LCR amplification cycle

then decomposes through a chemiexcitation process to emit blue light. The monomeric units were polymerized in the presence of the applicable capping head substrate to produce selfimmolative polycarbonates 6, 7, and 8 (Figure 7A). Polymer 6 was capped with a silyl-phenolic ether substrate suitable for activation by fluoride. In similar manner, polymer 7 was capped with an allyl substrate suitable for activation by palladium complex. Polymer 8 was designed for activation by hydrogen peroxide using phenyl boronic ester as the triggering substrate. The chemiluminescence kinetic profiles of the polymer 6 and related monomers, dimers, and trimers upon activation by TBAF (1 equiv) are presented in Figure 7. The signal produced upon disassembly of polymer 6 was about 20 times more intense than that obtained for the monomer. Signal intensities for trimer and dimer probes were 3-fold and 2-fold higher, respectively, than that of the monomer. We also observed significant chemiluminescence signal amplification upon disassembly of polymers 7 and 8 compared to their monomeric probes after activation by their corresponding analytes. These results indicate that chemiluminescent signal can be produced through activation of a polycarbonate selfimmolative polymer.

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MOLECULAR SCAFFOLDS FOR CHEMILUMINESCENCE AUTOINDUCTIVE AMPLIFICATION Use of self-immolative polymers is limited by practical synthetic concerns. To overcome these limitations, we have developed an amplification approach based on an autoinductive mode-of-action. Molecular probes designed for autoinductive amplification spontaneously release their end group molecules through domino-like reactions following a single activation event. The free end groups can trigger activation of another probe molecule. Therefore, a single activation event leads to a chain reaction, and all probe molecules are disassembled through an amplification progress. As an example of this type of probe, we recently reported probe 9 (Figure 8A), which functions as a molecular amplifier by producing a chemiluminescence signal. The amplification cycle is based on autoinductive regeneration of the analyte.48 The amplification cycle is demonstrated with fluoride as the analyte of interest. It is initiated by removal of the tertbutyldimethylsilyl triggering group by fluoride, to form a phenolate intermediate. This intermediate undergoes 1,6elimination to produce additional fluoride ion and quinonemethide 9a. The additional fluoride can now react with the triggering group of an additional probe, while, the quinonemethide reacts with an available nucleophile to regenerate a phenolate species. This phenolate can then undergo chemiexcitation to emit a blue photon and, thereby, to produce benzoate 9b (Figure 8A). A progressive increase in signal is continuously generated by this reaction sequence. Spectroscopic evaluation of probe 9 with various amounts of fluoride revealed that, in all concentrations of fluoride, signal increased to a maximum and then decayed to zero (Figure 8B). The signal obtained with probe 9, which results from the autoinductive amplification, was 219-fold stronger than that obtained using the classic Schaap dioxetane. The assimilation of chemiexcitation and analyte release processes in a single molecular probe has resulted in an effective chemiluminescence amplification cycle.

Figure 9. Autoinductive disassembly and chemiluminescence emission of probe 10 triggered by hydrogen peroxide.

of the probe is initiated by removal of the boronic ester by hydrogen peroxide under alkaline conditions. This reaction triggers the chemiexcitation of dioxetane, leading to the emission of blue light and the generation of phenolate intermediate 10c. The latter undergoes intramolecular transesterification to release a methanol molecule. The released methanol is then oxidized by alcohol oxidase (AOX) and molecular oxygen to generate formaldehyde and hydrogen peroxide; the latter can activate an additional probe molecule. Kinetic profiles of emission of probe 10 incubated with alcohol oxidase and different concentrations of hydrogen peroxide revealed signal increase to a maximum followed by decay to zero. The light emission signal generated in the absence of hydrogen peroxide was also amplified as a result of spontaneous side reactions. However, the observed background signal is substantially lower than that of a sample treated with 2.5 μM hydrogen peroxide. At the limit of our assay sensitivity, in the presence of 2.5 μM of hydrogen peroxide, the signal from probe 10 was about 19-fold brighter than that obtained using a classic probe that does not have an amplification mechanism of action.

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FLUORESCENT MOLECULAR SCAFFOLDS FOR MONITORING DRUG RELEASE The efficiency of chemotherapeutic drugs depends on the concentration of the agent in the cancerous tissue relative to that in healthy tissues.50,51 The relative small amount of chemotherapeutic drugs existing in tissues during usage are F


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Figure 10. (A) Disassembly of the of the theranostic prodrug 11. The triggering event initiates disassembly into active drug (red) and highly fluorescent 11c. PG, protecting group. (B) Structure of the prodrug 12. The dipeptide Phe-Lys is the triggering substrate for cathepsin B (pink). The disassembly yields active melphalan (green) and the fluorescent coumarin derivative (blue).

quantifying the emitted fluorescence, the amount of drug released can be directly calculated. Therefore, such theranostic prodrug design could enable prediction of the therapeutic effect. Using a different approach, we have designed a theranostic prodrug that can be monitored using Förster resonance energy transfer (FRET). In our design, prodrug 13 is composed of a dimeric self-immolative linker and a pair of fluorescein fluorophores, a phenyl acetamide triggering substrate, and the drug camptothecin drug (Figure 11). Since the two fluorophores are in close proximity to each other in prodrug 13, fluorescence is quenched through a FRET mechanism. Cleavage of the phenyl acetamide group by the enzyme PGA followed by 1,6-elimination of azaquinone-methide and subsequent decarboxylation yields the amino intermediate 13a. The latter rapidly undergoes two sequential 1,6azaquinone-methide eliminations that separate the two fluorescein molecules and release the free camptothecin drug. Upon linker fragmentation, the FRET quenching effect is terminated, and the measured fluorescent signal is used to monitor the drug release. Linear correlation between the fluorescence signal and drug release was observed when the prodrug was incubated with PGA in aqueous physiological solution. The theranostic prodrugs 12 and 13 produce fluorescence in the blue and green regions. For in vivo use, fluorescent reporters with red-shifted wavelengths are necessary in order to achieve better light penetration.54 Therefore, we incorporated the near-infrared dye QCy7 as a latent fluorophore linker for the prodrug (Figure 12).55 Prodrug 14 is composed of phenyl boronic ester as a triggering substrate attached through a QCy7 fluorophore to the drug molecule camptothecin. Removal of the triggering substrate yields phenolate intermediate 14a, which then undergoes 1,4-elimination to release the active drug through formation of quinone-methide

almost impossible to monitor in real time, but this is necessary in order to personalize treatment. Thus, there is an obvious demand for methods that can enable, in real time, imaging of drug biodistribution.52 Therefore, we sought to develop drug delivery systems that instantaneously report on the release of the active drug through a noninvasive imaging mode-of-action. Latent fluorophores are composed of fluorogenic dyes masked by a triggering group. Thus, such compounds can properly act as noninvasive imaging reporters. Real-time information about the release process can be straightforwardly gained, by coupling latent fluorophore activation to a drug-release event in an appropriate delivery system. Following the notion described above, we have developed a distinct prodrug system composed of a 7-hydroxycoumarin scaffold as a fluorescence reporter unit (Figure 10A).53 The phenolic hydroxyl group of 11 is masked by a protecting group that serves as the triggering substrate, and the hydroxymethyl substituent acts as a self-immolative linker for attachment of a drug molecule. The release of the drug is triggered by enzymatic cleavage of the head-substrate of molecule 11, resulting in formation of phenolate 11a. The drug is then released through spontaneous 1,8-elimination that generates the coumarin−quinone−methide intermediate 11b. Reaction of a nucleophilic water molecule with the electrophilic coumarin−quinone−methide results in formation of the fluorescent coumarin molecule 11c (Figure 10B). To prove this hypothesis, we prepared prodrug 12, with the Phe-Lys dipeptide, a known substrate of cathepsin B, and with the chemotherapeutic drug, melphalan (Figure 10). The disassembly cascade is initiated by enzymatic hydrolysis of the amide bond at the C-terminal side of the lysine. This event is leading to the release of free melphalan molecule and the generation of fluorescent coumarin derivative. A direct correlation was observed between the emitted fluorescence and the growth inhibition of the MOLT-3 tumor cells.53 By G


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Figure 11. Activation of a self-immolative theranostic prodrug by PGA enzyme. The prodrug disassembles to release two fluorescein moieties as reporters (green) and the active drug camptothecin (blue). The triggering substrate is shown in pink.

luminophores in water.56−58 Remarkably, this was achieved by simply improving the emissive nature of the excited species formed during the chemiexcitation of Schaap’s adamantyldioxetanes. A phenoxy-dioxetane probe bearing a conjugated acrylate substituent at the ortho position releases a benzoate derivative during chemiexcitation, which is highly emissive under aqueous conditions.59,60 The self-immolative chemiluminescence scaffold shown in Figure 6, with an addition of an acrylate substituent, was used to prepare a theranostic prodrug based on the clinically used cytotoxic agent monomethyl auristatin E (MMAE).61 Prodrug 15 is composed of a phenoxy-dioxetane molecular unit, which serves as a chemiluminescent reporter, attached to MMAE via a carbamate linkage and masked with the triggering substrate designed for activation by the β-galactosidase (Figure 13). The activation of prodrug 15 is initiated by enzymatic removal of the β-galactose group by β-galactosidase, following by 1,6elimination to release the phenoxide intermediate 15a. The latter undergoes an additional 1,6-elimination to release the MMAE drug and quinone-methide 15b, which then reacts with a water molecule to generate intermediate 15c. Electron transfer from the phenoxy-anion to the dioxetane results in

derivative 14b. Addition of a water molecule to quinonemethide generates dye 14c and results in a turn-ON response of the QCy7 fluorophore (Figure 12). There was a linear correlation between the light emitted and the drug released from prodrug 14 upon reaction with hydrogen peroxide in aqueous solution. The camptothecin prodrug 14 was also evaluated for its ability to undergo activation by endogenous hydrogen peroxide produced in tumor cells in vitro and in vivo. The prodrug activation was imaged effectively in real time in mice bearing tumors caused by injection of the glioblastomaderived U-87 MG cells.55

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CHEMILUMINESCENT MOLECULAR SCAFFOLDS FOR MONITORING OF DRUG RELEASE Monitoring of drug release through analysis of a chemiluminescence modality offers advantages over fluorescence techniques: Chemiluminescent probes have higher signal-tonoise ratios than fluorescent probes, and an external light source is unnecessary with chemiluminescent agents.47 Two years ago, we developed a new methodology to significantly improve light emission intensity of phenoxy-dioxetane H


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Figure 12. Disassembly pathway of turn-ON theranostic prodrug 14, which contains QCy7 as a latent central linker (gray), phenylboronic ester as a triggering substrate for hydrogen peroxide (pink), and camptothecin as the chemotherapeutic drug (blue).

fragmentation to release 2-adamantanone and benzoate 15d in its excited state. Radiative decay of the excited benzoate to its ground state is accompanied by the emission of a green photon. A linear correlation was observed between the chemiluminescence signal and drug release. In addition, when the prodrug was incubated with HEK293 cells engineered to express β-galactosidase, a bright chemiluminescence signal and high cytotoxicity was observed. A weak signal and only a slight effect on viability were observed in the control HEK293 cells that do not express the activating enzyme. Furthermore, the prodrug was successfully used to image of CT-26 tumors in mice.61 Most of our molecular scaffolds used to monitor a release event by an optical signal are based on the 1,6- and 1,4quinone-methide elimination. In a previous publication, we have reported a comparison study of a system that can disassemble through para- and ortho-quinone-methide elimination.62 The disassembly kinetics was evaluated with molecules that undergo single 1,6- or 1,4-elimination and with molecules that undergo double-elimination. The 1,6elimination was shown to occur only slightly faster than the 1,4-elimination under physiological conditions. The examples described above that employed a chemiluminescence signal, produced through a dioxetane chemiexcitation, are based on the domino-like disassembly of a selfimmolative scaffold. The self-immolative quinone-methide elimination was embedded into the chemiexcitation mechanism of Schaap’s adamantylidene-dioxetane by design of an exceptional building block. Such distinct molecular structure elegantly enables the dual function of quinone-methide elimination and chemiexcitation of the dioxetane. This selfimmolative scaffold exhibited complexed kinetic behavior of light emission that may be elucidated in the following explanation: A triggering event generates a phenolate species that undergoes 1,6-elimination to form a dioxetane quinone-

methide intermediate, which cannot undergo chemiexcitation in its current state. In a following step a nucleophilic water molecule can react with the quinone-methide to generate a phenolate ion, which only then undergoes chemiexcitation through electron transfer from the phenoxy-ion to the peroxide-dioxetane bond. Therefore, the light emission kinetics is considerably affected by the different concentrations of water molecules presented in the solution.

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CONCLUDING REMARKS In this account, we described the development of functional self-immolative molecular scaffolds and their use for signal amplification and for real-time monitoring of drug release. The mounting number of publications on this topic clearly indicates the interest of the scientific community in such molecular structures. In recent years, scaffolds that disassemble through the quinone-methide elimination mechanism have been used for development of drug delivery systems, optical probes, and other macro-molecular systems with self-immolative disassembly properties. The self-immolative quinone-methide-based disassembly mechanism was assimilated in various scaffolds to generate molecular adaptors that could almost simultaneously release a target molecule with accompanied emission of a light signal. This light emission signal, produced by either a fluorescence or chemiluminescence mode of action, is linearly correlated with the amount of target molecule released. We believe that further studies in this direction will lead to selfimmolative scaffolds with unique optical outputs, that are more stable under organic and aqueous conditions. Such linkers could further enhance the detection limit of the probes by improving the signal-to-noise ratio. We anticipate that the activity-based self-immolative scaffolds will continue to play a major role as functional linkers for drug delivery, theranostic, material science, polymeric chemistry, and molecular sensors. I


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Figure 13. Activation of the self-immolative chemiluminescence prodrug 15, which is composed of a chemiluminescent reporter (blue), attached to the drug MMAE via a carbamate linkage (red), and masked with the triggering substrate (pink). β-gal, β-galactosidase.

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AUTHOR INFORMATION

Israeli Council for Higher Education postdoctoral scholarship (2019− 2020).

Corresponding Author

Doron Shabat studied chemistry at the Technion-Israel Institute of Technology between 1987 and 1990. After obtaining his B.Sc. degree, he continued toward his Ph.D. degree under the supervision of Prof. Ehud Keinan in the field of catalytic antibodies. Upon the completion of his Ph.D. thesis in 1997, he joined a group led by Profs. Richard A. Lerner and Carlos F. Barbas, III, at The Scripps Research Institute in La Jolla, California as a postdoctoral fellow. There he continued to work in the area of catalytic antibodies. In 2000, he returned to Israel to start his independent career in the School of Chemistry at Tel Aviv University as a senior lecturer. He was promoted to the rank of associate professor in 2005 and to full professor in 2008. Since 2016, he is holding the Emerico Letay Chair of Chemical Processes. His research is focused in bioorganic chemistry with particular interests in self-immolative molecular systems, long-wavelength fluorescent dyes and chemiluminescence probes for in vitro and in vivo imaging. He is the recipient of the Juludan Prize for 2005, administered by the Technion-Israel Institute of Technology; the Israel Chemical Society’s Prize for Outstanding Young Chemists (2005); the Frost Fellowship administrated by Scripps Research (2012 and 2014); the ICS-Adama Prize for Technological Innovation (2018) and the Kolthoff prize for 2019.

*Mailing address: Department of Organic Chemistry, School of Chemistry, Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: +972 (0) 3 640 8340. Fax: +972 (0) 3 640 5761. E-mail: [email protected]. ORCID

Doron Shabat: 0000-0003-2502-639X Notes

The authors declare no competing financial interest. Biographies Samer Gnaim was born in 1991 in Baqa El-Garbia, Israel. He received his Ph.D. in organic chemistry in 2019 from Tel Aviv University under the supervision of Prof. Doron Shabat. His research focused on the development of targeted drug delivery systems and the development of self-immolative chemiluminescence probes for diagnostic and theranostic purposes. Samer is currently a postdoctoral fellow at Scripps Research Institute, La Jolla, in the laboratory of Prof. Phil Baran. Samer is the recipient of several scholarships and awards, among them: the Jortner Prize as a distinguished Ph.D. student awarded by the Israel Chemical Society (2018), Zvi Yanai Ph.D. excellence scholarship awarded by the Ministry of Science and Technology (2015−2018), the Fulbright postdoctoral fellowship (2019), the Rothschild postdoctoral fellowship (2019−2020), and the

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REFERENCES

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Activity-Based Optical Sensing Enabled by Self-Immolative

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